Muhammad Asad Ullah

Duangjai Tungmunnithum

Laurine Garros

Christophe Hano

Bilal Haider Abbasi
email: bhabbasi@qau.edu.pk

Laurine Garros B D E

Christophe Hano B E , Bilal

Haider Abbasi

Monochromatic lights-induced trends in antioxidant and antidiabetic polyphenol accumulation in in vitro callus cultures of Lepidium sativum L

Keywords: Polyphenols, Lepidium sativum, Monochromatic light quality, Chlorogenic acid, Quercetin, Kaempferol

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

For centuries, medicinal plants have been recognized for their wide pharmaceutical and therapeutic applications. Almost every culture has documented their use either in the form of root, seeds and leaves to fight against many diseases [START_REF] Li | Demystifying traditional herbal medicine with modern approach[END_REF]. Medicinal plants are rich source of phytochemicals, having wide range of anti-microbial, anti-cancerous and anti-tumor applications in medical field [START_REF] Jawdat | Essential oil profiling in callus of some wild and cultivated Daucus genotypes[END_REF][START_REF] Shebaby | Daucus carota pentane-based fractions arrest the cell cycle and increase apoptosis in MDA-MB-231 breast cancer cells[END_REF].

Lepidium sativum L., a herbaceous medicinal plant commonly known as garden cress, belongs to family Brassicaceae with average height of 30 to 50 cm. L. sativum is also an edible plant, especially its seeds provide enormous health promoting benefits [START_REF] Gokavi | Chemical composition of garden cress (Lepidium sativum) seeds and its fractions and use of bran as a functional ingredient[END_REF][START_REF] Paranjape | A study on clinical efficacy of Lepidium sativum seeds in treatment of bronchial asthma[END_REF][START_REF] Al Hamedan | Protective effect of Lepidium sativum L. seeds powder and extract on hypercholesterolemic rats[END_REF]. Biological activities including antimicrobial, anticancer, bronchodilator and allelopathic activity have previously been reported from aqueous extract of this plant [START_REF] Carbajal | Pharmacological screening of plant decoctions commonly used in Cuban folk medicine[END_REF][START_REF] Hasegawa | Isolation and identification of lepidimoide, a new allelopathic substance from mucilage of germinated cress seeds[END_REF]. Phytochemical profiling revealed the existence of important polyphenolic (phenolic acids and flavonoids) constituents including sinapic acid and its derivative, caffeic acid, quercetin, kaempferol, p-coumaric acid, and other phenolic compounds as well as glucosinolates that could play vital role in plant defense and antioxidant potential [START_REF] Fahey | The chemical diversity and distribution of glucosinolates and isothiocyanates among plants[END_REF][START_REF] Chandel | Biodiversity in medicinal and aromatic plants in India[END_REF]. L. sativum seeds contains pale yellow, semi-dry oil 20-25%, while main fatty acid in it are linolenic acid, comprises 32-34 % [START_REF] Diwakar | Physicochemical properties of garden cress (Lepidium sativum L.) seed oil[END_REF]. Oil is identified to contain several important compounds like palmitic, oleic, linoleic, stearic, benzyl cyanide, sterols, behenic, sitosterol. L. sativum also carries natural anti-oxidant, and vitamin E and carotenoid which guard the oil from rancidity. Seven imidazole alkaloids, in which five lepidine (B, C, D, E and F) are dimeric and there are two monomeric semi lepidinoside A and B alkaloid were previously testified in seeds [START_REF] Agarwal | Antioxidant activity-guided fractionation of aqueous extracts from Lepidium sativum and identification of active flavonol glycosides[END_REF].

Secondary metabolites have previously been studied for their anticancer potential. Aqueous extracts of leaves and seeds of L. sativum were investigated, and the results pointed out that the extract can fight against HEp2 cells and MCF-7 human breast cancer cell lines [START_REF] Mahassni | Apoptosis and necrosis of human breast cancer cells by an aqueous extract of garden cress (Lepidium sativum) seeds[END_REF]. Multiple studies have been conducted on mouse model for hepatoprotective [START_REF] Al-Asmari | Efficacy of Lepidium sativum against carbon tetra chloride induced hepatotoxicity and determination of its bioactive compounds by GC MS[END_REF], antihypertensive and diuretic activity [START_REF] Maghrani | Antihypertensive effect of Lepidium sativum L. in spontaneously hypertensive rats[END_REF], cardio-protective action [START_REF] Mohamed | Evaluation of cardioprotective activity of Lepidium sativum seed powder in albino rats treated with 5-fluorouracil[END_REF] and hypoglycemic activity [START_REF] Eddouks | Study of the hypoglycaemic activity of Lepidium sativum L. aqueous extract in normal and diabetic rats[END_REF] of L. sativum extracts. The type 2 diabetes mellitus occurs mainly because the body cannot take up and use insulin effectively that leads to hyperglycemic condition [START_REF] Prasad | Oxidative stress as a mechanism of diabetes in diabetic BB prone rats: effect of secoisolariciresinol diglucoside (SDG)[END_REF]. There are two key enzymes, αglucosidase and α-amylase, involve in proper carbohydrate digestion [START_REF] Kim | Inhibition of alpha-glucosidase and amylase by luteolin, a flavonoid[END_REF]. Inhibition of these enzymes can delay the starch digestion process, thus controlling the glycemic index in the body [START_REF] Srinivasan | Partial protective effect of rutin on multiple low dose streptozotocin-induced diabetes in mice[END_REF]. Different inhibitors have been used in clinical practices which sometimes cause gastrointestinal abnormalities [START_REF] Van De Laar | α-Glucosidase inhibitors for patients with type 2 diabetes: results from a Cochrane systematic review and meta-analysis[END_REF]. Plant's active ingredients have potential to act as natural inhibitors with minimum side effects [START_REF] Heacock | Effects of a medical food containing an herbal α-glucosidase inhibitor on postprandial glycemia and insulinemia in healthy adults[END_REF]. Antioxidant term usually applies to sum up huge class of compounds that act as reducing agents by giving up electrons by employing various modes of actions [START_REF] Aruoma | Free radicals, oxidative stress, and antioxidants in human health and disease[END_REF]. There are two major mechanistic approaches through which antioxidants inactivate free radicals i.e. ET (electron transfer) and HAT (hydrogen atom transfer) [START_REF] Prior | Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements[END_REF]. Different in vitro assays to determine antioxidant potential of an individual compound have been established and perfected in past few years. These assays provide extensive information on mechanistic approach involved in antioxidant potential of compounds. Excess of free radicals in the body can generate a cascade of reactions that later play a role in development of cancer and other neurodegenerative diseases. Advanced glycation end product (AGEs) formation in response to free radical oxidative stress is perfect example of its deleterious actions which results in cardiovascular disease and Alzheimer's disease development due to protein dysfunction [START_REF] Dzib-Guerra | Anti-Advanced glycation end-product and free radical scavenging activity of plants from the yucatecan flora[END_REF].

Plant phytochemicals responsible for such medical applications have a huge demand in pharmaceutical industry. Development of these pharmaceutical products is difficult due to low availability and high variability of phyto-content in their natural environment and not to mention the lack of appropriate extraction procedures [START_REF] Khan | Temporal variations in metabolite profiles at different growth phases during somatic embryogenesis of Silybum marianum L, Plant Cell, Tissue and Organ Culture[END_REF]. In vitro cultures establishment provides unlimited availability of plant phytochemicals with easy harvesting, irrespective to ever changing environmental conditions [START_REF] Matkowski | Plant in vitro culture for the production of antioxidants-a review[END_REF][START_REF] Davies | Prospects for the use of plant cell cultures in food biotechnology[END_REF].

For industrial scale manufacture of these active secondary metabolites, multiple strategies in last few decades have been utilized. For in vitro derived cultures, elicitation has shown promising results for enhanced production of phyto-content [START_REF] Yang | Trends for diverse production strategies of plant medicinal alkaloids[END_REF][START_REF] Yue | Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites[END_REF]. Elicitors primarily activate physiological cascades, resulting in stimulation of plant defense mechanism and production of phytochemicals [START_REF] Zhao | Elicitor signal transduction leading to production of plant secondary metabolites[END_REF][START_REF] Zhang | Nitric oxide elicitation for secondary metabolite production in cultured plant cells[END_REF]. Light is a considerable factor in optimum regulation of multiple growth related pathways. Intensity and wavelength variations may influence phytochemical accumulation [START_REF] Ellis | Towards a rational basis for testing seed quality[END_REF][START_REF] Senger | Blue light responses: phenomena and occurrence in plants and microorganisms[END_REF]. Light of multiple spectra significantly changes plant morphology when applied in a controlled environment as previously been studied on various plant species [START_REF] Okamoto | Development of plant growth apparatus using blue and red LED as artificial light source[END_REF][START_REF] Nhut | Growth of banana plantlets cultured in vitro under red and blue light-emitting diode (LED) irradiation source[END_REF].

The primary focus of current study is the establishment of in vitro callus culture of L. sativum for sustainable production of commercially suitable anticancer, antioxidant and anti-diabetic phytochemicals and to explore the potential influence of different monochromatic lights under controlled conditions on morphology and biochemical characteristics of L. sativum callus culture.

Material and Methods

. In vitro germination of plant

Seeds of L. sativum were taken from Department of Biotechnology, Plant Cell Culture Laboratory (PCCL), Quaid-i-Azam University Islamabad, Pakistan. Viability of L. sativum seeds was examined by employing water float test and the seeds were further subjected to surface sterilization using mercuric chloride (0.1 %) solution for 30 seconds, followed by 40 seconds of treatment with ethanol (70%). To eliminate contamination, sterilized seeds were further thoroughly washed 3 times using sterilized autoclaved distilled water. Murashige and Skoog medium (MS0) [START_REF] Murashige | A revised medium for rapid growth and bio assays with tobacco tissue cultures[END_REF] was used to inoculate sterilized seeds containing agar (0.8% w/v) as solidifying agent and sucrose as source of carbon (3%). Media with adjusted pH 5.6 -5.7 were autoclaved for 20 min at 121°C temperature prior to inoculation and flasks were under fixed photoperiod cycle (16/8 light/dark) in growth room at optimum temperature of 25±2°C.

. Callus culture establishment from in vitro derived plantlets

In vitro derived plantlets (21 days old) were used as source of explants. Leaf and stem (0.5 cm 2 , 1.0 cm) were excised from plantlets and inoculated in MS media along with multiple concentrations of thidiazuron (TDZ) and α-naphthalene acetic acid (NAA) alone and in combination ( Supplementary Table 1). Explants (4-5) of both stem and leaf were cultured in each flask. The whole experiment was performed in triplicates and placed in photoperiod cycle (16h light/8h dark) at 25 ± 1°C temperature. Data on callus initiation days and callus induction frequency was collected on weekly basis. Callus was sub-cultured on respective hormonal fresh media after every 4 weeks. Harvesting of callus was done after 4 th week of 2 nd subculture and data on fresh weight (FW) and dry weight (DW) production was obtained respectively.

. Monochromatic Lights Treatment on callus culture

Different source of monochromatic lights were employed as elicitor on explant derived callus including Red Light (24 h, wavelength 660 nm), Green Light (24 h, wavelength 510 nm), Blue Light (24 h, wavelength 460 nm), Yellow Light (24 h, wavelength 570 nm), Dark (24 h), White Light (24 h, wavelength 400-700 nm) and photoperiod cycle (16/8 h light/dark). Based on higher biomass accumulation, stem derived callus (1g) was used to inoculate 30ml MS media flask supplemented with optimized hormonal concentration and placed under different sources of monochromatic light of intensity of 40-50 μMol m -2 s -1 as previously described, recorded using Lux meter (SU10, Jeio-tech). Experiment was performed in triplicates and harvested after 28 days of inoculation for determination of FW, DW and phytochemical analysis.

. Analytical Scheme

Sample Extraction

For evaluation of phenolic, flavonoid content and DPPH activity of calli samples, extraction procedure was done according to Zahir et al [START_REF] Zahir | Synergistic effects of drought stress and photoperiods on phenology and secondary metabolism of Silybum marianum[END_REF] with some changes. Briefly, 100mg callus powder (dried at 60°C for 24h) was thoroughly mixed with 500μl of methanol (1:5), vortexed (10 min) and sonicated (30min). Whole procedure for phytochemical extraction was repeated twice and centrifuged (15000rpm, 10min). Supernatant of each sample was removed and stored (4 °C) for further analysis.

In order to determine peroxidase (POD) as well as superoxide dismutase (SOD) activities, fresh samples of calli were subjected to extraction according to Nayyar and Gupta [START_REF] Nayyar | Differential sensitivity of C3 and C4 plants to water deficit stress: association with oxidative stress and antioxidants[END_REF] with minor adjustments. Briefly, fresh calli samples (100 mg) were mixed with 1 ml KH2PO4 buffer (50 mM, pH7.0) containing 1% PVP. Homogenization of sample mixture was performed and centrifuged at 15000 rpm for half an hour. After centrifugation of sample, supernatant was stored (4 °C) for further analysis.

Estimation of secondary metabolites contents

For determination of total phenolic contents (TPC), Singleton and Rossi [START_REF] Singleton | Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents[END_REF] protocol was used by employing Folin-Ciocalteu reagent with minor modifications. Methanol extracted sample (20 µL) was mixed in FC reagent (90 µL) and sodium carbonate (90 µL) in reaction mixture.

Absorbance of mixture was taken at 630nm wavelength by micro-plate reader (Thermo Scientific Multiskan GO). TPC were expressed in equivalent of standard (gallic acid, GAE/g).

Aluminum chloride colorimetric method as previously performed by [START_REF] Ahmad | Efficient regeneration and antioxidant potential in regenerated tissues of Piper nigrum L, Plant Cell, Tissue and Organ Culture[END_REF] was employed in order to estimate total flavonoid content (TFC). Aluminum chloride and potassium acetate both added (10 µL each) in reaction mixture along with 20 µL of methanol extracted sample and raised the total volume up to 200 µL with distilled water. Absorbance of reaction mixture was taken after 30 min incubation at 415 nm using micro-plate reader. TFC were expressed in equivalent of standard (quercetin, QE/g).

DPPH Scavenging Activity

Antioxidant potential of callus extracts was determined using DPPH reagent as studies by Abbasi et al. [START_REF] Abbasi | Shoot regeneration and free-radical scavenging activity in Silybum marianum L, Plant Cell, Tissue and Organ Culture[END_REF]. The reaction mixture included sample extract (20 µl) and DPPH solution (180 µl), incubated for approximately 60 min in dark, followed by absorbance measurement at 517nm. Here AE used to express mixture absorbance with sample addition and AD expresses the DPPH absorbance without sample addition.

Peroxidase (POD) and Superoxide Dismutase (SOD) Activity

For estimation of antioxidant enzyme activity, extract from fresh samples was used. Lagrimini [START_REF] Lagrimini | Plant peroxidases: under-and over-expression in transgenic plants and physiological consequences[END_REF] method was employed with slight adjustments for POD activity. The reaction mixture was prepared for each sample using 20 µl calli extract (fresh), 0.1 ml distilled H2O, 20 µl hydrogen peroxide (27.5 mM; 10x), 20 μl guaiacol (100 mM, 10×) and 40 µl KH2PO4 buffer (50 mM; pH 7). Same reagent amounts were employed as control without adding any extract of sample.

Microplate reader was used to perform sample's absorbance at wavelength of 470 nm and the enzymatic activity expressed by employing following formula: A = ELC

A used to express absorbance of sample, C = presence of enzyme concentration expressed in terms of nM/min/mg FW), L= Length of wall (0.25 cm) and E is used to express Extinction coefficient (6.39 mM -1 cm -1 ).

Protocol proposed by Giannopolitis and Ries [START_REF] Giannopolitis | Superoxide dismutases: I. Occurrence in higher plants[END_REF] was used to perform SOD activity. Mixture was prepared by adding EDTA (20µl, 1 mM), methionine (20µl, 130 mM), calli extract (60µl, fresh), 2µl riboflavin (0.02 mM), phosphate buffer (78µl, 50 mM, pH 7), and 20µl NBT (0.75 mM). Absorbance at 660nm wavelength was measured by micro-plate reader after 7 min of incubation under fluorescent light.

High Performance Liquid Chromatography Quantification

Metabolites quantification was done by HPLC using HPLC grade solvents and standards (Sigma Aldrich. Separation was performed on Hypersil PEP 300 C18, 250 x 4.6 mm, 5 µm particle size equipped with a guard column Alltech, 10 x 4.1 mm was utilized at 35 °C and Varian highperformance liquid chromatography system (equipped with Varian Prostar 230 pump Meta chem Degasit, Varian Prostar 410 autosampler and Varian Prostar 335 Photodiode Array Detector (PAD) and driven by Galaxie version 1.9.3.2 software was used in detection of compounds at 280 and 320 nm wavelength. The mobile phase of HPLC system was using two solvents (A: acetonitrite and B: acidified formic acid (0.1%v/v) ultrapure water). Mobile phase composition range varied during the 60 min run from 5:95 to 100:0 (solvent A: B v/v) according to linear gradient with 0.8ml/min flow rate. Re-equilibration time of 10 min was applied after each individual run. Quantification was done based on retention time compare to commercial reference standards (Sigma Aldrich). Examination of given samples was done three times and outcomes were denoted as mg/g dry weight.

Ferric Reducing Antioxidant Power (FRAP) Assay

To evaluate FRAP potential of callus cultures, protocol proposed by Benzie and Strain [START_REF] Benzie | The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay[END_REF] was utilized. Briefly, extracted samples (10 μl) were mixed with of FRAP (190 μl) solution [composed of acetate buffer (300 mM, pH 3.6); FeCl3.6H2O (20 mM) and TPTZ (10 mM); ratio 10:1:1 (v/v/v)]. Reaction mixtures were then stored at 25 ± 1 °C for approximately fifteen min.

Absorption was measured with Microplate Reader (BioTek ELX800, Bio-Tek Instruments) at 630 nm. Antioxidant capacity of samples was expressed in terms of TEAC (Trolox C equivalent antioxidant capacity) and whole procedure was repeated three times.

Antioxidant ABTS assay

The method of Tagliazucchi et al. [START_REF] Tagliazucchi | In vitro bio-accessibility and antioxidant activity of grape polyphenols[END_REF] was used to evaluate ABTS antioxidant potential.

Briefly, ABTS (2,2-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) salt (7mM) was thoroughly mixed with 2.45mM of potassium persulphate in equal proportion to prepare ABTS solution and the solution was placed in dark for 16h. The absorbance was recorded at 734 nm and adjusted to 0.7 and then mixed with the extracts and placed in dark at 25 ± 1°C for 15 min. Microplate reader (ELX800, BioTek Instruments) was used to record absorbance of mixture at 734nm. Antioxidant capacity of samples was expressed in terms of TEAC and whole procedure was repeated three times.

Anti-AGEs Formation Activity

The inhibitory capacity of formation of AGEs (advanced glycation end products) was determined as described by Kaewseejan and Siriamornpun [START_REF] Kaewseejan | Bioactive components and properties of ethanolic extract and its fractions from Gynura procumbens leaves[END_REF].. Extracts were prepared at a concentration of 50µg/ml in DMSO mixed with 20 mg/ml BSA (Sigma Aldrich) and 0.5 M glucose (Sigma Aldrich) solution, both were prepared in phosphate buffer and 1 ml of 0.1 M phosphate buffer containing 0.02 % (w/v) sodium azide at pH 7.4. The amount of fluorescent AGE formed was measured after 5 days incubation of reaction mixture in dark at 37°C by using a fluorescent spectrometer (Bio rad Versa Fluor) with an excitation wavelength set at 330 nm and emission wavelength set at 410 nm. For each sample extract, anti-AGEs formation was showed as % inhibition with respect to the relative control (same volume addition of DMSO).

α-glucosidase inhibition

The α-glucosidase was partially purified from rat intestinal acetone powder (Sigma). Protocol used for immobilization of α-glucosidase was previously described by hano et al. [START_REF] Hano | Flaxseed (Linum usitatissimum L.) extract as well as (+)-secoisolariciresinol diglucoside and its mammalian derivatives are potent inhibitors of α-amylase activity[END_REF] on CNBractivated Sepharose 4B. To determine immobilized enzymatic activity, chromogenic method [START_REF] Hano | Flaxseed (Linum usitatissimum L.) extract as well as (+)-secoisolariciresinol diglucoside and its mammalian derivatives are potent inhibitors of α-amylase activity[END_REF] was performed using polyethylene filter (0.45 µm) end-capped column. In brief, the assay was performed using intestinal fluid (1mL) consisting of 4-nitrophenyl-α-D-glucopyranoside (5mM, 4NPG; Sigma). The reaction mixture was stopped after 30 min incubation time at 37°C by column filtration and further adding sodium carbonate (1M) solution. The activity of enzymes was determined against blank by measuring the increased absorbance value at 405nm wavelength. Percentage inhibition was determined by calculating difference in absorbance value in presence as well as in absence of extracts. Experiment was repeated four times.

α-amylase inhibition

The α-amylase from porcine pancreas was purchased from Sigma. A chromogenic method as previously described [START_REF] Hano | Flaxseed (Linum usitatissimum L.) extract as well as (+)-secoisolariciresinol diglucoside and its mammalian derivatives are potent inhibitors of α-amylase activity[END_REF] was used to measure the activities of soluble a-amylase. Briefly, enzyme was prepared in phosphate buffer (0.1 M, pH6.8) with 1u/mL concentration and thoroughly mixed in 5mM of 4-nitrophenyl-α-D-maltopentaoside (4NPM; Sigma). Furthermore, the reaction mixture was incubated in presence and absence of the extract at 37°C for 30 min.

Sodium carbonate (1M) solution was used in same volume to stop the reaction and the activity of enzymes was determined against blank by measuring the increased absorbance value at 405nm wavelength. Percentage inhibition was determined by calculating difference in absorbance value in presence as well as in absence of extracts. Experiment was repeated four times. .

Statistical analysis

All the experiments were conducted in synchronized way in triplicates. Mean value and standard error for each experiment was calculated and graphs were generated using Origin software (8.5).

Data were shown as mean ± SE. One-way analysis of variance (ANOVA) with significant difference P < 0.05 was used to compare the means of different treatments.

3.

Result and Discussion

Hormonal treatment for callogenesis:

Callus induction from both stem and leaf explants was carried out for an optimal accumulation of secondary metabolites and biomass in response to various concentrations of plant growth regulators (PGRs), applied alone and/or in combination. Callus induction was observed against all treatments of PGRs from both explants (Supplementary Table 1). Callus induction frequency largely varied with respect to type of explant and concentrations of PGR's used. Highest callus induction frequency (100%) was observed at NAA (3.0, 4.0, 5.0 mg/L) and TDZ + NAA (2.5 + 1.0, 5.0 + 1.0 mg/L) in case of leaf and stem explants. Results indicated that TDZ showed lower callus induction potential when applied alone compared to its combination with NAA. Similar observation was reported by Younas et al. [START_REF] Younas | Differential accumulation of silymarin induced by exposure of Silybum marianum L. callus cultures to several spectres of monochromatic lights[END_REF] for Silybum marianum L. callus culture in which TDZ along with NAA showed more callogenesis potential compared to TDZ alone. A positive trend was observed between NAA concentrations and callus induction frequency whereas in case of TDZ and TDZ+NAA, higher concentrations inhibited the callogenesis in both leaf and stem explants (Supplementary Table 1). This could likely be due to suppression of endogenously produced hormones [START_REF] Lisowska | In vitro propagation of Catalpa ovata G. Don[END_REF][START_REF] Mathur | Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside[END_REF]. Morphological variations were also recorded in present study which indicated that compact callus was produced for TDZ and TDZ+NAA unlike callus produced against NAA alone presented as friable (Fig. 2 a,b,c). Overall, stem explant was observed more responsive towards callogenesis as compared to leaf explant as previously studied by Anjum et al [START_REF] Anjum | Trends in accumulation of pharmacologically important antioxidantsecondary metabolites in callus cultures of Linum usitatissimum L, Plant Cell, Tissue and Organ Culture[END_REF] for Linum usitatissimum L.

Trend in Biomass Accumulation:

Different PGRs were employed on leaf and stem explants from L. sativum for optimum production of biomass in in vitro callus culture. Optimum biomass (FW; 106.88 g/L, DW; 9.233 g/L) accumulation was observed with 2.5 mg/L TDZ + 1.0 mg/L NAA for stem explant (Fig. 1b). However, leaf explant showed relatively different response by accumulating high biomass (FW; 74.333 g/L, DW; 7.69 g/L) at 4.0 mg/L TDZ (Fig. 1a). This differential response could be due to biochemical and physiological potential of different explant type and tissues. Multiple factors influences on callus formation including plant genotype, growth conditions, explant type and optimum PGR concentration [START_REF] Mathur | Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside[END_REF]. Higher concentrations of TDZ and NAA, when applied alone, promoted callus biomass in both explant types. Our results are similar to limited study of Abdellatef et al. [START_REF] Abdellatef | In Vitro callogenesis and proliferation from different explants of garden cress (Lepidium sativum. Linn)[END_REF] which employed various concentrations of 2, 4-D, TDZ and NAA to evaluate in vitro callus formation potential of L. sativum from different explants. Similar trend of biomass production was observed for TDZ in Artemisia absinthium L. callus culture [START_REF] Ali | Thidiazuron-induced changes in biomass parameters, total phenolic content, and antioxidant activity in callus cultures of Artemisia absinthium L[END_REF].

However, no previous study is available on combined effect of TDZ and NAA on L. sativum. In case of stem explant, TDZ + NAA significantly lower the biomass production at higher and lower concentration except optimum level (Fig. 1b). This suggest that auxins and cytokinins in combination at optimum level, produces synergistic effect for callus formation as studied in previous reports [START_REF] Nin | Agrobacterium-mediated transformation of Artemisia absinthium L.(wormwood) and production of secondary metabolites[END_REF][START_REF] Zia | Effect of growth regulators and amino acids on artemisinin production in the callus of Artemisia absinthium[END_REF][START_REF] Zia | Hormonal regulation for callogenesis and organgenesis of Artemisia absinthium L[END_REF]. Furthermore, hormonal optimized callus culture was placed under different spectral lights to investigate the potential effect on biomass accumulation. Optimum biomass production (FW; 166.28 g/L, DW; 15.36 g/L) was recorded in cultures placed in continuous light (24h) followed by yellow light (FW; 146.32 g/L; DW; 12.87 g/L) as compared to control (Fig. 1c). Cultures placed under dark conditions produced lowest biomass (FW; 110.16 g/L, DW; 8.7 g/L) compared to rest of lights. Maximum biomass production in cultures exposed to continuous white light could possibly be due to more energy level than other lights, playing vital role in plant's physiological and chemical processes. Tariq et al. [START_REF] Tariq | Morphogenic and biochemical variations under different spectral lights in callus cultures of Artemisia absinthium L[END_REF] previously concluded growth enhancing effect of white light compared to other lights in A. absinthium callus culture.

Similar results of high biomass accumulation in cell culture under white light were previously reported [START_REF] Ali | Light-induced fluctuations in biomass accumulation, secondary metabolites production and antioxidant activity in cell suspension cultures of Artemisia absinthium L[END_REF][START_REF] Adil | Interactive effects of melatonin and light on growth parameters and biochemical markers in adventitious roots of Withania somnifera L, Plant Cell[END_REF]. Influence of different spectral lights varies significantly with plant species, intensity and quality of applied light [START_REF] Ahmad | Light-induced biochemical variations in secondary metabolite production and antioxidant activity in callus cultures of Stevia rebaudiana (Bert)[END_REF]. Morphological changes were also recorded as cultures grown under dark conditions appeared to be pale yellow unlike those grown under continuous white light, blue and yellow (Fig. 2d-i).

Accumulation of Phenolic and Flavonoid Contents

Plant secondary metabolites play vital role in defense against environmental stress and regulation of plant growth and development [START_REF] Balasundram | Phenolic compounds in plants and agri-industrial byproducts: Antioxidant activity, occurrence, and potential uses[END_REF]. Optimum level of these metabolites (phenolics and flavonoids) in response to different hormones, explant source and spectral lights was also investigated in this study. Overall, TDZ in combination with NAA produced the best results in case of both leaf and stem derived callus cultures compare to their sole treatments. Highest TPC value (stem; 36.47 mg/g, leaf; 34.10 mg/g) were recorded in callus cultures at TDZ (2.5 mg/L) + NAA (1.0 mg/L) (Supplementary Fig. 1a). Optimum total phenolic production (TPP 232.6 mg/L) was observed at TDZ (1.0 mg/L) + NAA (1.0 mg/L) in case of leaf derived cultures whereas stem derived cultures showed optimum TPP (336.83 mg/L) at TDZ (2.5 mg/L) + NAA (1.0 mg/L) (Supplementary Fig. 1b). Similarly, optimum TFC (7.47 mg/g) level was recorded in stem derived culture at TDZ (1.0 mg/L) + NAA (1.0 mg/L) whereas maximum TFP level (52.53 mg/L) was recorded at TDZ (2.5 mg/L) + NAA (1.0 mg/L) in stem derived culture (Supplementary Fig. 1c,d). Overall, a positive correlation of secondary metabolites production and callus biomass accumulation was found which is in accordance with previous study of Ali and Abbasi [START_REF] Ali | Production of commercially important secondary metabolites and antioxidant activity in cell suspension cultures of Artemisia absinthium L[END_REF].

Based on highest biomass accumulation, total phenolic and flavonoids production, stem derived callus culture of L. sativum at TDZ (2.5 mg/L) + NAA (1.0 mg/L) hormonal concentration was used to determine potential influence of multi spectral lights on secondary metabolites. Light is considered a very effective abiotic elicitor that can stimulate biosynthesis of plant's precious metabolic compounds [START_REF] Fazal | Correlation of different spectral lights with biomass accumulation and production of antioxidant secondary metabolites in callus cultures of medicinally important Prunella vulgaris L[END_REF]. Various callus cultures have been tested in the past for enhanced metabolites biosynthesis upon exposure to multi spectral lights [START_REF] Shohael | Effect of light on oxidative stress, secondary metabolites and induction of antioxidant enzymes in Eleutherococcus senticosus somatic embryos in bioreactor[END_REF][START_REF] Exonomou | Light treatments to improve efficiency of in vitro propagation systems[END_REF]. Cultures grown under white light (24h) showed maximum TPC (47.43 mg/g), followed by cultures grown in dark (44.05 mg/g) and blue light (40.54 mg/g) and similar trend was observed in TFC accumulation under influence of all lights (Fig. 3a). Total phenolic and flavonoid production (TPP, TFP) were found in positive correlation with biomass accumulation of cultures grown under different lights (Fig. 3b). Different studies have been reported on beneficial influence of white light in in-vitro derived cultures. Maximum level of anthocyanin in hairy root cultures was reported under white light in Echinacea purpurea [START_REF] Zhong | Effect of light irradiation on anthocyanin production by suspended culture of Perilla frutescens[END_REF][START_REF] Beckwith | Relationship of light quantity and anthocyanin production in Pennisetum setaceum cvs. Rubrum and Red Riding Hood[END_REF]. Khan et al. [START_REF] Khan | Synergistic effects of melatonin and distinct spectral lights for enhanced production of anti-cancerous compounds in callus cultures of Fagonia indica[END_REF] reported enhanced production of anticancer compounds under exposure of white light in callus culture of Fagonia indica.

Adventitious root cultures of Withania somnifera L. produced optimum results when placed under white light [START_REF] Adil | Interactive effects of melatonin and light on growth parameters and biochemical markers in adventitious roots of Withania somnifera L, Plant Cell[END_REF]. Contrarily, blue and red lights have also been reported to have significant influence in biosynthesis of plant metabolic compounds in different studies [START_REF] Younas | Differential accumulation of silymarin induced by exposure of Silybum marianum L. callus cultures to several spectres of monochromatic lights[END_REF][START_REF] Fazal | Correlation of different spectral lights with biomass accumulation and production of antioxidant secondary metabolites in callus cultures of medicinally important Prunella vulgaris L[END_REF][START_REF] Nadeem | LED-enhanced biosynthesis of biologically active ingredients in callus cultures of Ocimum basilicum[END_REF]. Effects of monochromatic lights have previously been tested for enhanced metabolites synthesis in in vitro cultures of various plant species [START_REF] Szopa | Production of bioactive phenolic acids and furanocoumarins in in vitro cultures of Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams. under different light conditions[END_REF][START_REF] Szopa | Targeted lignan profiling and anti-inflammatory properties of Schisandra rubriflora and Schisandra chinensis extracts[END_REF][START_REF] Szopa | The importance of monochromatic lights in the production of phenolic acids and flavonoids in shoot cultures of Aronia melanocarpa, Aronia arbutifolia and Aronia× prunifolia[END_REF][START_REF] Szopa | Phytochemical studies and biological activity of three Chinese Schisandra species (Schisandra sphenanthera, Schisandra henryi and Schisandra rubriflora): Current findings and future applications[END_REF]. Such anomalies suggest that influence of light intensity and quality on active ingredients of plant can vary from plant's species to species.

Free radical scavenging activity

Plants under stress conditions tend to produce large quantities of reactive oxygen species (ROS) that has harmful effect on growth and development of cells by damaging the DNA [START_REF] Moran | Drought induces oxidative stress in pea plants[END_REF][START_REF] Alexieva | The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat[END_REF][START_REF] Yu | Reactive oxygen species-induced DNA damage and its modification: a chemical investigation[END_REF].

Plants are naturally equipped with defense mechanism to mitigate harmful effects of ROS by utilizing antioxidant enzymes [START_REF] Mittler | Oxidative stress, antioxidants and stress tolerance[END_REF]. Natural occurrence of phenolics, flavonoids, carotenoids and terpenoids in plants act as antioxidant compounds by providing protection against oxidative damages [START_REF] Ashry | Impact of secondary metabolites and related enzymes in flax resistance and or susceptibility to powdery mildew[END_REF][START_REF] Samuolienė | The effect of red and blue light component on the growth and development of frigo strawberries[END_REF]. In current study, optimum scavenging free radical activity (92.1 %) was observed in stem derived callus culture in response to TDZ (2.5 mg/L) + NAA (1 mg/L), followed by 91.9 % and 91.5 % at TDZ (0.1 mg/L) + NAA (1 mg/L) and TDZ (5.0 mg/L) + NAA (1.0 mg/L) respectively whereas maximum scavenging activity in leaf derived callus culture (90.5%) was noted at TDZ (5.0 mg/L) + NAA (1.0 mg/L) (Supplementary Fig. 2). The lowest antioxidant activity for both leaf (61.5 %) and stem (72.5 %) derived cultures was recorded at NAA (0.1 mg/L). Scavenging activity of hormonally optimized calli was also recorded under different spectral lights after 28 days exposure. Maximum antioxidant activity (95.1%) was observed under white light (24h), followed by dark (94.2%) and blue light (93.2%) whereas yellow light had a least effect on antioxidant activity (85.2%) (Fig. 4). A positive correlation was observed between enhanced phytochemicals biosynthesis (phenolic and flavonoids) and high free radical scavenging activity under white light (24h). A similar correlation was observed in a study of Khan et al. [START_REF] Khan | The interplay between light, plant growth regulators and elicitors on growth and secondary metabolism in cell cultures of Fagonia indica[END_REF] which implies that scavenging activity of callus culture is directly proportional to amount of phytochemical produced. Current results are in accordance with different studies [START_REF] Ali | Light-induced fluctuations in biomass accumulation, secondary metabolites production and antioxidant activity in cell suspension cultures of Artemisia absinthium L[END_REF][START_REF] Beckwith | Relationship of light quantity and anthocyanin production in Pennisetum setaceum cvs. Rubrum and Red Riding Hood[END_REF] stating the importance of light on biosynthesis of phenolic compounds and their antioxidant activity.

Lights effect on peroxidase and superoxide dismutase activity

Potential influence of different lights on POD and SOD activities was also investigated in callus culture of L. sativum at optimized hormonal concentration (TDZ: 2.5 mg/L + NAA: 1.0 mg/L).

Maximum POD enzyme activity (0.501 nM/min/mg FW) was observed in green light, followed by control (0.46 nM/min/mg FW) and white light (0.43 nM/min/mg FW) respectively whereas blue light significantly reduced the POD enzymatic activity (0.308 nM/min/mg FW) compared to other lights (Fig. 5). Similarly, highest SOD activity was also observed in green light followed by continuous white light (Fig. 6). These antioxidant enzymatic activities play vital role in sustaining cell viability during metabolic reactions by protecting them from free radicals toxicity [START_REF] Suzuki | Reactive oxygen species and temperature stresses: a delicate balance between signaling and destruction[END_REF] and spectral lights have a significant role in regulation of these enzymatic activities. Hina et al. [START_REF] Fazal | Correlation of different spectral lights with biomass accumulation and production of antioxidant secondary metabolites in callus cultures of medicinally important Prunella vulgaris L[END_REF] studied the influence of different lights on SOD and POD activities on callus culture of Prunella vulgaris in which highest antioxidant enzymatic activities were observed under yellow light. A study conducted by Tariq et al. [START_REF] Tariq | Morphogenic and biochemical variations under different spectral lights in callus cultures of Artemisia absinthium L[END_REF] on callus culture of A. absinthium in which red light showed prominent impact on POD enzymatic activities. Different spectral lights also influence micronutrients like Zn or Mn in plant cells that act as inorganic co-factor for antioxidant enzymes [START_REF] Pedas | Golgi localized barley MTP8 proteins facilitate Mn transport[END_REF].

Phytochemical Analysis

Plant phytochemicals play vital role in plant defense, survival and growth regulation and are widely distributed among plant species [START_REF] Balasundram | Phenolic compounds in plants and agri-industrial byproducts: Antioxidant activity, occurrence, and potential uses[END_REF]. Application of an elicitor such as light can enhance biosynthesis of these precious metabolites which possess huge pharmaceutical value. These metabolites can be easily quantified using high performance liquid chromatography (HPLC).

Current study includes quantitative analysis of 9 phenolic compounds from L. sativum callus cultures grown under different spectral light. Higher level of total secondary metabolites was observed in white light (39.19 mg/g), followed by dark (35.14 lens of diabetic people, erythrocytes and in plasma [START_REF] Grillo | Advanced glycation end-products (AGEs): involvement in aging and in neurodegenerative diseases[END_REF]. Therefore, the application of these therapeutic metabolites as inhibitors can prove to be useful in fight against complicated diseases.

Anti-diabetic potential in lights treated callus extracts of L. sativum was evaluated by measuring immobilized α-glucosidase and solubilized α-amylase inhibitory activities. Results indicated that callus extracts were more effective in α-amylase inhibition compared to membrane bound αglucosidase (Fig. 8). Extract of white light treated callus showed maximum α-amylase inhibitory activity (67.55 %), followed by dark (66.42 %) compared to control (41.63%). Highest inhibitory activity of α-glucosidase was recorded in dark (55.20 %), followed by white light (54.55%) whereas yellow light showed lowest anti-diabetic potential (28.41%). Finding of this assay revealed that anti-diabetic potential in L. sativum is largely due to its inhibition ability toward pancreatic α-amylase rather than α-glucosidase as previously studied by Hano et al. [START_REF] Hano | Flaxseed (Linum usitatissimum L.) extract as well as (+)-secoisolariciresinol diglucoside and its mammalian derivatives are potent inhibitors of α-amylase activity[END_REF] in determining the anti-diabetic activity of flax seeds. Eddouks et al. [START_REF] Eddouks | Study of the hypoglycaemic activity of Lepidium sativum L. aqueous extract in normal and diabetic rats[END_REF] previously studied the L. sativum seed's extract on diabetic rats for possible hypoglycemic effects. This is the first study determining the effect of lights on enhanced anti-diabetic activity in L. sativum callus extracts. In general, we cannot exclude the fact that phytochemicals (other than the one's quantified in current study) could play a role for observed biological activities.

Conclusion

In current study, optimized callus culture establishment of L. sativum was achieved using various concentrations of TDZ and NAA alone and in combination for sustainable production of industrially important secondary metabolites. Further, we conclude that light an excellent abiotic elicitor for increased biosynthesis of plant metabolites. Among treatment of different monochromatic lights, white light (24h) enhanced the level of phenolic profile and antioxidant potential of plant. White light significantly increased caffeic acid, chlorogenic acid, quercetin and kaempferol level after 28 days of exposure. Blue light had a potential effect on p-coumaric acid compared to rest of lights. This study revealed the potential applications of multi spectral lights on sustainable production of metabolites from in vitro derived callus cultures of L.

sativum. Columns with similar alphabets are not significantly different (P < 0.05). Columns with similar alphabets are not significantly different (P < 0.05).

  The final concentrations of ascorbic acid (5, 10, 20 and 40 µg/ml) and DPPH (180 µl) with DMSO (20 µl) were considered as negative control. Following formula was used to calculate FRSA activity, % DPPH activity = 100 × (1-AE/AD)

3. 7 .

 7 Effect of multispectral lights on antioxidant and anti-diabetic potential ofL. sativum callus culturesAntioxidant activity of L. sativum callus cultures placed under different monochromatic lights was evaluated by callus extract using FRAP, ABTS assay and inhibition of vesperlysine-like AGEs and pentosidine-like AGEs formation. Results of FRAP antioxidant assay suggested that white light grown cultures produced optimum antioxidant activity (629.78 uM), followed by blue light (576.41 uM) and dark (552.36 uM) (Fig.7.a). Similar trend was recorded for ABTS assay which showed highest activity in white light (401.44 uM), followed by dark (377.78 uM) (Fig.7.b). The antioxidant potential of both assays was expressed in TEAC (Trolox C equivalent antioxidant capacity). Results indicate a positive correlation between antioxidant potential and phenolic contents of callus cultures when exposed multispectral lights. Enhanced production of phenolic compounds could possibly be the reason behind significant increase in antioxidant activities of extract samples under white light. Direct correlation of antioxidant potential and secondary metabolites has previously been documented in different studies. [89, 90]. Furthermore, inhibition of formation of AGEs (pentosidine-and vesperlysine-like) was recorded in treatment of callus extracts of L. sativum. Significant increase in inhibition activity was shown by white light treated cultures (65.83 %, 56.47 %) against pentosidine and vesperlysine like AGEs formation respectively (Fig.7.c). Minimum activity was recorded in cultures treated with yellow light for both type of anti-AGEs formation. Trend in polyphenolic accumulation and anti-AGEs formation in response to various lights indicate the therapeutic potential of these secondary metabolites to protect the human body. Formation of AGEs is usually observed in eye

Fig. 1 a

 1 Fig. 1 a) Fresh and dry callus biomass from leaf explant b) Fresh and dry callus biomass from stem explant culture on MS medium supplemented with NAA and TDZ alone and in combination c) Fresh and dry callus biomass under different monochromatic lights. Values are means of triplicates with the standard deviation. Columns with similar alphabets are not significantly different (P < 0.05).

Fig 2 .

 2 Fig 2. Effect of PGRs and different monochromatic lights on Calli biomass and morphology from stem explant after 4 weeks of culture (a) NAA 5mg/l (b) TDZ 4mg/l (c) TDZ + NAA (2.5mg/l, 1mg/l) (d) Dark (e) Continuous light (f) Blue (g) Red (h) Green (i) Yellow

Fig. 3

 3 Fig. 3 Effect of different monochromatic lights on a) Total Phenolic Content (TPC, mg/g DW) and Total Flavonoid Content (TFC, mg/g DW) b) Total Phenolic Production (TPP, mg/L) and Total Flavonoid Production (TFP, mg/L) at optimized hormonal conditions. Values represent means ± standard errors from triplicates. Columns with similar alphabets are not significantly different (P < 0.05).

Fig. 4 DPPH

 4 Fig. 4 DPPH Free radical scavenging Activity (%) in response to Monochromatic lights treatment at optimized hormonal concentration. Values represent means ± standard errors from triplicates. Lines with similar alphabets are not significantly different (P < 0.05).

Fig. 5

 5 Fig. 5 Influence of different lights treatment on peroxidase (POD) activity of callus samples. Values represent means ± standard errors from triplicates. Columns with similar alphabets are not significantly different (P < 0.05).

Fig. 6

 6 Fig. 6 Influence of different lights treatment on superoxide dismutase (SOD) activity of callus samples. Values represent means ± standard errors from triplicates. Columns with similar alphabets are not significantly different (P < 0.05).

Fig. 7 .

 7 Fig.7. In vitro antioxidant (a, b) and anti-AGEs formation (c) potential of stem derived callus culture place under multispectral lights. (TEAC: Trolox C equivalent antioxidant activity, expressed in µM).

Fig. 8 .

 8 Fig.8. In-vitro anti-diabetic potential of L. sativum callus extracts in response to multispectral lights.

Table 1 :

 1 Effect of multi spectral lights on accumulation of polyphenolic metabolites ((mg/g DW)) in callus culture of Lepidium sativum.

	Marker			Different Lights Treatment
	Compounds (mg/g DW)	Control	Red	Green	Blue	Dark	White Light	Yellow
	caffeic acid	0.641 ± 0.01	0.684 ± 0.04	0.726 ± 0.065	0.74 ± 0.03	0.880 ± 0.06	0.988 ± 0.024	0.578 ± 0.03
	ferulic acid	0.435 ± 0.009	0.463 ± 0.03	0.492 ± 0.021	0.50 ± 0.051	0.593 ± 0.07	0.665 ± 0.044	0.393 ± 0.011
	vanillic acid	0.135 ± 0.004	0.131 ± 0.005	0.153 ± 0.0033	0.140 ± 0.028	0.169 ± 0.016	0.181 ±0.0075	0.120 ± 0.077
	p-coumaric	0.137 ±	0.162 ±	0.208 ±	0.220 ±	0.170 ±
	acid	0.006	0.007	0.005	0.047	0.063	0.168 ± 0.0054	0.130 ± 0.086
	sinapic acid	0.064 ± 0.0018	0.061 ± 0.005	0.071 ± 0.0093	0.073 ± 0.0065	0.074 ± 0.0034	0.077 ± 0.0038	0.059 ± 0.0033
	protocatechuic	0.049 ±	0.046 ±	0.053 ±	0.050 ±	0.054 ±
	acid	0.0064	0.002	0.0019	0.0044	0.0065	0.055 ± 0.0025	0.045 ± 0.006
	chlorogenic	4.99 ±		5.56 ±	5.61 ±
	acid	0.855	5.20 ± 0.822	1.209	0.99	6.52 ± 1.66	7.20 ± 1.555	4.55 ± 0.66
	Quercetin	14.55 ± 1.776	15.45 ± 2.05	16.41 ± 1.999	16.74 ± 2.44	19.73 ± 3.055	22.08 ± 1.877	13.15 ± 1.363
	Kaempferol	5.10 ± 1.055	5.42 ± 0.79	5.76 ± 0.622	5.89 ± 0.883	6.94 ± 1.22	7.77 ± 1.22	4.61 ± 0.92
	Total	26.11	27.63	29.45	29.96	35.14	39.19	23.64

Conflict of Interest

The authors declare no conflict of interest.