Pr Badran Bassam

Dr Hasan Rammal

Pr M Bassam Badran
email: bassam.badran@ul.edu

Pr Directeur M Bertrand Liagre

Dr Directeur M David Leger

Limoges

M Hasan

Dr Rammal Lebanese

Pr M Philippe Billiald

Pr M Mohammad Mroueh

FAOUR Rapporteur M Wissam

Pr Lebanese

L Abcdef

Hany Kallassy

Mohammad Fayyad-Kazan

Rawan Makki Ade

Yolla El-Makhour

Ade

Eva Hamade Cde

Rammal Hasan

Cde

David Y Leger Cde

Vincent Sol Bcde

Hussein Fayyad-Kazan

Acdefg

ABCDEFG Bertrand Liagre

Bassam Badran
email: bassam.badran@ul.edu.lb.

Rawan Makki

David Y Leger

Vincent Sol

Phytochemistry and biological activities of selected Lebanese plant species (Crataegus azarolus L. and Ephedra

Keywords: Anti-Inflammatory Agents, Antioxidants, Chemical Fractionation

Background:

In the present study, phytochemical screening, antioxidant, anti-inflammatory, and antiproliferative capacities of 3 extracts from leaves of Lebanese Crataegus azarolus L. were evaluated.

Material/Methods:

Fresh leaves were dissolved in 3 different solvents: distilled water, ethanol, and methanol. The chemical composition was determined using high-performance liquid chromatography (HPLC) and the content of essential oil of this plant was examined by gas chromatography (GC) coupled with mass spectrometry (MS). The antioxidant potential was evaluated using DPPH radical scavenging and Fe 2+ chelating activity assays. Anti-inflammatory effect was investigated by measuring the secreted amounts of the proinflammatory mediator PGE 2 using ELISA technique, as well as by assaying the mRNA levels of the proinflammatory cytokines (IL-a, IL-b, and Il-6), chemokines (CCL3 and CCL4) and inflammation-sensitive COX2 and iNOS enzymes using quantitative real-time PCR (qRT-PCR). The antiproliferative effect was evaluated using the XTT viability assay.

Results:

The obtained results show that alcohol (methanol and ethanol) extracts were rich in bioactive molecules with medical relevance and exerted substantial antioxidant, anti-inflammatory, and antiproliferative capacities. On the other hand, aqueous extract contained fewer chemical components and exhibited less therapeutic efficiency.

Conclusions:

Our observations indicate that Crataegus azarolus L. could be used for treating diseases related to oxidative stress, inflammatory reactions, and uncontrolled cell growth.

ABSTRACT

Plants have long been known for their natural arsenal, serving as an important source of nutrients and therapeutic components. Since about 600,000 years ago, humans used plants as medicines. Plant medicines correspond to the preparations issued from those plants. Today, plant medicines are widely worldwide where about 80% of the world's population uses herbs as primary medicines. This medicinal value is mainly attributed to the fact that plants are rich in bioactive phytochemicals. Lebanon, due to its geographical location and important environmental characteristics, is endowed with a rich flora. Hundreds of Lebanese plants have been defined in terms of their chemical composition and medicinal value where many other species are yet to be characterized.

In this study, we aimed at characterizing the phytochemical content and therapeutic value of two Lebanese plant species, Crataegus azarolus L and Ephedra campylopoda. Fresh leaves, derived from each plant species, were dissolved in three different solvents distilled water, ethanol, and methanol. The phytochemical composition of different extracts issued from the two plant species was examined using high performance liquid chromatography (HPLC) and the essential oil content was determined by gas chromatography (GC) coupled with mass spectrometry (MS). DPPH radical scavenging and Fe 2+ chelating activity assays were used to assess the antioxidant potential. Anti-inflammatory potential was evaluated by measuring the secreted amounts of the pro-inflammatory mediator PGE2 using ELISA technique, as well as by assaying the mRNA levels of the pro-inflammatory cytokines (IL-α, IL-β and IL-6), chemokines (CCL3 and CCL4) and inflammation-sensitive COX-2 and iNOS using quantitative RT-PCR (qRT-PCR). XTT viability assay was carried out to determine the anti-proliferative effect of each extract. For both plant species, we observed an important phytochemical content with the alcoholic (methanol and ethanol) extracts being more rich in bioactive molecules. In parallel, the two plant species exhibited significant biological activities with the alcoholic extracts exerting important, in vitro, antioxidant, anti-inflammatory and anti-proliferative effects.

Collectively, our observations suggest a promising potential for Crataegus azarolus L and Ephedra campylopoda during treatment of diseases associated with oxidative stress, aberrant inflammatory responses or uncontrolled cell proliferation. However, further in vivo characterization of these effects is indispensable.

List of abbreviations

List of Figures

List of Tables

Introduction 1.1 Overview about traditional medicine

Thousands of years ago, herbs and plant products were used in folk medicine for treating a broad spectrum of ailments and diseases. Folk remedies are usually prepared as powders, poultices, ointments, baths, decoctions, infusions and teas. The interest in examining the biological activities of traditional medicinal plants as well as isolating their bioactive components for therapeutic applications has been increasing worldwide and comprehensive screening programs have been established [1]. Floristic analysis showed that there are about 500,000 plant species on our planet. Out of these, about 120,000 plant species can be used to create biologically active products, which, in turn, might be used in disease treatment [2]. Today, a great number of different medicinal teas and other plant products are available on market including cosmetics and pharmaceuticals, which contain biologically active substances [3]. Recently, some products of plant origin were shown to be effective sources of chemotherapeutic agents showing strong biological activity without exerting undesirable side effects. This drew the attention of many scientists and encouraged them to screen the constituents of plants and study their biological activity and pharmacological benefits [4]. New systematic methods for separation, identification and determination of chemical constituents have developed, in parallel to different biological activity tests. Most of the new phytochemical studies follow similar methodologies including plant collection, chemical components extraction, biological tests, chemical analysis and statistical analysis [5].

Phytochemicals in plants

Plants have evolved secondary biochemical pathways for the synthesis of chemicals in response to environmental stimuli such as herbivore, pathogen deterrence and nutrient shortage. These secondary metabolites, which are called phytochemicals, do not interfere with primary metabolism, have relatively low or nontoxic nature and play a significant therapeutic importance such as to reduce or prevent some types of cancer and inhibit the development and spread of inflammatory diseases [6]. Those chemical compounds include flavonoids and quinones.

Phenolic compounds -Flavonoids

Phenolic compounds are widely distributed in the plant kingdom with over 10,000 types identified so far. These compounds share at least one aromatic hydrocarbon ring along with one or more hydroxyl groups in their structure (Table 1). The three most abundant phenolic compounds are phenolic acids, flavonoids and polyphenols. For instance, phenolic acids are the esters of polyols such as glucose. Caffeic acid is a phenolic acid compound normally found in esterified forms such as chlorogenic acid in coffee and vegetables (Figure 1). Tannins are derivatives of phenolic acids [7], since they are a heterogeneous group of high molecular weight polyphenolic compounds with the capacity to form reversible and irreversible complexes with proteins (mainly), polysaccharides (cellulose, hemicellulose, pectin, etc.), alkaloids, nucleic acids and minerals. On the basis of their structural characteristics it is therefore possible to divide the tannins into four major groups: gallotannins, ellagitannins, complex tannins, and condensed tannins (Figure 2). Tannins are commonly found in fruits such as grapes, persimmon, blueberry, tea, and chocolate. properties [8].

Flavonols

Major sources of dietary flavonols (Figure 4) are fruits, vegetables, tea and red wine. However, dietary intake of flavonols is influenced by species, growth rate, season, light availability, ripeness, food preparation and processing [9]. Decreased cardiovascular disease mortality has been reported in free living populations having high dietary intake of flavonols [10]. Quercetin is an important flavonol which is recommended at a daily dose of 1 g but normal dietary intake remains 10-100 mg [11]. Other important flavonols include kaempferol and myricetin. Quercetin and kaempferol inhibit calcium channel blockers such as nifedipine and felodipine and this was clinically significant, since patients experienced increased heart rate, decreased diastolic blood pressure and headaches Luteolin (5,7,3',4'-tetrahydroxyflavone) is usually found in parsley, celery, peppers, onion leaves, cabbage, broccoli, and apple skin. It is used to treat hypertension, inflammatory conditions, and cancer because of its anti-inflammatory, anti-carcinogenic, antioxidant, apoptosis promoting, anti-angiogenesis and anti-metastasis properties [12].

Chrysin (5,7-dihydroxyflavone) is a potent aromatase inhibitor and thus enhances testosterone levels in men. As such, chrysin is very poorly bioavailable but its methylated forms are stable and are highly bioavailable [13].

Flavanones

The primary sources of flavanones are citrus fruit, which contain naringenin, eriodictyol and hesperidin. Naringin is citrus flavonoid-glycoside, which produces the bitter taste in grapefruit. Naringin is hydrolyzed into naringenin by gut microflora.

Naringenin possess antioxidant, antitumor and radioprotective activities and thus affects cancer, cardiovascular diseases, hyperlipidemia, neurological disorders and skin disorders [14]. Hesperidin exhibits antioxidant, anti-inflammatory, anticarcinogenic, antimicrobial and radioprotective activities [15].

Isoflavones

Isoflavones are the predominant constituent of the diet. Most common in human consumption are genistein (4',5,7-trihydroxyisoflavone), diadzein (4',7dihydroxyisoflavone), and glycitein (7,4'-dihydroxy-6-methoxyisoflavone). These chemicals possess estrogenic activity upon binding the estrogen receptors ER-β.

Genistein exhibit significant antiproliferative and apoptotic activities and thus acts as chemopreventive agent [16]. Genistein is described to improve blood flow by affecting vasodilation and this effect can be reversed by NO synthase inhibition [17].

Flavan-3-ols

Flavan-3-ols like catechin, gallocatechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate, are found in grapes and other food sources. These compounds can enhance the release of nitric oxide (NO) and decrease superoxide production during platelet aggregation and thus limit this process. These phytochemicals also reduce the release of pro-inflammatory mediators from platelets and thus inhibit inflammation in arteries. These components also suppress LDL / HDL oxidation [11]. LDL are the major carrier of cholesterol in the blood; if LDL level is increased, those compounds can promote atherosclerosis upon oxidizing the LDL in artery walls and narrowing it with cholesterol particles. On the other hand, HDL act to reverse cholesterol transport and inhibit LDL-induced cytotoxicity.

Anthocyanidin

Anthocyanidins exhibit a range of pro-health effects including antioxidant, antiinflammatory, antimicrobial, anti-carcinogenic, neuroprotective, vision improving, and apoptosis inducing properties. Anthocyanidins such as pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvidin 3-glucoside, and malvidin 3,5-diglucosides are found to be strong inhibitors of NO production in vitro without exerting harmful effects [18]. Anthocyanins, cyanidins and delphinidins potently inhibit platelet derived growth factor (PDGF)-induced vascular endothelial growth factor (VEGF) expression in vascular smooth muscle cells by preventing p38 mitogen activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways. Thus, the ability of the active anthocyanin compounds to maintain endothelial cells in a quiescent state by preventing the vascular expression of VEGF might contribute to a decreased rate of development of atherosclerotic lesions [19].

Alkaloids

Alkaloids are a diverse class of cyclic nitrogenous compounds found in about 20% of plant species. Alkaloids exist in many categories such as indole, benzylisoquinoline, tropane, pyrrolizidine, or purine alkaloids (Table 2). For over 5000 years, alkaloids have been used as poisons, neurotoxins, and psychedelics. Caffeine, cocaine, ephedrine, morphine, nicotine, atropine, physostigmine, scopolamine are common alkaloids that affect many health conditions including behaviour [6]. Bioprotective alkaloids in plants and plant-endophyte associations include pyrrolizidines, ergot alkaloids, indole diterpenes, pyrrolopyrazines. Such alkaloids increase fitness of plants and host plants (which supplies food resources and acts as a substrate for commensalist insects or other fauna) against the exploitation by animals and make them more resistant and competent to drought. Ergot alkaloids are the main sources of toxicities to livestock [20].

The majority of polyhydroxylated alkaloids such as pyrrolidine, piperidine, and pyrrolizidine inhibit glycosidic bond hydrolysis by interfering with glycosidases, which have highly important functions in primary metabolism. Various polyhydroxylated alkaloids are used for therapeutic uses such as being anticancer, antiviral, and antidiabetic agents, immune stimulants and are also used in treating glycosphingolipid lysosomal storage diseases [21].

Table 2: Chemical classification of alkaloids [22].

Quinones

Quinones are aromatic compounds that are usually derived from benzene or naphthalene (Figure 5). The major groups of natural 2-methyl-3-oligoisoprenyl quinones are vitamin K, coenzyme Q (ubiquitones), and tocopherol quinones which are produced from the oxidation of vitamin E. Ubiquitone is a prenylated benzoquinone, which plays basic role in electron shuttle in the electron transport chain. It also participates in the mitochondrial oxidation of dihydroorotate as well as in the biosynthesis of pyrimidines [23]. Antiproliferative and cytotoxic properties of many quinones and hydroquinones derived from sponges have created promising opportunities for the development of antitumor agents and many quinones exhibit significant anti-tuberculosis, antimalarial, antifungal and cardiotonic activities [24]. studies as a multitude of factors can alter the path of bioactive compound under test [16].

Preliminary studies have provided impetus for well controlled and randomized trials to evaluate the efficacies and as more evidence gathers, scientific and medical knowledge of the therapeutic potentials of phytochemicals will become conclusive. Natural products are well known, nowadays, to exhibit an extensive spectrum of biological activities such as stimulation of the immune system, antibacterial, antiviral, anti-hepatotoxic, anti-ulcer, anti-inflammatory, antioxidant, anti-mutagenic, anti-cancer effects, and induction of apoptosis [25] (Table 3), and medicinal plants are still used despite the advantages of modern synthetic drugs. Moreover, medicinal plants are more natural and more accessible than manufactured drugs, so people believe that the use of the medicinal plants for the treatment of the diseases is more safe [26].

More than 70% of the developing world's population depends, today, on traditional medicinal system, otherwise known as complementary or alternative systems of medicine [27,28]. For instance, 30-75% of cancer patients in the USA uses the medicinal plants as complementary and alternative medicine [29]. This encourage the researchers to search for possible anticancer agents from plants in different countries.

Indeed, the use of medicinal plants by people in their food systems will open the window for many important new pharmaceuticals. In the last 20 years, more than 25% of drugs have been directly derived from plants, while other 25% have been chemically altered natural products [30]. Pharmacologically active chemicals present in medicinal plants are now proving potential for use in treating inflammatory diseases as well as cancer [28]. among the world's population being attributed to this disease [31][32][33]. The body maintains a system of checks and balances on cell growth so that cells divide to produce new cells only when new cells are needed or when normal cells grow old or get damaged. Disruption of this system results in an uncontrolled division and proliferation of cells that are build up an extra cells often forms a mass of tissue known as a tumor, which grow or proliferate throughout the tissues of the body and it may progress and cause death [34].

Tumors can be benign or malignant, where the term "cancer" refers usually to malignant tumors. Benign tumors can usually be removed and do not spread to other parts of the body and usually don't need to be removed. However, malignant tumors, grow aggressively and invade other tissues of the body and often can be removed but may grow back, allowing entry of tumor cells into the bloodstream or lymphatic system and then to other sites and organs in the body such as bone, brain and liver, where they overwhelm these sites by consuming their oxygen, nutrients, and space. This process of spread is termed metastasis, where the areas of tumor growth at these distant sites are called metastases. There are different causes of cancer, among which are chemicals, radiation, smoking, viral infection, dietary factors, and environmental factors. Physicians and researchers need to find a comprehensive cancer treatment that is based on the increased awareness of the role of traditional and complementary medicine [35].

According to number of global deaths, the most frequent types of cancer worldwide are lung, stomach, liver, colon (colorectal) and prostate, among men, while among women, they are breast, lung, stomach, colorectal and cervical [36].

There are many strategies to treat cancer, among which is a process called chemotherapy. Chemoprevention is defined as the use of non-toxic chemical substances or their mixtures to inhibit, retard or delay the overall process of multi-stage carcinogenesis. A wide array of compounds, of both synthetic and natural origin, have been reported to exert anti-mutagenic and anti-carcinogenic effects in numerous animal and cell culture systems. Chemotherapy may be used alone, with radiation therapy, or after surgery. Chemotherapy uses drugs to kill cancer cells. When radiation therapy and chemotherapy are given at the same time, the side effects may be worse. Cancer chemotherapy has faced dramatic problems. Poor selectivity of anticancer agents, kills both malignant and normal cells [37]. Contentious treatment with chemotherapy may lead to drug-resistant [38]. This in turn justifies the interest in search of possible anticancer agents from the flora of different countries [39]. Some products of plant origin have strong biological activity and can be used as an effective sources of chemotherapeutic agents without side effects. This attracted the attention of many scientists to screen plants and to study their chemical, pharmacological and biological activity [40]. In studies conducted in the Middle East, during chemotherapy treatment [41], about half of the cancer patients uses the complementary and alternative medicine (CAM) in Turkey [42],

and 35% in Jordan uses CAM [START_REF] Afifi | The use of herbal preparations as complementary and alternative medicine (CAM) in a sample of patients with cancer in Jordan[END_REF]. CAM is also used by patients with hematological, malignancies, gynecological [START_REF] Yildirim | The use of complementary and alternative medicine (CAM) therapies by Turkish women with gynecological cancer[END_REF] and pediatric disorders [START_REF] Genc | Complementary and alternative medicine used by pediatric patients with cancer in western Turkey[END_REF].

Many nutritional agents are believed to be critical in carcinogenesis [START_REF]Committee on Diet and Health implications for reducing chronic disease risk[END_REF].

Evidences from epidemiologic studies indicate that diets that contain high fruits and vegetables such as cabbage, broccoli, tomatoes, apples and grapes [START_REF] Vainio | Fruit and vegetables in cancer prevention[END_REF], are associated with a lower risk of different cancers [START_REF]Food, nutrition and the prevention of cancer: a global perspective[END_REF], such as prostate, oral cavity, lung, breast and colon [START_REF] Institute | Diet, nutrition, and cancer prevention: a guide to food choices[END_REF][START_REF] Amin | Overview of major classes of plant-derived anticancer drugs[END_REF]. For instance, a high consumption of soybean products in Asian countries reduce the incidence of colon cancer [START_REF] Zhu | Effects of soybean extract on morphology and survival of Caco-2, SW620, and HT-29 cells[END_REF], a high consumption of vegetables reduces the risk of colon cancer mortality [START_REF] Rijken | Effect of vegetable and carotenoid consumption on aberrant crypt multiplicity, a surrogate end-point marker for colorectal cancer in azoxymethane-induced rats[END_REF], and recently, medicinal plant extracts have been demonstrated to be able to control the proliferation of prostate cancer cells [START_REF] Hryb | The effect of extracts of the roots of the stinging nettle (Urtica dioica) on the interaction of SHBG with its receptor on human prostatic membranes[END_REF].

Moreover, several organizations, such as National research council of the national academy of sciences [START_REF]Committee on Diet and Health implications for reducing chronic disease risk[END_REF], the national cancer institute [START_REF]Guidelines on diet, nutrition, and cancer[END_REF], and the American cancer society [START_REF] Giovannucci | Tomato-Based Products, Lycopene, and Cancer[END_REF], encourage the increase intake of citrus fruits, green and yellow vegetables.

1.5 Anti-oxidant properties of phytochemicals 1.5.1 Free radicals, endogenous defense and the role of oxidative stress in human 1.5.1.1 Definition of free radicals "Any species (atoms, molecules or ions) that contain one or more unpaired electrons in their orbitals are known as free radicals [START_REF] Halliwell | Tell me about free radicals, doctor: a review[END_REF]. In other words, they possess an open shell configuration [START_REF] Ni | Seasonal variations of the antioxidant composition in ground bamboo Sasa argenteastriatus leaves[END_REF], where an open shell is a valence shell [START_REF] Carey | Advanced Organic Chemistry Part A: Structure and Mechanisms[END_REF], which is not completely filled with electrons, or all the electrons contained in it are not in chemical bonding with other atoms or molecules. Free radicals may carry positive, negative or zero charge [START_REF] Lennarz | Biochemical and Biophysical Research Communications[END_REF]. The free electrons present in the open shells make these radicals highly reactive species. These free radicals play a considerable role in the processes of polymerization [START_REF] Burnett | [END_REF] and combustion [START_REF] Lomnicki | Copper oxide-based model of persistent free radical formation on combustion-derived particulate matter[END_REF] and many chemical reactions of plasma chemistry, atmospheric chemistry [START_REF] Hurley | Atmospheric chemistry of n-C(x)F(2)(x)(+1)CHO (x = 1, 2, 3, 4): fate of n-C(x)F(2)(x)(+1)C(O) radicals[END_REF] and biochemistry [START_REF] Boveris | Biochemistry of free radicals: from electrons to tissues[END_REF]. In living organisms, intermediary metabolism [START_REF] Ramakrishnan | Intermediary metabolism of mycobacteria[END_REF] of many biological compounds takes place via these reactive species. In a process referred to as redox signaling [START_REF] Shah | Free radicals and redox signalling in cardiovascular disease[END_REF], free radicals serve as messengers.

Free radicals play a very important role in a number of chemical reactions, which occur in our body. Usually, our body exploits a number of free radical physiological and biochemical reactions, and keeps them under tight control [START_REF] Halliwell | Tell me about free radicals, doctor: a review[END_REF]. However, unfortunately, these processes may go out of control leading to a chain of reactions that are damaging to surrounding tissue. They, therefore, could play constructive as well as destructive roles in the body.

Origins and targets of free radicals

Each day, our body produces, during metabolism, numerous amount of free radicals which in turn react with other molecules in several of ways [START_REF] Halliwell | Tell me about free radicals, doctor: a review[END_REF][START_REF] Halliwell | Free radicals, antioxidants, and human disease: curiosity, cause, or consequence?[END_REF].

Mitochondria is the most important cellular source for free radicals and deal with regulatory and toxic reactions accomplishing different physiological processes including cell cycle, proliferation and cell death. Mitochondria is the major site of generation of primary free radicals such as superoxide anion (O2 -) and nitric oxide (•NO) along with hydrogen peroxide (H2O2) and peroxynitrite (ONOO -) as termination products (Figure 6) [START_REF] Cadenas | Mitochondrial free radical production and cell signaling[END_REF]. Electron transport chain directly produces superoxide anion radical and then hydrogen peroxide which in turn gives rise to hydroxyl radical [START_REF] Cadenas | Mitochondrial free radical generation, oxidative stress, and aging[END_REF]. As free radicals contain unstable or free electrons in their shells, they tend to be more reactive than nonradical species, although they possess a wide range of reactivity. When two radicals come together, they share their unpaired electrons through the formation of a covalent bond.

Hydroxyl free radicals are highly reactive and can attack any of the molecules found in the living cells causing a damage to the system. A hydroxyl radical can give birth to a chain of reactions in the cell and if it attacks DNA, it causes chemical changes in the DNA bases and even breaks the linkages between these bases, activating oncogenes and carcinogenesis.

In addition to DNA, proteins are considered as the initial cell targets of free radicals [START_REF] Du | Proteins are major initial cell targets of hydroxyl free radicals[END_REF]. Upon exposure to hydroxyl free radicals, proteins are described to be oxidized before lipids [START_REF] Siesjo | Free radicals and brain damage[END_REF].

Figure 6: Generation of primary free radicals by mitochondria.

Source of free radicals

As mentioned above, the source of free radicals is our body itself as well as the environment. Mitochondrial electron transport chain is an adventitious source of free radicals during normal metabolism [START_REF] Harman | Aging: a theory based on free radical and radiation chemistry[END_REF] in addition to other physiological factors [72].

Thus, sources of free radicals can be either internal or external.

Enzymatic reactions occurring in the body serve as a source of free radicals.

These reactions are involved in the electron transport chain, phagocytosis, cytochrome P450 systems and prostaglandin synthesis. Mitochondria, phagocytes, peroxisomes, xanthine oxidase, arachidonate pathways, ischemia, inflammation and exercise are described as internal sources of free radical generation [START_REF] Liu | The isoprostanes: novel prostaglandin-like products of the free radical-catalyzed peroxidation of arachidonic acid[END_REF].

Similarly, non-enzymatic reactions of oxygen with organic compounds and the reactions initiated by ionizing radiations serve as an external source of free radicals.

Additionally, cigarette smoke, environmental pollutants, ozone, drugs, pesticides, UV light, industrial solvents and electronic pollutants (extra low frequencies from computers, power lines, television, microwaves, ...) are some other external sources of free radicals [74]. Physiological factors like stress, emotions, anxiety and other disease conditions add to the generation of free radicals and put the body under oxidative stress [START_REF] Mal'tsev | Effect of emotional painful stress on affinity of blood to oxygen, on the antioxidant system and physical properties of the hepatocyte microsomal membrane[END_REF].

Formation of reactive oxygen species and action in vivo

Reactive oxygen species (ROS) are chemically highly reactive molecules with one unpaired electron in their valance shell derived from molecular oxygen [START_REF] Turrens | Mitochondrial formation of reactive oxygen species[END_REF]. ROS are generated during normal metabolism of oxygen by endogenous systems as well as exposure to physiochemical and patho-physiological conditions, and play a key role in cell signaling and homeostasis. ROS include superoxide, hydroxyl radical, hydrogen peroxide, peroxyl radical, organic hydro peroxide, singlet oxygen and ozone [START_REF] Turrens | Mitochondrial formation of reactive oxygen species[END_REF]. These reactive oxygen species are produced in the body with controlled mechanisms. Enzymes such as  -1-microglobulin, catalase, superoxide dismutase (SOD), glutathione peroxidase and lactoperoxidase defend living cells against oxidative damage by ROS [START_REF] Ray | Oxidants, antioxidants and carcinogenesis[END_REF] (Table 4). If these mechanisms get out of control, free radicals can adversely affect proteins, lipids, DNA and may lead to a number of human diseases [START_REF] Nishigori | Role of reactive oxygen species in skin carcinogenesis[END_REF].

Table 4: Reactive oxygen species (ROS) [START_REF] Stohs | Oxidative mechanisms in the toxicity of metal ions[END_REF][START_REF] Birben | Oxidative stress and antioxidant defense[END_REF].

Among ROS, the hydroxyl radical is the most reactive and potent damaging species which can affect each and every molecule found in the living cells. In vivo, it has been shown that hydroxyl radical can lead to formation of DNA-protein linkages, base damages, DNA strand breaks, protein fragmentation and lipid peroxidation [START_REF] Stohs | Oxidative mechanisms in the toxicity of metal ions[END_REF].

Antioxidants

Antioxidants are defined as the substances which at low concentration can significantly inhibit or delay the oxidative process, while often being oxidized themselves [START_REF] Valko | Free radicals and antioxidants in normal physiological functions and human disease[END_REF]. Endogenous and exogenous antioxidants are used to neutralize free radicals and protect the body from free radicals by maintaining redox balance [START_REF] Pham-Huy | Free radicals, antioxidants in disease and health[END_REF]. The human body is equipped with a variety of endogenous antioxidants that serve to counterbalance the effect of oxidants. These are divided into two categories: enzymatic and non-enzymatic antioxidants.

Enzymatic endogenous antioxidants

The major enzymatic antioxidants are superoxide dismutase, catalase, GSH (glutathione) peroxidase and GSH reductase. In addition to these major enzymes, other antioxidants, including glutathione S-transferase (GST), heme oxygenase-1, and redox proteins, such as thioredoxin (TRX), peroxiredoxins (PRXs) and glutaredoxins, are also described to play crucial roles during antioxidant defenses [START_REF] Birben | Oxidative stress and antioxidant defense[END_REF][START_REF] Ye | Oxidative stress, redox regulation and diseases of cellular differentiation[END_REF].

Non-enzymatic endogenous antioxidants

Non-enzymatic antioxidants include glutathione (GSH), uric acid, bilirubine and others.

1.5.2.2.1. Glutathione GSH is a tripeptide (L-g-glutamyl-L-cysteinyl-Lglycine) and the major soluble antioxidant being highly abundant in all cell compartments. It also serves as a cofactor for several detoxifying enzymes, such as GSH-Px and GSH transferase, and has a role in converting vitamin C and E back to their active forms. GSH protects cells against apoptosis by interacting with pro-apoptotic and anti-apoptotic signaling pathways. In addition, reduced glutathione donates protons to membrane lipids and protects them from oxidant attacks [START_REF] Birben | Oxidative stress and antioxidant defense[END_REF][START_REF] Sousa | Role of H(2)O(2) in hypertension, renin-angiotensin system activation and renal medullary disfunction caused by angiotensin II[END_REF].

Uric acid

It is a highly abundant aqueous antioxidant, considered as the main contributor for the antioxidant capacity in the plasma. It is able to quenche HO• and ONOO-and prevent lipid peroxidation [START_REF] Sousa | Role of H(2)O(2) in hypertension, renin-angiotensin system activation and renal medullary disfunction caused by angiotensin II[END_REF]. The scavenging of ONOO-by uric acid is critically enhanced in the presence of vitamin C and cysteine. Uric acid can also serve as a chelator of iron in extracellular fluids [START_REF] Sousa | Role of H(2)O(2) in hypertension, renin-angiotensin system activation and renal medullary disfunction caused by angiotensin II[END_REF] Chemically, polyphenols are compounds bearing one or more hydroxyl groups attached to a benzene ring. They can be classified as simple phenolics, phenolic acids (derivatives of cinnamic and benzoic acids), coumarins, flavonoids, stilbenes, tannins, lignans and lignins. Among these, flavonoids have the most potent antioxidant potential [START_REF] Fraga | Basic biochemical mechanisms behind the health benefits of polyphenols[END_REF][START_REF] Fadel | The natural antioxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidation in situ[END_REF].

Anti-inflammatory properties of phytochemicals 1.6.1 Inflammation

Inflammation is a process being implicated in the pathophysiology of an increasing number of diseases including cardiovascular disease, cancer, diabetes, agerelated macular degeneration, Parkinson's disease, Alzheimer's disease, and possibly depression [START_REF] Kulmatycki | Drug disease interactions: role of inflammatory mediators in depression and variability in antidepressant drug response[END_REF].

Overview over the inflammatory process

Inflammation is a rapid and non-specific response to cellular injury or infection.

The inflammatory response is produced and controlled by complex interactions between different cellular and plasma protein components. The cellular components involve intercellular communication effected by a range of cytokines. The causes of inflammation are apparently causes of diseases; it can be induced upon bacterial infections by compounds including lipopolysaccharides (LPS), as well as by viruses, which are detected by Toll-like receptors (TLRs), expressed by immune cells like macrophages.

Besides, inflammation can be triggered by physical injuries (i.e., UV) or chemical compounds (i.e., reactive oxygen species). It is characterized by five cardinal signs: redness, heat, swelling, pain, and loss of function [START_REF] Punchard | The Journal of Inflammation[END_REF].

Acute and chronic inflammation

Inflammation is classified crudely based on duration of the lesion and histological appearances into acute and chronic inflammation. Acute inflammation usually takes place first in response to the attack of the risk factor (s). As soon as the risk factor (s) is removed, the acute inflammatory response will stop. However, when the risk factor (s) persist, the acute inflammation will progress to chronic inflammation. It is the chronic inflammation that plays the major role in the pathogenesis of all inflammatory diseases.

By monitoring soluble markers corresponding to the sequential events of both acute and chronic inflammation it is possible not only to determine whether an individual is currently exposed to risk factor (s) of inflammation but also the extent of overall inflammation reaction.

All phases of inflammatory reaction are sustained and controlled by a number of extracellular mediators and regulators including cytokines, chemokines, growth factors, eicosanoids (prostaglandins, leukotrienes, etc), complement and peptides. These extracellular events are matched by equally complex intracellular signaling control mechanisms, with the ability of cells to assemble and disassemble an almost bewildering array of signaling pathways as they move from inactive to dedicated roles within the inflammatory response and site (Table 5) [START_REF] Punchard | The Journal of Inflammation[END_REF] .

Table 5: Different types of Cytokines found in acute and chronic inflammations [START_REF] Punchard | The Journal of Inflammation[END_REF].

The inflammatory response commences with a brief constriction of arterioles followed by vasodilatation and exudation of protein-containing plasma and blood cells into the injured tissue. This results in swelling and edema. Meanwhile, leukocytes adhere to vessel walls and cause the endothelial cells to contract, creating enough space between these cells, thus allowing leukocytes to enter the extra-vascular tissue. Increased vascular permeability is maintained until the inflammatory state is resolved, and it is the interplay between blood cells and plasma proteins in the affected tissue that controls the inflammatory response and interacts with part of the immune response (Figure 7).

Inflammation is orchestrated by different cell types including mast cells, endothelial cells, phagocytic leukocytes (polymorph nuclear neutrophils, macrophages, and eosinophils), and platelets which secrete different regulatory molecules such as cytokines, chemokines, prostaglandins and NO. PGI2 (prostacyclin) is the predominant PG in the vascular endothelium, where it inhibits platelet aggregation and causes vasodilation. PGE2 is the prevailing PG in the kidney [START_REF] Needleman | Arachidonic acid metabolism[END_REF]. PGE2 has several pro-inflammatory activities, including vasodilation and increasing vascular permeability, inducing fever, and enhancing pain and edema caused by other mediators such as bradykinin and histamine. Large amounts of PGE2 and PGF2 can be produced by stimulated monocytes and macrophages, whereas stimulated neutrophils produce moderate amounts of PGE2 [START_REF] Calder | Polyunsaturated fatty acids and inflammation[END_REF].

PGs are products of arachidonic acid metabolism. Arachidonic acid is released from the cell membrane by phospholipase enzymes, in particular phospholipase A2, and the free fatty acid can be metabolized to eicosanoids by cyclooxygenase (COX) and lipoxygenase (LOX). Metabolism catalyzed by COX gives rise to PGs of the 2-series as well as thromboxanes, while LOX metabolism leads to the formation of leukotrienes (Figure 8).

COX exist in two isoforms; these are known as COX-1 and COX-2 and their respective protein products show differential distribution. COX-1 is constitutively expressed in most tissues, where it catalyzes the biosynthesis of eicosanoids (PGs and thromboxanes) that regulate numerous cellular processes. In contrast, COX-2 activity is generally undetectable in most tissues, but the expression of COX-2 can be rapidly induced in inflammatory cells in response to stimulation by pro-inflammatory cytokines or by growth factors [START_REF] Calder | Polyunsaturated fatty acids and inflammation[END_REF] (Figure 9). Figure 9: Cyclooxygenase isoforms and their corresponding functions [START_REF] Lipsky | Specific COX-2 inhibitors in arthritis, oncology, and beyond: where is the science headed?[END_REF] .

A new class of anti-inflammatory and analgesic agents that are selective COX-2 inhibitors were developed in the 1990s (e.g. celecoxib, rofecoxib). The rationale for this drug development was that selective COX-2 inhibitors were not expected to interfere with homoeostatic physiological processes and should therefore be less likely than nonselective COX inhibitors to cause the unwanted side effects typical of traditional NSAIDs [START_REF] Lipsky | Specific COX-2 inhibitors in arthritis, oncology, and beyond: where is the science headed?[END_REF]. NO can be generated by three different forms of nitric oxide synthases (NOS):

inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS). These enzymes catalyze L-arginine into L-citrulline and nitric oxide in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) (Figure 10). eNOS is present in endothelium, and is involved in the dynamic control of vascular tone. nNOS is mainly present in neural tissue. iNOS is induced by either bacterial lipopolysaccharide (LPS) or a number of cytokines including TNF-α and interferon-γ (IFN-γ) in macrophages, hepatocytes and endothelial cells. iNOS and nitric oxide are involved in host defense and immunity and modulate the inflammatory response [START_REF] Lipsky | Specific COX-2 inhibitors in arthritis, oncology, and beyond: where is the science headed?[END_REF]. Key similarities and dissimilarities among the three NOS isoforms (nNOS, eNOS and iNOS) are summarized in Table 6. chemokines, all of which have two to four highly conserved cysteine residues (Table 8)

as well as a number of virally encoded chemokine-like proteins. The chemokine superfamily can be separated into four sub-families based on the presence and relative positioning of the first two cysteine residues at the N-terminus. The cysteine residues in the CXC (or α) family are separated by a non-conserved amino acid, while in the CC (or β) family, these cysteine residues are adjacent to each other. The XC (or γ) chemokines have only a single cysteine residue, while the CX3C (or δ) chemokine, CX3CL1, has three non-conserved amino acids between the first two cysteine residues.

Chemokines are also functionally classified depending on whether they are constitutively produced or are inducible. Constitutive, or homeostatic, chemokines are involved in basal leukocyte migration and development, whereas inducible, or inflammatory, chemokines control the recruitment of effector leukocytes during an immunological insult [START_REF] Proudfoot | Chemokine receptors: multifaceted therapeutic targets[END_REF]. It is noteworthy that this classification is not absolute, given that several chemokines cannot be assigned unambiguously to either one of the two functional categories. CXCL9, for instance, is a "dual-function" chemokine that is upregulated under inflammatory conditions, but also participates in T cell lymphopoiesis [START_REF] Moser | Chemokines: multiple levels of leukocyte migration control[END_REF]. Most chemokines are secreted from the cell, with the exception of CXCL16 and CX3CL1, which are membrane-bound proteins [START_REF] Bazan | A new class of membrane-bound chemokine with a CX3C motif[END_REF]. These proteins can also exist as soluble glycoproteins upon protease cleavage of their trans-membrane stalks. Thus far, 47 human chemokines have been described, many of which bind to several of the 18 described human chemokine receptors. Although there is considerable redundancy in the chemokine system, chemokine ligand-receptor binding does not usually cross the CC versus CXC chemokine boundaries. This redundancy also ensures sufficient levels of robustness within the system such that essential processes are not compromised by chance mutations. ) and hydrogen peroxide (H 2 O 2 ), as well as reactive nitrogen species (RNS), including nitric oxide (NO) and nitric dioxide (NO 2 ), play a dual role as both beneficial and deleterious chemical components. When present in moderate amounts, ROS and RNS could play a beneficial role upon serving as signaling messengers regulating a number of physiological processes, including gene expression, cell growth, and orchestration of immune responses [1][2][3][4]. However, excess of these reactive molecules generates oxidative stress, a harmful process that can damage all biological macromolecules and cell structures [4][5][6][7]. Oxidative stress is generally considered as a risk factor triggering the development of various critical pathologies, including cancer, arthritis, atherosclerosis, diabetes, autoimmune disorders, and cardiovascular and neurodegenerative diseases [8,9]. Endogenous antioxidants (fabricated by the body), as well as exogenous ones (supplied through diet), are chemicals that interact with and neutralize the ROS and RNS molecules, thus preventing their toxic effect [8,10]. Historically, plants are well known for their medicinal value, mainly related to their phytochemical component content, including phenolic compounds, flavonoids, alkaloids, tannins, and other stress-responsive products [11][12][13][14]. Indeed, plant-derived antioxidants, especially polyphenolic compounds, have proven success in minimizing the levels of toxic free radicals and relieving different oxidative stress-mediated diseases [13,15,16]. In addition, daily intake of natural antioxidants has been correlated with reduced occurrence of different diseases, including cancer, diabetes, and cardiovascular diseases [17]. Moreover, the phenolics and flavonoids of various medicinal plants exhibit potent anti-inflammatory and antiproliferative capacities [12,[18][19][20]. Crataegus (Hawthorn), belonging to the Rosacea family, comprises about 280 species that are mainly distributed in the northern temperate zones of North America, East Asia, Central Asia, and Europe [21]. Interestingly, hawthorn fruits have long been used in traditional medicine to treat different health concerns, mainly those related to the heart and blood vessels [22]. The pharmacological potential of hawthorn has been attributed to its important chemical composition, including proanthocyanidins, flavonoids, tannins, vitamin C, and glycosides [21,23]. Although the chemistry of different Crataegus species has already been described, the chemical composition of many other species is yet to be characterized. Crataegus, known as Zaarour in Lebanon, is represented by 3 species in the Lebanese flora: C. azarolus L., C. monogyna (Jacq), and C. sinaica (Boiss). So far, neither analytical nor biological studies have been performed on the Lebanese Crataegus species. In this study, we characterized the phytochemical component content, antioxidant, anti-inflammatory, and antiproliferative capacities of 3 extracts (water, ethanolic, and methanolic) prepared from fresh leaves of Crataegus azarolus L. grown in Lebanon.

Material and Methods

Plant collection and preparation of powders

Fresh leaves were gathered from southern Lebanon at 350 m altitude in spring season between March and May in 2011, and the biological authentication was carried out by Professor George Tohme, president of CNRS of Lebanon. After harvesting, they were well washed, cut into small pieces, and dried in the shade at room temperature, away from sunlight. After this period, the dried leaves were crushed and ground to a homogeneous fine powder by use of a grinder and then kept in the dark at room temperature until use in different studies.

Apparatus and chemicals

All of the used chemicals were of analytical grade. Absolute ethanol, methanol, n-hexane, sodium hydroxide, ethyl acetate, and dichloromethane were purchased from BDH England. Aluminium chloride and FeSO 4 •7H 2 O, silica gel was purchased from Merck Germany. Sodium carbonate and hydrogen peroxide were purchased from Unichem India. Ascorbic acid, gallic acid, rutin, Folin-Ciocalteau reagent, EDTA, ferrozine, and DPPH were purchased from Sigma Aldrich, USA. PBS was purchased from Gibco, UK. MS spectra were recorded on an Agilent series device and MSMS spectra were recorded on a Shimadzu series device.

Preparation of crude extracts using water, ethanol, and methanol as solvents

Powdered leaves (100 g) were deposited into a flask with 500 ml of the selected solvent (distilled water, ethanol, or methanol). After a period of maceration and stirring for 1 week at room temperature, the macerate was collected and filtered using filter paper. Extracts were then concentrated using a rotary evaporator at 40°C under reduced pressure (for ethanol and methanol extracts). The aqueous extract was prepared using the same steps as the alcoholic extraction except the temperature of the extraction was 60°C and the filtrates were then frozen before being lyophilized to obtain powders.

Phytochemical screening

To determine the chemical composition of the different extracts from leaves of C. azarolus, qualitative tests were done to detect the presence of primary and secondary metabolites as shown in Table 1. These tests are useful to estimate biological activities that might be due to the presence of secondary metabolites in the leaves of this plant.

Gas chromatography -mass spectrometry (GC/MS) analysis

The GC/MS analysis was performed on an Agilent 7890A-GCMS device. In the separation and identification by GC/MS technique, components were identified on the basis of the retention time and spectral index from the NIST and WILEY library. The instrument specifications and analysis conditions adjusted are given below in Tables 2 and3.

Liquid chromatography -mass spectrometry (LC/MS/MS) analysis

The LC/MS/MS analysis was performed on a Shimadzu-AB Sciex LCMSMS device for the detection. In the separation and identification by LC/MS/MS technique, components were identified on the basis of the retention time and mass spectral characteristics. The instrument specifications and analysis conditions adjusted are given below in Table 4.

Biological activities

DPPH radical scavenging assay

The antioxidant activity was assessed according to the method of Farhan et al. [24] using free radical DPPH. Increasing concentrations of extracts (0.05, 0.1, 0.2, 0.4, and 0.5 mg/ml) were prepared. We added 1 ml of each prepared dilution of each extract to 1 ml of DPPH reagent [0.15 mM]. The solutions were incubated in the dark at room temperature for 30 min and the absorbance was measured at 517 nm using a Gene Quant 1300 UV-Vis spectrophotometer. The DPPH-scavenging ability of leaf extracts was calculated according to the following equation:

% scavenging activity=[(Abs control-Abs sample)/

Abs control]×100 The control was prepared by mixing 1 ml DPPH with 1 ml of selected solvent. The blank was composed of 1 ml of the selected solvent.

Metal chelating activity

The chelation of ferrous ions by extracts was estimated by the method of Dinis et al. [24]. Briefly, 50 µl of FeCl 2 (2 mM) was added to 1 ml of different concentrations of the extract (500, 750, 1000, 1250, and 1500 µg/ml). The reaction was initiated by the addition of 0.2 ml of ferrozine solution (5 mM).

The mixture was vigorously shaken and left to stand at room temperature for 10 min. The absorbance of the solution was thereafter measured at 562 nm.

Anti-inflammatory activity RAW 264.7, a murine monocyte/macrophage cell line, was grown in DMEM medium supplemented with 10% defined FBS and 1% penicillin G-streptomycin in an atmosphere containing 5% CO 2 /95% air at 37°C. The macrophages were seeded in 12-well plates (1×10 6 cells/well) using fresh medium. After preincubation for 24 h, plates were cotreated with LPS at 100 ng/ml and 2 different concentrations of the drugs (100μg/ml and 50 μg/ml) in DMEM without FBS for 24 h (for RNA extraction and COX-2 activity).

PGE 2 immunoassay

PGE 2 amounts in culture medium were quantified in supernatants by enzyme immune assay using ELISA kits (R&D Systems), following manufacturer's guidelines.

Cell viability

Jurkat cells, corresponding to human leukemic T cell line, were seeded in 96-well plates (8×10 ranging from 5 to 200 μg/ml for 24, 48, and 72 h and cell viability was detected using Cell Proliferation Assay, XTT (Gentaur, Belgium) as previously described [25]. The XTT (sodium 3'-1 (phenylaminocarbonyl)-3,4-tetrazolium-bis (4-methoxy-6-nitro) benzene sulfonic acid) cell proliferation assay is an effective method to measure cell growth and drug sensitivity in tumor cell lines.

XTT is a colorless or slightly yellow compound that when reduced becomes bright orange. Briefly, XTT is cleaved by the mitochondrial dehydrogenase in metabolically active living cells to form an orange formazan dye. The absorbance of each sample was measured with a spectrophotometer at a wavelength of 450 nm.

Quantitative real-time PCR

Total RNA was extracted with Trizol reagent according to the manufacturer's guidelines (Invitrogen, Merelbeke, Belgium) and first-strand cDNAs were synthesized by reverse transcription (Superscript First-strand Synthesis System for RT-PCR kit; Invitrogen, Merelbeke, Belgium). Quantitative mRNA expression for the different genes was measured by real-time PCR with the PRISM 7900 sequence detection system (Applied Biosystems, Gent, Belgium), and the SYBR Green Master mix kit with b-actin mRNA was used as an internal control. The primers used for the amplification of each of the genes are indicated in Table 5. The program used for amplification was: 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. All qPCR reactions were performed in triplicate.

The expression levels (2 -DDCt ) of mRNAs were calculated as described previously [26].

Statistical analysis

The data are presented as means ±SEM of at least 3 independent experiments and analyzed using Student's t-test to determine any differences between group means, using SPSS for

GC/MS Analysis of essential oil obtained from the C. azarolus. L leaf extracts

The GC spectrum of the water, ethanolic, and methanolic extracts are shown in Figures 123, respectively. A total of 11 compounds present in the water extract, 7 compounds present in the ethanolic extract, and 8 compounds present in the methanolic extract were determined by the chromatographic method with the help of NIST and WILEY library as shown in Tables 789, respectively. In the case of water extract, pluchidiol compound was found to be in the highest concentration (33.62%) and other compounds were found in trace amounts (Table 7). In the case of ethanolic extract, g-tocopheryl methyl compound was found to be in the highest concentration (43.73%) followed by phytol isomer (20.47%), and other compounds were found in trace amounts (Table 8). In the case of methanolic extract, a tocopherol-beta-d-mannoside (21.87%), and ethyl linolinate (18.79%) compounds were found to be in the highest concentration and other compounds were found in trace amounts (Table 9).

The LC/MS/MS analysis for C. azarolus L leaf extracts

The LC spectrum results of the C. azarolus leaf extracts are shown in Table 10. A total of 2, 6, and 6 compounds were present in the water, ethanolic, and methanolic extracts, respectively, were determined by the chromatographic method based on the retention time and mass characteristics.

Antioxidant activity of C. azarolus L leaf extracts

The antioxidant activity of the aqueous, methanolic and ethanolic crude extracts was evaluated using 2 different assays: (1) DPPH free radical scavenging assay and (2) an assay assessing the iron (II) chelating ability. Our obtained results demonstrated that the different extracts displayed significant antioxidant activities and their scavenging effects on DPPH radical were in the following order: ethanolic extract (IC 50 =50±5.2 µg/ml) >methanolic extract (IC 50 =55±2.8 µg/ml) > water extract (IC 50 =60±2.2 µg/ml) (Table 11). Ferrous ion (Fe 2+ ) is a major preoxidant that upon interaction with hydrogen peroxide can lead to the generation of highly reactive hydroxyl radicals. The Fe 2+ chelating assay is based on the principle that ferrozine can quantitatively form colored complexes with Fe 2+ . However, when other chelating agents are present, the complex formation is disrupted and the extent of color reduction allows determination of the chelating activity of the coexisting chelator. Using the Fe 2+ chelating assay, the antioxidant potential of aqueous, methanolic, and ethanolic crude extracts derived from fresh leaves was assessed upon determination of their abilities to bind Fe 2+ in the presence of ferrozine. Methanolic extract had the highest Fe 2+ chelating capacity (IC 50 =0.5±0.08 mg/ml), followed by ethanolic extract (IC 50 =1±0.08 mg/ml) and then aqueous extract (IC 50 =1.5±0.07 mg/ml) (Table 11). Our observations indicate that the alcoholic extracts show more efficient antioxidant capacity than aqueous extract.

Anti-inflammatory activity of C. azarolus leaf extracts

Inflammatory response is a host's defensive mechanism against pathogens and is triggered by various microbial products such as lipopolysaccharide (LPS) [27]. Among the most important immune cells involved in this process are macrophages. Indeed, LPS can stimulate macrophages to produce large amounts of proinflammatory cytokines (such as IL-1a, IL-1b, and IL-6) and chemokine (including CCL3 and CCL4) [27] as well as other proinflammatory mediators including nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) that are fabricated by the inflammation-inducible isoforms of NO synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes [28,29]. or 100 µg/ml) of aqueous, methanolic, and ethanolic crude extracts derived from fresh leaves. In a first step, and upon using quantitative real-time PCR (qRT-PCR), relative iNOS and COX-2 mRNA levels in leaf extract-treated RAW264.7 cells versus nontreated control cells were determined. In the case of COX-2, 100 µg/ml of aqueous extract were required to trigger about 50% reduction in mRNA levels (Figure 4A). Ethanolic and methanolic extracts were more potent in terms of inhibition of COX-2 transcription since 50 µg/ml of either extract was sufficient to reduce COX-2 mRNA levels by about half (Figure 4A). Interestingly, COX-2 transcription was nearly lost upon treating cells with 100 µg/ ml of either ethanolic or methanolic extract (Figure 1A). In the case of iNOS, the 3 different extracts exhibited high efficiency in terms of impairing iNOS transcription, with ethanolic extract being the most potent, followed by methanolic extract and then aqueous extract (Figure 4B).

In a second step, and upon using ELISA technique, relative PGE 2 amounts present in the cell culture media were evaluated. Interestingly, all extracts were highly potent in terms of impairing PGE 2 production, with methanolic extract being the most efficient (Figure 4C). In a third step, and upon using qRT-PCR, the relative expression of the proinflammatory cytokines IL-1a, IL-1b, and IL-6 was assessed. In the case of IL-1a, neither of the 2 utilized aqueous extract concentrations was able to significantly impair IL-1a transcription (Figure 4D). On the other hand, 100 µg/ml of either ethanolic or methanolic extract dramatically reduced IL-1a mRNA levels (Figure 4D). For IL-1b, aqueous extract was again not efficient in terms of reducing the mRNA levels (Figure 4E). On the other hand, 100 µg/ml of ethanolic extract reduced IL-1b mRNA levels to less than half their level in control cells (Figure 4E). Methanolic extract was more efficient in impairing IL-1b transcription, since minimal IL-1b mRNA levels were detected in cells treated with 100 µg/ml of methanol extract (Figure 4E). In the case of IL-6, no striking alteration in mRNA levels was detected in cells treated with 50 µg/ml or 100 µg/ml aqueous extract (Figure 4F). IL-6 mRNA levels were not largely reduced in cells treated with 50 µg/ml of ethanolic extract but was lost following treatment with 100 µg/ml (Figure 4F). The effect of methanolic extract was more robust since about 75% of IL-6 mRNA amount was lost in cells treated with 50 µg/ml (Figure 4F). IL-6 transcription was lost in cells treated with 100 µg/ml of methanolic extract (Figure 4F).

In a fourth step, qRT-PCR was carried out to determine the transcription profiles of the proinflammatory chemokines CCL3 and CCL4. These 2 chemokines exhibited comparable transcription profiles under the different studied conditions (Figure 4G,4H). Neither CCL3 nor CCL4 transcription was lowered following treatment with either concentration of aqueous extract (Figure 4G,4H). Where no reduction in neither CCL3 nor CCL4 transcription was detected upon treating cells with 50 µg/ml of either ethanolic or methanolic extract, 100 µg/ml of the ethanolic extract reduced the mRNA levels of either chemokine by about 30%, and 100 µg/ml of methanolic extract decreased the mRNA levels by about 75% (Figure 4G,4H).

Antiproliferative activity of Crataegus azarolus L. leaf extracts

To determine if C. azarolus L leaf extracts affect cell viability, the XTT assay was carried out. This is a colorimetric assay during which the yellow water-soluble substrate XTT is reduced to a highly colored formazan product by succinate dehydrogenase enzymes in metabolically active cells. This conversion takes place only in viable cells; therefore, the amount of the formed formazan is proportional to the concentration of viable cells in the sample. Jurkat cancer cells were treated with different concentrations (5-200 µg/mL) of either aqueous, methanolic, or ethanolic crude extracts for different periods of time (24,[START_REF]Food, nutrition and the prevention of cancer: a global perspective[END_REF], or 72 h). The aqueous extract exerted no significant effect on cell viability in all tested conditions (Figure 5A), while ethanolic and methanolic extracts exerted dose-and time-dependent inhibitory effects (Figure 5B,5C).

In the case of ethanolic extracts, the IC 50 value (dose required to inhibit cell growth by 50%) corresponded to 150±4.4 µg/ml after 24 h (Figure 5B), and prolonged treatment for 48 h and 72 h caused a more striking inhibition of cell growth, as the IC 50 values were 25±1.6 µg/ml and 15±2.88 µg/ml, respectively (Figure 5B). Methanolic extract showed a more potent inhibitory effect than ethanolic extract; after 24 h of treatment with methanolic extract, the IC 50 value corresponded to only 50 µg/ml and prolonged treatment reduced this value to 22±1.6 µg/ml (after 48 h) and 13±1.8 µg/ml (after 72 h) (Figure 5C).

Discussion

Plants have been used throughout history to cure human diseases. Worldwide, people still use medicinal plants for healing and relieving physical suffering. Many modern medicines have been derived either directly or indirectly from medicinal plants [30].

Many studies still focus on identifying new medicinal plants.

In this study, we identified the chemical content of Crataegus azarolus L leaf extracts and assessed their therapeutic value. Interestingly, our phytochemical analysis showed the presence of various important medicinal components in the tested plant, including alkaloids, resins, phenol, quinones, diterpenes, lignin, sterols/steroids, flavonoids, carbohydrates, cardiac glycosides, reducing sugars, phlobatannins, and flavones. In agreement with previous observations [31][32][33], our data showed that the alcoholic solvents (ethanol and methanol) were more efficient than the aqueous one in extracting those bioactive compounds.

Identifying such important medicinal components in C. azarolus species is not surprising since various Crataegus species studied so far contained valuable therapeutic chemical compounds and are widely used in clinical applications [23]. Despite the identified chemical components, the Lebanese Crataegus azarolus L plant could carry much more compounds in them. In this study, different parameters account for the limited number of identified components. For instance, the output of the GC/ MS analysis, aimed to identify essential oils, is affected by the type of solvent used during the extraction method. Moreover, the limited number of chemical elements identified by LC/MS/ MS analysis is due to the fact that this analysis was targeted towards only 11 flavonoid compounds, and the presence/absence of many other elements was not checked. Further, the chemical composition of this plant could also be affected by environmental and geographical parameters, including the year and the season of harvest, exposure to sunlight, the altitude, and the region where the plant was harvested.

To characterize the medicinal potential of the Lebanese C. azarolus species, we determined their antioxidant, anti-inflammatory, and antiproliferative effects.

Oxidative stress occurs when the number of free radicals and reactive biomolecules in a body exceeds the body's ability to neutralize and eliminate them. Oxidative stress has deleterious effects on human health since it forms the basis for a variety of critical diseases affecting the heart, brain, kidney, liver, lungs, eyes, blood, skin, and joints [34]. Identifying new natural antioxidants is therefore highly valuable. In this study, 2 different methods, DPPH-scavenging ability and Fe 2+ -chelating activity assays, identified an antioxidant potential of C. azarolus extracts, with varied efficiency according to the type of used solvent. Indeed, and in both assays, extracts prepared using the alcoholic solvents showed more potent antioxidant activity than aqueous extracts. This could be related to the prominent chemical content in the alcoholvs. water-prepared extracts.

In addition to their cytotoxic effect, the reactive biomolecules might also trigger the initiation and/or amplification of inflammation via upregulation of different genes encoding for proinflammatory cytokines and molecules. Inflammation is believed to be associated with nearly all known chronic diseases, including heart diseases, diabetes, neurodegenerative disorders, autoimmune pathologies, and cancer [27,35]. Suppression of the inflammatory responses is therefore indispensable for treating these diseases. Interestingly, in the present study we evaluated the inhibitory potential of the indicated plant on both the secreted amounts of PGE 2 and the transcription levels of the proinflammatory cytokines (IL-a, IL-b and Il-6) and chemokines (CCL3 and CCL4), as well as COX-2 and iNOS enzymes. The different plant extracts showed varied anti-inflammatory capacities in a manner dependent on the type of solvent. Although the aqueous extract showed only moderate anti-inflammatory capacity, the alcoholic extracts strongly suppressed all of the mentioned proinflammatory mediators, with methanolic extract being more efficient than ethanolic extract. This robust anti-inflammatory potential is in agreement with their chemical arsenal and antioxidant activity. Crataegus azarolus L leaves could be then used to treat inflammatory diseases or serve as a promising resource for developing inflammatory-suppressive drugs.

In this study, the antiproliferative activity of the different extracts from leaves of Crataegus azarolus L was investigated in Jurkat cancer cells by XTT viability assay. In contrast to the aqueous extract, which failed to inhibit Jurkat cells proliferation, the alcoholic extracts substantially suppressed cell growth, with methanolic extract showing a more potent suppressive capacity than ethanolic extract. This inhibitory effect was time-and dose-dependent. The rich phytochemical arsenal identified in the alcoholic extracts might explain their robust antiproliferative potential. However, the molecular mechanisms accounting for this cytotoxicity are still unclear. Whether components of the apoptotic pathway are involved remains to be investigated. Moreover, whether Crataegus azarolus L leaf extracts could suppress the proliferation of different cancer cell types will be addressed in our future work.

Conclusions

In conclusion, the present study revealed the presence of different medicinal compounds in the leaves of Lebanese Crataegus azarolus L. Due to its substantial antioxidant, anti-inflammatory, and antiproliferative activities, this plant might offer a novel promising therapy that is beneficial for general health.

Ephedra corresponds to a genus of gymnosperms including over 40 species of tropical and subtropical, small, much-branched shrubs founds in the dry regions of both hemispheres. Ephedra is the only member of the family Ephedraceae. These plants, somewhat similar to the horsetails in appearance, are evergreen and their branches remain green for several seasons [116]. In Mediterranean region, Ephedra campylopoda is mainly found in Palestine and Jordan. In Lebanon, it is especially localized in Aandqet, Thoum, Monsef, Jiddayel, Chouane, Hazmieh, Saadiyat. [110].

Ephedra campylopoda is a clumbing shrubby tree in bushes beinvery ramified (Figure 13). The branches are long and distorted, the twigs are whorled often retrorse.

The male and female plants are independent: male flowers have 5-6 anthers in globulous For at least 5,000 years, the Ephedra species have been dispensed in traditional

Chinese medicines (TCM) [117], where dried stems are used to alleviate symptoms caused by common cold, influenza, asthma, bronchitis, nasal congestion and hay fever.

They were also used for a treatment of arthritis, fever, hives, lack of perspiration, headache, aching joints and bones, wheezing, and low blood pressure [118]. It is noteworthy that the tissue used in TCM is the dried green stem of one of three Ephedra species (Ephedra sinica, E. equisetina and E. intermedia), which are usually boiled in water and administered as a hot tea. In contrast to the diaphoretic uses and properties of ma-huang (stem part), the root and rhizome of Ephedra species, called mahuanggen, have antiperspirant property and are employed to treat spontaneous and night sweating [119].

In Western medicine, ephedrine is used for the treatment of nasal congestion due to hay fever, allergic rhinitis, asthma, and common cold [120]. Ephedrine salts are also prescribed in the form of nasal sprays to relieve congestion and swelling. When injected subcutaneously, ephedrine prevents hypotension during anesthesia. Orally administered ephedrine has been used in treating certain forms of epilepsy, nocturnal enuresis, myasthenia gravis, and urticaria accompanying angioneurotic edema. Pseudoephedrine, taken orally, is an effective nasal decongestant [117].

The major active ingredients of Ephedra are alkaloids that constitute 0.5 to 2.5 percent of the total mass, and are referred to as ephedrine-type alkaloids [22]. The six optically active alkaloids that have been isolated from Ephedra species are (-)-ephedrine and (+)-pseudoephedrine, (-)-N-methylephedrine, (+)-N-methylpseudoephedrine, (-)norephedrine, (+)-norpseudoephedrine. Usually, (-)-ephedrine is the major isomer that comprises 30 to 90 percent of total alkaloid fraction accompanied by (+)pseudoephedrine, with trace amount of other ephedrine-type alkaloids [22]. The total content of ephedrine-type alkaloids depends on the species of Ephedra, time of year of harvest, weather conditions, and altitude where the plant grows This variation according to environmental conditions explains why some Ephedra containing dietary supplements of the same brand often show alkaloid content markedly differing from label claims and also variation among lots when analyzed chemically [121].

Till date, neither chemical content nor the therapeutic potential of Lebanese

Ephedra campylopoda has been characterized. In this study, we assessed the chemical composition and the biological activities of this plant.

Background

Oxidative and inflammatory processes are described to be associated with a number of chronic diseases including atherosclerosis, Alzheimer's disease, cardiovascular diseases, neurodegenerative disorders as well as various human cancers [1]. Inflammatory response is a major defensive mechanism against infection, during which, inflammatory cells, such as macrophages, produce reactive oxygen species (ROS) and nitric oxide (NO) [2]. At low concentrations, these molecules serve important physiological role upon acting as second messenger in cell signaling. However, at higher amounts, these components can damage cellular lipids, proteins and DNA, leading to cell death [3][4][5][6]. To neutralize the toxic effect of these bioactive molecules, human body has evolved different defense mechanisms including the generation of antioxidants [7,8]. Oxidative stress is a result of imbalance between ROS formation and endogenous antioxidant capacity due to excessive ROS production and/or impaired antioxidant system. Identifying exogenous sources of antioxidants and antiinflammatory molecules is therefore of great importance. Plants have traditionally been used, for thousands of years, for treating inflammation and oxidative stress related disorders. Plants' medicinal value is mainly attributed to their phytochemical component content especially phenolic compounds and flavonoids that can exert potent antioxidant and anti-inflammatory effects [9][10][11][12][13][14]. In the Middle East region, herbal medicines are extensively used and a growing interest in identifying medicinal plants has been noted during the past years. Indeed, more than hundered of plant species, known for their medicinal value, have been isolated from this region [15,16] where many other plants are yet to be characterized. Lebanon, thanks to its geographic location, varied topography, distinct soil types and climatic variations, is characterized by a relatively large flora consisting of about 2607 species distributed over 783 genera. Among these, few hunderd species are used to treat various diseases including D gastrointestinal disorders, kidney and urinary diseases, cardiovascular diseases, diabetes, asthma, sexual disoders, hair problems and various tumours [17,18]. Nowadays, as many studies are focused on characterizing the therapeutic value of Lebanese plants, the list of Lebanese medicinal plants is expected to enlarge. The genus Ephedra Campylopoda belongs to the Ephedraceae family of plants. Those plants are small, leafless, highly branched shrubs, distributed in the dry regions of both hemisphers [19]. In Lebanon, Ephedra Campylopoda is found in different regions mainly rocky ones. In this study, we screened the phytochemical component content and characterized the antioxidant, anti-inflammatory and antiproliferative capacities of three extracts from the stems of Ephedra Campylopoda.

Materials and Methods

Plant collection and preparation of powders

Fresh plant was gathered from South of Lebanon at 350 m of altitude on spring season between March and May in 2011 and the biological authentication was carried out by Professor George Tohme, president of C.N.R.S of Lebanon. After that, they were well washed, cut into small pieces and dried in the shade at room temperature, away from sun light. After this period, the dried stems were crushed up and ground to get homogeneous fine powder by a grinder and then kept in a dark place at room temperature till use in different studies.

Apparatus and chemicals

All of the used chemicals were of analytical grade. Absolute ethanol, methanol, n-hexane and sodium hydroxide, ethyl acetate, dichloromethane were purchased from BDH England.

Aluminium chloride and FeSO4•7H2O, silica gel was purchased from Merck Germany.

E

Sodium carbonate and hydrogen peroxide were purchased from Unichem India. Ascorbic acid, gallic acid, rutin, Folin-Ciocalteau reagent, EDTA, Ferrozine and DPPH were purchased from Sigma Aldrich, USA. PBS was purchased from Gibco, UK. MS spectra were recorded on Agilent series, MSMS spectra were recorded on Shimadzu series.

Preparation of crude extracts using aqueous, ethanol and methanol as solvents

Powdered stems (100 g) were deposited into a flask with 500 ml of the selected solvent (distilled water, ethanol or methanol). After a period of maceration and stirring for 1 week at room temperature, the macerate was collected and filtered using a filter paper. Extracts were then concentrated using a rotary evaporator at 40 °C under reduced pressure (for ethanol and methanol extracts). The aqueous extract has been prepared using the same steps of the ethanolic extraction except the temperature of the extraction should be 60 °C and the filtrates were then frozen before being lyophilized to obtain powders.

Phytochemical Screening

In order to study the chemical composition of the different extracts from the stems of the studied plant, qualitative tests have been done to detect the presence or the absence of primary and secondary metabolites as shown in table 1. These tests are useful to estimate some biological activities that might be due to the presence of some secondary metabolites in the stems of this plant.

Gas chromatography-mass spectrometry (GC/MS) Analysis

The GC/MS analysis was performed on Agilent 7890A-GCMS. In the separation and identification by GC/MS technique, components were identified on the basis of the retention time and spectral index from the NIST and WILEY library.

F

Liquid Chromatography-Mass Spectrometry (LC/MS/MS) Analysis

The LC/MS/MS analysis was performed on Shimadzu-AB Sciex LCMSMS for the detection. In the separation and identification by LC/MS/MS technique, components were identified on the basis of the retention time and mass spectral characteristics.

Biological analysis

DPPH radical scavenging assay

The antioxidant activity was practiced according to the method of Farhan et al. [20] using free radical DPPH. Increasing concentrations of extracts (0.05, 0.1, 0.2, 0.4, 0.5 mg/ml) were prepared. 1 ml of each prepared dilution of each extract was added to 1 ml of DPPH reagent.

The solutions were incubated in the dark at room temperature for 30 minutes and the absorbance was measured at 517 nm by a Gene Quant 1300 UV-Vis spectrophotometer. The DPPH scavenging ability of peels extracts was calculated according to the following equation:

% scavenging activity = [(Abs control-Abs sample) / Abs control] ×100

Control was prepared by mixing 1 ml DPPH with 1 ml of selected solvent.

The blank was composed of 1 ml of the selected solvent.

Metal chelating activity

The chelation of ferrous ions by extracts was estimated by method of Dinis et al [21]. Briefly, 50 µl of FeCl2 (2 mM) was added to 1 ml of different concentrations of the extract (500, 750, 1000, 1250, 1500 µg/ml). The reaction was initiated by the addition of 0.2 ml of ferrozine solution (5 mM). The mixture was vigorously shaken and left to stand at room temperature for 10 minutes. The absorbance of the solution was thereafter measured at 562 nm.

Anti-inflammatory evaluation of the extracts G RAW 264.7, a murine monocyte/macrophage cell line, was grown in DMEM medium supplemented with 10% defined FBS and 1% penicillin G-streptomycin under the atmosphere containing 5% CO2/95% air at 37 °C. The macrophages were seeded in 12 well plates (1×10 6 cells/well) using fresh medium. After preincubation for 24 h, plates were cotreated with LPS at 100 ng/ml and two different concentrations of the drugs (100 μg/ml and 50 μg/ml) in DMEM without FBS for 24h (for RNA extraction and COX-2 activity).

PGE2 immuno assay

PGE2 amounts in culture medium were quantified in supernatants by enzyme immune assay, ELISA kits (R&D Systems), following manufacture's guidelines.

Cell viability

Jurkat cells, corresponding to human leukaemic T cell line, were seeded in 96-well plates (8 × 10 3 cells/well). The following day, cells were treated with the different extracts at concentrations ranging from 5 to 200 μg/ml for 24, 48, and 72 hours and cell viability was detected using Cell Proliferation Assay, XTT (Gentaur, Belgium) as previously described [22]. The XTT (sodium 3'-1 (phenylaminocarbonyl)-3,4-tetrazolium-bis (4-methoxy-6-nitro) benzene sulfonic acid) cell proliferation assay is an effective method to measure cell growth and drug sensitivity in tumor cell lines. XTT is a colorless or slightly yellow compound that when reduced becomes brightly orange. Briefly, XTT is cleaved by the mitochondrial dehydrogenase in metabolically active living cells to form an orange formazan dye. The absorbance of each sample was measured with a spectrophotometer at a wavelength of 450 nm.

H

Quantitative Real-Time PCR

Total RNA was extracted with Trizol reagent according to the manufacturer's guidelines (Invitrogen, Merelbeke, Belgium) and first-strand cDNAs were synthesized by reverse transcription (Superscript First-strand Synthesis System for RT-PCR kit; Invitrogen, Merelbeke, Belgium). Quantitative mRNA expression for the different genes was measured by real-time PCR with PRISM 7900 sequence detection system (Applied Biosystems, Gent, Belgium), and the SYBR Green Master mix kit with β-actin mRNA used as an internal control. The program used for amplification was: 10 min at 95°C followed by 40 cycles of 15 s at 95°C, 1 min at 60°C. All qPCR reactions were performed in triplicate. The expression levels (2 -ΔΔCt ) of mRNAs were calculated as described previously [23].

Statistical analysis

The data are presented as means ± SEM of at least three independent experiments and analyzed using Student's t-test to determine any differences between group means, using SPSS for Windows (Version 21

GC/MS Analysis of essential oil obtained from the Ephedra Campylopoda stem extracts

The GC spectrum of the aqueous, ethanolic and methanolic extracts are shown in figures 1, 2 and 3, respectively. A total of 7 compounds present in the aqueous extract, 6 compounds present in the ethanolic extract and 7 compounds present in the methanolic extract were J determined by the chromatographic method with the help of NIST and WILEY library as shown in Tables 3, 4 and 5, respectively. In the case of ethanolic extract, Linolenic acid methyl ester compound was found to be in the highest concentration (77.97%) whilst other compounds were found in trace amount (Table 4). In the case of methanolic extract, transphytol compound was found to be in the highest concentration (39.17%) whilst other compounds were found in trace amount (Table 5).

The LC/MS/MS Analysis of the Ephedra Campylopoda stem extracts

The LC spectrum results of the E. Campylopoda stem extracts are shown in the Table 6. A total of 1, 6, and 6 compounds present in the aqueous, ethanolic and methanolic extracts respectively were determined by the chromatographic method based on the retention time and mass characteristics.

Antioxidant activity of Ephedra Campylopoda stem extracts

To investigate the antioxidant activities of the aqueous, methanolic and ethanolic crude extracts-derived from fresh stems, DPPH free radical scavenging assay was carried out in a first step. As antioxidants can react with the violet coloured stable free radical DPPH, and convert it into a yellow coloured α,α-diphenil-β-picrylhydrazine, this assay is based on quantifying the change of the reaction mixture color as a readout of the scavenging capacity of antioxidants towards DPPH. The different extracts showed varied antioxidant potential and their DPPH scavenging capacities were in the following order: ethanolic extract (IC50=125 ± 4.4 µg/ml) > methanolic extract (IC50=150 ±5.1 µg/ml) > aqueous extract (IC50=300 ± 4.4 µg/ml) (Table 7).

Despites its beneficial roles as being required for oxygen transport, respiration and enzyme activity, iron is a highly reactive metal that can cause oxidative changes in proteins, lipids and K other structural components. Accordingly, in a second step, we characterized the antioxidant activities of the aqueous, methanolic and ethanolic crude extracts of E. Campylopoda stem by performing ferrous (Fe 2+ ) chelating activity assay. As Ferrozine can combine with Fe 2+ to form coloured complex and since other chelating agents, when present, can disrupt the complex formation and thus reduces the extent of color, the ferrous (Fe 2+ ) chelating assay is based on determination of the change of the reaction mixture color as a readout of the chelating activity of the coexisting chelator. Our results showed that methanolic extract was the most efficient Fe 2+ chelating capacity (IC50=1 ±1.2 mg/ml) in comparison to both the ethanolic and the aqueous extracts which presented IC50 values of more than 1.5 mg/ml (Table 7).

Anti-inflammatory activity of Ephedra Campylopoda stem extracts

Inflammation is one form of host's defense strategies to combat against pathogenic intruders.

Inflammation occurs when different immune cells, mainly macrophages, detect pathogenassociated molecular patterns (PAMPs) such as microbial lipopolysaccharide (LPS) [24].

Once activated, macrophages can then induce the expression of different pro-inflammatory cytokines (including IL-1α, IL-1β and IL-6), chemokine (such as CCL3 and CCL4) and other pro-inflammatory mediators including nitric oxide (NO) and prostaglandin E2 (PGE2) that are respectively synthesized by the induced isoforms of NO synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes [24]. To characterize the anti-inflammatory capacity of E. Campylopoda stem extracts, we used RAW 264.7 murine macrophage cells, which upon being stimulated with LPS, can produce PGE2. Cells were treated, for 24 h, with either LPS (100ng/ml) alone (control), or LPS together with varying concentrations (50 or 100 µg/ml) of either aqueous, methanolic or ethanolic crude extracts-derived from fresh stems. In a first step, quantitative Real-Time PCR (qRT-PCR) was used to assay the relative iNOS and COX-L 2 mRNA transcription in extract-treated RAW264.7 cells versus non-treated control cells. In the case of COX2, the mRNA levels were not significantly lowered in response to either of the utilized aqueous extract concentrations (Figure 4, Panel A). Interestingly, about 50-60% of COX2 mRNA levels were lost in response to either of the added ethanolic extract concentrations (Figure 4, Panel A). However, the methanolic extract was less efficient as only about 15% of COX2 mRNA levels were absent in response to 50 µg/ml but not 100 µg/ml of extract (Figure 4, Panel A). In the case of iNOS, the three different extracts were efficient in terms of impairing iNOS transcription with methanolic extract being the most efficient, followed by ethanolic extract and finally aqueous extract (Figure 4, Panel B).

In a second step, ELISA technique was used to assess the relative PGE2 amounts present in the cell culture media. Intriguingly, the different extracts showed prominent capacity to dampen PGE2 production (Figure 4, Panel C). Indeed, about 75%, 60% and 78% reduction in secreted PGE2 levels were observed upon treating cells with 50 µg/ml of aqueous, ethanolic and methanolic extracts, respectively (Figure 4, Panel C). These levels were further increased to reach 85%, 99% and 95% in response to 100 µg/ml of aqueous, ethanolic and methanolic extracts, respectively (Figure 4, Panel C).

In a third step, qRT-PCR was applied to assay the relative expression of the proinflammatory cytokines IL-1α, IL-1β and IL-6. In the case of aqueous extract, neither of the two utilized concentrations was able to significantly reduce IL-1α (Figure 4, Panel D), IL-1β

(Figure 4, Panel E) or IL-6 (Figure 4, Panel F). Interestingly, the ethanolic extract was highly efficient in impairing the transcription of the three tested cytokines. Indeed, treating cells with 50 µg/ml of ethanolic extract caused about 75% reduction in mRNA levels of either cytokine whereas 100 µg/ml of extract resulted in complete loss of transcription of the different cytokines (Figure 4, Panels D, E and F). Methanolic extract also showed efficiency, eventhough less than that of ethanolic extract, in lowering IL-1α, IL-1β and IL-6 transcription M levels. For instance, upon treating cells with 50 µg/ml of methanolic extract, the mRNA levels of IL-1α, IL-1β and IL-6 decreased by 60%, 10% and 60% respectively (Figure 4, Panels D, E and F). However, only minimal transcription of either cytokines was detected upon treating cells with 100 µg/ml of methanolic extract (Figure 4, Panels D, E and F).

In a fourth step, and upon performing qRT-PCR, the mRNA levels of the pro-inflammatory chemokines CCL3 and CCL4 were evaluated. In the case of CCL3, 50 µg/ml of either extract was enough to loose about 75% of mRNA levels (Figure 4, Panel G). Although this % was only slighltly increased to reach about 78% upon treating cells with 100 µg/ml of aqueous extract, it was more strikingly enhanced to reach about 99% and 95% in the case of ethanolic and methanolic extracts, respectively (Figure 4, Panel G). In the case of CCL4, aqueous extract moderately reduced CCL4 transcription as about 45% of mRNA levels were lost upon exposure of cells to 50 but not 100 µg/ml of extract (Figure 4, Panel H). On the other hand, both ethanolic and methanolic extracts exhibited prominent efficiency in terms of impairing CCL4 trasncription. Indeed, about 75% and 70% of CCL4 mRNA levels were absent following cell treatment with 50 µg/ml of ethanolic and methanolic extracts, respectively (Figure 4, Panel H). Moreover, only residual transcription was observed upon treating cells with 100 µg/ml of either extract (Figure 4, Panel H).

Antiproliferative activity of Ephedra Campylopoda stem extracts

In order to evaluate the antiproliferative capacity of Ephedra Campylopoda stem extracts, XTT assay was performed. This colometric assay measures cell viability upon determining mitochondrial activity. Indeed, XTT, yellow water-soluble substrate, can be converted, by mitochondrial succinate dehydrogenase enzymes, to a highly colored formazan product.This reaction occurs in viable but not dead cells and thus, the quantity of the generated formazan is proportional to the amount of viable cells in the sample. Jurkat cells were treated with various N fresh stem-derived aqueous, methanolic or ethanolic extract concentrations (in range from 5 to 200 µg/mL) for different times (24, 48 or 72 h). Neither of the utilized aqueous extract concentrations exerted a significant effect on cell viability at any of the indicated time periods (Figure 5, Panel A). However, a dose-dependent inhibitory effect was detected in the case of ethanolic and methanolic extracts (Figure 5, Panels B and C). Time-impact on the observed in a manner dependent on the type of utilized solvent. In fact, the alcoholic solvents appeared to exert more potent antioxidant effect than the aqueous extract. This observation parallels the observed more important chemical content in the alcoholic-than water-derived extracts.

Nowadays, a tight association is established between oxidative stress and inflammation since free radicals can be a result or a cause of inflammation. Inflammation has been identified as a primary cause of numerous chronic diseases including arthritis, atherosclerosis, Alzheimer's, diabetes, heart disease and cancer [30,31]. Immune signaling via proinflammatory cytokines and mediators is the major mechanism for inflammation initiation and amplification.

Inhibiting the expression of these pro-inflammatory molecules, is therefore, a requisite to suppress the inflammatory responses. In this study, we evaluated the ability of the mentioned plant extracts to inhibit PGE2 secretion and suppress the transcription of the proinflammatory cytokines (IL-α, IL-β and IL-6), chemokines (CCL3 and CCL4) and inflammation-responsive COX-2 and iNOS enzymes. Varied plant extract-mediated suppressive capacities on these P different pro-inflamatory components was observed. Indeed, alcoholic extracts were more efficient than aqueous one in impairing PGE2 secretion and inhibiting IL-α, IL-β, IL-6, CCL3, CCL4, COX-2, and iNOS transcription. The prominent anti-inflammatory effect exerted by alcoholic extracts parallels their rich chemical arsenal as well as their substantial antioxidant activity. These observations highlight Ephedra Campylopoda as a putative promising resource for designing novel inflammatory suppressive drugs and treating inflammatory disorders.

In the present work, we assessed, using XTT viability assay, the ability of Ephedra

Campylopoda stem extracts to inhibit Jurkat cancer cell proliferation. In line with their low phytochemical content and moderate antioxidant and anti-inflammatory capacities, aqueous extract was inefficient in terms of suppressing cell growth. However, the alcoholic extracts exerted prominent cytotoxic effect that appeared to be dose-dependent. This robust antiproliferative potential could be attributed to the prominent phytochemical content detected in the alcoholic solvents. So far, a mechanistic understanding of this cytotoxic effect is still lacking. A possible involvement of the apoptotic pathway in the observed impaired cell growth should be addressed in future studies. Moreover, whether Ephedra Campylopoda stem extracts are cytotoxic for cancer cells and thus can be used in tumour therapy is the objective of our ongoing work.

In conclusion, the present study identified the presence of various medicinal constituents in Ephedra Campylopoda stem extracts. Moreover, this plant exhibited prominent antioxidant, anti-inflammatory and antiproliferative capacities, thus can be suggested as a novel promising therapy for a wide array of human diseases.

Tables

General discussion

Approximately 80% of the world population depend exclusively on plants for their health and healing. In the developed world, reliance on surgery and pharmaceutical medicine is more usual but in the recent years, more and more people are complementing their treatment with natural supplements. Furthermore, motivation of people towards herbs is increasing due to their concern about the side effect of drugs, especially those In this study, we identified an important phytochemical arsenal in the two studied plants. This chemical composition was associated with important biological activities where each of these two plants exhibited significant antioxidant capacity, antiinflammatory effect and antiproliferative potential. The molecular mechanisms underlying the observed anti-inflammatory effect are yet to be determined. In future work, we will try to evaluate the involvement of different signaling pathways such as the NF-B pathway in mediating this effect. Moreover, the signaling events leading to the anti-proliferative effect are still vague. Accordingly, we will also address the requirement of different signaling cascades, such as ones involved during apoptosis, to ensure this behavior. Further, we will evaluate the anti-proliferative effect of these extracts on other cancer types including breast and colon cancer. Since the observed anti-inflammatory and anti-proliferative effects are observed in vitro, it will be indispensable, before claiming any therapeutic value, to examine the in vivo potential of these plant extracts. Further, other biological activities such as anti-bacterial, anti-fungal and anti-viral effects of these plant extracts are still to be addressed.

Finally, a survey conducted on medicinal herbs used by Lebanese herbalists revealed that 128 plant species were used for treating a number of diseases. Most plants were used to treat more than one medical condition.

This study highlights a medicinal potential for the two studied plant species and the list can be even more enlarged upon further and deep investigations of different Lebanese species for assessing their chemical and biological effects.

Figure 1 :

 1 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 2 :

 2 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 3 :

 3 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 4 :

 4 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 5 :

 5 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 6 :

 6 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 7 :

 7 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 8 :

 8 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 9 :

 9 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 10 :

 10 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 11 :

 11 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 12 :

 12 Figure 1: Structures of the important naturally occurring phenolic acids ......................................Figure 2: Classification of tannins .............................................................................................Figure 3: Basic structures of flavonoid subclasses. ....................................................................Figure 4: Different flavonol structures. ......................................................................................Figure 5: Different quinones structures. .....................................................................................Figure 6: Generation of primary free radicals by mitochondria. .................................................Figure 7: Inflammation response steps ......................................................................................Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) .....................................................................Figure 9: Cyclooxygenase isoforms and their corresponding functions ......................................Figure 10: Synthesis of nitric oxide by NOS .............................................................................Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits .......................Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits. ..................................

Figure 1 :

 1 Figure 1: Structures of the important naturally occurring phenolic acids

Figure 2 :

 2 Figure 2: Classification of tannins

Figure 3 :

 3 Figure 3: Basic structures of flavonoid subclasses.

Figure 4 :

 4 Figure 4: Different flavonol structures.1.2.3 FlavonesFlavones, which have a very close structural relationship to flavonols (Figure3), are generally less abundant in fruits and vegetables as compared to flavonols. Parsley and celery are the primary sources of flavones. Important flavones include luteolin and apigenin.

Figure 5 :

 5 Figure 5: Different quinones structures.

Figure 7 :

 7 Figure 7: Inflammation response steps [90].

Figure 8 :

 8 Figure 8: Schematic representation of the arachidonic acid metabolism catalysed by cyclooxygenase (COX) and lipoxygenase (LOX) [93].

1. 6 . 5

 65 Nitric oxide (NO) and nitric oxide synthase (NOS) NO, short-lived free radical, is a small gaseous signaling molecule that diffuses freely within cells from site of formation to site of action. NO is implicated in distinct physiological and pathophysiological conditions and can have detrimental or beneficial effects in the human body. During inflammation, NO modulates various vascular and cellular responses. It regulates vasorelaxation and platelet aggregation. NO also regulates neutrophil activation and cell growth and induces apoptosis. NO has also an important role in host defense mechanisms. It mediates the toxicity of natural killer cells and regulates T cell activation. It acts as a neurotransmitter in NANC (non adrenergic, non cholinergic) nerves and in the central nervous system. Nitrosylation of proteins by NOdriven radicals regulates protein activity and function.[START_REF] Lipsky | Specific COX-2 inhibitors in arthritis, oncology, and beyond: where is the science headed?[END_REF]

Figure 10 :

 10 Figure 10: Synthesis of nitric oxide by NOS [95].

Figure 11 :

 11 Figure 11: Different parts of C. azarolus L.: A: leaves, B: flowers and C: fruits

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. GC chromatogram of the water extract of C. azarolus L. leaf.

Figure 4 .

 4 Figure 4. Effects of C. azarolus leaf extracts on LPS-Induced iNOS, COX-2, PGE 2 , IL-1a, IL-1-b, IL-6, CCL3, and CCL4 levels in RAW 264.7 cells. Cells were treated for 24 h with 100 ng/ml LPS in the absence or presence of 50 or 100 µg/ml of either aqueous (A), ethanol (E), or methanol (M) extract. Total RNA was prepared and qRT-PCR was performed to quantify the mRNA levels of COX2 (A), iNOS (B), IL-1a (D), IL-1b (E), IL-6 (F), CCL3 (G), and CCL4 (H). The presented data correspond to the relative mRNA levels (values obtained in: RAW 264.7 cells treated with both LPS and extract/RAW 264.7 cells treated with only LPS). (C) Cellfree supernatants were collected and assayed for PGE 2 content via ELISA. The data correspond to the relative percentage of PGE 2 . Reported values represent the averages ±SEM of 3 independent experiments (n=3) each done in triplicate. * p<0.05; ** p<0.01, *** p<0.001 vs. control untreated cells (Student's t-test).

Figure 5 .

 5 Figure 5. Effects of C. azarolus L leaf extracts on Jurkat cancer cell proliferation. Cells were treated with various concentrations (0, 5, 25, 50, 100, 200 µg/ml) of extracts for 24, 48, and 72 h and antiproliferative activities were measured by XTT assay. Each value represents a mean ± SEM for 3 independent experiments (n=3) each done in triplicate. Fresh leaf-derived aqueous extract (A), ethanol extract (B), and methanol extract (C). * p<0.05; ** p<0.01, *** p<0.001 vs. control untreated cells (Student's t-test).

Figure 12 :

 12 Figure 12: Different parts of Ephedra campylopoda: A: stems, B: fruits.

Figure 1 .

 1 Figure 1. GC chromatogram of the water extract of E. Campylopoda stem.

Figure 2 .

 2 Figure 2. GC chromatogram of the ethanol extract of E. Campylopoda stem.

Figure 3 .

 3 Figure 3. GC chromatogram of the methanol extract of E. Campylopoda stem.

Figure 4 .

 4 Figure 4. Impact of Ephedra Campylopoda stem extracts on LPS-Induced iNOS, COX-

Figure 5 .

 5 Figure 5. Impact of Ephedra Campylopoda stem extracts on Jurkat cells proliferation.

  that are prepared from synthetic materials[122].Phytochemicals are biologically active, naturally occurring chemical compounds found in plants, and are involved in protecting plant cells from environmental hazards such as pollution, stress, drought, UV exposure and pathogenic attack. Besides protecting plants from disease and damage, phytochemicals contribute to the plant's color, aroma and flavor[123]. Remarkably, phytochemicals were identified to serve important roles in the protection of human health, when their dietary intake is significant. More than 4,000 phytochemicals have been catalogued and are classified by protective function, physical characteristics and chemical properties where 150 phytochemicals have been studied in details [124]. Phytochemicals can exist in different parts of the plants, such as in the roots, stems, leaves, flowers, fruits or seeds [125]. Many phytochemicals, particularly the pigment molecules, are often concentrated in the outer layers of the various plant tissues. Phytochemical levels vary from plant to another depending upon the variety, processing, cooking and growing conditions [126]. Phytochemicals compounds are known as secondary plant metabolites and have biological properties such as antioxidant activity, antimicrobial effect, modulation of detoxification enzymes, stimulation of the immune system, decrease of platelet aggregation and modulation of hormone metabolism and anticancer property.In the Middle East, a growing interest in medicinal plants has been noted. Several ethnopharmacological surveys on the medicinal herbs used in the region, mainly in Jordan, have been conducted[127, 128]. They showed that more than 100 plant species are used in herbal medicine and that there is still a flourishing and big demand on traditional drugs, namely of plant origin[127]. However, such extensive ethnopharmacological studies have not been conducted in Lebanon, which has the richest flora in the Middle East, and there is little information about which medicinal plants are still used by Lebanese herbalists who are decreasing in number. In order to preserve this heritage of plant usage in Lebanon, we selected during this study the following two plants: Crataegus azarolus, and Ephedra campylopoda. We then screened the phytochemical component content and characterized the antioxidant, anti-inflammatory and antiproliferative capacities of three extracts (water, ethanol, methanol) from both the stems of Ephedra campylopoda and the leaves of Crataegus azarolus.

Table 1 :

 1 The major classes of phenolic compounds in plants .......................................................

Table 2 :

 2 Chemical classification of alkaloids .............................................................................

Table 3 :

 3 Medicinal plants used to treat distinct ailments based on traditional Arab medicine. .....

Table 4 :

 4 Reactive oxygen species (ROS). ..................................................................................

Table 5 :

 5 Different types of Cytokines found in acute and chronic inflammations .......................

Table 6 :

 6 Biological comparison of three known isoforms of nitric oxide synthases ....................

Table 7 :

 7 Different cytokines involved in inflammation. .............................................................

Table 8 :

 8 The chemokine superfamily and classification .............................................................

Table 1 :

 1 The major classes of phenolic compounds in plants

	S.N.	Number of	Basic skeleton	Class
		carbon atom
	1.	6	C6	Simple phenols
				benzoquinones
	2.	7	C6-C1	Phenolic acids
	3.	8	C6-C2	Acetophenones
				tyrosine
				derivatives
	4.	9	C6-C3	Hydroxycinnamic
				acid, coumarins
	5.	10	C6-C4	Naphthoquinones
	6.	13	C6-C1-C6	Xanthones
	7.	14	C6-C2-C6	Stilbenes
	8.	15	C6-C3-C6	Flavonoids
	9.	18	(C6-C3)2	Lignans
	10.	30	(C6-C3-C6 ) 2 Bioflavonoids
	11.	N	(C6-C3-C6)n	Condensed
				tannins

Table 3 :

 3 Medicinal plants used to treat distinct ailments based on traditional Arab medicine.

	Plant species	Preparation		Uses
	Allium cepa	Bulb juice	Diabetes, liver diseases, and
			coughing
	Arum palaestinum	Foliage decoction	bacterial infection, poisoning,
			and circulatory system
	Peganum harmala	Roots and seeds	anti-bacterial	activity,
			cytotoxicity	antitumoral
			activity, and anti-oxidant
			activity.
	Crataegus azarolus	Fruit and flower decoction Cardiovascular	diseases,
			diabeties, and sexual diseases
	Quercus calliprinos	Fruit and bark decoction	Ulcer, diabetes, and skin
			diseases
	Zea mays	Kernel and fiber decoction Blood	pressure,	joint
			inflammation, and weight loss
	Triticum aestivum	Shoot decoction	Anemia, and skin diseases
	1.4 Anti-cancer properties of phytochemicals
	Cancer is a major health problem worldwide, with more than 20% of all deaths

Table 6 :

 6 Biological comparison of three known isoforms of nitric oxide synthases and 30 kD, and can be secreted by immune and non-immune cells in response to various stimuli. The cytokine family of regulatory peptides includes interleukins, tumor necrosis factors, interferons, lymphokines, colony stimulating factors, transforming growth factors, chemotaxins, and some growth factors. Table7lists different cytokines that play an important role in inflammation.

	Characteristics	nNOS	eNOS	iNOS
	Primary tissue or	Brain, peripheral	Vascular endothelial	Macrophages
	cellular source	(PNS) and central	cells, cardiomyocytes
		nervous system
		(CNS), and skeletal
		muscles.
	Known functions	Neuromodulator in	Vasodilator regulation	Activation of
		brain	of blood flow and	APCs against
		neurotransmittor in	pressure	infections and
		PNS regulation of		microbes cell
		smooth muscle		death
		activity and
		neuroendocrine
		functions in skeletal
		muscles
	Expression	Constitutively	Constitutively present Inducible
		present
	Concentration	In picomolar range	In picomolar range	In nanomolar
		Calcium dependence	Ca 2+ dependent	range Ca2+
			Utilized only small	dependent
				Quantities of Ca 2+ .
				Mostly considered
				as Ca 2+
				independent
	Sub-cellular	Cytosol (not yet	Found in caveolae of	Cytoplasm
	location	comfirmed)	membrane bound to
			calveolin-1
	Association of	Dysregulation	Hypertension,	Infections,
	dysregulated levels	associated with	hypercholesterolemia	rheumatoid,
	of nitric oxide with	neurodegenerative	diabetes, heart failure.	arthritis, Crohn's
	disorders	disorders	At low concentrations	disease, asthma,
			prevents apoptosis of	septic shock
			endothelial cells,
			inhibits platelet
			aggregation, and
			smooth muscle
			proliferation
	1.6.6. Cytokines
	Cytokines control a broad range of physiological processes including immune

responses, inflammation, haemopoiesis, cell proliferation, bone formation, wound healing, and the response to injury

[START_REF] Clemens | Cytokines[END_REF]

. They are peptides of low molecular weight, typically between 15

Table 7 :

 7 Different cytokines involved in inflammation.

	Cytokine	Source	Main actions relevant to
			inflammation
	IL-1	Mainly macrophages	Inflammatory mediator;
			increases prostaglandin
			production
	IL-6	Monocytes, macrophages, T	Stimulates inflammatory
		and helper T cells	response
	IL-9	Helper T cells	T cell and mast cell growth
			factor
	IFN-γ (IL-18)	T and helper T cells, NK	Activates macrophages
		cells
	TNF-α	Macrophages, mast cells,	Increases other cytokines;
		lymphocytes	increases inflammatory and
			immune responses
	Macrophage colony	Inflamed endothelial cells,	Macrophage growth factor
	stimulating factor	monocytes, lymphocytes,
		fibroblasts
	Transforming growth	Macrophages, platelets,	Chemotactic for
	factor-β	lymphocytes	macrophages; increases IL-
			1 production
	1.6.7. Chemokines
	Chemokines are soluble, low molecular weight (8-14 kDa) chemotactic cytokines
	that bind to their cognate seven trans-membrane G-protein coupled receptors (GPCRs) in
	order to mediate cellular migration. To date, there are over 40 characterized human

Table 8 :

 8 The chemokine superfamily and classification types, and a variety of climatic zones as well as microclimates ranging from dry and hot to temperate and humid [103, 105]. All of these factors resulted in a relatively small geographical area with a very diverse flora adapted to several habitats. Being at the "meeting point of three continents" [103], Lebanon has had an important role in the Middle East as a trade center, and as a result obtained good knowledge and experience in herbal medicine. Old Lebanese generations relied heavily on herbal medicine, and herbs such as sage, thyme, rosemary, lavender, mint and chamomile, were commonly grown in gardens [106]. A report from Abou Chaar [107] listed the use of 138 plants commonly used in herbal medicine in Lebanon. Although a number of Lebanese plants have been described for their medicinal value, many other plants are yet to characterized for their biological activities and therapeutic potential.

	Systematic	Alternate Names	Receptor(s)	Expression
	Names			Profile
	CXC
	Chemokines
	CXCL1	Groα/MGSAα	CXCR2, CXCR1	Inducible
	CXCL2	Groβ/MGSAβ	CXCR2	Inducible
	CXCL3	Groγ	CXCR2	Inducible
	CXCL4	PF4	CXCR3b	Inducible

1.7. Medicinal plants in Lebanon Long time ago, plants have been the source of medicines for the treatment of many diseases. Nowadays, plants remain an important part of the health care in many countries, mainly the developing ones [100]. In 2000, the World Health Organization reported that a big percentage of the world's population depends on plants as the main source for the treatment of many diseases in the primary health care [101]. In the last years, herbal medicine has been gaining interest in the scientific research, specifically, regarding cancer prevention or treatment [102]. It is still highly used in the Mediterranean region, where a high percentage of individuals collect and consume wild edible plants as part of their traditional source of food with low health risks species, out of which 78 are endemic [103, 104]. Its geographical location, eastern to the Mediterranean Sea, has contributed to a varied topography with different elevations, a diversity of soil

Table 1 .

 1 Detection of primary and secondary metabolites in leaves of Crataegus azarolus L.

	Metabolites	Added reagent	Expected result
	Alkaloids [36]	Dragendorff reagent	Red or Orange precipitate
	Tanins [36]	FeCl 3 (1%)	Blue coloration
	Resines [36]	Acetone + water	Turbidity
	Saponines [37]	Agitation	Formation of foam
	Phenols [36]	FeCl 3 (1%) + K 3 (Fe(CN) 6 ) (1%)	Green-blue coloration
	Terpenoids [37]	Chloroform + H 2 SO 4 conc	Reddish brown coloration
	Flavonoids [38]	KOH (50%)	Yellow coloration
	Carbohydrates [37]	a-naphtol + H 2 SO 4	Purple ring
	Reducing sugars [37]	Fehlings (A+B)	Brownish-red precipitate
	Quinones [39]	HCl conc	Yellow precipitate
	Sterols & steroids [37,38]	Chloroform + H 2 SO 4 conc	Red color (surface) + fluorescence Greenish-yellow
	Cardiac glycosides [37,38]	Glacial acetic acid + FeCl 3 (5%) + H 2 SO 4 conc	Ring
	Diterpenes [36]	Copper acetate	Green coloration
	Anthraquinones [38]	HCl (10%) + chloroform + Ammonia (10%) Pink coloration
	Proteins & aminoacids [40]	Ninhydrin 0.25%	Blue coloration
	Lignines [40]	Safranine	Pink coloration
	Phlabotannins [41]	HCl (1%)	Blue coloration
	Anthocyanines [42]	NaOH (10%)	Blue coloration
	Flavanones [42]	H 2 SO 4 conc	Bluish-red Coloration
	Fixed oils and fats [37]	Spot Test	Oil stain

Table 2 .

 2 The instrument specifications and analysis conditions for water extracts.

	GC Program			GC Program
	Oven Maximum Temperature	325	°C	Oven Temperature Set point	35°C
	Hold time	1 min	Hold time	2 min
	Post Run	50 c	Post Run	60 c
	Program	8c/min -290 c -11 min	Program	3 c/min -320 c -1 min
	Equilibration Time	3 min	Equilibration Time	0.5 min
	Injection volume	1 µl	Injection volume	1 µl
	Front SS Inlet Mode	Split	Back SS Inlet Mode	Split
	Injector temperature	280	°C	Injector temperature	300	°C
	Pressure	52.76 psi	Pressure	11.192 psi
	Total Flow	6 ml/min	Total Flow	504 ml/min
	Split Ratio	5: 1	Split Ratio	500: 1
	Split Flow	5 ml/min	Split Flow	500 ml/min
	Column	DB-5MS: 30 m×250 µm×0.25 µm	Column	TG-5MS: 30 m×250 µm×0.25 µm
	MS Source	230 c maximum 250 c	MS Source	230 c maximum 250 c
	MS Quad	150 c maximum 200 c	MS Quad	150 c maximum 200 c
	Acquisition Mode	Scan	Acquisition Mode	Scan
	Solvent Delay	2.5 min	Solvent Delay	2 min
	Low Mass	33	Low Mass	1.6
	High Mass	500	High Mass	450

3

cells/well). The following day, cells were treated with the different extracts at concentrations

Table 3 .

 3 The instrument specifications and analysis conditions for methanol and ethanol extracts.

Table 4 .

 4 The instrument specifications and analysis conditions.

	HPLC/Pump	Shimadzu/LC20AD
	Mass spectrometer	API 4000/AB Sciex instruments
	Component Name	Triple Quadrupole LC/MS/MS Mass Spectrometer
	Source Temperature (at set point)	300°C
	LC system Equilibration time	2 min
	LC system Injection Volume	10 µl
	Pumping Mode	Low pressure Gradient:
		Time (min) Module Events Parameter
		0.01 Pumps ACN+ 0.1% Formic acid 0.0
		0.10 Pumps ACN+ 0.1% Formic acid 20
		6.00 Pumps ACN+ 0.1% Formic acid 90
		9.00 Pumps ACN+ 0.1% Formic acid 90
		9.50 Pumps ACN+ 0.1% Formic acid 0
		12.00 System Controller Stop
	Total Flow	0.3 ml/min
	Autosampler model	SIL-20A/HT
	Column	C18 (15 cm*0.2 mm*3.5 um)
		Primers	Sequence (5'-3')
		IL-1a-FO	GAATGACgCCCTCAATCAAAGT
		IL-1a-RE	TCATCTTGGGCAGTCACATACA
		IL-1b-FO	CCTTCCAGGATGAGGACATGA
		IL-1b-RE	TGAGTCACAGAGGATGGGCTC
		IL-6-FO	GAGGATACCACTCCCAACAGACC
		IL-6-RE	AAGTGCATCATCGTTGTTCATACA
		CCL3-FO	CAGCCAGGTGTCATTTTCCT
		CCL3-RE	CTGCCTCCAAGACTCTCAGG
		CCL4-FO	AAAACCTCTTTGCCACCAATACC
		CCL4-RE	GAGAGCAGAAGGCAGCTACTAG
		COX2-FO	CAGACAACATAAACTGCGCCTT
		COX2-RE	GATACACCTCTCCACCAATGACC
		iNOS-FO	GCAGAATGTGACCAT CATGG
		iNOS-RE	ACAACCTTG GTGTTGAAG GC

Table 5 .

 5 List of primers used in this study.

	Windows (Version 21). P-Values <0.05 (*), <0.01 (**), <0.001 ( ***)
	were considered significant.
	Results
	Phytochemical screening of the leaves of Crataegus
	azarolus L.
	Phytochemical screening of C. azarolus L. fresh leaf crude ex-
	tract indicated the presence of some important bioactive com-
	ponents, which are listed in Table 6. The aqueous crude extract
	showed high concentrations of saponins, phenols, terpenoids,
	flavonoids, amino acids, reducing sugars, and lignin; moderate
	concentrations of cardiac glycosides; low concentrations of res-

ins, carbohydrates, phlobatannins, and flavones; and absence of alkaloids, tannins, quinone, coumarin, sterols/steroids, diterpenes, anthraquinones, anthocyanin, and fixed oils and lipids. On the other hand, the methanolic crude extract showed high abundance of alkaloids, resins, phenol, quinones, diterpenes, and lignin; moderate abundance of sterols/steroids; low abundance of flavonoids, carbohydrates, cardiac glycosides,

Table 6 .

 6 Phytochemical screening of C. azarolus. L. leaf extracts. Key: -, absent; +, low in abundance; ++, moderate in abundance; +++, high in abundance.

Table 7 .

 7 Results of the GC-MS analysis of the water extract of the C. azarolus. L. leaf.

	Peak#	RT	Name	MW		Structure			Molecular formula	Area %
					HO
	1	10.507	Epoxylinalol	170.25						C 10 H 18 O	4.73
						O
	2	13.324	Isophytol	296.54	HO					C 20 H 40 O	3.03
					O
	3	13.584	Syringol	154.163	H 3 C	HO	O	CH 3		C 8 H 10 O 3	0.58
					CH 3	OH
	4	13.754	8-Hydroxylinalool	170.24	HO				CH 2	C 10 H 18 O	1.46
							CH 3
					H 3 C	CH 3
					H 3 C
	5	16.106	2,4-DI-T-Butylphenol	206.324						C14H 22 O	0.85
					H 3 C
							OH
					H 3 C	CH 3
							CH 3
	6	19.240	4-Oxo-Beta-Isodamascol	208.297	H 3 C H 3 C HO	CH 3 CH 2		C 13 H 20 O	3.04
							O
	7	19.422	Gamma-Hydroxyisoeugenol (Coniferol)	180.201	HO		O	OH		C 10 H 12 O	2.35
					O		O
	8	19.811	Gallic acid trimethyl ether	212.2	HO		O			C 10 H 12 O	2.82
							O
	9	20.204	Pluchidiol	208		-----			C 13 H 20 O	33.62
					O
							O
	10	20.483	Beta-Hydroxypropiovanillone	196.202	O					C 10 H 12 O	2.16
					H
								O	H
							O
								H
						O
								O
	11	27.663	Trichothecin	332.39		O				C 19 H 24 O	5.67
								O

Table 8 .

 8 Results of the GC-MS analysis of the ethanol extract of the C. azarolus. L. leaf.

	Peak#	RT	Name	MW			Structure				Molecular formula	Area%
					O
	1	52.169	Palmitic acid	256.42	HO								C 16 H 32 O 2	3.99
					H 3 C							OH
	2	56.691	Phytol Isomer	296.53	CH 3	CH 3			CH 3		CH 3	C 20 H 40 O	20.47
						H 3 C	O
						O
	3	57.440	9,12,15-Octadecatrienoic acid	278.436						CH 3			C 18 H 30 O 2	7.44
												O
	4	58.077	Stearic acid	284.48								OH	C 18 H 36 O 2	0.95
	5	81.175	Gamma-Tocopheryl methyl	416.69	O	O							C 28 H 48 O 2	43.73
											CH 3
	6	84.651	Gamma-Sitosterol	414.71		CH 3	H	H 3 C CH 3	H	CH 3	CH 3	C 29 H 50 O	19.2
							H		H
					HO
						H 3 C			CH 3
	7	85.572	Cedryl acetate	264.41		H 3 C			O	CH 3		C 17 H 28 O 2	0.72
						H			CH 3	O

To assess the potential anti-inflammatory properties of C. azarolus. L leaf extracts, RAW 264.7 murine macrophage cells were used. These are capable of producing PGE 2 upon stimulation with LPS. Cells were treated for 24 h with either LPS (100 ng/ml) alone (control) or LPS together with different concentrations

[START_REF] Amin | Overview of major classes of plant-derived anticancer drugs[END_REF]

Table 9 .

 9 Results of the GC-MS analysis of the methanol extract of the C. azarolus. L.leaf.

	Peak#	RT	NAME	MW								Structure	Molecular formula	Area%
													O
	1	6.859	Oxalic acid dimethyl ester	118.09								O	O	C 4 H 6 O 4	1.58
												O
													OH
	2	30.027	Syringol	184.19								O	C 9 H 12 O 4	1.62
												HO	O
												H 3 C	CH 3
												H 3 C
	3	36.597	2,4 di-tert-butylphenol	206.324									C 14 H 22 O	1.51
												H 3 C	OH
												H 3 C	CH 3
						O
	4	52.103	Cetylic acid	256.42	HO								CH 3	C 16 H 32 O 2	2.56
													H
	5	56.738	Phytol	296.53									O H	C 20 H 40 O	13.3
							O
	6	57.388	Ethyl linolinate	308.50	H 3 C	O							CH 3	C 20 H 34 O 2	18.79
										H	O
	7	61.917	4,4'-biguaiacol	246.26					O			O	C 14 H 14 O 4	1.37
													O	H
	8	81.133	alpha-tocopherol-beta-d-mannosid	592.858		O H	H	O	O	O	O	C 35 H 60 O 7	21.87
						H	O	O H

Table 10 .

 10 Results of LC/MS/MS technique of C. azarolus. L. leaf.

	C. azarolus L.	Compounds names	Retention time	Q1 Mass (Da)	Q3 Mass (Da)	CE	DP (V)
		Vitexin	4.96	431	341	-30	-40
	Water
		Hyperoside	4.97	463	301	-38	-40
		Prunin	5.23	433	271	-20	-40
		Quercetin	5.87	301	179	-35	-40
		Rutin	4.89	609	301	-35	-40
	Ethanol
		Vitexin	4.94	431	341	-30	-40
		Hyperoside	4.99	463	301	-38	-40
		Isoorientin	4.88	447	429	-30	-40
		Prunin	5.23	433	271	-20	-40
		Quercetin	5.88	301	179	-35	-40
		Rutin	4.89	609	301	-35	-40
	Methanol
		Vitexin	4.94	431	341	-30	-40
		Hyperoside	4.99	463	301	-38	-40
		Isoorientin	4.88	447	429	-30	-40
					DPPH assay	Fe 2+ chelating assay
			Extract		IC 50 , μg/ml	IC 50 , mg/ml
			Aqueous		60±2.28	1.5±0.07
	Fresh leaves		Methanol		55±2.886	0.5±0.08
			Ethanol		50±5.207	1±0.08

Table 11 .

 11 DPPH free scavenging capacity (IC 50 , μg/ml) and Ferrous-ion (Fe 2+ ) chelating ability (IC 50 , mg/ml) of aqueous, methanol or ethanol extracts derived from fresh C. azarolus leaves. IC 50 value corresponds to the effective concentration of sample required to scavenge DPPH radical or Ferrous-ion by 50%. Each value represents a mean ±SD (n=3).

  inhibition was not striking since, for both extracts, the IC50 values (dose required to inhibit cell growth by 50%) calculated at the different time intervals were comparable. For instance, in the case of ethanolic extract, the IC50 values obtained after 24, 48 and 72 h were 40±2.88, 38 ± 1.54 and 35 ±1.54 µg/ml, respectively (Figure5, Panel B) whilst in the case of methanolic extract, the IC50 values corresponded to 40±1.45, 48 ±3.51 and 50 ±7.73 µg/ml after 24, 48 and 72 h, respectively (Figure5, Panel C). In the present study, we aimed at screening the chemical content of Ephedra Campylopoda stem extracts and characterizing their pharmaceutical value. Our phytochemical analysis identified the presence of various important medicinal compounds, such as phenols, flavonoids, carbohydrates, proteins, diterpenes and lignins in stem extracts being prepared using aqueous, ethanolic or methanolic solvents. Coumarins, sterols/steroids and flavones were found in both of the alcoholic solvents but not the aqueous one. Alkaloids were detected only in the ethanolic extracts whilst tannins, resin, terpenoids, reducing sugars, phlobatannins and anthocyanin were detected in the methanolic extracts, specifically. Consistently with O previous reports[26][27][28], our data indicate that the methanolic and ethanolic solvents extract are more rich, than aqueous one, in plant bioactive components. As the medicinal value of Ephedra Campylopoda species is yet not fully defined, we therefore assessed their antioxidant, anti-inflammatory and anti-proliferative activities.Although they can mediate beneficial physiologic roles, overload of free radicals and reactive biomolecules that cannot be balanced or destroyed generates oxidative stress. This process is harmful for humans since it plays a major role in the initiation and development of chronic and degenerative pathologies such as cardiovascular and neurodegenerative diseases, autoimmune disorders, aging and cancer[29]. Identifying plants with potent antioxidant potential is therefore of great interest. Here, we assessed the antioxidant capacity of Ephedra Campylopoda extracts upon evaluating their ability to scavenge free DPPH radicals or chelate Fe 2+ ions. Our data showed a varied antioxidant potential of Ephedra Campylopoda extracts,

Discussion

Humans have used plants as medicine throughout the history. People all around the world still rely on herbs to relieve pain and heal sickness. Today, medicinal plants play an important role in modern medicine development since a large number of modern drugs are simple copies or synthetic modifications of natural chemical substances found in plants

[25]

. Nowadays, substantial research investments are skewed toward identifying and characterizing new medicinal plants.

Table 1 :

 1 Detection of primary and secondary metabolites in stems of Ephedra Table. 2. Phytochemical screening of Ephedra Campylopoda stem extract using aqueous, methanol or ethanol as extraction solvents. Key: -, absent; +, low in abundance; ++, moderate in abundance; +++, high in abundance.

	Campylopoda
	Metabolites	Added reagent	Expected result
	Alkaloids [32]	Dragendorff reagent	Red or Orange precipitate
	Tanins [32]	FeCl 3 (1%)	Blue coloration
	Resines [32]	Acetone + water	Turbidity
	Saponines [33]	Agitation	Formation of foam
	Phenols [32]	FeCl 3 (1%) + K 3 (Fe(CN) 6 ) (1%)	Green-blue coloration
	Terpenoids [33]	Chloroform + H 2 SO 4 conc	Reddish brown coloration
	Flavonoids [34]	KOH (50%)	Yellow coloration
	Carbohydrates [33]	α-naphtol + H 2 SO 4	Purple ring
	Reducing sugars [33]	Fehlings (A+B)	Brownish-red precipitate
	Quinones [35]	HCl conc	Yellow precipitate
	Sterols & Steroids (Khandelwal, 2005 ; [33]	Chloroform + H 2 SO 4 conc	Red color (surface) + fluorescence Greenish-yellow
	Cardiac glycosides (Khandelwal, 2005 ; [33]	Glacial acetic acid + FeCl3 (5%) + H2SO4 conc	Ring
	Diterpenes [32]	Copper acetate	Green coloration
	Anthraquinones [34]	HCl (10%) + chloroform + Ammonia (10 %)	Pink coloration
	Proteins & aminoacids [36]	Ninhydrin 0.25%	Blue coloration
	Lignines [36]	Safranine	Pink coloration

Table 3 .

 3 Results of the GC-MS analysis of the water extract of the E. Campylopoda stem

	Peak#	RT	Name	MW	Structure	Molecular formula	Area%
	5	12.981	Vinyl guaiacol	150.17		C9H10O2	1.47
	7	13.591	Syringol	154.163		C8H10O3	3.48
	8	16.107	di-tert-butylphenol	206.324		C14H22O	0.62
	9	17.558	Antiarol	184.2		C9H12O4	3.28
			N-
	11	20.038	Butylbenzenesulfonamide 213.30		C10H15NO2S	0.83
	12	20.153	Pluchidiol	208	-----	C13H20O2	7.85
	13	22.149	Cetylic acid	256.42		C16H32O2	4.8

Table 4 .

 4 Results of the GC-MS analysis of the ethanolic extract of the E. Campylopoda stem

	Peak#	RT	NAME	MW	Structure	Molecular formula	Area%
	2	52.165	Palmitic acid	256.42		C16H32O2	2.17
	3	56.680	Phytol	296.53		C20H40O	5.04
	4	57.222	Linoleic	280.452		C18H32O2	0.7
	5	57.450	Linolenic acid methyl ester	294.479		C19H34O2	6.33
	6	81.142	α-tocopherol-β-d-mannosid	592.858		C35H60O7	2.82
	7	84.636	γ-sitosterol	414.71		C29H50O	4.97

Table 5 .

 5 Results of the GC-MS analysis of the methanolic extract of the E. Campylopoda

		stem
	Peak#	RT	NAME	MW	Structure	Molecular formula	Area%
	2	24.327	Coumaran	120.15		C8H8O	6.04
	3	28.406	Vinylguaiacol	150.17		C9H10O2	4.85
	4	36.608	2,4-di-tert-butylphenol	206.324		C14H22O	4.15
	5	52.114	Palmitic acid	256.42		C16H32O2	4.19
	6	56.677	Trans-phytol	296.53		C20H40O	39.17
	7	57.383	Linolenic acid methyl ester	294.479		C19H34O2	9.6
	8	81.15	Vitamin E	430.717		C29H50O2	3.31

Table 6 .

 6 Results of LC/MS/MS technique of E. Campylopoda plant

	E. CAMPYLOPODA Compounds	RETENTION	Q1	Q3	CE	DP
		names	TIME	Mass	Mass		(V)
				(Da)	(Da)
	WATER	Vitexin	4.96	431		-30	-40
	ETHANOL	Prunin	5.22	433		-20	-40
		Quercetin	5.88	301		-35	-40
		Rutin	4.86	609		-35	-40
		Vitexin	4.93	431		-30	-40
		Hyperoside 4.98	463		-38	-40
		Isoorientin	4.74	447		-30	-40
	METHANOL	Prunin	5.24	433		-20	-40
		Quercetin	5.89	301		-35	-40
		Rutin	4.91	609		-35	-40
		Vitexin	4.96	431		-30	-40
		Hyperoside 5.02	463		-38	-40
		Isoorientin	4.79	447		-30	-40

Table . 7

 . . DPPH free scavenging capacity (IC50, μg/ml) and Ferrous-ion (Fe 2+ ) chelating ability (IC50, mg/ml) of aqueous, methanol or ethanol extracts derived from fresh stems of Ephedra Campylopoda. IC50 value represents the concentration of sample required to scavenge DPPH radical or Ferrous-ion by 50%. Each value represents a mean ± SD (n = 3).

		DPPH assay	Fe 2+ chelating assay
	Extract	IC50, μg/ml	IC50, mg/ml
	Fresh stems
	Aqueous	300 ±4.4	>1.5
	Methanol	150 ±5.1	1 ±1.2
	Ethanol	125 ±4.4	>1.5

Kallasy H. et al.: Pharmacological potential of Crataegus azarolus L. © Med Sci Monit Basic Res, 2017; 23: 270-284

This work is licensed under Creative Common Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

ACKNOWLEDGEMENTS

I am grateful to my supervisor, Professor Bassam Badran, Dean of the Faculty of Sciences at the Lebanese University, for his friendly support and guidance over the last four years of my research work. This thesis would have never been finished without the

Acknowledgments

We thank Professor George Tohme for his valuable help in the identification of this plant, and the Toxicology Department, Faculty of Health Sciences, American University of Science and Technology, Beirut Lebanon, VP Amer Saker for his technical support.

Acknowledgments

We do thank Professor George Tohme for his valuable help in the identification of this plant, and the Toxicology Department, Faculty of Health Sciences, American University of Science and Technology, Beirut Lebanon, VP Amer Saker for his technical support. R

This work is supported by the Lebanese University and the Lebanese National Council for Scientific Research (CNRS-L) This work is supported by the Lebanese University and the Lebanese National Council for Scientific Research (CNRS-L).

Chapter 2 Chemical composition, antioxidant, anti-inflammatory and antiproliferative activities of Lebanese

Ephedra campylopoda plant