Caroline Tokarski

Katell Bathany

Corinne Bure

Stéphane Chaignepain

Nicolas Desbenoit

Landry Blanc

Catherine Gilbert

Vaclav Krupicka

Michael Tuck

Anne-Marie Lomenech

Francesca Galluzzi

Rita Touti

Jean-William Dupuy

Myra O Villareal

Shinya Takahashi

Farhana Ferdousi

Kengo Iwata

Munkhzul Ganbold

Ahmed Salah

Mouad Sabti

Nozomu Iwata

May Sin

Younes Pr

Pr Badaoui Zaid

Pr Souad Bouabid

Pr Nezha Benaich

Pr Aicha Bouhaddou

Zakaria Madkour

Khadija Bernoussi

Siham Boualam

Sara Boumhaouad

Fadia Arahou Idrissi

Dr Laila Alem

Rania Alem

my grandmother Khadija MAHFOUD Jihane Alem

Meriem ➢ Khalfi

Leila My

Fatima Mghafri

Ajana Zahra

Mehdi Tazi

Massinissa Amara

Slim Mtibaa

Aymen Halleb

Meriem Mouaddine

Dr David Sokolowski

Karim Bellarabi

Dr Ayoub Badi Oumaima

Maamoun El

Fikri

Abdelmalek Papa

Alem

Keywords: Lawsonia inermis L, -naphthoquinones-Extraction-HPLC-spectrométrie de masse-mélanome Lawsonia inermis L, -naphthoquinones-Extraction-HPLC-Mass spectrometry -Melanoma

Extraction et analyse par chromatographie liquide couplée à la spectrométrie de masse de Lawsonia inermis et l'étude de son effet anti-mélanome Résumé : Lawsonia inermis L. communément appelée Henné appartient à la famille des Lythraceae. Elle est connue pour son importance traditionnelle à la fois comme agent cosmétique et médicinal, en particulier dans les régions indigènes de l'Afrique du Nord, de l'Asie du Sud et des pays du Moyen-Orient. Les différentes parties de cet arbuste ; les feuilles, les tiges, les graines et les racines comportent différents constituants chimiques comme les naphthoquinones, les polyphénols, les flavonoïdes, les tanins, etc., qui ont des activités biologiques diverses. L'intérêt de cette plante méditerranéenne en plus de son utilisation cosmétique, c'est son activité biologique antimicrobienne, antiinflammatoire, anticancéreuse et plusieurs d'autres. Ce travail de recherche s'est intéressé particulièrement à l'étude de son effet anticancéreux de plus particulièrement son potentiel anti-mélanome métastatique vue son utilisation traditionnelle souvent comme traitement topique de la peau irritée. Ce travail de recherche a permis, en premier lieu, la mise au point d'un plan d'expérience pour l'optimisation du rendement global d'extraction des molécules actives à partir du Henné (Lawsonia inermis L.). En seconde étape, l'identification et la quantification des composés actifs par chromatographie liquide à haute performance couplé à la spectrométrie de masse. Une cartographie géographie des cultures du henné au sud du Maroc, utilisant l'analyse en composantes principales et basée sur la nature du climat de chaque région.

Par la suite, l'effet de la plumbagin sur le mélanome métastatique a été étudié. L'étude de cette activité a été évaluée in-vitro sur des cellules murines et humaine respectivement B16F10 et SKMEL-28. La plumbagin s'est avérée inhiber de manière significative trois processus métastatiques importants : la migration, l'adhésion et l'invasion des cellules B16F10. Ces résultats ont été confirmés dans la lignée cellulaire humaine SK-MEL-28.

Publications and communications

Publications:

• Alem Fatima-Zahra, Meriem Bejaoui, Myra O. Villareal, Boutayna Rhourri-Frih, Isoda Hiroko (2020). "Elucidation of the effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via MAPK signalling pathway." Experimental Dermatology 29.4: 427-435.

• Alem Fatima-Zahra, Samira Ananthabothla Gita, Lise Cougnaud, Cedric Affnar, Issmail Nounah, Bakri Youssef, Alessandra Lopes de Oliveira, Zoubida Charrouf, Boutayna Rhourri-Frih (2020). "Lawsone quantification in Lawsonia inermis L. by HPLC-MS: How does the temperature and pluviometry affect lawsone concentration?" Industrial Crops and Products 158: 112960.

• Gita Samira Ananthabhotla, Fatima-Zahra Alem, Hedjila Ammour, Zoubida Charrouf, Alessandra Lopes de Oliveira and Boutayna Rhourri-Frih (2020).

Aknowledgments

Thanks to,

Pr Boutayna Rhourri-Frih, for believing in me since my master's year. Thank you for having been my thesis supervisor, my friend, the person to whom I turned for the slightest problem that happened to me. Thanks to your availability, your enthusiasm and your permanent positive attitude, I was able to accomplish what I accomplished, I was able to carry out my research despite all the obstacles I encountered. You have always been my source of motivation, my refuge when I needed security. Thanks for supporting my initiatives.

I cannot thank you enough for your excellent support, your involvement, your invaluable advice and your tireless encouragement. Your competence, your humility, your altruism, your human qualities and your desire to give the best to your students make you the best model to follow.

May God Bless you and your lovely family.

Pr Saaid Amzazi, for believing in me and believing in my dreams since my master's degree. Your encouragement and advices have been of great help to me. You have always been available and listened to me despite your responsibilities and your commitments. I am very honored to have benefited from your expertise and your kindness. Thank you for our warm scientific discussions, for teaching me your thoroughness, your modesty, your human qualities, your perseverance and your perfectionism.

Your work ethic and your involvement are admirable, I am lucky to have someone as dedicated and successful as you during my research journey! Pr Bakri Youssef, for trusting me and accepting me as a doctoral student in your team at BioPath laboratory, thanks for your support and your availability.

Pr Isoda Hiroko, for giving me chance to work as a researcher in your laboratory 'ISODA Lab' at the University of Tsukuba, Japan. I would like to express my deep gratitude for the honor you have done me by agreeing to entrust me with this work of great interest and guide me at each stage of its realization with rigor and kindness. Despite your professional obligations, you were always available and you always greeted me with sympathy, smile and cordiality. I dedicate this work to you as a testimony of my gratitude and my deep respect.

Thank you, Isoda Sensei.

Dr. Meriem Bejaoui, for being the best mentor and the best roommate ever, thanks for your help, your support, your guidance and for being my daily positive energy. Without you, my journey in Japan would have been very hard. You are the example of the young beautiful woman that combine ambition, creativity, perseverance and kindness. I cannot thank you enough for what you have done to me. Thanks also to your lovely family for inviting me very warmly to their home in Tunisia during the conference. Thank you, Tata Intidhar.

Pr. Catia Contado, for having accepted to be reporter for this thesis and to provide your expertise on the subject.

Pr. Amaral Joana Soares, for honoring me by agreeing to be reporter of this work.

Pr Abdelaziz Benjouad, Pr. Eddouks Mohammed, Pr. Rabii Ameziane Hassani, Pr. Waffo-Teguo Pierre, Pr. Nadia Dakka, Pr. Zoubida Charrouf, For having accepted to participate in this thesis jury and to examine this work .

Je t'aime Lalla Ghwita

A mes très chers grands-parents

A ma grand-mère Mama Kadija, ce travail est le fruit de ta bienveillance et ton amour. A mon grand-père parti très tôt, Elhajj Mohammed Khalfi, qui m'a vue grandir et a toujours été fier de moi. Je te dédie ce travail. Que ton ame repose en paix. A ma très chère défunte grand-mère, Zahra Alem et mon très cher défunt grand-père Si Ali Alem. Vous avez toujours été présents dans mon coeur même si je n'ai jamais eu l'occasion de vous voir. Je vous dédie ce travail à travers lequel je voudrais vous exprimer à quel point je vous aime. Que vos âmes reposent en paix. Plant material __________________________________________________________________ 3.

Figures List

Tables list

Abbreviations list

HPLC

Extraction of the plant material ___________________________________________________ 4.

HPLC-ESI-QTrap-MS conditions _________________________________________________ 5.

Standard addition method (SAM) _________________________________________________ 6.

Cell culture and treatment _______________________________________________________ 7.

Cell viability assay ______________________________________________________________ 8.

Data Analysis __________________________________________________________________

IV.

Results and discussion __________________________________________________

1.

Plumbagin decreased the viability of B16F10 murine melanoma cells. __________________ 2.

Quantification of lawsone, lapachol and plumbagin in Lawsonia inermis L. leaves. ________

3.

Discussion _____________________________________________________________________

General Conclusion _______________________________________________________

General Introduction

In In fact, approximately 65 to 80% of the world's population use traditional medicines to meet their primary health care needs, due to poverty and lack of access to modern medicine at the time. Nowadays, the use of plant derived medications in the treatment and prevention of disease and wound healing has become a good alternative to avoid chemical drugs, also, as a rule, herbs and natural products at a certain concentration lack much of the toxicity that is present in synthetic chemicals, thus, enhancing their appeal for long term preventive strategies.

Medical benefits from plant forms have been scientifically recognized.

Furthermore, traditional communities are now faced with a very difficult choice: to open up to progress without renouncing their culture and to take the path of socio-economic development without losing their soul and their roots.

In this context, researchers are becoming more and more interested in going deeper into the benefits of traditional herbal medicine and trying to understand their mechanisms of action on pathologies that they can treat to break the gap between traditions and novel discoveries in pharmaceutical industry. Therefore, the remain goturn to nature becomes an essential element in the life of the human being today.

This thesis is part of a collaboration between the University of Bordeaux in France, the University of Tsukuba in Japan and finally the Mohamed V University in Morocco. Its main goal was to study the effect of Lawsonia inermis L. on the skin and more specifically metastatic skin cancer.

Our interest in an alternative and non-invasive treatment solution (such as chemotherapy) based on natural products is one of the reasons for our development.

The objective of this research is, in one hand, the optimization of the overall yield of extraction of active molecules from Henna (Lawsonia inermis L.) in order to explore and characterize the molecules which are at the origin of its anti-cancer activity using the development of an experimental design. This identification is based on different techniques of extracting the plant; Pressurized Liquid Extraction (PLE), Supercritical Fluid Extraction (SFE)

and Soxhlet followed by the analysis by high performance liquid chromatography coupled with mass spectrometry (LC-MS). Secondly, and always for the sake of development and optimization of the use of this plant, the phytochemical profile of the same plant from different geographical areas in Morocco: Zagora, Errachidia, Foum Zguid and Tazzarine was studied and the differences were evaluated by Principal Component Analysis. A quantitative analysis by high performance liquid chromatography-electrosray ionization-Qtrap mass spectrometry (HPLC-ESI-Qtrap-MS) showed that the Foum Zguid region is characterized by the highest level of Lawsone (0.6%).

Subsequently, the effect of plumbagin, one of the major naphthoquinones present in Lawsonia inermis L., on metastatic melanoma, which is the most dangerous form of skin cancer, was studied. The study of this activity was evaluated in vitro on murine and human cells, B16F10 and SKMEL-28, respectively. Plumbagin has been shown to significantly inhibit three important metastatic processes: migration, adhesion, and invasion of B16F10 cells. These results were confirmed in the human cell line SK-MEL-28 with a marked decrease in their viability. The study of the molecular mechanism was also explored; A decrease in the expression of genes associated with cell migration, adhesion and invasion have been observed

during treatment with plumbagin. An under expression of matrix metalloproteinase (MMPs)

was observed after treatment of the cells with plumbagin. It should be remembered that MMPs are a class of protease enzymes involved in the degradation of the extracellular matrix (ECM) and, in cancer, they facilitate the process of invasion and metastasis and participate in tumor proliferation, to immunoresistance and angiogenesis.

In parallel, a significant overexpression of the genes involved in apoptosis was observed during treatment with plumbagin, with regulation of the adaptive immune and inflammatory response, and of the response to reactive oxygen derivatives (ROS).

In a nutshell, this thesis will allow us to broaden our understanding of this miraculous plant used for a very long time by our ancestors and that we sometimes 'underestimate'. this understanding will bring:

• On one hand, on the chemical composition which will guide us towards a study of its composition in naphthoquinones.

• On the other hand, on the effect of the environment on this chemical composition by its cultivation in different regions of Morocco.

• Finally, on the molecular mechanism and the signaling pathway where this molecule intervenes to inhibit the growth of cancer cells in the skin.

P.S: Each chapter of this thesis contains the information reported in individual papers: two papers that have been already published (chapter 3 and chapter 4) and one article is under review (chapter 2). The last article (Chapter 5) will be communicated soon by the time of the defense. Since ancient times, Lawsonia inermis L. has been used originally from Mesopotamia.

It spread to the east, arriving in northern India and some eastern and western regions of the peninsula, then westward into Syria, Palestine, and Egypt where it followed the migration of people [START_REF] Aubaile-Sallenave | Les voyages du Henné[END_REF] . Two forms of use of Henna are known since time: its fragrance provided by the flowers, and its coloring by its leaves

In Egypt, the mummy of Ramses II (1298-1235) has been analyzed, it has been reported that his hair, hands and feet were dyed with Henna (or pouker, name for Henna in Egyptian).

This information did not allow us to know if, at the time, the use of henna was common in all individuals or it was exclusive to the dead 2 .

The Hebrews also used Lawsonia inermis L. and took its benefits from their long stay with the Zoroastrian Persians. They glorified this plant and mentioned is in the Bible in the wedding songs:'song of songs' attributed to Salomon 970-932 BC): 'You are a closed garden, my sister, my fiancee, where is the kopher with the spikenard', 'My beloved is for me a bunch of kopher'. Kopher (Henna) flowers in white, pink or red clusters have a female sexual odor which became and linking the citation with its usage. Moreover, in order to stand out from other Semitic populations, the Hebrews did not use it as dyes but only as a perfume.

Romans as well used this plant in the hair colorings. They used to mix it with blood to obtain a red hair color, with orange highlights, or with indigo for black color and other natural products for more colors [START_REF] Aubaile-Sallenave | Les voyages du Henné[END_REF] .

Then came Islam which made this plant one of the most important plants. The Prophet Mohammad (sws) used Henna to heal his wounds and encouraged women to dye their hair, hands and feet as a sign of beauty.

Henna was then introduced to North Africa, Madagascar, Indo-China and Japan from Arabia following Islamic armies and traders. Then in the 16 th century, it was spread to Andalusia through North Africa. In France, Henna was introduced as hair natural dying until Nowadays, this plant is used worldwide in cosmetics and for its health benefits [START_REF] Ali | Anti-inflammatory, antipyretic, and analgesic effects of Lawsonia inermis L. (henna) in rats[END_REF][START_REF]Chemical Constituents, Antioxidant Activity and Cytotoxic Effects of Essential Oil from Strobilanthes crispus and Lawsonia inermis -SciAlert Responsive Version[END_REF] and its history allows us to study archaeological discoveries from Egypt, Sumer and Babylon but also to follow the trade routes between the East and the West.

Botanical classification

Lawsonia inermis L. commonly known as Henna in North Africa and Middle East of Mehendi in India belongs to the family Lythraceae. It is cultivated in tropical, subtropical and warm temperate areas in particular to North Africa and Asia. It is about 2-6 m in height, small shrub like with spiny branchlets. The leaves are elliptical, opposite, sub-sessile, smooth with clear depressed veins on the dorsal surface and , about 1.5 to 5 cm long, 0.5 to 2 cm wide, green in color and short-stalked (Figure 1C) [START_REF] Lemordant | Plantes médicinales des régions tempérées[END_REF] . The flowers contain four sepals and crumbled petals, the floral formula of Henna is: 4S+4P+(4+4) E+4C [START_REF] Lemordant | Identification, contrôle, fraudes, additifs[END_REF] . They have a diameter of about 6 millimeters. The fruits are small spherical capsules 5mm in diameter (Figure 1B). The seeds are angular with a thick seed coat (Figure 1A). Species Lawsonia inermis L.

Geographical conditions of cultivation

The economic life of Lawsonia inermis L. culture is on average five years. It is however preserved in culture for a dozen years. The plant does not grow in cold, heavy soils and brackish waters [START_REF] Chattaoui | Etude agropédologique du henné dans la région de Gabès[END_REF] . Contrary to what is believed, an important amount of water is required for a better culture of Henna; therefore, its water needs reach 20,000 m 3 per year / ha.

The plots intended for planting should be deep. It is then proceeded to space the transplanted saplings and making planks of 2 to 5 m 2 depending on the irrigation possibilities.

The period of plant sowing is usually in May. It takes 8 to 10 days to appear after snowing. The reproduction of Henna could be sexual and asexual [START_REF] Henné | Lawsonia inermis: vertus, usages et culture[END_REF] . The harvest of Henna could be done 3 to 5 times a year. The best crop is the early July one and the weakest is in February [START_REF] Phogat | [END_REF] . The first cut takes place about two months after transplanting (July-August). The second towards the end of September, beginning of October and the last during the month of November. Yields vary around 30 Qx / ha / year and harvest takes place after flowering [START_REF] Henné | Lawsonia inermis: vertus, usages et culture[END_REF] .

It is in the regions of the Middle East, Africa (especially North Africa), and South Asia that Henna is used by almost the entire population, hence the need for large production. It is therefore the regions where the cultivation of Lawsonia inermis L. is very abundant (Figure 2). protection metabolites. In addition, these secondary metabolites represent an important source of molecules that can be used by humans in different fields such as pharmacology or food industry.

Chemical composition of Lawsonia inermis L.

The composition of Lawsonia inermis L. has been widely studied. The report of its secondary metabolites are be classified based on their biosynthetic origins 12 a. Phenolic compounds • Lawsone (2-Hydroxy-1,4-naphthoquinone and also called Hennotannic acid) is the major active compound present in Lawsonia inermis L. [START_REF] Chaudhary | Lawsonia inermis Linnaeus: A Phytopharmacological Review[END_REF][START_REF] López López | NAPHTHOQUINONES: BIOLOGICAL PROPERTIES AND SYNTHESIS OF LAWSONE AND DERIVATIVES -A STRUCTURED REVIEW[END_REF] . It is responsible for the natural dyeing (red-orange) and present in the flower, leaves and branches of the plant. The highest content is in leaves and the lowest in the branches [START_REF] Oda | Comparison of lawsone contents among Lawsonia inermis plant parts and neurite outgrowth accelerators from branches[END_REF] (Figure 3A).

• Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) was isolated from Lawsonia inermis L. stem bark [START_REF] Gupta | A naphthoquinone from Lawsonia inermis stem bark[END_REF] (Figure 3B). It was reported that this naphtoquinone can suppress the invasion, migration and adhesion of breast cancer and melanoma skin cancer by downregulation of MMP9 and MMP2 [START_REF] Yan | Suppressive Effects of Plumbagin on Invasion and Migration of Breast Cancer Cells via the Inhibition of STAT3 Signaling and Down-regulation of Inflammatory Cytokine Expressions[END_REF][START_REF] Alem | Elucidation of the effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via MAPK signalling pathway[END_REF] .

• Lapachol (4-hydroxy-3-(3-méthylbut-2-ényl) naphtaléne-1,2-dione) is a derivative of Lawsone (Figure 3C).

• 1,2,4-trihydroxynaphthalene-2-O-β-D-glucopyranosid is a powerful antioxidant and inhibitor of Aβ 42 aggregation [START_REF] Dhouafli | 1,2,4-trihydroxynaphthalene-2-O -β-D-glucopyranoside: A new powerful antioxidant and inhibitor of Aβ 42 aggregation isolated from the leaves of Lawsonia inermis[END_REF] . It was isolated and identified from the leaves [START_REF] Takeda | New phenolic glucosides from Lawsonia inermis[END_REF] (Figure 3D).

• Flavonoids: Luteolin-7-J-b-D-glucopyranoside and Ascorbic acid (5-(1,2dihydroxyéthyl)-3,4-dihydroxyfuran-2-one) (Figure 3E and 3F) both isolated from the leaves and present an antioxidant activity [START_REF] Hsouna | Antioxidant constituents from Lawsonia inermis leaves: Isolation, structure elucidation and antioxidative capacity[END_REF][START_REF] Khazaeli | Identification of Luteolin in Henna (Lawsonia inermis) Oil, a Persion Medicine Product, by HPTLC and Evaluating Its Antimicrobial Effects[END_REF] as well as Apigenin(Figure 3G) and gallic acid (Figure 3H) [START_REF] Elansary | Antioxidant and Biological Activities of Acacia saligna and Lawsonia inermis Natural Populations[END_REF] .

• Tannins: (+)-catechin(Figure 3I ), 1,2,3,6-tetra-O-galloyl-~-D-glucose (Figure 3J) and 1,2,3,4,6-penta-O-galloyl-~-D-glucose (Figure 3K) were identified in hairy roots of Lawsonia inermis L. [START_REF]Tannin production in hairy root cultures of Lawsonia inermis[END_REF] . Tannic acid was also identified in the plant (Figure 3L) [START_REF]Corrosion inhibition of mild steel in 1M HCl solution by henna extract: A comparative study of the inhibition by henna and its constituents (Lawsone, Gallic acid, α-d-Glucose and Tannic acid)[END_REF] .

• Coumarins are also present in all parts of Lawsonia inermis L. [START_REF] Chaudhary | Lawsonia inermis Linnaeus: A Phytopharmacological Review[END_REF] . From the leaves, Lacoumarine (6-methyl-7-(allyloxy)coumarin) (Figure 3M), fraxetin (Figure 3N) and scolopetol (Figure3O) were identified [START_REF] Chaudhary | Lawsonia inermis Linnaeus: A Phytopharmacological Review[END_REF][START_REF]Lawsonia inermis -From traditional use to scientific assessment | PharmaTutor[END_REF] .

b. Terpenes

Lawsonia inermis L. racemes smell an extremely powerful and extremely diffusible scent which recalls that of several white flowers, privet or crataegus when diluted [START_REF] Gast | [END_REF] . This propriety of Lawsonia inermis L. was highly appreciated and used as fragrance. The main compounds responsible of this fragrance are Terpenes. They are mainly present in essential oil extracted from the flowers. There are two types of terpenes; volatiles and non-volatile ones.

• Volatile terpenes: Mainly β-ionone and its derivatives (Figure 4A); 48,6% in yellow flowers and 2.5% in red flowers [START_REF] Wong | Volatile Components of Lawsonia inermis L. Flowers[END_REF] . Volatile compounds were also found in the leaves of the plant: methyl-α-Dglucopyranoside, 2-hydroxy-1,4-naphthalenedione, n-Hexadecanoic acid, Benzene, (ethenyloxy) [START_REF] Hema | Gas Chromatography -Mass Spectroscopic analysis of Lawsonia inermis Leaves[END_REF] . Furthermore, 1,8-cineole (figure 4B), αpinene (figure 4C), p-cymene (figure 4D) [START_REF] Ogunbinu | Study on the Essential Oil of Lawsonia inermis (L) Lythraceae[END_REF] . All the above compounds were identified and quantified by Gas Chromatography-Mass Spectrometry (GC-MS). Moreover, a chemical investigation of essential oil extracted from Ethiopian Lawsonia inermis L.

leaves has revealed the presence of Linalool (Figure 4E), α-terpineol (Figure 4F), eugenol (Figure 4G) [START_REF] Kidanemariam | Chemical Investigation of Lawsonia inermis L. Leaves from Afar Region, Ethiopia[END_REF] .

• Non-Volatile terpenes: Lawnermis acid (Figure 4H) and Lawnermis acid methyl ester (Figure 4I) were identified from Lawosnia inermis seeds [START_REF] Handa | Lawnermis acid: a new anticomplementary triterpenoid from Lawsonia inermis seeds[END_REF][START_REF](Lawsonia+Inermis+L.)+from+the+Powdered+Leaves+of+Henna+Pla nt&rlz=1C1GCEA_enFR905FR905&oq=Phytochemical+Investigation+on+Isolation+an d+Characterization+of+2+Hydroxy1%2C+4-Naphthoquinone+(Lawsonia+Inermis+L[END_REF] . Lupeol (Figure 4J) and botulin (Figure 4K) were isolated from the leaves [START_REF] Singh | -A commercially important primaeval dying and medicinal plant with diverse pharmacological activity: A review[END_REF] . In the barks, 3β,30-dihydroxylup-20 (29)-ene (hennadiol) (Figure 4L) and (20 S)-3β,30-dihydroxylupane (Figure 4M) were found [START_REF] Chakrabartty | Isolation of dihydroxylupene and dihydroxylupane from the bark of Lawsonia inermis[END_REF] .

c. Alkaloids

TLC analysis showed the presence of two alkaloids in the mature seeds of Henna: Harmine (Figure 5A) and Harmaline (Figure 5B) [START_REF] Jacob | Two Harmala Alkaloids from Lawsonia inermis Seeds[END_REF] .

Biological effects of Lawsonia inermis L.

Lawsonia inermis L. have many biological effects depending on the organ used for the prevention or/and the treatment of the disease. Its flowers are soothing agents, liver tonic, cardiotonic, refrigerant, sedatives and also treats cough, fever, insomnia… The antifungal, antibacterial, antiparasitic and virucidal activity is concentrated on the corteccia. The leaves react differently; they are depurative, febrifuge, liver tonic [START_REF] Singh | -A commercially important primaeval dying and medicinal plant with diverse pharmacological activity: A review[END_REF] . Furthermore, it has been reported that this plant has an antimalarial [START_REF] Afolayan | Antimalarial actions of Lawsonia inermis, Tithonia diversifolia and Chromolaena odorata in combination[END_REF][START_REF] Singh | Fraxetin and ethyl acetate extract from Lawsonia inermis L. ameliorate oxidative stress in P. berghei infected mice by augmenting antioxidant defence system[END_REF] , anti-lipidemic [START_REF] Singh | Safety and efficacy of hydroalcoholic extract from Lawsonia inermis leaves on lipid profile in alloxan-induced diabetic rats[END_REF] , anti-diabetic [START_REF] Arayne | In vitro hypoglycemic activity of methanolic extract of some indigenous plants[END_REF] , hypothyroidic activities [START_REF] Zumrutdal | Lawsonia inermis -an alternative treatment for hyperthyroidism? Bratisl[END_REF] .

• Anti-cancer activity

Current research on cancer focuses on natural products as therapeutic tools. Lawsonia inermis L. has a long history of use and plays an important role as an effective anticancer agent. Priya R. found that ethanolic extract of Lawsonia inermis L. root exhibited significant antitumor activity; it increases the life span of Dalton's Lymphoma Ascites (DLA) bearing mice [START_REF] Priya | Effect of Lawsonia inermis on tumor expression induced by Dalton's lymphoma ascites in Swiss albino mice[END_REF] .

Moreover, naphthoquinone and anthraquinones present in Lawsonia inermis L. were more effective than benzoquinone against colon cancer by studying the proliferation of the cancer cells. The flow cytometric analysis showed that the S-phase of cell cycle was blocked by lawsone leading to inhibitory effect. It is proved that the plant can be exploited as a source of novel drug development for cancer treatment [START_REF] Kamei | Inhibition of cell growth in culture by quinones[END_REF][START_REF] Ishteyaque | In Vitro: Cytotoxicity, Apoptosis and Ameliorative Potential of Lawsonia inermis Extract in Human Lung, Colon and Liver Cancer Cell Line[END_REF][START_REF] Tsao | Discovery of Isoplumbagin as a Novel NQO1 Substrate and Anti-Cancer Quinone[END_REF][START_REF]Inhibitors of melanogenesis in B16 melanoma 4A5 cells from flower buds of Lawsonia inermis (Henna)[END_REF][START_REF]Chemoprevention of skin cancer: effect of Lawsonia inermis L. (Henna) leaf powder and its pigment artifact, lawsone in the Epstein-Barr virus early antigen activation assay and in two-stage mouse skin carcinogenesis models[END_REF][START_REF] Kumar | Antiproliferative and Apoptosis Inducing Effects of Non-Polar Fractions from Lawsonia inermis L. in Cervical (HeLa) Cancer Cells[END_REF][START_REF] Lozza | The Henna pigment Lawsone activates the Aryl Hydrocarbon Receptor and impacts skin homeostasis[END_REF][START_REF] Barani | Lawsone-loaded Niosome and its antitumor activity in MCF-7 breast Cancer cell line: a Nano-herbal treatment for Cancer[END_REF] .

• Anti-microbial activity

Lawsonia inermis L. seeds exhibited a significant inhibition with a MIC range of 2.5-10 mg/ml against Vibrio cholerae that causes cholera. This study has tested several other plants that showed less effect than Henna [START_REF] Sharma | Vibriocidal activity of certain medicinal plants used in Indian folklore medicine by tribals of Mahakoshal region of central India[END_REF] . Furthermore, Ethanolic extract of Lawsonia inermis L. leaves has an inhibitory effect against Staphylococcus aureus, Salmonella paratyphi, Candida albicans by four folds. It inhibits Escherichia coli and Staphylococcus dysenteriae by two folds [START_REF] Ahmad | Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens[END_REF] . Malassezia is a fungus that causes cutaneous infections such as Pityriasis versicolor and folliculitis. Aqueous extract of Lawsonia inermis L. inhibited Malassezia at 0.25, 0.5 and 4 (V/V%). This study reported the potential antifungal effect of Henna 53 .

A Harmine

• Anti-oxidant activity

The composition study of Henna showed a well-known natural compounds for their antioxidant potential: presence of oils, fats, phytosterols and terpenoids in petroleum ether and dichloromethane extracts and phenolics, tannins, saponins, and flavonoids are present in ethanol and water extracts [START_REF] Jacob | Free radical scavenging and reducing power of Lawsonia inermis L. seeds[END_REF][START_REF] El Babili | Lawsonia Inermis: Its Anatomy and its Antimalarial, Antioxidant and Human Breast Cancer Cells MCF7 Activities[END_REF] . It was delineated that alcoholic extract is potent radical scavenger.

Furthermore, in other studies, the most active chemical component in the DPPH free radical scavenging test is 1,2,4-Trihydroxynaphthalene-1-J-b-D-glucopyranoside (EC50 = 6.5 lg/ml) Therefore, Lawsonia inermis L. could be used as a potential source of new natural antioxidants [START_REF] Hsouna | Antioxidant constituents from Lawsonia inermis leaves: Isolation, structure elucidation and antioxidative capacity[END_REF] .

• Anti-inflammatory activity 1,5-diphenylpent-3-en-1-yne derivatives, lawsochylin A-C, 3-methyl naphthalene carboxylates and lawsonaphtoate A-C were isolated from the stem and leaves of henna utilizing a bioassayguided fractionation and were used to evaluate the anti-inflammatory effect of Lawsonia inermis L. [START_REF] Liou | 1,5-Diphenylpent-3-en-1-ynes and methyl naphthalene carboxylates from Lawsonia inermis and their anti-inflammatory activity[END_REF] . Two compounds lawsochylin A and lawsonaphtoate A showed potent antiinflammatory activity by inhibition of superoxide anion generation with an IC50=1.80 and 1.90 µg/ml, and elastase release with IC50= 1.58 and 3.17 µg/ml of human neutrophils in response to NFormylmethionyl-leucyl-phenylalanine or cytochalasinB. Furthermore, luteolin, apigenin, 4S-4-hydroxy-atetralone and 2-butoxysuccinic acid showed also important inhibition of superoxide anion generation with an IC50=0.75-1.78 µg/ml and elastase release with IC50=1.62-3.61µg/ml.

In other research, the in vivo anti-inflammatory activity of Lawsonia was compared with a reference drug phenylbutazone and it has been showed that the effect between the two treatment presents no difference in the activity with different treatment concentrations (500 mg/kg for lawsone-100mg/kg for reference drug) [START_REF] Ali | Anti-inflammatory, antipyretic, and analgesic effects of Lawsonia inermis L. (henna) in rats[END_REF] .

II. Phytotherapy 1. Definition of medicinal plants

Phytotherapy is part of traditional medicine also called medicine alternative or parallel.

According to French Pharmacopeia, medicinal plants are defined as plant drugs of which at least a part has drug properties. More than 25% of current medical pharmacopeia is derived from medicinal plants [START_REF] Dattner | From medical herbalism to phytotherapy in dermatology: back to the future[END_REF] . The use of medicinal plants has been known since ancient times. They were used in Mesopotamian and Egyptian times in the production of perfumes, vegetable and essential oils as well as waxes [START_REF] Bart | Extraction of Natural Products from Plants -An Introduction[END_REF] . They are now considered as a heritage for many countries. It is usually used for prevention and treatment of a wide range of diseases.

The medicinal plants continue to constitute an important need despite the influence growth of the modern health system. Their effects are due to individually or synergically to their secondary metabolites.

According to the World Health Organization (WHO), about 80% of people in developing countries employ traditional herbal medicine.

The main types of phytotherapy are as follows:

a. Aromatherapy:

It consists of using essential oils as treatment. They could be extracted from all the plant parts.

They are highly active compounds that should be used often through the skin [START_REF] Cooke | Aromatherapy: a systematic review[END_REF] .

b. Homeopathy

It is a therapeutic method developed by Samuel Hahnemann (1755-1843) and still practiced until now in all over the world [START_REF] Swayne | International Dictionary of Homeopathy[END_REF] . It is based on the idea that a substance that causes a symptom can be used to treat the same symptom of the disease [START_REF] Ernst | A systematic review of systematic reviews of homeopathy[END_REF] . This therapy remains one of the most controversial subjects in therapeutics.

c. Herbalism:

The preparation is based on simple methods, most often water-based: decoction, infusion, maceration of the plant bought from herbalists. It is the usual type used in medication to treat or prevent diseases in human and animals traditionally.

III. Chemoprevention

Definition

Chemoprevention is a pharmacological approach that consists generally in the administration of non-cytotoxic drugs, vitamins or other natural or synthetic substances or micronutrients to reduce the risk of developing cancer, delay its development or prevent a recurrence. Among numerous cases, doctors use chemoprevention when the person is at a high risk of developing cancer such as family history, or when the patient has had cancer before [START_REF] Benetou | Chemoprevention of cancer: current evidence and future prospects[END_REF][START_REF]Chemoprevention[END_REF][START_REF] Krishnan | Cancer chemoprevention drug targets[END_REF] . The term chemoprevention has been initiated in 1976 by Michael B. Sporn by using Vitamin A analogs for a potential epithelial cancer prevention during preneoplastic period [START_REF] Sporn | Approaches to Prevention of Epithelial Cancer during the Preneoplastic Period[END_REF] .

Chemoprevention has been classified in four principal categories: hormonal, medications, dietrelated agents, and vaccines [START_REF] Benetou | Chemoprevention of cancer: current evidence and future prospects[END_REF] . In this thesis, we will be focusing mainly on the effect of dietrelated agents that stand for natural products: more specifically the effect of naphthoquinones presents in Lawsonia inermis L. on the metastatic melanoma skin cancer in vitro.

Diet-related agents as chemo preventive drugs

Diet and lifestyle play a very important role in people's health. A good diet reduces the risk of attacking several diseases and / or slows the proliferation of cancer cells as well as prevents inflammation. Diet related compounds have recently attracted researcher's attention as potential therapeutics mainly for their low toxicity and their availability compared with regular drugs.

Researchers were more likely concerned about the antioxidant effect of several micronutrient as potential cancer-preventive agents. Flavonoids are polyphenolic compounds and possess various antioxidant properties that inhibit carcinogen-activating enzymes. Previous studies suggest that flavonoids could play a protective role on several cancer types: lung, breast, colon and prostate [START_REF] Romagnolo | Flavonoids and cancer prevention: a review of the evidence[END_REF][START_REF] Rossi | Flavonoids, proanthocyanidins, and cancer risk: a network of case-control studies from Italy[END_REF] . However, we should note that combinations of beta carotene and vitamin A may increase the risk of lung cancer as well as other cancers, like prostate and stomach [START_REF]Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease -PubMed[END_REF][START_REF]Effects of alpha-tocopherol and beta-carotene supplements on cancer incidence in the Alpha-Tocopherol Beta-Carotene Cancer Prevention Study -PubMed[END_REF] . As for Vitamin D, it plays an important role in calcium metabolism. Moreover, it has several other physiological functions; colorectal cells and others express receptors for vitamin D and their activation has been reported to have a potential antitumor effect [START_REF] Zhang | Calcium, vitamin D and colorectal cancer chemoprevention[END_REF] .

Chapter 2: Extraction and Chemical Analysis of Lawsonia inermis L. Use of whole or a part of plant extracts for several therapy dates back to centuries. It has attracted the curiosity of researchers to develop new extraction methods that make it possible to obtain a good yield of molecules of interest as well as to optimize the extraction process whether at the solvent or time level.

Techniques of extraction

a. Soxhlet

Soxhlet is a standard extraction method that has been practiced for over a century. It is as well the most widely used technique for the extraction of the natural products from solid phase.

This method requires small amounts of solvents, which is advantageous. Moreover, the solvent which condenses is always pure. The solubilization of the substance is therefore promoted thanks to better partition coefficients. However, it has some disadvantages: the extractions are quite long (consumes time) and requires the heat which could cause damage to the substances sensitive to heat.

The principle of this method (as shown in the Figure 6) consists in placing the solid matrix in the cellulose cartridge then in the Soxhlet tank and heating the solvent under reflux (Figure 6A).

The amount of solvent should be sufficient to take in account the volume that will be trapped in the tank during handling. Then, the extractor is equipped with a condenser that allows the solutes to be transferred from the extraction chamber into the reservoir (Figure 6B). The process is repeated several times (depends on the solubility of the sample) and continuously (Figure 6C). Finally, the extraction is complete and the extraction is fully done, the extract is ready to use (Figure 6D).

b. Ultrasound assisted extraction

Ultrasonic extraction is performed when high-power, low-frequency ultrasonic waves are coupled to a sample made of plant material in a solvent. High power ultrasonic waves are coupled to the mixture through a probe-type ultrasonic processor.

Ultrasound extraction process principle is mainly the disruption of cells and improvement of mass transfer in the boundary layer surrounding the solid matrix. Sonication, the perforation of cell walls and membranes, improves the permeability of cell walls and membranes and is often an intermediate step before cells are completely disrupted by sonication. The mechanical effects of ultrasound-induced cavitation, such as differences in heat and pressure, shock waves, shear forces, liquid jets and microflows, intensify the penetration of the solvent into the interior of the cell and improve the mass transfer between cell and solvent, so that intercellular materials are transferred into the solvent.

c. Supercritical fluid extraction (SFE)

It is a green method of extraction (Figure 7A) that involves circulating supercritical fluid, under pressure and temperature that is above its critical point. The most commonly used solvent in SFE method is Carbon Dioxide (CO2) (Figure 7B) due to its low critical temperature (TC = 31ºC) and pressure (PC = 74 bar), its safety, availability, low cost and non-toxicity.

This method consists in the circulation of the fluid under pressure and temperature, through a material, then decompression to recover the extract or contaminant. On depressurization, the supercritical fluid is released in gaseous form (reusable) and the desired compound in liquid or solid form. Supercritical CO2 as fluid used can have its polarity modified by the addition of a polar "dopant" commonly known as a co-solvent, thus increasing the spectrum of molecules extracted by the supercritical solvent. In addition, the development of units operating at high pressure (> 400 bar) makes it possible to recover increasingly polar molecules without the addition of co-solvent.

The advantage of this method, unlike the previous method, make it possible to preserve the chemical integrity of the thermolabile molecules (sensitive to heat) due to the low temperatures used (generally 40 ° C to 60 ° C).

d. Pressurized liquid extraction (PLE)

Pressurized liquid extraction is a sophisticated technology that involves extracting a solid sample with a solvent (or mixture of solvents) heated to high temperature in order to accelerate the extraction process. The extraction is therefore carried out under pressure to maintain the A B It is used in several fields of application, mainly for the estimation of pharmaceutical and biological samples. HPLC is used more specifically to analyze and purify natural products (which is the application used in this project), to measure active ingredients, impurities and degradation products and to characterize the metabolites.

It should be noted that this technique was not widely used until after it began an explosive growth in the 1990s which made it the most popular analytical method judged by instrument sales and scientific importance. Its current popularity results from its convenient separation of a wide range of sample types, exceptional resolving power, speed and nanomolar detection levels [START_REF] Bensakhria | Chromatographie Liquide Haute Performance (HPLC) » Analytical Toxicology. Analytical Toxicology[END_REF] .

HPLC needs a column that holds the stationary phase, a pump that moves the mobile phase through the column, and a detector that shows the retention times (Rt) of the monitored molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the eluents (solvents) chosen. The basic parameters of a chromatographic separation are considered in the Table 1: Greater is the length of the column, greater will be the number of theoretical plates and greater will be the efficiency of the column. N= 5.54 (t r /w h/2 ) 2

It represents the ratio of retention times of two substances in the stationary phase

α= (t r2 -t r1 )/(w 1 h/2 -w 2 h/2 )
It is the distance between the peaks at the peak bases, it corresponds to the degree to which one peak is separated from the other Total time spent by sample molecules in stationary and mobile phases respectively

Corresponds to retention time of the solvent molecule without interaction with the stationary phase => t 0 =0.7. π r 2 L/F

b. HPLC apparatus

HPLC instrument includes typically a solvent reservoir, pump, injector, column, and a detector (figure 9) [START_REF] Bensakhria | Chromatographie Liquide Haute Performance (HPLC) » Analytical Toxicology. Analytical Toxicology[END_REF] .

ii. Injector

Large numbers of samples can be automatically injected onto an HPLC system by the autosampler. The system of the injection loop allows to have a constant injected volume, which is important for the quantitative analysis.

iii. Column

It contains the stationary phase which is a porous support covered with a gel (grafted liquid) which has the properties desired to retain solute molecules. There are different types of column used as stationary phase in the HPLC analysis, the main used ones are:

• Normal phase HPLC:

This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and non-polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these. Polar samples are thus retained on the polar surface of the column packing longer than less polar materials.

• Reverse phase HPLC:

The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. It works on the principle of hydrophobic interactions hence the more nonpolar the material is, the longer it will be retained.

iv. Detector

There are several types of detectors:

• Spectroscopic detectors:

• Refractometry;

• Electrochemical detectors (dec):

• Conductimetry;

• UV diode array;

• Evaporative Light scattering Detector;

• Spectral detection with coupling: to mass spectrometry (ms). Examples: gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry; to atomic spectroscopy.

Mass spectrometry a. Definition

Mass spectrometry is an analytical method that measures mass/charge (m / z) ratios of ionized molecules as well as their fragmentation products. This analytical method can come up with very important information: the structure of the molecule and molecular mass and concentration (qualitative and quantitative information respectively). It consists in the separation of analytes according to their mass to charge ratio (Figure 10A). It is a destructive method; the analyte could not be recovered after the analysis.

The analyte molecules are first converted to ions in the ionization source and by acquiring positive or negative charges, then they go through the analyzer that separates ions according to their mass-to-charge ratio. Finally, when the ions contact the detector, it gives information to the computer in the form of signals which are processed by the software and give interpretable data. The Figure 10B represents a schema of the main components of a mass spectrometer.

b. Ionization methods

Different ionization methods exist, and can be organized in two main methods:

1. Direct Ionization method such as: the electron ionization and the chemical ionization

Molecular peak 188 g/mol fragments

Adapted from NIST Chemistry WebBook (https://webbook.nist.gov/chemistry)

A B

In this report we will be focusing on the electrospray ionization (ESI) which was used during our analysis.

i. Electrospray Ionization (ESI)

Electrospray ionization is now the most widely used as an ionization technique in analytical techniques. The main principle of this method is: volatilization (formation of microdroplets) of the compound and its ionization. The ionization is carried out at atmospheric pressure. After the injection and separation, the compound is introduced onto the ion source through a metallic capillary, then dispersed in microdroplets charged, under the joint action of a potential differential between two electrodes and the application of a spray gas. After that and during the movement of these microdroplets, their solvent evaporates and their diameter therefore become smaller. The charge density within each droplet increases until it becomes too large and a Coulomb explosion occurs, causing the gas phase ions to be released. Finally, the ions are transported from the source, which is at atmospheric pressure, to the high vacuum of the analyzer to be analyzed.

This method of ionization presents several advantages: it can extract polar molecules, operates at low temperature and at atmospheric pressure as said before, so that the sensitive compounds are not degraded. It also allows an accurate measurement of the molecular weight. However, some disadvantages are not to be neglected; electrospray ionization could not be carried out unless there is an MS/ MS, it is very sensitive to the presence of salts and/or additives and gives little information about the structure.

c. Mass analyzer:

The function of this part of the mass spectrometer is to separate ions according to their m/z ratio Many types of analyzers are used: quadrupoles (Q), ion traps (IT), time of flight (TOF), Fourier transform cyclotron ion resonance analyzers (FT-ICR), magnetic analyzers, orbitrap.

In our research, we used a combination of two types of analyzers: a quadrupole and an ion trap from Sciex (Q-Trap).

i. Quadrupole Trap analysis

The quadrupole is composed from two parallel bars where an electrical field is created thanks to a difference potential. This field will drive the ions from the analyzer entrance into the direction of the next analyzer making the separation possible. The second analyzer is a collision cell that is used with a gas beam that provokes the fragmentation of the ions and in the third analyzer which is a trap, the ions are trapped and we have separation of the fragments.

Figure 12: Quadrupole ion trap mass spectrometer. [START_REF] Santoiemma | Recent methodologies for studying the soil organic matter[END_REF][START_REF]Quadrupole Ion Trap -an overview[END_REF] The main advantage of this analyzer is its high specificity and sensitivity thanks to different scan modes that we can operate in tandem mass spectrometry and allows a full scan of all produced ions and gives complete information about the sample composition such as MRM and MS2 mode:

• MS2 is a tandem mass spectrometry scan mode where we can detect the product ion produced from one selected parent ion, it allows to increase signal to noise ratio and consequently the sensitivity.

• MRM is very specific mode where both analyzers are used to select one parent ion and one specific fragment, this mode offers a very high sensitivity by reducing completely the noise with high specificity with the double selective mode. As validation method all extracts were triplicated and the optimal conditions were applied to two different Henna leaves from two different regions in Morocco.

Comparative

Introduction

Considering their minimal side effects [START_REF] Agyare | An ethnopharmacological survey of medicinal plants traditionally used for cancer treatment in the Ashanti region, Ghana[END_REF] , nowadays, the use of natural products in traditional medicine for the treatment and prevention of disease and wound healing has become a good alternative to chemical drugs. Mediterranean as well as Asian countries have been developing

since centuries, effective treatment strategies based on natural products, to treat a large number of diseases.

Extraction is the first and the most crucial step to obtain active ingredients from natural products. The amount of the compounds of interest present in the natural product is in general moderately very low and recovery depends on the extraction method, the solvent used, time and temperature.

There are several extraction methods: conventional ones such as maceration, Soxhlet, percolation and unconventional methods like Ultrasound-assisted, supercritical fluid, pressurized liquid extraction and, microwave-assisted extraction [START_REF] Đukić | Conventional and unconventional extraction methods applied to the plant, Thymus serpyllum L. IOP[END_REF][START_REF] Zhang | Techniques for extraction and isolation of natural products: a comprehensive review[END_REF] .

Supercritical fluids (SCF) are advantageous to extract constituents from solid matrices of natural products, of food and pharmaceutical significance, efficiently above critical conditions of temperature and pressure but below the pressure required to attain condensation wherein liquid and gaseous phases form a single phase of supercritical fluid with the chemical properties of liquid and physical properties of gas (Figure 12), the properties of the fluid being dependent on its density as referred in Table 1. At the end of extraction SCFs escape out as gases leaving behind pure extract [START_REF] Abbas | A Review on Supercritical Fluid Extraction as New Analytical Method[END_REF][START_REF]Green Solvents I: Properties and Applications in Chemistry[END_REF][START_REF] Nalawade | Supercritical carbon dioxide as a green solvent for processing polymer melts: Processing aspects and applications[END_REF] . Solubility studies of the important chemical constituents from Henna in supercritical CO2 is a good support to the present study [START_REF] Sv | UV/Vis spectra and solubility of some naphthoquinones, and the extraction behavior of plumbagin from Plumbago scandens roots in supercritical CO2[END_REF] . 14), stems, seeds and, roots contain many bioactive secondary metabolites for diversified pharmacological activities. Research on henna (Lawsonia inermis L.) dates back to many decades to isolate and identify its constituents and many studies proved its pharmacological significance as an antimicrobial agent [START_REF] Hanke | The Biochemistry and Physiology of Henna (Lawsonia alba): its use as a Remedy for Intestinal Amoebiasis[END_REF] .

Review of literature reveals isolation of constituents like lawsone, lawsonicin, lawsonadeem, which is known for its traditional use as red-orange natural dye in cosmetics 108 .

Other naphthoquinone compounds like Plumbagin (Figure 16A), Lapachol (Figure 16B) have also been proven to have large and diversified pharmacological activities.

Plumbagin, chemically 5-hydroxy-2-methyl-1, 4-naphthoquinone is a medicinal plant derived quinoid originally found in the roots of Plumbago zeylanica and is a vitamin-K analogue. Other studies reported its effect on the amelioration of memory dysfunction in streptozotocin induced Alzheimer's disease 109 . In addition to that, it also inhibits NF-kappa B activation in lymphocytes that leads to an anti-inflammatory effect 110 .

Lapachol

Material and methods

a. Raw Material

Dried powdered samples of Henna leaves (Lawsonia inermis L.) from Tazarine (T) and Foum Zguid (FZ) regions of Morocco used for the study were kindly provided by women cooperatives (Ait Matan and La Batha) in Morocco and identified by Professor Zoubida Charrouf. The experimental run with higher yield was repeated in triplicate for confirmation.

Optimized conditions were applied to extract chemical constituents from FZ leaves.

For both PLE and SFE, α value was calculated presuming the rotatability of study domain around the central point. The extreme levels of α were determined using the formulae:

-α = 0-[(0-1) × 1.414] +α = 0+ [(+1-0) × 1.414] • Effect of co-solvent
Since supercritical carbon dioxide is non-polar and specific to non-polar compounds, it was Ionization was achieved using electrospray in the negative mode for Plumbagin and Lapachol respectively.

MRM mode was used to quantify the analytes of interest. The detection of precursor ion and major fragment ion of the standards and MRM acquisition was done monitoring the 187m/z➔159m/z transition for Plumbagin in negative mode and 241m/z➔186m/z transition for Lapachol in negative mode. The method was optimized in the infusion mode by injecting each standard.

ii. Standard Calibration Curves

Standard stock solutions of Plumbagin and Lapachol were prepared in the mobile phase at the concentration of 1 mg/ mL, and then several dilutions were done before the analysis: 0.005-0.05 mg/ mL.

iii. Preparation of extracts for analysis

Extracts from different methods of extraction under study were diluted to a final concentration of 0.05 mg/ mL in mobile phase for quantification.

f. Data Analysis

The analysis was done by extracting the value of the peak area for m/z of Plumbagin and Lapachol (187 and 241 respectively) analyzed in triplicate, then the standard calibration curves were established. The final content of Plumbagin and Lapachol were estimated in each sample using the calibration curve equation after validation.

Results and discussion

a. Moisture Analysis

Analysis of moisture percentage of the samples is important especially during supercritical fluid extraction owing to non-polarity of supercritical CO2 that could interfere and exert impact on the global extraction yield, due to alterations in surface tension and contact angle of solvent and sample thereby inhibiting its penetration to internal pores of sample matrix. Also, presence of moisture in the extracts shall lead to microbial growth. The results confirm slightly high amount of moisture in FZ leaves than T leaves (Table 4) due to high average temperatures and drier climate of Foum Zguid region than Tazarine region. Thus, it can be explained that henna leaves adapt to climate modifications by controlling transpiration and conserving its own water.

c. Pressurized Liquid Extraction

Experimental matrix with response is represented in Table 5. PLE was initially operated at 5 static cycles at 5 minutes' static time, 100% volume for leaves from the two regions to construct experimental matrix for CCD. The proposed experimental design based on the interpretation of Pareto chart (Figure 17A) depicted better yield when operated under less cycles at higher volumes of solvent at -1, +1 condition of factorial region (Experimental run 1). The negative influence of static cycles and interaction effect with solvent volume could be due to the hindrance of non-polar constituents like lipid components that effect the extraction yield % and depleted the recovery of the chemical constituents. Also, as observed, higher volume of ethanol derived more extract owing to the solute-sample interactions to expel compounds of interest that are soluble in ethanol. The optimum conditions were applied to henna leaves from two regions of Morocco (Tazarine and Foum Zguid), and the results depicted a higher yield (+5%) in Tazarine leaves.

Since pressure of the equipment is constant, extraction time could be taken as another factor to study its impact on the extraction yield since the considered factors did not show significant influence on the response. Also, experimental runs with initial removal of lipophilic constituents with solvents like methylene chloride shall be interesting research to assess analytes of interest. Considering other polar solvents similar to ethanol for extraction could also be an interesting research. The Standard deviation (SD) of central point is considered to be uniformly distributed around the design space as calculated by the software. Average flow rate of CO2 was 1.22 × 10 -2 kg/ s. Adjunction of co-solvent yielded obvious results with higher yields (Table 7) due to the extracting capability of wide range of chemical constituents and enhanced solvent strength of ScCO2 by ethanol. However respecting laboratory conditions, only Supercritical CO2 was used for optimization. The two factors-Pressure and temperature attributes to the solvent strength of Supercritical fluid extraction as well its density and hence were chosen for the circumscribed central composite design. Pareto chart of effects (Figure 17B) confirm significant positive influence of pressure on the response. Response surface curves (Figures 18A and18B) of both linear and quadratic models depict that higher pressure leads to higher extraction yield at almost all levels of temperature. However, in the given range of temperature and the interaction effects of two factors do not elicit significant influence while temperature being negative factor on the recovery of the extract from henna samples. Statistical analysis by One-way ANOVA (Tables 89) show significance of both linear and quadratic models (Fstat > Ftheoretical, 95% significance level). The coefficients of determination for both linear and quadratic models interpret good fit of linear model for predicting the response (global extraction yield %).

d. Supercritical Fluid Extraction

e. Comparison of extraction yield of Soxhlet with Supercritical Fluid Extraction

Table 10 has drawn interesting results. Soxhlet extraction with hexane, yielded higher lipid content than SFE. Even though the polarity indices of both CO2 and hexane are similar and both solvents are non-polar, due to the selectivity constraints of ScCO2 under specific conditions of temperature and pressure which alters its solvating power, resulted in high global yields by hexane. On the other hand, addition of modifier resulted in increased solvent strength of ScCO2 yielded higher amount of extract than hexane. In contrast to global yields, selectivity of ScCO2 towards naphthoquinones of interest with non-polar methyl group of plumbagin and 3-methyl-2butenylic group of Lapachol resulted in higher content of by SFE than Soxhlet extraction.

f. Global yield % of different samples of henna leaves

Based on the results (Figure 19) it was decided to focus on the extracts of Tazarine leaves owing to high global yield with respect to all the extracts from different techniques, to perform the quantification of the two naphthoquinones based on the compounds' interesting activity on melanoma 112,113 .

Figure 19 : Summary of global extraction yield of different extraction techniques

Global yield of Supercritical Fluid Extraction at +α, 0 axial level, global yield of Pressurized Liquid Extraction at (-1, +1) based on Central Composite Design which showed better yield.

g. Results of quantification by UHPLC-ESI-MS-MS

The method was optimized and the following conditions were normalized for the two compounds in Tazarine leaves.

i. Mass spectrometry fragmentation

Deprotonation of the parent molecule (-OH group) of both compounds in negative mode led to molecular ion peak of one mass unit less than molecular mass.

The product ion [M-H-C3H4

] m/z 185.9 appears as the highest and the most stable ion product in the mass spectrum of Lapachol with molecular formula C15H14O3 ([M-H] -, m/z) in MS2 mode. This is explained by the two consecutive fragmentation steps and loss of 55 mass units (Figure 20A and Figure 20B).

ii. Calibration curve validation

The regression equations from the standard calibration curves used for the quantification of Plumbagin and Lapachol in Lawsonia inermis L. were y = 2 × 10 6 x + 4089.5 and y = 4 × 10 6

x + 18107 respectively.

iii. Quantification results

Results of the analyses showed significant difference in the concentration of Plumbagin and

Lapachol in the same sample and obtained by different methods of extraction. PLE and SFE results were of the better conditions based on global yield % from circumscribed Central Composite Design.

Thus, multivariate analysis using design of experiments demonstrated influence of interaction effects rather than single factor experiments to optimize the extraction and quantitation.

Conclusion

In a nutshell it can be concluded that Pressurized Liquid Extraction yielded high global yield of both samples from FZ and T regions. Supercritical Fluid Extraction resulted with lesser global yields than Soxhlet extraction using hexane drawing attention towards analysis of different compounds like lipids.

Central Composite Design, a robust statistical approach, projected influence of the significant factor on the desired output for both SFE and PLE.

Geographical divergence has impact on the global extract % with T region deriving significant Industrial Crops and Products, 158, 112960.

Chapter 3 Geographical study of

Lawsonia inermis L. from different Moroccan regions using Principal component analysis

Abstract

Lawsonia inermis L, commonly known as Henna belongs to the family Lythraceae. It is cultivated in tropical, sub-tropical and warm temperate areas in particular to North Africa and Asia. It's a plant with a wide spectrum of biological activities; it's widely used in Moroccan traditions especially for cosmetic purpose in all special occasions such as weddings, birth or circumcisions. This usage is related to its brown dark color and the main compound responsible of this coloration is lawsone a naphthoquinone.

Generally, the humidity and temperature as well as the nature of the soil influence the accumulation of secondary metabolites in a plant. In this work, it was demonstrated for the first time that a small change in temperature alone induces a large change in the concentration of lawsone in henna in geographically very close regions.

This study aimed to evaluate the concentration of Lawsone in planted Henna (Lawsonia inermis L) from four different cities in the South Eastern Morocco, namely Tazzarine, Foum Zguid, Zaroga and Errachidia. Quantitative analysis by High Performance Liquid Chromatography-Electrosray Ionisation-Qtrap-Mass Spectrometry (HPLC-ESI-Qtrap-MS) showed that Foum Zguid is characterized by the highest level of Lawsone (0.6%). Errachidia has the lowest amount of Lawsone. After statistical analysis, important correlation between temperature and lawsone concentration was demonstrated. In particular 2 locations showed a very high pourcentage of lawsone; Foum Zguid and Zagora that were more in the south. In the opposite, there was no significant correlation with the precipitation. Samples with humid climate and high pluviometry showed lower content of Lawsone.

The analysis method was validated and principal component analysis on quantitative data was performed, correlation between, temperature, pluviometry and Lawsone content was established.

Key words:

Lawsonia inermis L, Lawsone, HPLC-ESI-QTRAP, Mass spectrometry, MRM, Principal, Component analysis, Classification, Correlation, Morocco.

Introduction

Morocco, country of North Africa, spans from the Mediterranean Sea and Atlantic Ocean. It includes a rich and large nature including mountains areas in the interior body of the kingdom to the Sahara Desert in the south east and the extreme south. This richness of its nature offers a great biodiversity of medicinal and aromatic plants. This biodiversity has opened the field to the development of phytotherapy and aromatherapy in Morocco but also in other countries using these plants. additionally, this natural richness has contributed to the development of new therapeutic and or preventive strategies with less toxicity.

Mediterranean plants have been used for treatment of several diseases since time immemorial 114 . Artemesia herba alba, Nigella sativa, Thymus vulgaris and others were considered as remedies for diabetes mellitus 115,116 . Moreover, different plants were found to have an anti-cancerous activity, such as Olea europae, Salvia officinalis L, Cicer arietinum and Vicia faba that were used for the treatment of lung cancer 117-120 .

Among the large list of plants growing in Morocco, Lawsonia inermis L, has drown our attention by its wide spectrum of biological activities. It's a plant widely used in Moroccan traditions especially for cosmetic purpose in all special occasions such as weddings, birth or circumcisions where women get henna on their hair, hands and feet 121 .

The origin of this plant is the Babylon region (the Mesopotamia) 3000-1500 before J-C. It was used as a sign of richness in the noble families and especially for the preparation of the bride 1 .

Later it was migrated to south Asia especially India and in Egypt where the pharaohs used it as a cosmetic agent for dying their feet and hands. The Egyptian Jewish was also using this plant as perfume mixed with oils from other plants.

After the Islam era, the plant was becoming widely used in the Middle East and North Africa.

It has several appellations based on the region: in North Africa, it is Henna or Hinna; in India, it is Mehendi; in ancient Egypt, it was pouker, in Hebrew it is kopher, but the common purpose of it usage is its desired dark color used in cosmetic [START_REF] Aubaile-Sallenave | Les voyages du Henné[END_REF] . As previously reported, the responsible compound of the coloring is Lawsone 122,123 .The concentration of Lawsone in L. inermis L varies from a region to another depending on the conditions of the culture and weather. Lawsone (Figure 24) is the major compound present in the leaves of L. inermis L responsible for its specific color used for dying hair, skin and nails [START_REF] Chaudhary | Lawsonia inermis Linnaeus: A Phytopharmacological Review[END_REF] . In addition to the cosmetic use, purified

Lawsone is also widely used in the coloration of cotton and silk fabric 124 . phenolics, and phenolic glycosides [START_REF] Liou | 1,5-Diphenylpent-3-en-1-ynes and methyl naphthalene carboxylates from Lawsonia inermis and their anti-inflammatory activity[END_REF]106,[125][126][127] . Among this wide variety of secondary metabolites, several studies focused on naphthoquinone derivatives. This interest stands for the reported anti-microbial effect of Lawone; 2-hydroxy-1,4-naphthoquinone 128 , as well as the leishmanicidal and antiviral agent of Lapachol; 4-hydroxy-3-(3-méthylbut-2-ényl) naphtaléne-1,2-dione 129 and finally the antimetastatic melanoma in vitro effect of Plumbagin; 5-hydroxy-2-methyl-1,4-naphthoquinone [START_REF] Alem | Elucidation of the effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via MAPK signalling pathway[END_REF] .

In parallel, its biological activities have been widely described in the literature 13,120,125,125,130- 132 . Its antioxidant potential has been found to be higher than the Vitamin E and α-tocopherol 120 . The same study showed that L. inermis L extract displayed a cytotoxic effect against liver cancer cell line. Furthermore, this species constitutes a high antibacterial candidate against several pathogens: Staphylococcus aureus, Bacillus cereus, Corynebacterium pseudotuberculosis, Klebsiella pneumonia, Escherichia coli and Salmonella enterica serovar typhi 128,132 .

The aim of this work is to analyze and quantify Lawsone in Moroccan L. inermis L that grows specifically in the south of Morocco and compare its chemical profile with other reported.

Nevertheless, the difficulty in quantifying lawsone is mainly due to the presence of several quinones concurrently in the extract, which leads to an error in estimating the accurate quantity of this compound. Moreover, several works had reported this difficulty and tried to estimate the relative error encountered in the quantification of lawsone using LC-UV-VIS 133 as an analytical technique or HTPLC 134,135 . Another problem in the literature dealt with the quality of the plant after the bleaching operation carried out before use in cosmetics and which can seriously affect the concentration of Lawsone 104 .

In this work, a simple and unique specific method is used for the first time to quantify lawsone in Lawsonia inersmis L. from four regions renowned to be the leading ones in the production 25) and to compare them using principal component analysis. The use of tandem mass spectrometry has enabled the development of a specific and sensitive method for the quantification of lawsone in the different samples from the four regions and the correlation of these data with the geographic data that we were able to collect. Generally, the humidity and temperature as well as the nature of the soil influence the accumulation of secondary metabolites in a plant. In this work, it was demonstrated for the first time that a small change in temperature alone induces a large change in the concentration of lawsone in henna in geographically very close regions.

Material and methods

a. Chemical and reagents

Analytical grade ethanol and methanol purchased from Sigma Aldrich were used throughout the extraction and analysis process. Lawsone was purchased from Sigma Aldrich Chemical Corp. (Saint-Quentin-Fallavier, France). Ultra-pure water( was obtained with an Elga apparatus.

b. Plant material and geographical zone of culture

Lawsonia inermis L. was kindly provided fresh from certified Moroccan cooperatives: The Tazarine henna was purchased from Ait Matan cooperative, Foum Zguid, Er Rachidia and Zagora samples were purchased from La Batha cooperative. The harvest and drying process A: Foum Zguid B: Er-Rachidia C: Tazarine D: Zagora

were same for all the samples. The choice of geographic areas for growing henna was made based on the popularity of its quality by locals. It is known that these 4 regions are the most producers of the best quality of Henna in Morocco.

c. Climate data collection

Climate data (temperature and rainfall) were collected from website climate data .org. and distance from ocean was calculated using the web calculator 136 . The collected data are showed in table 12 and figure 27.

d. Extraction

The henna leaves were freshly harvested, dried and grounded. The ultrasound-assisted extraction was performed at room temperature in an ultrasonic bath device with 46KH3 transducers annealed to the bottom. However, a slight increase of the temperature to reach 37°C was registered due to the ultrasound's frictions. 10 g of dried henna leaves powder were placed in an Erlenmeyer flask (500 mL) and mixed with 100 mL of ethanol and sonicated for 45 minutes. This process was done simultaneously for all the samples to get the same extraction conditions and to avoid the influence of temperature variation. The suspension was recovered and a second extraction was carried out for 45 additional minutes with 100 ml ethanol. Then, the extracts obtained were filtered and evaporated with a rotavapor for 4 hours. Finally, the extracts were lyophilized. The lyophilizate was recovered with mobile phase prior to LC-MS-MS analysis.

e. HPLC-ESI-QTrap-MS conditions

A Shimadzu gradient LC system (CBM-20A) interfaced to a Qtrap 5500 mass spectrometer equipped with a turboV electrospray source (SCIEX API 5500) was used for LC-ESI-MS/MS analysis. The separation of the compound was carried out on a C18 Phenomenex 100x1mm column with 3 µm particles, 110 Å. The flow rate was 100 µl/min. Henna extract was dissolved in 10/90 (H2O/Meth OH). Afterward, 5 µl of sample volume was injected to the HPLC system.

f. Standard preparation

Standard stock solutions of lawsone were prepared in the mobile phase at the concentration of 1mg/ml, and then several dilutions were done before the analysis: 2.5-5-10-20 µg/ml.

g. Data Analysis

The analysis was done by extracting the value of area under curve for m/z (175) analyzed in triplicate, then the standard calibration curves were established.

The final content of lawsone was estimated in each sample using the calibration curve equation after validation.

h. Statistical analysis

A one-way analysis of variance (ANOVA) followed by the Tukey post hoc test were used to analyse statistical significance (p < 0.05) using Xlstat software (Addinsoft 2020. XLSTAT statistical and data analysis solution. NEW York, USA). The analysis was carried out at least in triplicate. Principal component analysis (PCA) was performed on lawsone concentration calculated from every individual sample, temperature of the four regions (°C), rainfall parameters (mm) and distance from ocean (km).

Results and discussion

a. Geographical analysis:

Morocco is characterized by a very marked spatial and temporal variability in precipitation. The northwest is on average more watered than the rest of the Kingdom. Even in this region, the average annual cumulative precipitation varies considerably. For example, it can reach more than 800 mm on the reliefs, while it does not exceed 300 mm on the surrounding plains.

The Moroccan climate is mostly arid. Two exceptions are recorded: the far north where the climate is subhumid, and the Atlas region where the climate is humid. The further we go to the south and south-east of Morocco, the drier the climate is.

This variability in temperature and precipitation is the main source of plant wealth found there.

Additionally, we can notice the direct effect that this variability in temperature and humidity can have on the chemical composition, mainly the secondary metabolites of its plant kingdom.

Secondary metabolites are the main source of the pharmaceuticals and additives in all the aromatic and medicinal plants. They also play an important role in the plant defense from biotic and abiotic stress. The biosynthesis of this metabolites is related to the conditions of the growth.

Among the influencers on this production there are high temperature, cold, salinity, light, phytohormones 138 .

The four regions selected for the quantification of lawsone in Lawsonia inermis L, all belong to the central-east region of Morocco (figure 26). Despite this, they all have different mean temperatures and pluviometry; these data were considered to study the influence of the climate on the lawsone content in the different samples.

The climate data for the 4 regions are summarized in Figure 26 and table 12.

The four regions show very close temperature in the summer season but a very different precipitation during the year. Tazarine is by far the region with the highest precipitation attempting a mean of 70 mm in April while Foum Zguid, Er Rachidia and Zagora the mean precipitation value doesn't exceed 20 mm Foum Zguid is the hottest region with a mean value of 35°C (95 °F) in the summer, while Er Rachidia has registered the lowest temperature around 30°C in the summer.

b. Stability

Before performing the quantitative analyzes in LC-MS-MS, the lawsone stability study was carried out by preparing standard lawsone solutions in an ethanol / water mixture. The solutions were exposed to daylight at room temperature during four days. Meanwhile, daily analysis using the developed method in HPLC-UV (244 nm) indicated that the recoveries of the target peak of lawsone decreased over the time. We noted a decrease in peak area of 25% after 48 hours and 40% after 4 days (Figure 28). The validation results indicated that the developed method can provide accurate quantification results within 36 hours post-preparation. This stability test allowed us to develop the quantification analysis conditions afterwards with the use of shaded vials and thermostatically controlled autosampler in the HPLC system.

c. Liquid chromatography optimization

Plant extracts are always very rich in various compounds, including a large number of isomers.

Despite the use of a very specific mass spectrometry method, the development and optimization of a separation method is essential to fill a quantitative analysis. To optimize the analytical method, we have worked first on HPLC-UV (244 nm) using Lawsone standard solutions.

Reverse Phase Chromatography was the preferred mode because of the low polarity of Lawsone. Initially a column of 50 mm was used for the separation, but the shape and retention time of lawsone peak was unsatisfactory (2.5 min), we decided to use a 100 mm column where retention time of lawsone peak was registered at 4.8 minutes.

Following a comparison of the chromatographic efficiency and asymmetry of the lawsone peak, the best compromise between chromatographic resolution, signal intensity, and time of analysis was achieved using a gradient elution composed of MeOH and Water with a gradient slope of 7.8%/min of organic solvent. This program led to narrower peak of Lawsone with higher efficiency than the one that was obtained with a slope of 2,5% (Figure 29). The gradient elution was set as the following: 0-1 min: 70% MeOH and 30% H2O; 1-8 min:100% MeOH; 8-14 min; 14-15 min: 70% MeOH and 30% H2O and 15-20 min: 70% MeOH and 30% H2O.

d. Mass spectrometry optimization ions

Due to its high specificity and sensitivity, liquid chromatography coupled to mass spectrometry was used for the quantitative analysis of lawsone using an ESI-QTrap-MS instrument.

To optimize mass spectrometer source parameters, declustering potential, collision energy, and collision cell exit potential (table 13), Lawsone standard solution was injected in the ESI-Qtrap-MS at the concentration of 1, 25 µg/ml. A simple mass screening, followed by a MS2 analysis was performed to determine different fragments in the positive ionization mode (figure 30).

The multi-reaction monitoring mode (MRM) was also performed and different transition were followed in order to increase the LOD and LOQ.

The signal to noise ratio was calculated experimentally from both chromatogram in MS2 and MRM mode at the lowest concentration from the calibration curve (1, 25 µg/ml), the S/N of the peak at 4, 8 min was found equal to 16 for MS2 and to 23 for MRM (Figure 31).

e. Calibration curve

The calibration curves were established to quantify lawsone in the four samples of henna leaves harvested from different regions of Morocco.

Three independent calibrators for each concentration level were prepared (1.25-2.5-5-10-25 µg/ml). The linearity of response of the LC/MS system across the selected concentration range was determined by linear regression analysis in MS2 and MRM modes.

In all cases linear regression analysis of the curves afforded determination coefficients r 2 > 0.96 indicating excellent linearity of the method within the concentration ranges. Also, student test on the slope and the origin was validated.

Fisher test on both curves showed a value higher than F0, proving the significance of regression.

f. Quantification

The LOD and LOQ were determined experimentally using the S/N Ratio, we estimated the LOQ to 0, 76 µg/ml in MS2 and 0, 54 µg/ml in MRM.

The calibration curve obtained with the MS2 mode was used for the calculation of the concentration in the four regions. Foum Zguid has shown the highest (0.6%) amount of Lawsone in the extract analyzed (Figure 32).

The quantification results are expressed as the mean of content ratio mass/mass calculated with the calibration curve obtained with the developed HPLC-MS-MS method

Our results are in accordance with previous study that quantified Lawsone in Henna leaves in Egypt. Six samples were quantified and the range of Lawsone concentration was found between 0.004 and 0.608 g/100 g of dried material obtained using high performance thin layer chromatography 139,140 .

g. Principal component analysis

The quantitative data were again analyzed using Principal Component Analysis (PCA) with the aim to differentiate the samples from each other and evaluate which variables among temperature precipitation or distance from ocean, influences their location in the 2D space. As evidenced, samples are separated along first component in two main groups: first group, (in the highest negative values), is composed by samples collected in Errachidia and Tazarine, the second one, (in the highest positive values), by those collected in Zagora and Foum Zguid (Figure 34A).

In order to evaluate the influence of each variable on the classification of the samples, the loading plot obtained for the same dataset was then studied and results are shown in Figure 33B.

As shown, along the highest positive values of PC1 are located the regions with high mean temperature in the space corresponding to the samples collected in Foum Zguid and Zagora that showed. By contrast, the main information able to discriminate samples from Errachidia and Tazarine is due to the variable pluviometry which is located through the direction of the samples.

Conclusion

In this work, the discrimination of lawsone content in Lawsonia inermis L. in four regions from South east of Morocco was presented.

A LC-MS/MS targeted quantitative analysis for lawsone estimation on Lawsonia Inermis L.

samples collected in the different areas. After statistical analyses, important correlation between temperature and lawsone concentration was demonstrated. In particular two regions showed a very high % of lawsone; Foum Zguid and Zagora that were more in the south. In the opposite there was no significant correlation with the precipitation. Samples with very humid climate and high pluviometry showed lower content of Lawsone. • Melanocytes, which produce the melanin responsible for the pigmentation of the skin.

Chapter 4 Elucidation of the effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via MAPK signaling pathway

Synthetic melanin turnover accumulates in granules called melanosomes. These accumulate near the nucleus of keratinocytes and form a pigmentary shield that protects the nucleus against ultraviolet rays (Figure 35).

• Langheran cells, which participate in the immune system of the skin.

The epidermis also consists of five layers (Figure 36) which are as follows 142,143 :

• Corneal layer (stratum corneum), composed of corneocytes, results of the final phase of mutation of keratinocytes that progressively rise from the basal layer, and epidermal lipids. Its lower third is a real protective barrier against exogenous factors (pollution, sun, cold) and endogenous water loss.

The dermis

Connective tissue, which supports the epidermis, protects the vascular network and nerve fibers.

The dermis is divided into two layers 143 :

• Papillary dermis (superficial dermis): an intermediate layer rich in nerve endings and in permanent symbiosis with the epidermis, from which it is separated by the dermal-epidermal junction.

• Reticular dermis (deep and medium dermis): a dense connective tissue composed of a several elastic fibers.

It has different types of cells:

• Fibroblasts: cells that synthesize collagen, a protein essential to the elasticity of tissues.

• Histiocytes and mast cells: play an important role in the immune reactions of the skin.

The hypodermis

Adipose tissue lying under the dermis. It is crossed by the vessels and the nerves arriving in the dermis. It plays several roles:

• Protector, it serves as a buffer between the dermis and bones.

• Thermal isolator.

• Morphological, it shapes the silhouette according to the age, the sex, the nutritional state of the individual.

• Energetic, by storing fat.

II. Skin pigmentation

It is the mixture of pigments that gives a color of the skin. Thus, derivatives of hemoglobin or the abnormal presence of pigment of endogenous or exogenous origin modify the hue of the cutaneous tissue. Thickening of the epidermis can also cause color variations. However, most of the pigmentation of the skin, hair and eyes results from quantitative and qualitative variations of the melanic pigment.

Melanic pigmentation is genetically predetermined. However, it can be regulated by ultraviolet (UV) radiation, as well as by many agents (hormones, peptides, chemical mediators) that are able to stimulate or inhibit skin pigmentation.

The main function of differentiated melanocytes is the synthesis of melanins or melanogenesis.

This process involves different enzymes that catalyze each of the reactions leading to the formation of melanin pigments in specialized organelles called melanosomes 144 .

Types of melanin: In general, melanins correspond in humans to a mixture of eumelanines and phaeomelanines in different proportions. Eumelanins are melanins of brown or black color, high molecular weight, insoluble in most solvents. Formed by the polymerization of several hundreds of phenol radicals oxidized to quinone, they cyclize to form a chemical body completely absorbing light, hence their black or dark color. Paleomelanins are characterized by their yellow-orange color, they are soluble in bases. They contain nitrogen and sulfur and come from the oxidative polymerization of cystéinyldopas.

The eumelanins and the phaeomelanins come from the enzymatic transformation of tyrosinase into dihydroxyphenylalanine (dopa) and then into dopaquinone under the action of tyrosinase.

Subsequently, synthetic pathways diverge, involving either the tyrosinase-related protein (TRP)

1 and TRP2 enzymes in eumelanogenesis, or the incorporation of sulfur derivatives for phaeomelanogenesis (figure 37).

III. Melanoma Cancer

A melanoma is a disease of skin cells called melanocytes. It develops from an initially normal cell that transforms and multiplies anarchically to form a lesion called malignant tumor Melanoma is a dangerous skin cancer when diagnosed at an advanced or metastatic stage because there is no effective treatment. At the metastatic stage, the survival of the patients does not exceed 9 months in half of the cases. Although it accounts for only 4% of all skin cancers, melanoma accounts for 80% of deaths from this type of cancer 146 .

Types of Melanoma :

Melanoma can invade nearby tissues and destroy them. It can also spread (metastasize) to other parts of the body. Melanoma is also called skin melanoma or malignant melanoma of the skin.

There are 4 main types of melanoma: Superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, and Acral lentiginous melanoma.

Superficial spreading melanoma: Extensive superficial melanoma usually occurs in the central part of the body (trunk), arms and legs. It tends to appear on the back of the man and on the legs of the woman, and the upper back in both. It could extend outward, horizontally (radial growth), over the entire surface of the skin. But it can also begin to descend deep into the skin (vertical growth). It is often flat and thin (less than 1 mm thick) with irregular contours.

Superficial spreading melanoma is of various colors and may have different shades of red, blue, brown, black, gray and white. Sometimes it starts from a mole already existing on the skin.

Nodular melanoma descends deeply into the skin. It grows and spreads faster than other types of melanoma. It is in the form of a raised mass that protrudes from the surface of the skin (polypoid), and may look like a mushroom with a stem or head (pedicled). Nodular melanoma is usually black, but it can be red, pink, or the same color as your skin. It usually occurs on the face, chest, or back. It can be observed on areas of the skin not exposed to the sun.

Lentigo malignant melanoma usually occurs in areas of skin that are regularly exposed to the unprotected sun, such as the face, ears, and arms. It usually appears in the form of a large flat plate of ocher or brown color, the contours of which are irregular. It tends to become darker when growing and has several shades of brown or black. It often originates from an in-situ tumor called lentigo maligna, which is an early form of tumor present only on the upper or outer layer of the skin (epidermis). Malignant lentigo-like melanoma usually extends outward over the entire surface of the skin for many years before it begins to sink deep into the skin.

Acral lentiginous melanoma: Lentiginous melanoma of the extremities occurs most often in dark-skinned people, such as those of African, Asian and Hispanic origin. It is not related to sun exposure. The Acral lentiginous melanoma appears as a small discolored flat spot that often has a dark brown or black color. It usually extends outwards, over the entire surface of the skin, for a long time before starting to descend deep into the skin. It usually occurs on the soles of the feet, on the palms of the hands, or under the nails.

Targeted therapies

Various genetic anomalies have been identified in melanoma (Figure 38) 147 Activating mutations or amplification of the c-KIT gene (10%)

• Activating mutations of the NRAS gene (20%)

• Activating mutations of the BRAF gene (50%)

• Mutations or deletions of the PTEN gene (50%)

• Activating mutations or amplification of the AKT gene (60%)

The identification of these anomalies led to conclude the existence of several "subtypes" of melanoma, for which specific inhibitors have been developed in more targeted action.

Mutations in the BRAF gene are found in 30-70% of melanomas in Caucasians. Their presence seems to be correlated with the phototype. They are more common in light-skinned individuals, younger people (under 55 years of age), while NRAS mutations are more common in older people 148 . In 90% of cases, this is the V600EBRaf mutation. This mutation leads to a constitutive activation of the MAP kinase pathway. The other mutations identified are V600k BRAF and V600D / V600R BRAF. They represent respectively 16 and 3% of all BRAF mutations. There is also a minor group of mutations occurring on BRAF but on a position other than 600. These mutated forms require the presence of CRAF to be active.

PI3K is a kinase composed of a complex of two heterodimer-forming proteins. One of the subunits has regulatory activity: p85. The other has a catalytic role: p110. They can be activated either by a tyrosine kinase receptor or by Ras. It displaces a phosphate of the molecule of ATP on the PIP2, one thus obtains PIP3. PTEN is a tumor suppressor gene. It negatively regulates the signaling path via PI3K. It can be inactivated and this is the case in 30% of cell lines and in 10% of melanomas. AKT is a proto-oncogene intervening downstream of mTOR. 50% of melanomas have a constitutive activity of AKT.

PTEN is a tumor suppressor gene. It negatively regulates the path of signaling via PI3K. It can be inactivated and this is the case in 30% of cell lines and in 10% of melanomas 149 .

Chemotherapy is reserved for melanoma with poor prognosis (stage IV). A large number of conventional chemotherapy agents have been tried without results on metastatic melanoma.

Among the substances tested, the platinum analogues (cisplatin and carboplatin) cause less than 10% of objective responses and the agents targeting microtubules (vinca alkaloids and taxanes) are part of poly-chemotherapies. Alkylating agents such as dacarbazine or temozolomide are currently the most widely used compounds despite very limited benefit for patients 150,151

Metastatic melanoma

Cutaneous melanoma is characterized by its high capacity for invasion and metastasis 152 .

Dissemination of tumor cells is the principal cause of mortality in tumor patients. Tumor cell invasion as well as metastasis formation is complex and multistep processes, in which tumor cells detach from the primary tumor, invade surrounding tissue and basement membranes, intravasate into the lymphatic or blood circulation, and, finally, adhere and extravasate in distant organs to form a secondary tumor.

The extracellular matrix (ECM) is the non-cellular component present within all tissues and organs, and provides not only essential physical scaffolding for the cellular constituents but also initiates crucial biochemical and biomechanical cues that are required for tissue morphogenesis,

IV. Elucidation of the effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via MAPK signaling pathway

Fatima-Zahra Alem, Meriem Bejaoui , Myra O. Villareal, Boutayna Rhourri-Frih, and Hiroko Isoda 1. Abstract Melanoma is the most dangerous form of skin cancer with a very poor prognosis. Melanoma develops when unrepaired DNA damage causes to skin cells to multiply and form malignant tumors. The current therapy is limited by the highly ability of this disease to metastasize rapidly.

Plumbagin is a naphthoquinone (5-hydroxy-2-methyl-1,4-naphthoquinone), isolated from the roots of medicinal plant Plumbago zeylanica, and it is widely present in Lawsonia inermis L. It has been shown that plumbagin has anti-proliferative and anti-invasion activities in various cancer cell lines, however, the anticancer and anti-metastatic effects of plumbagin are largely unknown against melanoma cells. In this study, we evaluated the effect of plumbagin on B16F10 murine melanoma cell line. Plumbagin decreased B16F10 cell viability as well as the migration, adhesion, and invasion. The molecular mechanism was studied, and plumbagin downregulated genes relevant in MAPK pathway, matrix metalloproteinases (MMP's), and cell adhesion. Furthermore, plumbagin elevated the expression of apoptosis and tumors suppressor genes, and genes significant in reactive oxygen species (ROS) response. Taken together, our findings suggest that plumbagin has an anti-invasion and anti-metastasis effect on melanoma cancer cells by acting on MAPK pathway and its related genes.

Keywords: Plumbagin, metastasis, MAPK pathway, invasion, migration, adhesion

Introduction

Melanoma a malignant neoplasm of melanocytes is responsible for the majority of skin cancer deaths despite including less than 5% of all cutaneous malignancies 154 . Even though invasive melanoma represents about 1% of all skin cancer, it results in most deaths and is considered to be the most aggressive type of cancer for which there are few effectives therapies 155,156 . The incidence of metastatic melanoma keeps increasing over the past years and according to the World Health Organization (WHO), the second leading cause of death in the world is cancer and around 132,000 melanoma skin cancers cases occur globally each year (4). Moreover, melanoma is one of the most deadly human cancers in western countries, and in the USA 96,480 new cases were diagnosed in 2019 and 7,230 deaths occurred 156,157 . Ultraviolet (UV) radiation is mostly the cause of the development of skin lesions that cause genetic abnormalities allowing the melanocytes, melanin producing cells to rapidly multiply and form malignant tumors. Genetic studies have supported the significance of the exposure to UV radiation in melanoma development and showed the presence of a very high somatic mutations comparing to other cancer 154,158 . It has been shown that mutations in the protein coding genes for cellular transduction pathways are involved in the progression, invasion, and survival of tumor cells in melanoma 159 . The most common mutations occurred during the progression of melanoma skin cancer are associated with mitogen-activated protein (MAP) kinase (MAPK) pathway which regulates cell proliferation and survival 160 . Mutations in MAPK in the gene encoding the serine-threonine protein kinase BRAF happens in 40 to 60% of melanoma cases, and around 7 to 8% of all cancers 161 . Mutations in PI3K and P53 was observed in patients with advanced stage of melanoma 160 . Matrix metalloproteinases (MMPs) play a relevant role in the metastasis of various types of cancer mainly melanoma by interacting with cell-matrix adhesion molecules and integrin promoting cell migration and metastasis formation 155,162 . MMP9 a matrix metalloproteinases, interfere in the resistance to anti-cancer drugs, and the activation of cell migration by forming a complex with the cell surface molecule CD44 163,164 . Hence, tumor metastasis is the leading cause of mortality in melanoma, however, despite being an aggressive disease with metastasis in the brain, lung, liver, and skin, there are few effective therapies 165 . The Food and Drug Administration (FDA) have approved two therapies to treat melanoma, a high-dose interleukin-2 and dacarbazine, yet a success rate of only 10 to 20% of the treated cases was observed 166,167 . Ipilimumab a monoclonal antibody that has a marketing authorization in the treatment of second-line metastatic melanoma improved overall survival in patients but the effect was associated with immune-mediated diarrhea 146,168 .

In this context, developing a drug with no undesirable side effects is becoming urgent in order to discover new cancer therapies. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) is an active compound found in several plants such as Plumbago zeylanica and Lawsonia inermis L. that has many biological properties. It has been reported that plumbagin ameliorated memory dysfunction in streptozotocin induced Alzheimer's disease and had as well an antiinflammatory effect by inhibiting NF-kappa B activation in lymphocytes 109,169 . Other studies found that plumbagin had a melanogenesis regulatory effect as it inhibited tyrosinase activity in B16F10 murine melanoma cells 170 . Nevertheless, plumbagin is well studied in cancer research because of its high activity against different cancer cell lines in vivo and in vitro 171,172 .

In this study we investigated the potential anti-metastatic effect of Plumbagin on B16F10 murine melanoma cells and SK-MEL-28 human melanoma cells by studying the adhesion, migration, and invasion in vitro. Moreover, global gene expression analysis was performed to elucidate the mechanism of action of plumbagin showing the regulation of MAPK pathway and the MMPs in B16F10.

Methods

a. Sample preparation

Plumbagin was obtained from Sigma Aldrich Chemical Corp (Saint-Quentin-Fallavier, France).

The sample was dissolved in dimethyl sulfoxide (DMSO) (stock solution: 1mg/ml) (SIGMA, Saint Louis, USA) and then diluted in the growth medium to obtain 0.1% of DMSO.

b. Cell culture and treatment

B16F10 murine melanoma cells were obtained from RIKEN, Tsukuba, Japan. Cells were cultured as a monolayer in Roswell Park memorial institute medium (RPMI 1640) (Gibco, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco, Karlsruhe, Germany) and 0.1% Penicillin/Streptomycin (Gibco, Karlsruhe, Germany). SK-MEL-28 human melanoma cell line were obtained from RIKEN, Tsukuba, Japan. Cells were cultured as a monolayer in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco, Karlsruhe, Germany) and 0.1% Penicillin/Streptomycin (Gibco, Karlsruhe, Germany).

The cells were kept under sterile conditions at 37 °C in 75-cm 2 flask (BD Falcon, England, UK) in a humidified atmosphere of 5% CO2. The viability of the cells was determined using trypan blue exclusion method.

c. Cell viability assay

Cell viability was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Dojindo, Kumamoto, Japan) or MTT assay. Cells were seeded into 96-well plates (3x10 4 cells/well) and incubated at 37 °C in a humidified atmosphere of 5% CO2 for 24 and 48 h. The medium was then replaced with a new one containing plumbagin at various concentrations (0-1-2-3-5 µM). 10 µL/well of MTT (5 mg/ml) was then added to the cells, and incubated for 8 h then 10% Sodium dodecyl sulfate (SDS) was added at 100 µL/well. The plates were incubated for overnight at 37 °C. The absorbance was measured at 570 nm using a microplate reader (Powerscan HT, NJ, USA). This experiment was run in triplicate.

d. Wound healing assay

Cells were seeded onto 6 well plates at a concentration of 5x10 5 cells/well. When a monolayer of cells is formed (90% confluency), a scratch was made in the middle of the well using sterile white tip. The cells were washed twice with phosphate buffered saline (PBS) to remove the unattached cells, and the medium was replaced with a new one containing plumbagin treatment (0-1-2 µM). Photos were taken before and after the treatment for 0, 6, 12, and 24 h using a contrast microscope (Leica Microsystems, Wetzlar, Germany). The results were validated by three times repetition.

e. Cell invasion assay

Invasion assay was performed using the 24-well Corning BioCoat Matrigel Invasion Chambers containing Falcon cell culture inserts (8 μm pore size PET membrane with a thin layer of Matrigel basement membrane matrix) (Corning, MA, USA), following the manufacturer's instructions. Cells (5×10 5 cells/ml), were seeded in a serum-free media onto the Matrigel-coated chambers while 10% FBS was added to the lower chamber as chemoattractant. After 24 h incubation, the lower chamber was stained with DAPI (Vector Laboratories, CA, USA), and the number of the cells that moved to the lower chamber was counted using Leica DMI-4000B fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Three fields in each sample were counted and the mean values from three independent experiments were used.

f. Cell adhesion assay

Cells were seeded onto 10 cm petri dish at a concentration of 3x10 5 cells/ml and incubated for 24 h at 37 °C. The medium was replaced with 2 µM plumbagin for 0, 12, 24, 48, and 72 h, then the cells were collected, trypsinized, and suspended (2x10 5 cells/ml) in a serum free RPMI 1640.

Human fibronectin cell ware plate (Corning Incorporated, New York, United States) were used where 100 µL of the cell suspension were seeded and incubated for 1 h at 37 °C. Medium was removed and the plates were washed three times with PBS to remove the unattached cells. MTT assay was then performed as described above. The experiment was run in triplicate.

g. RNA extraction

The total RNA was extracted using ISOGEN kit (Nippon Gene, Tokyo, Japan) following the manufacturer's instructions. Briefly, B16F10 was seeded onto 5 ml petri dish at a density of 5x10 5 per dish. Cells were allowed to attach for overnight, then the medium was removed and replaced with a fresh one with or without 2 µM plumbagin for 24 h. A washing with cold PBS was performed before the total RNA extraction. The total RNA was quantified using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Massachusetts, USA).

h. Microarray analysis

DNA microarray was performed using Affymetrix (Santa Clara, CA, USA) Gene Chip Mouse Genome 430 2.0 Array following the manufacturer's instructions. Total RNA was extracted as described above and the biotin-labeled aRNA was synthesized by in vitro transcription, then the purified aRNA (100 μg) was fragmented using the Gene Atlas 3' IVT Express Kit, and hybridized to the gene chip for 16 h at 45 °C. The chips were washed and stained in the Gene Atlas Fluidics Station 400 (Affymetrix, Santa Clara, USA) and the resulting image was scanned using the Gene Atlas Imaging Station (Affymetrix, Santa Clara, USA). Identification of the differentially expressed genes was performed using Transcriptome Analysis Console (TAC) Software (version 4.0.1) and the database for annotation, visualization and integrated discovery (DAVID) 173 .

i. Western blotting

B16F10 cells were seeded onto 5 cm petri dish at a concentration of 3x 10 5 cells/ ml and then treated with and without plumbagin (2 µM) for 12, 24, 48, and 72 h. Total protein was extracted using Radio-Immunoprecipitation Assay (RIPA) buffer (SIGMA, Saint Louis, USA) and protease inhibitor, following the manufacturer's instructions. The protein (10 µg) was resolved in 10% Sodium Dodecyl-Sulphate-Polyacrylamide Gel Electrophoresis (SDS PAGE) then transferred to a Polyvinylidene difluoride (PVDF) membrane (Merck Millipore, NJ, USA). The proteins were blotted with primary antibodies against MMP2, MMP9, and CD44 (Abcam, Cambridge, UK), at 4 °C for overnight, then washed and incubated with secondary antibody, IRDye 800CW Donkey anti-rabbit IgG (LI-COR, Inc., NE, USA) at room temperature (RT) for 40 min. The signal was detected using the Odyssey Fc Imaging System (LI-COR, Inc., NE, USA). All protein quantifications were normalized to GAPDH expression level (Santa Cruz, Biotechnology, Texas, USA). The experiment was done in triplicate.

j. Statistical analysis

Results were expressed as mean ± standard deviations (SD), and the statistical evaluation was performed using Student's t-test when two value sets were compared (control vs treated cells).

A P value of ≤0.05 was considered to be statistically significant.

Results

a. Plumbagin decreased the viability of B16F10 murine melanoma cells and SK-MEL-28 human melanoma cells

MTT assay was performed on B16F10 and SK-MEL-28 to check the cytotoxic effect of the sample. The results showed that plumbagin inhibited significantly the cell viability of B16F10 at dose and time-dependent manner (Figure 40A). A cytotoxic effect was observed after 24 and 48 h treatment with 5 µM plumbagin as the viability decreased up to 20%. For the following experiments, a lower concentration of the sample was chosen. Moreover, results showed the same tendency in SK-MEL-28 (Figure 40B). These results suggested that plumbagin decreased the viability of melanoma cell lines.

Figure 40 Effect of plumbagin on the viability of the cells in B16F10 murine melanoma cells (A), and in SK-MEL-28 (B).

Cell viability was assessed after 24 and 48 h treatment with various concentrations of plumbagin

b. Plumbagin inhibited the adhesion, migration and invasion of B16F10 murine melanoma cells and SK-MEL-28 human melanoma cells

Plumbagin metastasis effect was investigated in B16F10 and SK-MEL-28. A wound healing assay was carried out to oversee the cell-cell interaction, and the cell movement and migration.

The gap established in the middle of the confluent monolayer was completely closed in the untreated cells after 24 h, however after treatment with 2 µM plumbagin, an inhibition of the B16F10 cell migration was observed time-dependently (Figure 41). Moreover, an invasion assay was done to monitor the cell movement through the extracellular matrix (ECM) as invasiveness is a fundamental function of the metastatic cancer cells. In Figure 42A, DAPI staining showed that the number of treated cells in the lower chamber is significantly lower compared with the control. Results revealed as well that 1 and 2 µM plumbagin inhibited the invasiveness of B16F10 cells by 40% and 80%, respectively, after 12

A B

A B h and less than 20% after 24 h for both concentrations compared with the untreated cells (Figure 42B). Furthermore, in order to evaluate the adhesion ability of the ECM, an adhesion assay was performed. The cells were treated with 0 and 2 µM plumbagin for 0, 12, 24, 48, and 72 h.

Results showed that 2 µM plumbagin inhibited time-dependently the adhesion of the cells to the ECM-coated plates comparing with the control (untreated cells) (Figure 43).

Figure 43: Evaluation of the effect of plumbagin on the adhesion to the extracellular matrix by performing an MTT assay on B16F10 cells cultured in fibronectin-coated 96-well plates

Additionally, the effect of plumbagin on the metastasis of SK-MEL-28 melanoma cells was checked as well. Results showed that 2 µM plumbagin inhibited as well the migration, invasion, and adhesion of these cells (Figure 44).

B A

These results demonstrated the effectiveness of plumbagin on the inhibition of metastasis in mouse and human melanoma cell lines by decreasing the cell migration, adhesion, and invasion of B16F10 and SK-MEL-28.

c. Gene expression changes in B16F10 melanoma cells caused by plumbagin

To determine the molecular mechanism underlying the effect of plumbagin on the inhibition of the metastasis of B16F10 melanoma cells, a global gene expression analysis using DNA microarray after 24 h with 0 and 2 µM plumbagin was conducted. The data analysis revealed that the expression of 520 genes were regulated, out of which 232 genes were upregulated with a fold-change ≥ 1.1, while 288 were downregulated with a fold-change ≥-1.1 (Figure 45A). Genes relevant in apoptosis, ATP binding, regulation of inflammatory response, and cellular homeostasis were upregulated as shown in Figure 48C. On the other hand, genes involved in MAPK pathway, cell signaling, cell migration and motility were downregulated (Figure 45B).

Table 17 summarize the significantly modulated genes and their biological activities. Genes with tumor suppressor activity and genes involved in Wnt/β-catenin and MAPK pathway regulation such as Igfbp5, Pten, Axin1, and Dusp8, were upregulated after plumbagin treatment (Table 17). 17). Figure 47 illustrates the summary of the mechanism of action of plumbagin on the inhibition of the metastasis of B16F10 through the modulation of MAPK pathway and its related genes.

d. Effect of plumbagin on the expression of metastasis-associated proteins in B16F10 murine melanoma cells

The protein expression of CD44, MMP2, and MMP9 known to interact with MAPK pathway and responsible of the migration, the adhesion, and the invasion of metastatic cell, was checked.

Results showed that 2 µM plumbagin downregulated the expression of MMP2 and MMP9 compared with the control time-dependently (Figure 46A andB).

In addition, the expression of CD44 involved in cell migration after interaction with MMP9 was not detected in the treated cells at 12, 24, 48, and 72 h, however it was expressed at 0 h (untreated cells) (Figure 46A andB). These results indicated that plumbagin inhibited the migration of melanoma cells by downregulated the MMP's and their interaction with cell adhesion molecules.

Discussion

Malignant melanoma is a very aggressive form of skin cancer characterized by an uncontrolled growth of the melanocytes, an increasing rate every year, and a high risk of resistance to chemotherapy and radiation 174 .

A B

Phytochemicals from plants play a very prominent role in traditional and modern medicine, and have a variety of properties including anticancer activity 175 . In this study, we reported the antimetastatic effect of plumbagin a naphthoquinone against B16F10 murine melanoma cells. In addition, the effect of plumbagin in the inhibition of the metastasis of SK-MEL-28 was determined as well. Our results showed that plumbagin inhibited the viability of B16F10 known to be a highly metastatic malignant melanoma cell line at dose and time-dependent manner (Figure 40A). It has been described that melanoma metastasis is a complex series of cell movements, adhesion, invasion, and migration in blood and lymph 176 . This complex process depends on cell to cell, and cell to extracellular matrix (ECM) interactions, which are mediated directly by cell surface adhesion molecules and receptors 177 . The invasive primary tumors will pass through the ECM and penetrate the lymphatic or vascular circulation and eventually will attach to a new location and form secondary tumors 178 . Plumbagin was found to inhibit significantly three important metastatic processes: migration, adhesion, and invasion of B16F10 cells (Figure 40). The results obtained in B16F10 were further confirmed in SK-MEL-28 human cell line where plumbagin downregulated the cell viability and inhibited the migration, adhesion, and invasion of the cells (Figure 44). In addition, it has been reported that plumbagin exhibited as well an anti-proliferative, anti-invasive, and apoptotic property both in human cancers cell line and in vivo 179,180 .

The transition of melanoma cancer cells from a benign noninvasive stage to a metastatic, aggressive, and invasive tumors is due to the accumulation of genetic and epigenetic alterations and the dysregulation of signal transduction pathways 181,182 . An early stage of melanoma is often characterized by a BRAF (V600E; 66%) mutation or an ectopic activation of NRAS, leading to elevated MAPK pathway signaling and tumor growth 182,183 .Therefore, clinical trials targeting the MAPK pathway with BRAF and NRAS inhibitors is now an attractive target for pharmacologic intervention, however up to now, the success rate is still limited and not highly significant 184 . Global gene expression analysis revealed that plumbagin downregulated MAPKrelated genes including Map3k3, Mapk14, Braf, c-Myc, and Mapk1 (Table 17 and Figure 45B).

MAPK pathway regulates c-Myc expression and stability, and an overexpression of c-Myc drives melanoma metastasis 185,186 .

Moreover, a decrease in the expression of genes associated with cell migration, cell to cell interaction, and cell adhesion was observed upon treatment with plumbagin (Figure 46B). The gene expression of matrix metalloproteinase (MMP's) was downregulated as well (Table 17).

MMP's are a class of protease enzymes involved in the degradation of ECM, and in cancer they facilitate the invasion and metastasis process and participate in tumor proliferation, immune-resistance, and angiogenesis 187,188 . Proteolytically active MMP's can be localized on the cell surface after binding to membrane molecules which leads to a more directed ECM degradation, for instance MMP2 can bind to integrin αvβ3, and MMP9 can be associated with CD44 an adhesion molecule considered as a cancer maker 162 . Additionally, the interaction between MMP's and CD34 is regulated by the MAPK pathway, and MMP 9 seems to play a role in the resistance of the treatment with BRAF inhibitor 189 . In this current study, microarray analysis showed a downregulation in the gene expression of Cd34 and Mmp9, and a further investigation of the effect of plumbagin on the protein expression of CD34, MMP9, and MMP2 revealed that plumbagin inhibited significantly their expression (Table 17 and Figure 46). These results further confirm the effect of plumbagin on the inhibition of migration and invasion of melanoma cells by targeting the MMPs and cell adhesion molecule (Figure 47).

Furthermore, an upregulation of genes significant in apoptosis, regulation of adaptive immune and inflammatory response, response to reactive oxygen species (ROS), and ATP binding was observed upon plumbagin treatment (Figure 45C). Plumbagin is found to induce cytotoxicity in cancer cells through modulating the genes involved in the generation and regulation of ROS that will lead to cell cycle arrest and apoptosis 190 . Also based on previous work, plumbagin has a low cytotoxic effect on normal human skin cell (BJ cells), however other studies showed that plumbagin decreased human MCF-7 breast cancer cells viability and inhibited PI-5 kinase expression known to be involved in ROS generation 191,192 . In this work, we can conclude that plumbagin has a cytotoxic effect on B16F10 melanoma cells. In addition, in table 1 an enhancement of the gene expression of Igfbp5 and Pten was illustrated. Igfbp5 functions as a tumor suppressor in human melanoma cells by inhabiting MMP9 expression, cell migration, and MAPK pathway 193,194 . Pten on the other hand, is a tumor suppressor gene, often found mutated in melanoma patient which will promotes a further activation of MAPK pathway inducing tumor cell growth 195 .

Figure 47: Summary of the possible pathway involved in the effect of plumbagin on the inhibition of metastasis in melanoma cells

The use of plumbagin in combination with radiation in chemo and radio-resistance in melanoma cells appears to be more effective compared to chemo and radiation treatment alone 196 . As our data showed that plumbagin affected the MAPK pathway often mutated in cancer, and inhibited the invasion and the migration of melanoma cells, plumbagin can be a powerful candidate in the treatment of metastasis melanoma (Figure 47). Furthermore, plumbagin induced ROS in melanoma cells leading to the apoptosis, and upregulated genes that function as tumors suppressors, and involved in MAPK pathway inhibition.

Further investigation and in vivo studies would however be necessary in order to validate more the effect of plumbagin on the inhibition of the metastasis in melanoma cells and to establish plumbagin as a drug against metastatic cancer.

Chapter 5: Quantification of naphtoquinones present in Lawsonia inermis L. extract and its cytotoxic effect on B16F10 melanoma cells.

II. Introduction

According to World Health Organization, cancer is the second leading cause of death globally.

In 2018, one in six deaths are caused by cancer. The early identification of cancer could result to a positive respond to the treatment. Moreover, a prevention is the best way to anticipate and generate hope to avoid or change the behavior of the cancer cells before invasion.

Chemoprevention has taken the interest of many researchers in last decades. It is classified in four major categories: hormonal, medications, diet-related agents, and vaccines. Secondary metabolites or natural products have been classified into diet related agents as chemopreventive agents such as flavonoids [START_REF] Benetou | Chemoprevention of cancer: current evidence and future prospects[END_REF] .

There are several secondary metabolites that were reported to have anti cancerous properties including quinones. It consists to be highly interactive with biological target and play an important role in oxidation-reduction reactions by forming covalent bonds 197 .

This class of secondary metabolites contains three main molecules used in phytotherapy as alternatives: benzoquinones, anthraquinones and naphtoquinones.

Naphtoquinones are a class of quinones present in numerous families: Avicenniaceae, Bignoniaceae, Boraginaceae, Droseraceae, Ebenaceae, Juglandaceae, Nepenthaceae, Plumbagnaceae and Lythraceae, and have been classified as privileged structure in medicinal chemistry thanks to their biological activities summarized in the Table 1 198 . In addition to that, there is an important characteristic of naphtoquinones is that they are coloured and play a role in the attraction of pollinators. This color vary between yellow, orange and brown 199 .

Lawsonia Inermis L. commonly named "Henna" belongs to the family Lythraceae. It is known for its traditional use both as cosmetic and medicinal agent especially in the indigenous areas of Morocco, India and Middle East countries [START_REF] Chaudhary | Lawsonia inermis Linnaeus: A Phytopharmacological Review[END_REF]121,125,200 . The different parts of this shrub including leaves, stems, seeds and roots contain many active compounds like naphthoquinones, polyphenols and flavonoids.

Taken together, the presence of naphtoquinones in Lawsonia inermis L. There are several studies described in the literature for the quantification of the three naphtoquinones in Lawsonia inermis L. (table 1). In addition, it has been reported that Lawsonia inermis L. has a cytotoxic effect against several cell lines mainly cancerous ones like liver cells (HepG2), human breast (MCF-7) performed using MTT assay (134).

The table 14 summarizes the biological effect reported of the three naphthoquinones present in Lawsonia inermis L.and that are the focus of our research.

In this context, we were particularly interested in the optimization of the analysis using standard addition method for the preparation of the extract to analyze our samples using LC-MS-QTrap and also for more precision MRM mode in order to have an exact quantification. Moreover, cytotoxicity of Lawsonia inermis L. leaves extracted by Soxhlet was performed on B16F10 metastatic melanoma cell line. Growth inhibition of human K562 leukemia cells and Ehrlich carcinoma in a dose dependent manner.

Anti-proliferation effect on MCF-7 human breast cancer cell line, Herpes Simplex Virus type 2 infection and snake poison-induced mycotoxicity.

III. Material and methods

Chemical and reagents

Analytical grade ethanol and methanol purchased from Sigma Aldrich were used throughout the extraction and analysis process. Lawsone, plumbagin and lapachol were purchased from Sigma Aldrich Chemical Corp. (Saint-Quentin-Fallavier, France). Ultra-pure water was obtained with an Elga apparatus.

Plant material

Lawsonia inermis L. was purchased from certified Ait Matan cooperative: its harvest provenance is from Tazarine. The dried and grounded leaves of the plant were used in this study.

Extraction of the plant material

10 g of sample was weighed in a capsule and placed in Soxhlet apparatus and extraction was carried out for 8 hours using hexane as solvent. The extract was evaporated in rotavapor

HPLC-ESI-QTrap-MS conditions

A Shimadzu gradient LC system (CBM-20A) interfaced to a Qtrap 5500 mass spectrometer equipped with a turboV electrospray source (SCIEX API 5500) was used for LC-ESI-MS/MS analysis of the separation of the compound was carried out on a C18 Phenomenex 100x1mm column with 3 µm particles, 110 Å. The flow rate was 100 µl/min. Henna extract was dissolved in 10/90 (H2O/Meth OH), 5 µl of sample volume was injected to the HPLC system.

Standard addition method (SAM)

The Standard Addition Method (SAM) is the method used in our study to quantify our compounds of interest present in Lawsonia inermis L. which are: lawsone, lapachol and plumbagin of the naphthoquinone family (Figure 48, Table 15). (5 mg/ mL) was then added to the cells and incubated for 8 hours, and then 10% Sodium dodecyl sulphate (SDS) was added at 100 µl/well. The plates were incubated for overnight at 37°C. The absorbance was measured at 570 nm using a microplate reader (Powerscan HT). This experiment was run in triplicate.

Data Analysis

The analysis was done by extracting the value of area under curve for m/z (174, 188, 242 for lawsone, plumbagin and lapachol respectively) analyzed in triplicate, then the standard calibration curves were established.

IV. Results and discussion

1. Plumbagin decreased the viability of B16F10 murine melanoma cells.

Cytotoxic effect of Lawsonia inermis L. leaves was checked by performing MTT assay on B16F10 murine melanoma cells. The results showed that the extract inhibited significantly the proliferation of the cells at time-dose dependent-manner (Figure 49). After 48 h, the cell viability decreased to 60 % at the concentration of 25 µg/mL. Above this concentration, the extract was very toxic. Lawsonia inermis L. has been studied as a potential anti-proliferation agent against several cell lines including liver cells (HepG2), human breast (MCF-7) 202 . This plant was usually used since ages in cosmetics for dying hair, nails, hands and feet, but rarely used therapeutically. In this context, it was important to combine the two elements: cosmetic use with therapeutical one, by focusing, for the first time, on the melanoma cell lines. Plumbagin, naphthoquinone present in Lawsonia inermis L. presented a promising anti-cancer effect in vitro on B16F10 murine and SK-MEL-28 human melanoma cell lines [START_REF] Alem | Elucidation of the effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via MAPK signalling pathway[END_REF] .

2. Quantification of lawsone, lapachol and plumbagin in Lawsonia inermis L. leaves.

Due to the low amount of naphthoquinones present in Lawsonia inermis L. and also the high sensitivity of the LC-MS analysis optimized earlier, the analysis of the extract was performed using the standard addition method. It consists on adding standards (Figure 50) (lawsonelapachol-plumbagin) to the aliquots of analyzed samples. The range of concentrations used for the quantification was 0-1-2.5-5-7.5-10-12.5-15 µg/ml for the three standards.

Discussion

Natural products lead to an upcoming therapeutic approach to cancer. Lawsonia inermis L.

presented an anti-proliferative effect on several cancer cell lines including liver cells (HepG2), human breast (MCF-7) 202 . This plant was usually used since ages in cosmetics for dying hair, nails, hands and feet, but rarely used therapeutically. In this context, it was important to combine the two elements: cosmetic use with therapeutic one, by focusing, for the first time, on melanoma cell lines to study its effect on skin cancer.

Naphthoquinones were targeted in this study because of their anti-oxidant potential that leads to the generation of reactive oxygen species (ROS). Lawsone, Lapachol and Plumbagin were quantified using LC-MS-Qtrap-MS. A direct injection was performed first for a scanning of the compound at a low concentration and to detect the ionization mode (Figure 50). The results showed excellent correlation between the peak area and concentration. The fivepoint calibration curves for the three compounds showed a linear correlation between concentration and peak area.

For the optimization of the compounds, the use of multiple reaction monitoring (MRM) allowed us to select, for each compound, daughter ions after fragmentation for quantification (Table 16). This work is aiming to study the effect of the leaves of the plant in proliferation of murine melanoma cell lines B16F10 to take into consideration the synergic effect of the compounds presents in Lawsonia inermis L.: Lawsone, Lapachol and Plumbagin, since it has already been reported that each compound has an-proliferative effect on several cancer cell lines [START_REF] Alem | Elucidation of the effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via MAPK signalling pathway[END_REF]123,208 .

Standard

As previously mentioned, the final quantifications revealed that in one gram of Lawsonia In a nutshell, Lawsonia inermis L. with its three compounds quantified in our study could be a promising anticancerous agent that should be deeply regarded and explored for a potential drug.

General Conclusion

"

  Optimized supercritical fluid extraction and pressurized liquid extraction of Lapachol and Plumbagin from Henna Leaves (Lawsonia inermis L.)". submitted to Industrial Crops and Products. Participation in international Conferences: • Alem Fatima-Zahra, Samira Ananthabothla Gita, Hedjila Ammour, Zoubida Charrouf, Alessandra Lopes de Oliveira. Boutayna Rhourri-Frih 'Comparative Study of Three Extraction Methods of Henna (Lawsonia inermis L.) harvested from Two Different Regions in Morocco and their HPLC-MS Analysis'. 48TH International Symposium on High-Performance Liquid Phase Separations and Related Techniques 2019, Milan, Italy. Poster. • Alem Fatima-Zahra, Meriem Bejaoui, Myra O. Villareal, Boutayna Rhourri-Frih, Isoda Hiroko 'Anti-metastatic melanoma potential of plumbagin: compound of Lawsonia inermis L.'. The Tunisia-Japan Symposium on Science, Society and Technology 2019, Sousse, Tunisia, Oral Communication. • Alem Fatima-Zahra, Samira Ananthabothla Gita, Zoubida Charrouf, Alessandra Lopes de Oliveira. Boutayna Rhourri-Frih. 'Comparative study of four extraction methods of Lawsonia inermis L. harvested from two different regions in Morocco and their LC-ESI-Q-TOF-MS analysis' International Analytical Development Symposium 2019, Université de Bordeaux, France. Oral Communication.

  FIGURE 1 PRESENTATION OF LAWSONIA INERMIS L.: A.FLOWERS / B. FRUIT / C. LEAVES. ...................................... FIGURE 2 : GEOGRAPHICAL DISTRIBUTION OF HENNA CULTURE. ........................................................................................... FIGURE 3: PHENOLIC COMPOUNDS PRESENT IN LAWSONIA INERMIS L. .................................................................... FIGURE 4: VOLATILE AND NON-VOLATILE TERPENES PRESENT IN LAWSONIA INERMIS L. ........................................................... FIGURE 5: ALKALOIDS PRESENT IN LAWSONIA INERMIS L. ................................................................................................... FIGURE 6: SCHEMATIC ILLUSTRATION OF THE WORKFLOW OF SOXHLET EXTRACTOR. FIGURE ADAPTED FROM (1) .......................... FIGURE 7: SUPERCRITICAL FLUID EXTRACTION SCHEMA...................................................................................................... FIGURE 8: SCHEMATIC DIAGRAM OF PRESSURIZED LIQUID EXTRACTION .............................................................. FIGURE 9 : GENERAL CONCEPTION OF HPLC INSTRUMENTATION .......................................................................... FIGURE 10: A. EXAMPLE OF MASS SPECTRUM OF PLUMBAGIN ADAPTED FROM NIST. B.SCHEMATIC REPRESENTATION OF A MASS SPECTROMETER. ............................................................................................................................................... FIGURE 11: SCHEMATIC REPRESENTATION OF ELECTROSPRAY IONIZATION (61) .................................................... FIGURE 12: QUADRUPOLE ION TRAP MASS SPECTROMETER.(62,63) ....................................................................... FIGURE 13: PHASE DIAGRAM WITH SUPERCRITICAL FLUID REGION ........................................................................................ FIGURE 14: HENNA (LAWSONIA INERMIS L.) LEAVES ............................................................................................... FIGURE 15:RESEARCH FOCUS OF HENNA (LAWSONIA INERMIS L.) IN DIFFERENT FIELDS. ........................................ FIGURE 16 :CHEMICAL STRUCTURE OF PLUMBAGIN (A) AND LAPACHOL (B) STRUCTURE DRAWN USING ACD/ CHEMSKETCH SOFTWARE ..................................................................................................................................................................... FIGURE 17: PARETO CHART OF EFFECTS OF CCD-T LEAVES GENERATED BY LINEAR MODEL ................................ FIGURE 18: RESPONSE SURFACE CURVE OF CCD-SFE-T LEAVES. ....................................................................... FIGURE 19 : SUMMARY OF GLOBAL EXTRACTION YIELD OF DIFFERENT EXTRACTION TECHNIQUES ....................... FIGURE 20: A. NEGATIVE PRODUCT ION MASS SPECTRUM OF LAPACHOL AND ITS CHEMICAL STRUCTURE (M/Z 241) (B) : FRAGMENTATION PATTERN OF LAPACHOL IN ESI-MS WITH NEGATIVE ION MODE(109) ..................... FIGURE 21: NEGATIVE PRODUCT ION MASS SPECTRUM OF PLUMBAGIN AND ITS CHEMICAL STRUCTURE (M/Z 186.9) ..................................................................................................................................................................... FIGURE 22: CONCENTRATION OF PLUMBAGIN AND LAPACHOL IN THE SUPERCRITICAL FLUID EXTRACTS (T LEAVES) ....................................................................................................................................................... FIGURE 23: CONCENTRATION OF PLUMBAGIN IN HENNA LEAVES FROM TAZARINE REGION EXTRACTED BY DIFFERENT METHODS OF EXTRACTION. ......................................................................................................... FIGURE 24: CONCENTRATION OF LAPACHOL IN HENNA LEAVES FROM TAZARINE REGION EXTRACTED BY DIFFERENT METHODS OF EXTRACTION. ......................................................................................................... FIGURE 25 : LAWSONE TWO-DIMENSIONAL CHEMICAL STRUCTURE AND LAWSONIA INERMIS L LEAVES AND POWDER. ...................................................................................................................................................... FIGURE 26 : MAP SITUATION OF THE FOUR SOUTH EASTERN MOROCCAN REGIONS WHERE LAWSONIA INERMIS L (HENNA) WAS HARVESTED FOR THIS STUDY: TAZZARIN, FOUM ZGUID, ERRACHIDIA AND ZAGORA. ........... FIGURE 27 : WEATHER CONDITIONS OF FOUM ZGUID, TAZARINE, ZAGORA AND ER-RACHIDIA; PLUVIOMETRY AND TEMPERATURE: MEAN TEMPERATURE AND PLUVIOMETRY (125) .................................................................. FIGURE 28 : STABILITY TEST OF LAWSONE, EVOLUTION OF THE PERCENTAGE OF PEAK AREA OF LAWSONE REGISTERED DAILY, DURING 4 DAYS WITH HPLC-UV. ................................................................................ FIGURE 29: PEAKS COMPARISON OF THE CALIBRATION CONCENTRATIONS OF LAWSONE OBTAINED BEFORE AND AFTER OPTIMIZATION OF THE GRADIENT. ..................................................................................................... FIGURE 30: (A) SIMPLE MASS SPECTRUM OF LAWSONE IN POSITIVE MODE WITH THE ESI-QTRAP SPECTROMETER (B) TANDEM MASS SPECTRUM (MS2) OF LAWSONE IN POSITIVE MODE: FRAGMENTS OF 175 M/Z ............................................................. FIGURE 31 :TOTAL ION CHROMATOGRAM OF LAWSONE STANDARD (1.25 µG/ML), SIGNAL TO NOICE RATIO (A) OF MS2 OF THE PIC 175 M/Z(B) OF MRM MODE. ............................................................................................... FIGURE 32: QUANTITATIVE ANALYSIS OF LAWSONE IN FOUR HENNA CORPS LOCATIONS IN THE SOUTH EASTERN OF MOROCCO: ZAGORA, FOUM ZGUID, ERRACHIDIA AND TAZZARINE. ............................................................ FIGURE 33: SCORE PLOT OF DATASET OF 12 OBSERVATIONS: 4 SAMPLES IN BIOLOGICAL TRIPLICATE AND 3 VARIABLES (PRECIPITATION, MEAN TEMPERATURE AND QUANTIFICATION OF LAWSONE). ........................... FIGURE 34: LOADING 2D PLOT OF DATASET OF 12 OBSERVATIONS: 4 SAMPLES IN BIOLOGICAL TRIPLICATE AND 4 VARIABLES (DISTANCE FROM OCEAN, RAINFALL, MEAN TEMPERATURE AND QUANTIFICATION OF LAWSONE) .....................................................................................................................................................................FIGURE 35: SKIN ANATOMY 3D ILLUSTRATION -IMAGE COPYRIGHT : SCIENCEPICS / SHUTTERSTOCK .......................................... FIGURE 36: STRUCTURE OF THE EPIDERMIS ..................................................................................................................... FIGURE 37: SYNTHETIC PATHWAY OF MELANIN AND THE INVOLVEMENT OF MELANOGENIC ENZYMES(148). .......

FIGURE 8 :

 8 FIGURE 1 PRESENTATION OF LAWSONIA INERMIS L.: A.FLOWERS / B. FRUIT / C. LEAVES. ...................................... FIGURE 2 : GEOGRAPHICAL DISTRIBUTION OF HENNA CULTURE. ........................................................................................... FIGURE 3: PHENOLIC COMPOUNDS PRESENT IN LAWSONIA INERMIS L. .................................................................... FIGURE 4: VOLATILE AND NON-VOLATILE TERPENES PRESENT IN LAWSONIA INERMIS L. ........................................................... FIGURE 5: ALKALOIDS PRESENT IN LAWSONIA INERMIS L. ................................................................................................... FIGURE 6: SCHEMATIC ILLUSTRATION OF THE WORKFLOW OF SOXHLET EXTRACTOR. FIGURE ADAPTED FROM (1) .......................... FIGURE 7: SUPERCRITICAL FLUID EXTRACTION SCHEMA...................................................................................................... FIGURE 8: SCHEMATIC DIAGRAM OF PRESSURIZED LIQUID EXTRACTION .............................................................. FIGURE 9 : GENERAL CONCEPTION OF HPLC INSTRUMENTATION .......................................................................... FIGURE 10: A. EXAMPLE OF MASS SPECTRUM OF PLUMBAGIN ADAPTED FROM NIST. B.SCHEMATIC REPRESENTATION OF A MASS SPECTROMETER. ............................................................................................................................................... FIGURE 11: SCHEMATIC REPRESENTATION OF ELECTROSPRAY IONIZATION (61) .................................................... FIGURE 12: QUADRUPOLE ION TRAP MASS SPECTROMETER.(62,63) ....................................................................... FIGURE 13: PHASE DIAGRAM WITH SUPERCRITICAL FLUID REGION ........................................................................................ FIGURE 14: HENNA (LAWSONIA INERMIS L.) LEAVES ............................................................................................... FIGURE 15:RESEARCH FOCUS OF HENNA (LAWSONIA INERMIS L.) IN DIFFERENT FIELDS. ........................................ FIGURE 16 :CHEMICAL STRUCTURE OF PLUMBAGIN (A) AND LAPACHOL (B) STRUCTURE DRAWN USING ACD/ CHEMSKETCH SOFTWARE ..................................................................................................................................................................... FIGURE 17: PARETO CHART OF EFFECTS OF CCD-T LEAVES GENERATED BY LINEAR MODEL ................................ FIGURE 18: RESPONSE SURFACE CURVE OF CCD-SFE-T LEAVES. ....................................................................... FIGURE 19 : SUMMARY OF GLOBAL EXTRACTION YIELD OF DIFFERENT EXTRACTION TECHNIQUES ....................... FIGURE 20: A. NEGATIVE PRODUCT ION MASS SPECTRUM OF LAPACHOL AND ITS CHEMICAL STRUCTURE (M/Z 241) (B) : FRAGMENTATION PATTERN OF LAPACHOL IN ESI-MS WITH NEGATIVE ION MODE(109) ..................... FIGURE 21: NEGATIVE PRODUCT ION MASS SPECTRUM OF PLUMBAGIN AND ITS CHEMICAL STRUCTURE (M/Z 186.9) ..................................................................................................................................................................... FIGURE 22: CONCENTRATION OF PLUMBAGIN AND LAPACHOL IN THE SUPERCRITICAL FLUID EXTRACTS (T LEAVES) ....................................................................................................................................................... FIGURE 23: CONCENTRATION OF PLUMBAGIN IN HENNA LEAVES FROM TAZARINE REGION EXTRACTED BY DIFFERENT METHODS OF EXTRACTION. ......................................................................................................... FIGURE 24: CONCENTRATION OF LAPACHOL IN HENNA LEAVES FROM TAZARINE REGION EXTRACTED BY DIFFERENT METHODS OF EXTRACTION. ......................................................................................................... FIGURE 25 : LAWSONE TWO-DIMENSIONAL CHEMICAL STRUCTURE AND LAWSONIA INERMIS L LEAVES AND POWDER. ...................................................................................................................................................... FIGURE 26 : MAP SITUATION OF THE FOUR SOUTH EASTERN MOROCCAN REGIONS WHERE LAWSONIA INERMIS L (HENNA) WAS HARVESTED FOR THIS STUDY: TAZZARIN, FOUM ZGUID, ERRACHIDIA AND ZAGORA. ........... FIGURE 27 : WEATHER CONDITIONS OF FOUM ZGUID, TAZARINE, ZAGORA AND ER-RACHIDIA; PLUVIOMETRY AND TEMPERATURE: MEAN TEMPERATURE AND PLUVIOMETRY (125) .................................................................. FIGURE 28 : STABILITY TEST OF LAWSONE, EVOLUTION OF THE PERCENTAGE OF PEAK AREA OF LAWSONE REGISTERED DAILY, DURING 4 DAYS WITH HPLC-UV. ................................................................................ FIGURE 29: PEAKS COMPARISON OF THE CALIBRATION CONCENTRATIONS OF LAWSONE OBTAINED BEFORE AND AFTER OPTIMIZATION OF THE GRADIENT. ..................................................................................................... FIGURE 30: (A) SIMPLE MASS SPECTRUM OF LAWSONE IN POSITIVE MODE WITH THE ESI-QTRAP SPECTROMETER (B) TANDEM MASS SPECTRUM (MS2) OF LAWSONE IN POSITIVE MODE: FRAGMENTS OF 175 M/Z ............................................................. FIGURE 31 :TOTAL ION CHROMATOGRAM OF LAWSONE STANDARD (1.25 µG/ML), SIGNAL TO NOICE RATIO (A) OF MS2 OF THE PIC 175 M/Z(B) OF MRM MODE. ............................................................................................... FIGURE 32: QUANTITATIVE ANALYSIS OF LAWSONE IN FOUR HENNA CORPS LOCATIONS IN THE SOUTH EASTERN OF MOROCCO: ZAGORA, FOUM ZGUID, ERRACHIDIA AND TAZZARINE. ............................................................ FIGURE 33: SCORE PLOT OF DATASET OF 12 OBSERVATIONS: 4 SAMPLES IN BIOLOGICAL TRIPLICATE AND 3 VARIABLES (PRECIPITATION, MEAN TEMPERATURE AND QUANTIFICATION OF LAWSONE). ........................... FIGURE 34: LOADING 2D PLOT OF DATASET OF 12 OBSERVATIONS: 4 SAMPLES IN BIOLOGICAL TRIPLICATE AND 4 VARIABLES (DISTANCE FROM OCEAN, RAINFALL, MEAN TEMPERATURE AND QUANTIFICATION OF LAWSONE) .....................................................................................................................................................................FIGURE 35: SKIN ANATOMY 3D ILLUSTRATION -IMAGE COPYRIGHT : SCIENCEPICS / SHUTTERSTOCK .......................................... FIGURE 36: STRUCTURE OF THE EPIDERMIS ..................................................................................................................... FIGURE 37: SYNTHETIC PATHWAY OF MELANIN AND THE INVOLVEMENT OF MELANOGENIC ENZYMES(148). .......

  LOD: Limit of Detection LOQ : Limit of Quantification % : percentage SAM : Standard Analysis Method FBS : Fetal Bovine Serum RPMI: Roswell Park Memorial Institute medium MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide SDS: Sodium Dodecyl Sulfate MAPK: Mitogen-activated protein kinase MMP: matrix metalloproteinase RNA: Ribonucleic acid DNA: Deoxyribonucleic acid

  1978, during the 31st World Health Assembly, WHO has established an inventory of medicinal plants used in 91 different countries. It has estimated 21000 different species used for their medicinal effects.

1 .

 1 Origin of Lawsonia inermis L.

Figure 1

 1 Figure 1 presentation of Lawsonia inermis L.: A.Flowers / B. Fruit / C. Leaves. Source: Flora de Filipinas [Atlas I] 7 According to the Integrated Taxonomic Information System (ITIS), the taxonomic classification of Lawsonia inermis L. is as follow 8 : Kingdom Plantae Subkingdom Viridiplantae Infrakingdom Streptophyta Super division Embryophyta Division Tracheophyta Subdivision Spermatophytina Class Magnoliopsida Superorder Rosanae Order Myrtales

1 .

 1 Chemical composition of Lawsonia inermis L Lawsonia inermis L. originates from arid and semi-arid areas. It was then naturalized in tropical and subtropical regions (Africa, Asia, Australia) where it was reported to emerge the best dyeing qualities when grown in temperatures between 35 °C and 45 °C. This diversity in culture especially in arid area has promoted the pathways towards the production of plant

Figure 2 :

 2 Figure 2 : Geographical distribution of henna culture.

Figure 3 :Figure 4 :

 34 Figure 3: Phenolic compounds present in Lawsonia inermis L.

Figure 5 :

 5 Figure 5: Alkaloids present in Lawsonia inermis L.

Chapter 2 :I. Extraction techniques of plant material 1 .

 21 Extraction and Chemical Analysis of Lawsonia inermis L. Definition of extractionExtraction (as the term is pharmaceutically used) is the separation of medicinally active portions of plant (and animal) tissues using selective solvents through standard procedures.Such extraction techniques separate the soluble plant metabolites and leave behind the insoluble cellular residues. The products obtained from plants are relatively complex mixtures of metabolites, in liquid or semisolid state or (after removing the solvent) in dry powder form, and are intended for oral or external use. These include classes of preparations known as decoctions, infusions, fluid extracts, tinctures, pilular (semisolid) extracts or powdered extracts.

Figure 6 :

 6 Figure 6: Schematic illustration of the workflow of Soxhlet Extractor. Figure adapted from (1)

Figure 7 :

 7 Figure 7: Supercritical Fluid Extraction schema A. Operating principle of supercritical fluid extraction system. B. The phase diagram of carbon dioxide(46).

Figure 8 : 1 .

 81 Figure 8: Schematic diagram of Pressurized Liquid ExtractionThe extraction process takes place in several successive stages following the diagram (Figure8)[START_REF] Lundstedt | Analysis of PAHs and their transformation products in contaminated soil and remedial processes[END_REF] .The solid sample (powdered) is placed in the extraction cell (made of stainless steel to withstand high pressures). The solvent is pumped into the extraction cell, and is brought to the desired extraction temperature (the pressure in the cell is adjusted according to the temperature). After a given extraction time, the valve at the cell outlet is open: the liquid contained in the cell is transferred to a collection container. A nitrogen purge of the cell allows all the solvent and extracted compounds to be recovered and transferred to the collection vessel. II. Analytical methods: Separation and analysis 1. High performance liquid chromatography a. Definition High Performance Liquid Chromatography or High-Pressure Liquid Chromatography (HPLC) is an analytical technique of separation, identification and quantification of active compounds.

* t0 :

 t0 Dead time ** Tt: Retention time *** R: Resolution **** α : Selectivity ***** N of the chromatogram at 5% width T= W 0.05 /2f Asymmetry factor is denoted at 10% width of the peak.

Figure 9 :

 9 Figure 9 : General conception of HPLC instrumentationi. Solvent Reservoir and PumpIt is equipped with a gradient system making it possible to program the nature of the solvent to pump from the reservoir. It allows to work either in isocratic mode, that works with 100% of the same eluent throughout the analysis or in gradient mode, that means with a variation in the concentration of the constituents of the eluent mixture. Current pumps have a variable flow rate from a few µl to several ml/min.

(

  from Ayoub BENSAKHRIA)

2 .

 2 Soft ionization methods which are the most used and it contains the atmospheric pressure chemical ionization, photoionization at atmospheric pressure, the electro-spray, desorption ionization MALDI, secondary ion mass spectrometry with liquid target and fast atom bombardment.

Figure 10 :

 10 Figure 10: A. Example of mass spectrum of plumbagin adapted from NIST. B.Schematic representation of a mass spectrometer.

Figure 11 :

 11 Figure 11: schematic representation of Electrospray ionization[START_REF] Banerjee | Electrospray Ionization Mass Spectrometry: A Technique to Access the Information beyond the Molecular Weight of the Analyte[END_REF]

  supercritical fluid extraction and pressurized liquid extraction of Lapachol and Plumbagin from Henna Leaves (Lawsonia inermis L.)Submitted to Industrial Crops and ProductsGita Samira Ananthabhotla a,c , Fatima-Zahra Alem a,b , Hedjila Ammour a , Zoubida Charrouf b , Alessandra Lopes de Oliveira c *and Boutayna Rhourri-Frih a * Gita Samira Ananthabhotla and Fatima Zahra Alem have contributed equally to this work1. AbstractNaphthoquinones constitute important secondary metabolites, known for multitude pharmacological activities. Active biomolecules with substituted 1, 4-naphthoquinone moiety like Lawsone, Plumbagin and, Lapachol have been reported to possess significant antiproliferative and cytotoxic effects.The objective of this study is to enhance the content of naphthoquinones (Lapachol and Plumbagin) from Moroccan Henna leaves extracts.In order to optimize the extraction yield of these compounds, traditional methods such as soxhlet has been used and compared with modern greener methods known for their efficiency and their low impact on the environment, in particular Supercritical Fluid extraction (SFE) and Pressurized Solvent extraction (PLE).For both methods the optimization was performed using a circumscribed central composite design (2 factors, 5 levels). The effect of ethanol as co-solvent in conjunction with Supercritical Carbon dioxide was also considered to enhance global yield (%). Global extraction yield from SFE and PLE were compared with soxhlet extraction using hexane as solvent. A sensitive and specific Ultra High Performance Liquid Chromatography-ESI-MS-MS Method with Multiple Reaction Monitoring mode was used for Plumbagin and Lapachol quantification (with respectively transition of 187 m/ z => 159 m/ z and 241 m/ z => 186 m/ z).

Figure 13 :

 13 Figure 13: Phase diagram with supercritical fluid region

Figure 14 :

 14 Figure 14: Henna (Lawsonia inermis L.) leaves

  vomifoliol[START_REF] Siddiqui | Two New and a Known Compound from Lawsonia inermis[END_REF] in addition to quantification of polyphenols, sterols, flavonoids, saponins and, tannins using conventional techniques including Maceration, Soxhlet and advanced techniques like Microwave assisted techniques using different solvents like ethanol, water, methanol and their analysis by Thin Layer Chromatography, High Performance Thin Layer Chromatography and, High Performance Chromatography. Owing to adulteration of commercial samples with p-phenylenediamine, several methods have also been developed to identify counterfeit products[START_REF] Siddiqui | Two New and a Known Compound from Lawsonia inermis[END_REF][START_REF] Kharbade | Identification of natural red dyes in old Indian textiles: Evaluation of thin-layer chromatographic systems[END_REF][90][START_REF] Gallo | Henna through the centuries: a quick HPTLC analysis proposal to check henna identity[END_REF][START_REF] Wagini | Phytochemical Analysis of Nigerian and Egyptian Henna (Lawsonia Inermis L.) Leaves using TLC[END_REF][START_REF] Sukuroglu | Monitoring of Lawsone, p-phenylenediamine and heavy metals in commercial temporary black henna tattoos sold in Turkey[END_REF][START_REF] Zohourian | Polyphenolic Contents and Antioxidant Activities of Lawsonia Inermis Leaf Extracts Obtained by Microwaveassisted Hydrothermal Method[END_REF][START_REF] Gallo | Chemical fingerprinting of Lawsonia inermis L. using HPLC, HPTLC and densitometry[END_REF][START_REF] Almeida | Quantification of pphenylenediamine and 2-hydroxy-1,4-naphthoquinone in henna tattoos[END_REF][START_REF] Babulaa | -A commercially important primaeval dying and medicinal plant with diverse pharmacological activity: A review[END_REF] .Advanced research is focused on application of miniaturized and greener techniques like Matrix solid phase dispersion, Supercritical Fluid Extraction and, Ultrasonic Assisted Extraction (UAE) to extract and quantify henna extracts by hyphenated techniques like Gas Chromatography-Mass Spectrometry (GC-MS), Liquid Chromatography-Mass Spectrometry (LC-MS) and, Raman spectroscopy 98-101 . Recent publications on counterstaining capabilities of gram bacteria, hepatic cells, post blanching effect on the chemical constituents and, tissue engineering of Lawsonia inermis L. dug new paths towards novel research 102-105 . Statistics as shown in Figure 15 depict progressive research in the field of medicine. Studies have also been reported related to anticancer, antioxidant, antimicrobial and several other therapeutic effects of different constituents of henna extracts 56,106,107 .

Figure 15 :

 15 Figure 15:Research focus of Henna (Lawsonia inermis L.) in different fields. Aforementioned significance of Henna attributes to leading secondary metabolites like Naphthoquinones, Coumarins, Terpenoids and, Steroids etc. Hydroxy naphthoquinones possess naphthalene as basic moiety with carbonyl and double bonds in conjugation. They act on topoisomerases and generate Reactive Oxygen Species inside the cell, thus making them potent redox dyes. Hitherto research was focused on Lawsone (2-hydroxy-1, 4-naphthoquinone)

  , chemically 4-hydroxy-3-(3-methylbut-2-en-1-yl) naphthalene-1, 2-dione. It is a yellow crystalline powder naturally present in plant species of several families like Bignoniaceae, Leguminosae, Malvaceae, Plumbaginaceae, Lamiaceae etc. It is widely known for anti-cancer activity like other naphthoquinones 111 . However, the prevalence of Plumbagin and Lapachol in Henna is not evident till date despite rich amount of several naphthoquinones in the plant. In this context, it is found interesting to optimize and quantify Plumbagin and Lapachol extracts in Henna leaves. Hence the present research aims at extraction and analyses of the global extraction yields of henna by different techniques with emphasis on greener Supercritical fluid extraction (SFE) and Pressurized liquid extraction (PLE) comparatively to conventional Soxhlet extraction method imposing central composite experimental design (CCD) and using response surface methodology to optimize the global extraction yield. Furthermore, it is targeted to develop a sensitive and specific analytical method by Ultra High-performance liquid chromatography coupled to mass spectrometry using multiple reaction monitoring mode (MRM) to quantify Plumbagin and Lapachol.

Figure 16 :

 16 Figure 16 :Chemical Structure of Plumbagin (a) and Lapachol (b) Structure drawn using ACD/ Chemsketch software

  α = [No. of factorial runs] 1/4 = [4] 1/4 = 1.414

  initially decided before imposing CCD to study the influence of co-solvent ethanol on the extraction yield of both samples. 20% ethanol was utilized as co-solvent with CO2 at a flow rate of 5 g/ min respectively for each solvent on an automated extractor (Thar-Waters PH1503012) to study the impact on the global yield %. The extraction conditions were maintained at 200 bars, 50 o C for 7 hours with 20 minutes of static time.a) Soxhlet extractionSoxhlet extraction was performed using 10 g of powdered Lawsonia inermis L. leaves. The powder was transferred into a clean filter paper and inserted into a Soxhlet assembly. A volume of 250 ml of Hexane was added in the boiling flask and extraction was for 6 hours. After the extraction, the sample was recovered using a rotary evaporator.e. Quantification and Analysisi. UHPLC-ESI-MS/ MS conditionsLC-ESI-MS/ MS analysis was performed using a Shimadzu gradient UHPLC system (CBM-20A) interfaced to a Qtrap 5500 mass spectrometer equipped with a turboV electrospray source (SCIEX API 5500). The separation of the compounds contained in the extract was achieved using an inert sustain C18 column (3 µm; 1.0 x 150 mm, GL Sciences, Eindhoven, The Netherlands). The injection volume was 5 µL of each sample and was separated by an isocratic mobile phase composed of 0.1M Formic Acid in water-Methanol (10: 90 v/v), at a flow rate of 40 µL/ min. Mass spectrometry was operated over the m/z 50-500 at 1000 Da/sec scan speed. The ion spray voltage was set at -5500 V in the negative ion mode. The trap field time was set at 3 ms.

68 a

 68 Average of three readings b. Significance of Design of Experiments (DoE) Approach in the current study Statistica version 9.0 was used to run experimental design of both SFE and PLE. Initially screening of factors was done to study the significant influence of desired output (global yield %) by imposing factoring design. Then optimization of extraction was done with the experimental runs in the axial region along with the central point. Advantage of response surface methodology is the ease of interpretation and optimization through pareto charts and 3D response surface curve. The rate of change of desired response with respect to each factor (coefficient of variable) along with interaction effects of both linear and quadratic models is depicted in an understandable way. Curcumscribed Central Composite Experimental Design finds merit in comparison with other optimization techniques for considering points at the extreme levels in the axial region outside the study domain. Considering the nature of two naphthoquinones and their solubility in medium to low polar solvents, DoE helped to understand interaction behavior of compounds with respect to each technique and surpasses single factor experiments.

Figure 17 :

 17 Figure 17: Pareto chart of effects of CCD-T leaves generated by linear model Legend: A.Pareto Chart of circumscribed Central Composite Design of extraction henna leaves from Tazarine region of Morocco by Pressurized Liquid Extraction (PLE) depicting effects of each factor and their interaction. B. Pareto chart of circumscribed Central Composite Design of henna leaves from Tazarine region of Morocco by Supercritical Fluid Extraction (SFE) depicting effects of each factor and their interaction from the linear model.

Figure 18 :

 18 Figure 18: Response Surface curve of CCD-SFE-T leaves. A. Response Surface Curve of linear model on a 3D surface of circumscribed Central Composite Design of henna leaves from Tazarine region of Morocco depicting regions of optimum response. B. Response Surface Curve of Quadratic Model-CCD-SFE-T leaves Green pixel being low yield region and Red pixel being high yield region in both regions.

Figure 21 :Figure 20 :

 2120 Figure 21: Negative product ion mass spectrum of Plumbagin and its chemical structure (m/z 186.9)

Figure 22 :

 22 Figure 22: Concentration of Plumbagin and Lapachol in the Supercritical Fluid extracts (T leaves) Determination of concentration based on the standard calibration curve method using regression equation. Central points-5,6,7 is considered as SFE5, Axial point 8 is SFE6, Axial 9 is SFE7, Axial point 10 is SFE8, Axial point 11 is SFE9.

Figure 22

 22 Figure 22 illustrates the comparative results of Plumbagin and Lapachol concentrations in supercritical fluid extracts. Central point of CCD yielded highest amount of Plumbagin (250 bars, 60 o C) (72.26 µg/ mL). Experimental run in the axial region (0, -α) at 250 bars and 46 o C

Figure 23 :Figure 24 :

 2324 Figure 23: Concentration of Plumbagin in henna leaves from Tazarine region extracted by different methods of extraction. PLE and SFE results were of the better conditions based on global yield % from circumscribed Central Composite Design

  results than FZ region. However simultaneous quantification of Plumbagin and Lapachol in different extracts by specific method of Tandem Mass Spectrometry in MRM mode has clearly demonstrated the choice of extraction technique for each compound for optimization of quantification.Overall, it can summarize that relative concentration of Plumbagin is high in all SFE extracts owing to bulkier side chain of Lapachol than methyl group of Plumbagin, thus lowering interaction of Lapachol and decreased solubility in ScCO2 compared with plumbagin. Hence understanding kinetics of solubility can be an added advantage for the study. Screening other be of great interest to progress towards the evaluation of therapeutic efficacy.

Chapter 3 :

 3 Geographical study of Lawsonia inermis L. from different Moroccan regions using Principal component analysis I. Lawsone quantification in Lawsonia inermis L. by HPLC-Qtrap-MS: how does the temperature and pluviometry affect lawsone concentration? Alem, F. Z., Gita, S. A., Cougnaud, L., Affnar, C., Nounah, I., Youssef, B., ... & Rhourri-Frih, B. (2020).

Figure 25 :

 25 Figure 25 : Lawsone two-dimensional chemical structure and Lawsonia inermis L leaves and powder.

Figure 26 :

 26 Figure 26 : Map situation of the four South Eastern Moroccan regions where Lawsonia inermis L (Henna) was harvested for this study: Tazzarin, Foum Zguid, Errachidia and Zagora.

Figure 28 :

 28 Figure 28 : Stability test of Lawsone, Evolution of the percentage of peak area of lawsone registered daily, during 4 days with HPLC-UV.

Figure 29 :

 29 Figure 29: Peaks comparison of the calibration concentrations of Lawsone obtained before and after optimization of the gradient. (a) is the superposition of 5 standards solutions picks eluted with gradient of Methanol water with a slope of 2,5%, (b) is the superposition of 5 standards solutions picks eluted with gradient of Methanol water with a slope of 7,8%.

Figure 30 :Figure 31 :

 3031 Figure 30: (a) Simple mass spectrum of lawsone in positive mode with the ESI-QTrap spectrometer (b) Tandem mass spectrum (MS2) of Lawsone in positive mode: fragments of 175 m/z

Figure 32 :

 32 Figure 32: Quantitative analysis of Lawsone in four Henna corps locations in the south eastern of Morocco: Zagora, Foum Zguid, Errachidia and Tazzarine.

  The created dataset was composed by 12 observation (4 samples in biological triplicate) and 3 variables (precipitation, mean temperature and quantification of lawsone). Score Plot obtained is showed in Figure32, PC1 represents a variability of 72,1% and PC2: 25,5% consequently this PCA shows 97,6% of variability.

Figure 33 :

 33 Figure 33: Score plot of dataset of 12 observations: 4 samples in biological triplicate and 3 variables (precipitation, mean temperature and quantification of lawsone).

Figure 34 :

 34 Figure 34: Loading 2D plot of dataset of 12 observations: 4 samples in biological triplicate and 4 variables (Distance from Ocean, rainfall, mean temperature and quantification of lawsone) In (A) is plotted the variables distribution on the bidimensional plot, in (B) are represented the observations distribution along the two main components F1 and F2 and in (C) is plotted the co-distribution of observations (samples) and variables.

Chapter 4 :

 4 Elucidation of the effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via MAPK signaling pathway I. Skin structureThe skin represents 16% of the total weight of the human body; which gives it to be the largest organ. It forms a protective barrier for the body against the external environment; isolates the body and prevents heat loss. It is composed of three parts: the epidermis, the dermis and the hypodermis.1. The epidermisIt is an epithelial semi-permeable coating tissue. It is composed of three types of cells 141 :• Keratinocytes, filled with keratin (a protein also used in the composition of hair and nails) and lipids.

Figure 35 :

 35 Figure 35: Skin anatomy 3D illustration -Image Copyright : sciencepics / Shutterstock

Figure 36 :

 36 Figure 36: Structure of the Epidermis 143

Figure 37 :

 37 Figure 37: Synthetic pathway of melanin and the involvement of melanogenic enzymes 145 .

Figure 38 :

 38 Figure 38: genetic anomalies of melanoma skin cancer (Laurine Sacré et al;2010)

  differentiation and homeostasis.Matrix Metalloproteinases (MMPs) belong to a rapidly growing multigene family of zincdependent endopeptidases that are involved in the degradation of ECM components in both physiologic and pathologic processes. MMP expression and activation are highly regulated at several levels, and involve both transcriptional and post-transcriptional mechanisms. MMP mRNA expression is primarily regulated by many different factors including cytokines, growth factors, hormones, oncogenes, and tumor promoters (Figure39). MMP2 is associated with tumor invasion and formation of metastasis. MMP9 is associated with cell migration 153 .

Figure 39 :

 39 Figure 39: Structure of extracellular matrix (ECM) adopted from Theodora Stivarou et al; 2015.

Figure 41 :

 41 Figure 41: (A) Scratched cell monolayer images for the wound healing assay results after plumbagin treatment. (B) The effect of plumbagin on the migration of B16F10 cells was quantified at dose and time-dependent manner

Figure 42 :

 42 Figure 42: (A) Fluorescence micrographs of DAPI stained B16F10 cells after invasion using matrigel assay. (B) Cellinvasion assay quantification after 12 h and 24 h treatment with 0, 1, and 2 µM plumbagin.

Figure 44 :

 44 Figure 44: Effect of plumbagin on the metastasis process in SK-MEL-28 Human melanoma cancer cells

  520 genes were regulated: 232 genes were upregulated and 288 were downregulated. (A) The volcano plot represents the regulated genes between control and plumbagin-treated cells. The red color represents the upregulated genes, the green color the downregulated genes, and the grey color the unregulated genes. (B) Summary of the functional categories of downregulated genes in response to plumbagin treatment. (C) Summary of the functional categories of upregulated genes in response to plumbagin treatment.

Figure 45 :

 45 Figure 45: Transcriptome changes induced by plumbagin in B16F10 murine melanoma cells

(

  A) Protein expression of MMP2, MMP9, and CD34 after treatment with 2 µM plumbagin at 0, 12, 24, 48, and 72 h. (B) Band intensities was done using LI-COR system after 0, 12, 24, 48, and 72 h treatment. Results represent the mean ± SD of triplicate samples. *Statistically significant (P ≤0.05) difference between control and treated cells. **Statistically significant (P ≤0.01) difference between control and treated cells.

Figure 46 :

 46 Figure 46: The evaluation of the expression of matrix metalloproteinases (MMP's) and CD44 upon plumbagin treatment

  201 has taken our interest to deepen our knowledge by developing a method of quantification by High Performance Liquid Chromatography coupled to Mass spectrometry ESI Qtrap, particularly: Lawsone (2-hydroxy-1,4-naphthoquinone), Lapachol (2-hydroxy-3-(3-methyl-but-2-enyl)-1,4naphthoquinone) and plumbagin (5-hydroxy-2-methy-1,4-naphthoquinone) and using MRM mode in order to have an accurate quantification. Moreover, the cytotoxicity assay (MTT) was performed to explore the anti-proliferative response of Lawsonia inermis L. leaves extract.

  melanoma cell viability as well as cell migration, adhesion, and invasion resulting to a promoting antimetastatic melanoma agent. Inhibition of NF-kappaB activation in lymphocytes inducing an antiinflammatory effect Induction of apoptosis in human promyelocytic leukemia cells by generation of reactive oxygen species as a novel therapeutic agent for myeloid leukemia. Activation of JNK and down regulation of p53 and MDM2 induction cell cycle arrest (G2/M) and apoptosis of human nonsmall cell lung cancer cells. Anti-microbial effect on Streptococcus, Prevotella, Peptostreptococcus, Mycobacterium, Clostridium, Helicobacter and Escherichia Lawsone 2-hydroxy-1,4naphthoquinone Anti-inflammatory, analgesic, and antipyretic activity.

3

  Coloration of the skin, hair, nails and traditional use as body artInhibition effect on HCT-15 cells derived from human colon carcinoma by the blockage of the S phase.Similar anti-oxidant effect to ascorbic acid using free radical scavenging assay.Anti-microbial effect on Streptococcus, Prevotella, Peptostreptococcus, and anti-inflammatory effect evaluated in a murine model of inflammatory angiogenesis induced by subcutaneous implantation of polyether polyurethane sponge discs.

Figure 48 : 6 . 7 .

 4867 Figure 48: Standard curves of Plumbagin, Lapachol and Lawsone for quantification. Range of concentrations used: 0-1-2.5-5-7.5-10-12.5-15 µg/ml for the three standards.

Figure 49 :

 49 Figure 49: Effect of Lawsonia inermis L. leaves on the viability of B16F10 metastatic melanoma cells after 24 and 48 h treatment with various concentrations of the extract (0, 25, 50, 75, 100 µg/ml).

Figure 50 :

 50 Figure 50: Simple mass spectrum of three naphthoquinones quantified from Lawsonia inermis L. with the ESI-Qtrap spectrometer (A) Lawsone spectrum in positive mode, (B) Lapachol in negative mode, (C) Plumbagin in positive mode.

  Lawsone andPlumbagin were protonated to form [M+H] + ions in Q1 and have recorded respectively the and 188.9 in positive mode (ESI). Lawsone was deprotonated to form[M+H] -ion in Q1 and has recorded the molecular ion 241 negative mode (ESI).Linear calibration plots for Lapachol, Lawsone and Plumbagin were obtained over the calibration range at five concentration levels (0-15 µg/ml) in triplicate by HPLC-ESI-QTrap-MS. Linear regression analysis gave the equations respectively: Y=174226X +75122, Y=47402X +18424 and Y=16793X + 9646.7 and calibration data indicated the linearity of the detector response for the three compounds respectively: R² = 0.996, R²= 0.9975 and R²=0.9934.

  inermis L. leaves extracted by Soxhlet contains 431.1756 µg of Lapachol, 388.67 µg of Lawsone and 574.44 µg of Plumbagin. By extrapolation, the content of Lawsone, plumbagin and lapachol in the concentration of IC50 of treatment (75 µg/ml) used for the proliferation assay are respectively as follow: 29.1 ng, 43.05 ng, 32.32 ng. Adding to our results, a recent in vivo study has shown that a concentration of 500 and 1000 mg/kg body weight of Lawsonia inermis L. leaves orally administrated to C57BL/6 Mice has significantly suppressed the growth of B16F10 tumors on day 40 th . It has also reduced the amounts of free oxygen radicals generated in chemical system and played an antioxidant role 209 .

  ..... FIGURE 16 :CHEMICAL STRUCTURE OF PLUMBAGIN (A) AND LAPACHOL (B) STRUCTURE DRAWN USING ACD/ CHEMSKETCH SOFTWARE FIGURE 17: PARETO CHART OF EFFECTS OF CCD-T LEAVES GENERATED BY LINEAR MODEL ................................ FIGURE 18: RESPONSE SURFACE CURVE OF CCD-SFE-T LEAVES. ....................................................................... FIGURE 19 : SUMMARY OF GLOBAL EXTRACTION YIELD OF DIFFERENT EXTRACTION TECHNIQUES ....................... FIGURE 20: A. NEGATIVE PRODUCT ION MASS SPECTRUM OF LAPACHOL AND ITS CHEMICAL STRUCTURE (M/Z 241) FIGURE 22: CONCENTRATION OF PLUMBAGIN AND LAPACHOL IN THE SUPERCRITICAL FLUID EXTRACTS (T FIGURE 26 : MAP SITUATION OF THE FOUR SOUTH EASTERN MOROCCAN REGIONS WHERE LAWSONIA INERMIS L (HENNA) WAS HARVESTED FOR THIS STUDY: TAZZARIN, FOUM ZGUID, ERRACHIDIA AND ZAGORA. ........... FIGURE 27 : WEATHER CONDITIONS OF FOUM ZGUID, TAZARINE, ZAGORA AND ER-RACHIDIA; PLUVIOMETRY AND FIGURE 28 : STABILITY TEST OF LAWSONE, EVOLUTION OF THE PERCENTAGE OF PEAK AREA OF LAWSONE REGISTERED DAILY, DURING 4 DAYS WITH HPLC-UV. ................................................................................ FIGURE 29: PEAKS COMPARISON OF THE CALIBRATION CONCENTRATIONS OF LAWSONE OBTAINED BEFORE AND AFTER OPTIMIZATION OF THE GRADIENT. ..................................................................................................... FIGURE 30: (A) SIMPLE MASS SPECTRUM OF LAWSONE IN POSITIVE MODE WITH THE ESI-QTRAP SPECTROMETER (B) TANDEM MASS SPECTRUM (MS2) OF LAWSONE IN POSITIVE MODE: FRAGMENTS OF 175 M/Z ............................................................. FIGURE 31 :TOTAL ION CHROMATOGRAM OF LAWSONE STANDARD (1.25 µG/ML), SIGNAL TO NOICE RATIO (A) OF MS2 OF THE PIC 175 M/Z(B) OF MRM MODE. ............................................................................................... FIGURE 32: QUANTITATIVE ANALYSIS OF LAWSONE IN FOUR HENNA CORPS LOCATIONS IN THE SOUTH EASTERN OF MOROCCO: ZAGORA, FOUM ZGUID, ERRACHIDIA AND TAZZARINE. ............................................................ FIGURE 33: SCORE PLOT OF DATASET OF 12 OBSERVATIONS: 4 SAMPLES IN BIOLOGICAL TRIPLICATE AND 3 VARIABLES (PRECIPITATION, MEAN TEMPERATURE AND QUANTIFICATION OF LAWSONE). ........................... FIGURE 34: LOADING 2D PLOT OF DATASET OF 12 OBSERVATIONS: 4 SAMPLES IN BIOLOGICAL TRIPLICATE AND 4 FIGURE 35: SKIN ANATOMY 3D ILLUSTRATION -IMAGE COPYRIGHT : SCIENCEPICS / SHUTTERSTOCK .......................................... FIGURE 36: STRUCTURE OF THE EPIDERMIS ..................................................................................................................... FIGURE 37: SYNTHETIC PATHWAY OF MELANIN AND THE INVOLVEMENT OF MELANOGENIC ENZYMES(148). ....... FIGURE 38: GENETIC ANOMALIES OF MELANOMA SKIN CANCER (LAURINE SACRÉ ET AL;2010) .................................................. FIGURE 39: STRUCTURE OF EXTRACELLULAR MATRIX (ECM) ADOPTED FROM THEODORA STIVAROU ET AL; 2015. ........................ FIGURE 40 EFFECT OF PLUMBAGIN ON THE VIABILITY OF THE CELLS IN B16F10 MURINE MELANOMA CELLS (A), AND IN SK-MEL-28 (B). .............................................................................................................................. FIGURE 41: (A) SCRATCHED CELL MONOLAYER IMAGES FOR THE WOUND HEALING ASSAY RESULTS AFTER PLUMBAGIN TREATMENT. (B) THE EFFECT OF PLUMBAGIN ON THE MIGRATION OF B16F10 CELLS WAS QUANTIFIED AT DOSE AND TIME-DEPENDENT MANNER ................................................................................. FIGURE 42: (A) FLUORESCENCE MICROGRAPHS OF DAPI STAINED B16F10 CELLS AFTER INVASION USING MATRIGEL ASSAY. (B) CELL INVASION ASSAY QUANTIFICATION AFTER 12 H AND 24 H TREATMENT WITH 0, 1, AND 2 µM PLUMBAGIN. ................................................................................................................................ FIGURE 43: EVALUATION OF THE EFFECT OF PLUMBAGIN ON THE ADHESION TO THE EXTRACELLULAR MATRIX BY PERFORMING AN MTT ASSAY ON B16F10 CELLS CULTURED IN FIBRONECTIN-COATED 96-WELL PLATES .... FIGURE 44: EFFECT OF PLUMBAGIN ON THE METASTASIS PROCESS IN SK-MEL-28 HUMAN MELANOMA CANCER CELLS ................. FIGURE 45: TRANSCRIPTOME CHANGES INDUCED BY PLUMBAGIN IN B16F10 MURINE MELANOMA CELLS ................................... FIGURE 46: THE EVALUATION OF THE EXPRESSION OF MATRIX METALLOPROTEINASES (MMP'S) AND CD44 UPON PLUMBAGIN TREATMENT ................................................................................................................................................... FIGURE 47: SUMMARY OF THE POSSIBLE PATHWAY INVOLVED IN THE EFFECT OF PLUMBAGIN ON THE INHIBITION OF METASTASIS IN MELANOMA CELLS ........................................................................................................................................... FIGURE 48: STANDARD CURVES OF PLUMBAGIN, LAPACHOL AND LAWSONE FOR QUANTIFICATION. RANGE OF CONCENTRATIONS USED: 0-1-2.5-5-7.5-10-12.5-15 µG/ML FOR THE THREE STANDARDS............................ FIGURE 49: EFFECT OF LAWSONIA INERMIS L. LEAVES ON THE VIABILITY OF B16F10 METASTATIC MELANOMA CELLS AFTER 24 AND 48 H TREATMENT WITH VARIOUS CONCENTRATIONS OF THE EXTRACT (0, 25, 50, 75, 100 µG/ML). FIGURE 50: SIMPLE MASS SPECTRUM OF THREE NAPHTHOQUINONES QUANTIFIED FROM LAWSONIA INERMIS L.

..................................................................................................................................................................... (B) : FRAGMENTATION PATTERN OF

LAPACHOL IN ESI-MS WITH NEGATIVE ION MODE(109) ..................... FIGURE 21: NEGATIVE PRODUCT ION MASS SPECTRUM OF PLUMBAGIN AND ITS CHEMICAL STRUCTURE (M/Z 186.9) ..................................................................................................................................................................... LEAVES) ....................................................................................................................................................... FIGURE 23: CONCENTRATION OF PLUMBAGIN IN HENNA LEAVES FROM TAZARINE REGION EXTRACTED BY DIFFERENT METHODS OF EXTRACTION. ......................................................................................................... FIGURE 24: CONCENTRATION OF LAPACHOL IN HENNA LEAVES FROM TAZARINE REGION EXTRACTED BY DIFFERENT METHODS OF EXTRACTION. ......................................................................................................... FIGURE 25 : LAWSONE TWO-DIMENSIONAL CHEMICAL STRUCTURE AND LAWSONIA INERMIS L LEAVES AND POWDER. ...................................................................................................................................................... TEMPERATURE: MEAN TEMPERATURE AND PLUVIOMETRY (125) .................................................................. VARIABLES (DISTANCE FROM OCEAN, RAINFALL, MEAN TEMPERATURE AND QUANTIFICATION OF LAWSONE) ..................................................................................................................................................................... ................................................................................................................................................ WITH THE ESI-QTRAP SPECTROMETER ................................................................................................................

TABLE 1 :

 1 COMPARISON OF PROPERTIES OF GAS, LIQUID, AND SCF .......................................................................

TABLE 2 :

 2 CENTRAL COMPOSITE DESIGN OF T LEAVES BY PLE .............................................................................

TABLE 3 :

 3 CENTRAL COMPOSITE DESIGN OF T LEAVES BY SFE .............................................................................

TABLE 4 :

 4 MOISTURE ANALYSIS DATA OF SAMPLES ...............................................................................................

TABLE 5 :

 5 RESULTS OF CENTRAL COMPOSITE DESIGN-PRESSURIZED LIQUID EXTRACTION OF T LEAVES .............

TABLE 6 :

 6 RESULTS OF CENTRAL COMPOSITE DESIGN-SUPERCRITICAL FLUID EXTRACTION OF T LEAVES ...........

TABLE 7 :

 7 CO-SOLVENT EFFECT ON EXTRACTION YIELD OF SUPERCRITICAL FLUID EXTRACTION .........................

TABLE 8 :

 8 STATISTICAL ANALYSIS-ONE WAY ANOVA OF LINEAR MODEL-CENTRAL COMPOSITE DESIGN-SUPERCRITICAL FLUID EXTRACTION ............................................................................................................

TABLE 9 :

 9 STATISTICAL ANALYSIS-ONE WAY ANOVA OF QUADRATIC MODEL-CENTRAL COMPOSITE DESIGN-SUPERCRITICAL FLUID EXTRACTION ............................................................................................................

TABLE 10 :

 10 COMPARISON OF EXTRACTION YIELD BY SOXHLET AND SFE ...............................................................

TABLE 11 :

 11 SUMMARY OF STANDARD CALIBRATION CURVES OF PLUMBAGIN AND LAPACHOL .............................

TABLE 12 :

 12 CLIMATE DATA OF THE FOUR CORPS LOCATIONS COLLECTED FROM THE WEBSITE: CLIMAT MARRAKECH: PLUVIOMÉTRIE ET TEMPÉRATURE MOYENNE MARRAKECH, DIAGRAMME OMBROTHERMIQUE

POUR MARRAKECH -HTTPS://FR.CLIMATE-CLIMATE-DATA.ORG. ................................................................

TABLE 13 :

 13 OPTIMIZED VALUES OF MASS SPECTROMETER IONIZATION SOURCE PARAMETERS IN MULTI REACTION MONITORING MODE FOR THE THREE MAIN TRANSITIONS OF LAWSONE: 175 → 101; 175→ 77; 175→ 70;

WHERE DP: DECLUSTERING POTENTIAL, CE: COLLISION ENERGY, CXP: COLLISION CELL EXIT POTENTIAL. .....................................................................................................................................................................

TABLE 17 :

 17 TOP REGULATED GENES IN PLUMBAGIN-TREATED B16F10CELLS (VS CONTROL) *. ..............................

TABLE 14 :

 14 BIOLOGICAL EFFECTS OF LAWSONE, LAPACHOL AND PLUMBAGIN PREVIOUSLY REPORTED ................. TABLEAU 15: STANDARD CALIBRATION EQUATION OF LAWSONE, LAPACHOL AND PLUMBAGIN .................................................

TABLE 16 :

 16 FRAGMENTATION OF NAPHTHOQUINONES STUDIED AND THEIR MRM ANALYSIS MODE ...........................................

Table of contents Résumé : __________________________________________________________________ Abstract: __________________________________________________________________ Publications and communications ______________________________________________ Figures List ________________________________________________________________ Tables list ________________________________________________________________ Abbreviations list __________________________________________________________ General Introduction _______________________________________________________ Chapter 1 : Lawsonia inermis L : chemopreventive agent __________________________

 of

	I.	Lawsonia inermis L. _______________________________________________________
	1.		Origin of Lawsonia inermis L. ______________________________________________________
	2.		Botanical classification ___________________________________________________________
	3.		Geographical conditions of cultivation ______________________________________________
	4.		Chemical composition of Lawsonia inermis L. ________________________________________
		a.	Phenolic compounds ___________________________________________________________
		b.	Terpenes _____________________________________________________________________
		c.	Alkaloids _____________________________________________________________________
	5.		Biological effects of Lawsonia inermis L. _____________________________________________
	II. Phytotherapy_____________________________________________________________
	1.		Definition of medicinal plants ______________________________________________________
		a.	Aromatherapy: _______________________________________________________________
		b.	Homeopathy __________________________________________________________________
		c.	Herbalism: ___________________________________________________________________
	III.		Chemoprevention _______________________________________________________
	1.		Definition _______________________________________________________________________
	2.		Diet-

related agents as chemo preventive drugs _______________________________________ Chapter 2: Extraction and Chemical Analysis of Lawsonia inermis L. _______________ I. Extraction techniques of plant material _______________________________________ 1. Definition of extraction ___________________________________________________________ 2. Techniques of extraction __________________________________________________________ a.

	III.	Optimized
			Soxhlet ______________________________________________________________________
	b.		Ultrasound assisted extraction ___________________________________________________
	c.		Supercritical fluid extraction (SFE) ______________________________________________
	d.		Pressurized liquid extraction (PLE) ______________________________________________
	II. Analytical methods: Separation and analysis __________________________________
	1.	High performance liquid chromatography ___________________________________________
	a.		Definition ____________________________________________________________________
	b.		HPLC apparatus ______________________________________________________________
		i.	Solvent Reservoir and Pump __________________________________________________
		ii.	Injector ___________________________________________________________________
		iii. Column ___________________________________________________________________
		iv. Detector ___________________________________________________________________
	2.	Mass spectrometry _______________________________________________________________
	a.		Definition ____________________________________________________________________
	b.		Ionization methods ____________________________________________________________
	c.		Mass analyzer: ________________________________________________________________

supercritical fluid extraction and pressurized liquid extraction of Lapachol and Plumbagin from Henna Leaves (Lawsonia inermis L.) ___________________________

	1.	Abstract ________________________________________________________________________
	2.	Introduction ____________________________________________________________________
	3.	Material and methods ____________________________________________________________
	a.		Raw Material _________________________________________________________________
	b.		Chemicals and Reagents ________________________________________________________
	c.		Moisture Analysis _____________________________________________________________
	d.		Extraction ____________________________________________________________________
		i.	Pressurized Liquid Extraction (PLE) __________________________________________
		ii.	Supercritical Fluid Extraction (SFE) ___________________________________________
	e.		Quantification and Analysis _____________________________________________________
		i.	UHPLC-ESI-MS/ MS conditions _____________________________________________
		ii.	Standard Calibration Curves _________________________________________________
		iii. Preparation of extracts for analysis ____________________________________________
	f.		Data Analysis _________________________________________________________________
	4.	Results and discussion ____________________________________________________________
	a.		Moisture Analysis _____________________________________________________________
	b.		Significance of Design of Experiments (DoE) Approach in the current study ___________
	c.		Pressurized Liquid Extraction ___________________________________________________
	d.		Supercritical Fluid Extraction ___________________________________________________
	e.		Comparison

of extraction yield of Soxhlet with Supercritical Fluid Extraction __________ f. Global yield % of different samples of henna leaves

________________________________ g.

Results of quantification by UHPLC-ESI-MS-MS ________________________________ i.

Mass spectrometry fragmentation _____________________________________________ ii.

Calibration curve validation __________________________________________________ iii.

Quantification results ________________________________________________________ 5. Conclusion ______________________________________________________________________ Chapter 3: Geographical study of Lawsonia inermis L. from different Moroccan regions using Principal component analysis ___________________________________________ I. Lawsone quantification in Lawsonia inermis L. by HPLC-Qtrap-MS: how does the temperature and pluviometry affect lawsone concentration?__________________________ 1. Abstract ________________________________________________________________________ 2. Introduction ____________________________________________________________________ 3. Material and methods ____________________________________________________________ a. Chemical and reagents _________________________________________________________ b. Plant material and geographical zone of culture ____________________________________ c. Climate data collection _________________________________________________________ d.

		Extraction ____________________________________________________________________
	e.	HPLC-ESI-QTrap-MS conditions _______________________________________________
	f.	Standard preparation __________________________________________________________
	g.	Data Analysis _________________________________________________________________
	h.	Statistical analysis _____________________________________________________________
	4.	Results and discussion ____________________________________________________________
	a.	Geographical analysis: _________________________________________________________
	b.	Stability ______________________________________________________________________
	c.	Liquid chromatography optimization ____________________________________________
	d.	Mass spectrometry optimization ions _____________________________________________
	e.	Calibration curve _____________________________________________________________
	f.	Quantification ________________________________________________________________
	g.	Principal component analysis ___________________________________________________
	5.	Conclusion ______________________________________________________________________
	Chapter 4:

Elucidation of the effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via MAPK signaling pathway _____________________________

	I.	Skin structure ____________________________________________________________
	1.	The epidermis ___________________________________________________________________
	2.	The dermis _____________________________________________________________________
	3.	The hypodermis _________________________________________________________________
	II. Skin pigmentation ________________________________________________________
	III.	Melanoma Cancer ______________________________________________________
	1.	Types of Melanoma : _____________________________________________________________
	2.	Targeted therapies _______________________________________________________________
	3.	Metastatic melanoma _____________________________________________________________
	IV.	Elucidation of the

effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via MAPK signaling pathway ______________________________________

	1.	Abstract ________________________________________________________________________
	2.	Introduction ____________________________________________________________________
	3.	Methods ________________________________________________________________________
	a.	Sample preparation ____________________________________________________________
	b.	Cell culture and treatment ______________________________________________________
	c.	Cell viability assay _____________________________________________________________
	d.	Wound healing assay __________________________________________________________
	e.	Cell invasion assay _____________________________________________________________
	f.	Cell adhesion assay ____________________________________________________________
	g.	RNA extraction _______________________________________________________________
	h.	Microarray analysis ___________________________________________________________
	i.	Western blotting ______________________________________________________________
	j.	Statistical analysis _____________________________________________________________
	4.	Results _________________________________________________________________________
	a.	Plumbagin

decreased the viability of B16F10 murine melanoma cells and SK-MEL-28 human melanoma cells ______________________________________________________________ b. Plumbagin inhibited the adhesion, migration and invasion of B16F10 murine melanoma cells and SK-MEL-28 human melanoma cells

___________________________________________ c.

Gene expression changes in B16F10 melanoma cells caused by plumbagin ________________ d.

Effect of plumbagin on the expression of metastasis-associated proteins in B16F10 murine melanoma cells____________________________________________________________________ 5. Discussion _____________________________________________________________________ Chapter 5: Quantification of naphtoquinones present in Lawsonia inermis L. extract and its cytotoxic effect on B16F10 melanoma cells. _________________________________ I. Abstract ________________________________________________________________ II. Introduction ____________________________________________________________ III. Material and methods __________________________________________________

	1.	Chemical and reagents __________________________________________________________
	2.

Table 1 :

 1 Significant parameters of HPLC

  results of global extraction yields hoisted Pressurized Liquid Extraction as Thus, the current study forms a source to amplify the prospects of the anticancer potential of henna plant based on the optimization of each method.

	Key Words: Green extraction method, Lapachol; Lawsonia inermis L; Multiple Reaction
	Monitoring; Plumbagin; Pressurized Liquid Extraction; Supercritical Fluid Extraction; Tandem
	Mass Spectrometry.
	superior technique (22.80%) amongst other extraction methods. Highest concentrations of
	Plumbagin were reported in PLE optimized extract (96.59 µg/ mL) and Lapachol in SFE
	optimized extract (46.09 µg/ mL) with respect to independent factors along with interactions of
	the response surface design and relative solubilities of the two naphthoquinones in the solvents

under study (Plumbagin 5 mg/ mL and Lapachol 10 mg/ mL respectively in ethanol and supercritical CO2.

Table 1 :

 1 Comparison of Properties of Gas, Liquid, and SCFPressurized Liquid Extraction (PLE), also called Accelerated Solvent Extraction is an efficient surpasses the demerits of One Variable at a Time (OVAT) approach wherein OVAT ignores interaction effects and does not lead to optimization. DoE, on the other hand, is an efficient way to achieve optimization through few runs, considering multiple factors and interactions, thus eliminating conventional Trial and Error strategy[START_REF]Chemometrics -an overview[END_REF] .

	Extraction of active metabolites from natural products is complex due to wide range of chemical
	constituents in the extract thus effecting the specificity of the extraction method. Several factors
	are associated to achieve extraction-time, temperature, static cycles and, solute/ solvent ratio
	in PLE and Pressure, Temperature, Static time and, flow rate in SFE. Monitoring significant
	factors can lead to optimum extraction of biomolecules of interest.

and highly selective greener technique associated with pressurized liquids that operates in static and dynamic modes. The advantage of this technique is that solvent of interest exists in liquid state above its atmospheric boiling point at higher temperatures ranging from room temperature to 200 o C, under high pressure (35-200 bars) with the aid of inert gas like Nitrogen, thus enhancing penetration of solvent into the sample matrix and solid-mass transfer into the solvent. Choice of solvent depends solely on the chemical nature of analyte of interest. Significant factors associated with the optimization of extraction by PLE are pressure, temperature, extraction time, number of cycles and, volume of solvent. Wide range of solvents employed include single solvents like water where the technique is called Pressurized Hot Water Extraction, ethanol, methanol and binary solvents like methanol: water, ethanol: water in different ratios depending on the chemical properties of bioactive principles. With respect to these unique properties, PLE is achieved at short time with enhanced recovery of active biomolecules in comparison with conventional methods of extraction. However, operating under high temperature is a demerit for thermolabile compounds 79,84,85 . Chemometrics is the chemical discipline that uses mathematical and statistical methods to improve the understanding of chemical information and to correlate quality parameters or physical properties or select optimal measurement procedures and experiments to analytical instrument data. Design of Experiments (DoE) is one of the chemometric approaches for Lawsonia Inermis L. commonly named "Henna" belongs to the family Lythraceae. It is known for its traditional use both as cosmetic and medicinal agent particularly in the indigenous areas of Morocco, India and Middle East countries. The different parts of this shrub including leaves (Figure

Table 2 :

 2 Central Composite Design of T leaves by PLEFor the experiment initially 20.0 g of T leaves were taken into a 300 mL extraction cell of Supercritical extractor (Sulab, Campinas, BR) operated manually under same experimental conditions but with a static time of one hour.Since pressure and temperature exhibit prime effect on the solvent strength of ScCO2 and ultimately on the extraction of constituents, central composite design with those 2 factors each at 5 levels had been framed as tabulated in Table3 witha total of 11 experiments. Based on previous experimental observations, it was decided to carry on the experiment including 4 hours of dynamic extraction and one hour of static time in an extraction vessel. Since it is manual equipment, the flow rate was calculated towards the end of extraction from the initial and final volumes of CO2 consumed/utilized.

	Factor/Level	-α	-1	0	+1	+α
	Static Cycles	1	2	3	4	5
	Volume %	72	80	100	120	128

Table 3 :

 3 Central Composite Design of T leaves by SFE

	Factor/Level	-α	-1	0	+1	+α
	Pressure (bars)	179	200	250	300	321
	Temperature ( o C)	46	50	60	70	74

Table 4 :

 4 Moisture Analysis Data of samples

	Sample	Sample average weight (g) a	Moisture% ± SD
	T leaves	0.508	7.17 ± 0.18
	FZ leaves	0.506	9.56 ± 0.

Table 5 :

 5 Results of Central Composite Design-Pressurized Liquid Extraction of T leaves

	Block	Experimental	Sample	Static	Volume %	Response
		Run	weight g	Cycle	(x2) b	Yield %
				(x1) a
	Factorial	1	5.0012	-1	+1	22.8005
		2	5.0007	+1	-1	20.8051
		3	5.0023	-1	-1	18.7354
		4	5.0008	+1	+1	20.3787
	Central Point	5	4.9992	0	0	19.6551
		6	5.0018	0	0	19.8589
		7	4.9995	0	0	19.2179
	Axial	8	5.0081	0	-α	18.7356
		9	4.9995	-α	0	16.0236
		10	5.0015	0	+α	19.1263
		11	5.0039	+α	0	21.8010

a x1 corresponds to Factor 1 b x2 corresponds to Factor 2

Table 6

 6 depicts experimental matrix with response of Central Composite Design of T leaves with pressure and temperature as two factors each at 5 levels. Extraction time was chosen at 4 hours with one-hour static time based on the preliminary observations.

Table 6 :

 6 Results of Central Composite Design-Supercritical Fluid Extraction of T leaves

	Block	Experimental	Sample	Pressure (x1) a Temperature	Response
		Run	weight g		(x2) b	Yield %
	Factorial	1	20.0016	-1	+1	1.0039
		2	20.0016	+1	-1	1.7459
		3	20.0017	-1	-1	1.2059
		4	20.0025	+1	+1	1.8133
	Central Point	5	20.0017	0	0	1.2879
		6	20.0011	0	0	1.4244
		7	20.0033	0	0	1.3453
	Axial	8	20.0031	0	-α	1.1353
		9	20.0036	-α	0	0.9733
		10	20.0554	0	+α	1.1219
		11	20.0416	+α	0	1.8477
			a x1 corresponds to Factor 1
			b x2 corresponds to Factor 2

Table 7 :

 7 Co-solvent effect on Extraction yield of Supercritical Fluid Extraction

	Sample	Sample weight (g) Yield % a
	T leaves	20.0085	11.1038
	FZ leaves	20.0475	8.2858
		a 200 bars, 50 o C

Table 8 :

 8

	Variation	SS	dF	MS	F stat	F theoretical
	Regression	0.477893	3	0.159298 20.65478	9.28
	Error	0.023137	3	0.007712
	Total	0.501031	6

Statistical Analysis-One Way ANOVA of Linear model-Central Composite Design-Supercritical Fluid Extraction

ANOVA; Var.:Y (%); R-sqr=.95382; Adj:.90764

Table 9 :

 9 Statistical Analysis-One Way ANOVA of Quadratic model-Central Composite Design-Supercritical Fluid ExtractionIt can be criticized that higher pressures enhance the ability of supercritical carbon dioxide to non-selectively extract compounds from the sample on the solute phase with higher solvent strength. Thus, higher pressures can contribute to non-specific extraction due to enhanced penetrability of the solvent. With increase in temperature, the density of Supercritical CO2

		ANOVA; Var.:Y (%); R-sqr = .9287; Adj:.85739
	Variation	SS	dF	MS	F stat	F
						theoretical
	Regression 0.936678	5	0.187336 13.02446	5.05
	Error	0.071917	5	0.014383
	Total	1.008594	10

owing to existence as glycosides. Cleavage of aglycone portion is associated with several principles like hydrolysis to cleave O-glycosidic linkage. Using greener ScCO2 under critical conditions could extract compounds of interest through enhanced diffusion of the solute onto the solid phase associated with mass transfer to the solvent. Considering Sovova model of the solute-solvent interactions and applying the model to the practical conditions could give a better understanding of the extraction process. Since pressure has significant positive impact, several single factors experiment at fixed temperature and varying pressures could be interesting with suitable mathematical modeling to study kinetics of each experiment.

Table 10 :

 10 Comparison of Extraction yield by Soxhlet and SFE

	Sample	Yield%
		Soxhlet	SFE a
	T Leaves	4.0937	1.8477
	FZ leaves	3.6519	1.5366
	a Extraction yield at +α, 0 condition

Table 11 :

 11 Summary of Standard Calibration Curves of Plumbagin and Lapachol

	Plumbagin	Lapachol

Table 11 .

 11

Table 12 :

 12 Climate data of the four corps locations collected from the website: Climat Marrakech: Pluviométrie et Température moyenne Marrakech, diagramme ombrothermique pour Marrakech -https://fr.climate-Climate-Data.org.

	Extract	Distance from	Temperature	Rainfall	Lawsone content
		the ocean (km)	(°C)	(mm)	(%)
	Zagora	468.2	22.9	61	0.44 +/-0.007
	Tazzarine	102.4	20.9	74	0.40 +/-0.019
	Errachidia	743	19.2	127	0.09 +/-0.006
	Foum Zguid	407.9	22.1	106	0.61+/-0.027

Figure 27 : Weather conditions of Foum zguid, Tazarine, Zagora and Er-rachidia; pluviometry and temperature: mean temperature and pluviometry

137 Foum Zguid Tazarine Zagora Er Rachidia

Table 13

 13

	: Optimized values of mass spectrometer ionization source parameters in Multi Reaction Monitoring mode for the
	three main transitions of Lawsone: 175 → 101; 175→ 77; 175→ 70; where DP: Declustering Potential, CE: Collision
	Energy, CxP: Collision Cell exit Potential.
	Compound	Molecular weight Ionisation mode (ESI)	Parent ion	Product ion
	Lawsone
	2-hydroxy-1,4-	174,15 g/mol	Positive	175	101-77-70
	naphthoquinone

Table 14 :

 14 Top regulated genes in plumbagin-treated B16F10cells (vs control) *.

	Gene	Gene name			Biological function		Fold-change P value
	symbol									**
	Upregulated genes
	Igfbp5	Insulin-like growth factor	Downregulated in ovarian	1.84	0.011
		binding protein 5		cancer; tumor suppressor in
						human melanoma cells;
						apoptosis
	Dusp8	Dual specificity phosphatase 8 Negative regulator of MAPK 1.4	0.016
	Pten	Phosphatase	and	tensin	Tumor suppressor; mutated	1.39	0.015
		homolog			in melanoma,
	Axin1	Axis inhibition protein 1	Negative regulation of Wnt	1.38	0.027
						β-catenin;	reduction
						correlates	with	tumor
						progression
	Akt1s1	Akt1 substrate 1			Inhibition of mTORC1 in	1.26	0.043
						cancer cells
	Downregulated genes
	Map3k3 Mitogen-activated	protein	Positive regulation of p38;	-1.4	0.023
		kinase kinase kinase 3		MAPK	cascade;	cell
						migration; tumorigenesis
	Mapk14 Mitogen-activated	protein	MAPK	pathway;	cell	-1.3	0.003
		kinase 14			adhesion;	transmembrane
						receptor
	Braf	B-Raf	proto-oncogene,	MAPK pathway; mutated in	-1.27	0.006
		serine/threonine kinase	melanoma and other cancer
	C-myc	Myelocytomatosis oncogene	MAPK pathway; melanoma	-1.26	0.001
						metastasis
	Mapk1	Mitogen-activated	protein	ERK1 and ERK2 cascade	-1.2	0.0034
		kinase 1
	Cd44	CD44 antigen			Cell adhesion; regulation of	-1.2	0.006
						ERK; interaction with MMPs
	Mmp14	Matrix metallopeptidase 14	Cell growth and migration;	-1.2	0.0017
						Positive regulation of NF-kB
	Mmp9	Matrix metallopeptidase 9	Cell	migration;	drug	-1.11	0.0002
						resistance
	Rassf1	Ras	association	domain	Ras	protein	signal	-1.11	0.017
		family member 1		transduction ;
						MAPK signaling

*Genes functions were obtained from Mouse Genome Informatics (MGI). **ANOVA was performed to assess the level of significance between groups (untreated cells vs plumbagin-treated cells).

  Fatima-Zahra Alem, Hedjila Ammour, Meriem Bejaoui, Hiroko Isoda, Boutayna Rhourri-Nowadays, natural products are used as substances to stop cancer cells from developing or to lower the risk of cancer recurrence, this is chemoprevention. Studies have shown that naphtoquinones such as Lawsone, Lapachol and Plumbagin are a potential agent that could be studied clinically as potential chemopreventive agents. These three compounds are present in abundance is several plants, including Lawsonia inermis L. In this study we have performed an extraction from Lawsonia inermis L. and quantified lawsone, lapachol and plumbagin using standard addition method in the preparation of the samples by LC-ESI-Qtrap analysis with MRM mode. Then we have studied the effect of the extract on the proliferation of murine melanoma B16F10 cells. Results showed that in one gram of Henna leaves extract, there is 574.44 µg of plumbagin, 431.17 µg of lapachol and 388.67 µg of lawsone. MTT assay showed

	Frih
	I. Abstract
	a decrease of the proliferation of B16F10 cells in time-dose dependent manner. The IC50 for
	24 h treatment with Henna leaves is 75 µg/ml and for 48 h treatment is 30 µg/ml. the results are
	promising and encouraging to deepen the research on Henna leaves as a potential anti cancerous
	agent.

Key words: Lawsonia inermis L.-Lawsone-Lapachol-Plumbagin-LC/ESI/Qtrap/MS-Melanoma.

Table 15 :

 15 biological effects of lawsone, lapachol and plumbagin previously reported

  Plumbagin was monitored by the transition 188/159 and 188/130, lawsone by the transition 175/66 and 175/77 and finally lapachol by the transition 241/186 and 241/121.

		Precursor ion	Fragment ion 1	Fragment ion 2	Analysis
		(m/z)	(m/z)	(m/z)	mode
	Plumbagin	187	159	130	Negative
	Lawsone	175	66	77	Positive
	Lapachol	241	186	121	Negative

Table 17 :

 17 fragmentation of naphthoquinones studied and their MRM analysis mode

  98. Zhong, Z., Li, G., Wu, Y., Luo, Z. & Zhu, B. Ultrasound-assisted matrix solid-phase dispersive liquid extraction for the determination of intermediates in hair dyes with ion chromatography. Anal. Chim. Acta 752, 53-61 (2012). 99. Guerra, E., Lamas, J. P., Llompart, M. & Garcia-Jares, C. Determination of oxidative hair dyes using miniaturized extraction techniques and gas chromatography-tandem mass spectrometry. Microchem. J. C, 308-318 (2017). 100. Zohourian, T. H., Quitain, A. T., Sasaki, M. & Goto, M. Extraction of Bioactive Compounds from Leaves of Lawsonia inermis by Green Pressurized Fluids. Sep. Sci. Technol. 47, 1006-1013 (2012). 101. Heo, J.-Y., Cho, C.-H., Jeon, H.-S., Cheong, B.-S. & Cho, H.-G. Enhanced Raman spectrum of lawsone on Ag surface: vibrational analyses, frequency shifts, and molecular geometry. Spectrochim. Acta. A. Mol. Biomol. Spectrosc. (2011) doi:10.1016/j.saa.2011.08.057. 102. Adisa, J. O., Musa, K. K., Egbujo, E. C. & Uwaeme, I. M. A study of various modifications of Lawsonia inermis (Henna) leaf extract as a cytoplasmic stain in liver biopsies. Int. J. Res. Med. Sci. 5, 1058-1065 (2017). 103. Chukwu O. O. C. Application of extracts of Henna (Lawsonia inamis) leaves as a counter stain. Afr. J. Microbiol. Res. 5, (2011). 104. Jyotshna, Gaur, P., Singh, D. K., Luqman, S. & Shanker, K. Validated method for quality assessment of henna (Lawsonia inermis L.) leaves after postharvest blanching and its cosmetic application. Ind. Crops Prod. C, 33-42 (2017). 105. Yousefi, I. et al. An investigation of electrospun Henna leaves extract-loaded chitosan based nanofibrous mats for skin tissue engineering. Mater. Sci. Eng. C Mater. Biol.

Appl. 75, 433-444 (2017).

Acknowledgments

The authors acknowledge the support of Professor Zoubida Charrouf, Department of Chemistry, Mohammed V University, for the identification of the plant (Henna) given kindly by women cooperatives (Ait Matan and La Batha) in Morocco.

Chapter 5 Quantification of naphtoquinones present in Lawsonia inermis L. extract and its cytotoxic effect on B16F10 melanoma cells.

Research on Lawsonia inermis L. goes back years to extract and isolate the different compounds using conventional techniques like maceration, ultrasonic assisted extraction and Soxhlet and more advanced techniques like supercritical fluid extraction, or assisted by microwave and using different solvents such as ethanol, water, methanol.

The interest of this Mediterranean plant in addition to its cosmetic use is its biological antimicrobial, anti-inflammatory, anticancer activity and many others.

Melanoma skin cancer was the target of this research project. The objective was to identify whether Lawsonia inermis L. and its constituents (mainly naphthoquinones) could be used as a chemo preventive agent against this type of cancer that is able to spread very quickly to the other organs.

The use of modern analytical methods has made it possible to obtain a very precise quantification of the naphthoquinones present in the plant as well as to identify the extraction method which allows the best extraction. Thus, the experimental design used in the optimization of the extraction allowed us to avoid a large number of analyzes while saving the solvent, the plant and the time (chapter 2).

Then we proceeded to the quantification of lawsone in the plant as a major compound (compound allowing the desired color on the skin in addition to its therapeutic virtues) in different regions of morocco: four regions which are traditionally known by the production of henna. This study was carried out using principal component analysis to identify the variant responsible for this difference in composition of henna (chapter 3)

Then, based on the literature, the choice of naphthoquinone present in henna for the in vitro study of its anti-metastatic melanoma effect was made on the plumbagin. This choice was made on the basis of what was reported; plumbagin has been proven to be a potentially effective against several cancers including the lungs, colon but which was never performed on melanoma at the beginning of our research (chapter 4).

All this allowed us subsequently to identify the extraction method that gives the best yield and the best growing area for henna in Morocco, in order to be able to test the toxicity of this miraculous plant on B16F10 murine melanoma cell line (chapter 5).

Further in-depth studies could be carried out for the continuity of this project. First, a study of the effect of the whole plant on reconstituted skin before moving on to the in vivo study on mice with tumors in the skin that we can cause.

Extraction and analysis by liquid chromatography coupled with mass spectrometry of

Lawsonia inermis L. and the study of its anti-melanoma effect

Abstract: The interest of this Mediterranean plant, in addition to its cosmetic use, is its biological activity antimicrobial, anti-inflammatory, anticancer and many others.

This research work has been particularly interested in studying its anticancerous effect and more particularly its potential anti-melanoma metastasis given its traditional use often as a topical treatment for irritated skin.

This research work allowed, first of all, the development of an experimental plan for the optimization of the global yield of extraction of active molecules from Henna (Lawsonia inermis L.). In the second stage, the identification and quantification of active compounds by high performance liquid chromatography coupled with mass spectrometry have been performed. A geographical mapping of henna crops in southern Morocco, using principal component analysis and based on the nature of the climate of each region was carried out.

Subsequently, the effect of plumbagin on metastatic melanoma was studied. The study of this activity was evaluated in-vitro on murine and human cells respectively B16F10 and SKMEL-28. Plumbagin was found to significantly inhibit three important metastatic processes: migration, adhesion and invasion of B16F10 cells. These results were confirmed in the human cell line SK-MEL-28.

Keywords