Investigation of Morinda citrifolia Activities through Pinoresinol and α-EG Related Gene Expression

α-EG is a unique substance that was first found in the leaves and fruits of Morinda citrifolia (Mc) growing in Thailand using GC-MS at 52.33% and 54.12%. It was then concentrated and its abundance quantified, along with that of pinoresinol, via GC, compared to the standards in leaves, ufp, rfp, rawfs, and seeds. α-EG and pinoresinol, which have collagen stimulating, skin whitening, and an inhibitory effect on wrinkle formation, were found in different concentrations and amounts. Three different concentrations of the five Mc part extracts were tested on NHDF for gene expression related to the aforementioned activities, COL1A1, COL1A2, and COL3A1, FGF1 and FGF7 by qRT-PCR. The results showed various expression levels, both stimulatory and inhibitory, with different concentrations of plant parts and genes. Similar results were revealed when the experiments were performed with Morus alba (Ma), which was found to contain 20.48 g protein p/100 g leaves at concentrations of 3.11 mg/mL. The studied Mc parts seem to have advantages based on the stated objectives, gene type and level of activity of each plant part. Rawfs and leaves supplemented with Ma samples were selected for toxicity tests with PBMCs. The lack of both cell and DNA toxicity from the rawfs indicated that they can be used safely.


Introduction
Plants benefit humanity in many ways. For example, Morinda citrifolia (Mc) is a useful pantropical species supporting human life as an edible fruit; it is also used to create drinks, supplements, etc., and has medicinal value. There are many publications on it in the literature. Various aspects of the species have been studied, including its chemical and medicinal properties, and new reports and reviews are being published all the time. Some important Mc studies have indicated the the plant's fruit has anti-inflammatory, anticancer, and antioxidant properties. Additionally, a 50% ethanolic noni seed extract showed more potent in vitro inhibition of elastase and tyrosinase, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than leaf extract [1]. Xeronine, a small alkaloid which is one of the bioactive compounds of noni fruit, is believed to be capable of modifying the molecular structure of specific inactive proteins, thereby regulating the folding of active enzymes [2]. It has also been reported that the plant has anti-inflammatory effects which may be useful in the treatment of inflammatory bowel disease [3]. Furthermore, the consumption of noni fruit juice does not cause any significant disturbances to the liver or kidney outside of immune modulation [4]. The main alkaloids present in noni are Plants 2022, 11,1985 2 of 17 xeronine, scopolenin, dammacanthol; it also contains vitamins A and C, Mg, K, Fe, alizarin, and ursolic acids, and therefore, may be able to slow the growth of cancer. Additionally, it may be a viable therapeutic resource for the treatment of obesity and obesity-related metabolic problems or indigestion [5]. Drinking 40 mL per day of noni wine was shown to effectively prevent high-fat diet-induced oxidative stress and obesity in mice [6]. Noni fruit water extract and noni fruit polysaccharide were shown to alleviate oxidative stress and inflammation in mice on a high-fat diet, and the noni fruit polysaccharide at 100 mg per kg bodyweight had a better effect than noni fruit water extract with a similar polysaccharide dosage, illustrating that noni fruit polysaccharide may be an important component in the water extract [7]. M. citrifolia fruit juice contains large groups of secondary metabolites including polyphenols, reducing compounds, mucilage and terpenoids. The antimicrobial activity of these compounds was found to inhibit the growth of Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, S. epidermidis, Proteus vulgaris, Streptococcus oralis, Enterococcus faecalis and Escherichia coli [8]. Additionally, four publications reported important data about the species. Noni seeds contain 3,3'-bisdemethylpinoresinol and americanin A, which have been found to inhibit tyrosinase activity. Inhibited α-melanocyte activity stimulates melanogenesis, resulting in a skin whitening effect, wrinkle inhibition and enhanced blood fluidity, platelet aggregation and fibrinolytic activity [9]. Several phytochemical constituents which contain the most valuable compounds for industrial and pharmacological activities have been described [10]. Additionally, the phenolic acids, lignans, flavonoids, flavones, flavans-3-ol, anthocyanins, phytosterols, alkaloids, vitamins and minerals present in the noni plant have been shown to affect obesity and obesityassociated metabolic dysfunction by various mechanisms [11]. In [12], the traditional uses, phytochemicals, phytotherapic properties, toxicological properties and recent advances in the processing and standardization of products derived from noni fruit were reported. The advantages of this species seem to be attributable to the abundance of chemicals that give rise to its therapeutic properties. One other plant species with high nutritional value, i.e., mulberry, Morus alba (Ma), is a well-known plant food for animals such as silkworm. Its leaves and fruits are also widely consumed by humans and have reported medicinal properties. The authors of [13] studied the safety of consuming the leaves and proposed a pharmacological dosage of a leaf extract, which might be a valuable drug [13]. Currently, mulberry leaves are authorized as a food resource with high protein, carbohydrate, vitamin, microelements and dietary fiber contents. They are also rich in phenolic acids, flavonoids, alkaloids, and γ-aminobutyric acid [14]. As such, their use has been proposed as a food supplement.
Another area of ongoing research is how different environments may produce different substances and properties in M. citrifolia, since the plant grows all over the tropics and has a myriad of uses. As such, researchers need to undertake further study on the substances, activities and genes that determine these characteristics in the leaves, raw and ripe fruit, and seeds to confirm and augment the data that have already been published. In this research, the protein matter in Ma will be studied to clarify whether it supports gene expression.

Macronutritional Components
According to a proximate analysis, 100 g plant leaf powder of Ma leaves comprised 16.91 g ash, 316.46 Kcal energy, 11.10 g moisture, 20.48 g protein, 45.81 g total carbohydrate and 5.70 g total fat.

Phytochemical Constituent Evaluation by Gas Chromatography-Mass Spectrometry
Mature Ma leaves and the leaves and fruits of Mc were analyzed. The various types and quantities of detected phytochemicals are listed in Table 1, and chromatograms showing retention time and peak areas are shown in Figure 1. The major chemical in Mc parts is ethylα-d-glucopyranoside or α-EG, which made up 54.12% and 52.33% of the ethanol extracts of leaves and fruits, respectively. This compound was not found in the Ma leaf extract. Mature Ma leaves and the leaves and fruits of Mc were analyzed. The various types and quantities of detected phytochemicals are listed in Table 1, and chromatograms showing retention time and peak areas are shown in Figure 1. The major chemical in Mc parts is ethyl-α-d-glucopyranoside or α-EG, which made up 54.12% and 52.33% of the ethanol extracts of leaves and fruits, respectively. This compound was not found in the Ma leaf extract.

Gas Chromatography (GC) Compared to the Pinoresinol and α-EG Standards
The abundances of 3,3 -bisdemethylpinoresinol (pinoresinol) and ethyl-α-d-glucoside (α-EG) were measured in five Mc parts, i.e., the leaves, ufp, rfp, rawfs, and seeds. Pinoresinol was found in all studied plant parts, with the highest abundances in both concentrations of rfp being 0.14 mg/mL and 0.38 mg/g (Table 2), based on a peak area in the extract chromatograms ( Figure 2). The plotted graph of the peak areas and the pinoresinol standard concentration created a linear equation, i.e., y = 749.07x − 18.13, and a correlation coefficient (R 2 ) of 0.9991. α-EG was also found in all studied plant parts, with the highest concentration in leaves, i.e., 0.30 mg/mL, and in rawfs, i.e., 3.77 mg/g plant sample (Table 2, Figure 2). The plotted graph of the peak areas and the α-EG standard concentration created a linear equation, i.e., y = 239.75x − 10.004, and the correlation coefficient (R 2 ) was 0.9965.

Gene Expression Quantification
The results of the gene expression research are divided into two parts. The first part presents the results regarding genes which should be expressed in high percentages in plants and which are effective at stimulating type I collagen, COL1A1 and COL1A2, type III collagen and COL3A1, as well as angiogenesis-stimulating and fibroblast proliferation genes FGF1 and FGF7, which were quantified by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR). Three concentration levels of the samples were tested. The results indicated that all studied plant parts and their extract concentrations affected all studied genes expressed in various high percentages compared to a control (without the plant extracts). Graphs of various relative gene expression and the studied plant parts with various concentrations are shown in Figure 3. The observed increases in expression percentages were as follows: FGF1 and FGF7 genes increased by 970% from 5.06 mg/mL rawfs, followed by 960% from 0.07 mg/mL ufp, 4870% from 0.65 mg/mL ufp and 2700% from 1.04 mg/mL seeds. The expression percentages of collagen stimulation genes were 4160% and 3480% from 0.65 mg/mL and 0.07 mg/mL ufp, respectively, and 3380% from 0.01 mg/mL seeds for COL1A1 collagen type I; 2930%, 2370%, and 1070% from 1.04, 0.10, and 0.01 mg/mL seeds, respectively, and 960% from 6.50 mg/mL ufp for COL1A2 collagen type I; finally, 2870% from 5.06 mg/mL rawfs and 1180% from 10.30 mg/mL rfp for COL3A1 collagen type III.
The second part contains the results regarding genes which should be expressed in low percentages but with high inhibitory efficacies, as follows: TYR, an essential enzyme in melanin synthesis; α-MSH, an α-melanocyte-stimulating hormone gene which stimulates melanogenesis; and NRF2, a transcription factor for stress response. These factors cause dull skin and aging, and therefore, should not be highly expressed. The results indicated that all studied plant parts and concentrations affected all of these genes compared to a control (without the plant extracts). Graphs of various relative gene expression levels and the studied plant parts with various concentrations are shown in Figure 4. The lowest and second lowest percentage expression which showed high

Gene Expression Quantification
The results of the gene expression research are divided into two parts. The first part presents the results regarding genes which should be expressed in high percentages in plants and which are effective at stimulating type I collagen, COL1A1 and COL1A2, type III collagen and COL3A1, as well as angiogenesis-stimulating and fibroblast proliferation genes FGF1 and FGF7, which were quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Three concentration levels of the samples were tested. The results indicated that all studied plant parts and their extract concentrations affected all studied genes expressed in various high percentages compared to a control (without the plant extracts). Graphs of various relative gene expression and the studied plant parts with various concentrations are shown in Figure 3. The observed increases in expression percentages were as follows: FGF1 and FGF7 genes increased by 970% from 5.06 mg/mL rawfs, followed by 960% from 0.07 mg/mL ufp, 4870% from 0.65 mg/mL ufp and 2700% from 1.04 mg/mL seeds. The expression percentages of collagen stimulation genes were 4160% and 3480% from 0.65 mg/mL and 0.07 mg/mL ufp, respectively, and 3380% from 0.01 mg/mL seeds for COL1A1 collagen type I; 2930%, 2370%, and 1070% from 1.04, 0.10, and 0.01 mg/mL seeds, respectively, and 960% from 6.50 mg/mL ufp for COL1A2 collagen type I; finally, 2870% from 5.06 mg/mL rawfs and 1180% from 10.30 mg/mL rfp for COL3A1 collagen type III. for the TYR gene; 1.29% from 0.01 mg/mL and 1.33% from 0.10 mg/mL seeds, followed by 2.33% from 0.51 mg/mL rawfs for the α-MSH gene; 3.84% from 3.90 mg/mL leaves, followed by 4.70% from 10.30 mg/mL rfp for the NRF2 gene; 0.20% from 0.51 mg/mL rawfs, and 3.90 mg/mL leaves, followed by 0.30% from 0.01 mg/mL seeds, 0.065 mg/mL ufp and 0.39 mg/mL leaves for the HLE gene  The second part contains the results regarding genes which should be expressed in low percentages but with high inhibitory efficacies, as follows: TYR, an essential enzyme in melanin synthesis; α-MSH, an α-melanocyte-stimulating hormone gene which stimulates melanogenesis; and NRF2, a transcription factor for stress response. These factors cause dull skin and aging, and therefore, should not be highly expressed. The results indicated that all studied plant parts and concentrations affected all of these genes compared to a control (without the plant extracts). Graphs of various relative gene expression levels and the studied plant parts with various concentrations are shown in Figure 4. The lowest and second lowest percentage expression which showed high efficiency were 0.10% from 5.06 mg/mL rawfs, followed by 0.20% from 3.90 mg/mL leaves for the TYR gene; 1.29% from 0.01 mg/mL and 1.33% from 0.10 mg/mL seeds, followed by 2.33% from 0.51 mg/mL rawfs for the α-MSH gene; 3.84% from 3.90 mg/mL leaves, followed by 4.70% from 10.30 mg/mL rfp for the NRF2 gene; 0.20% from 0.51 mg/mL rawfs, and 3.90 mg/mL leaves, followed by 0.30% from 0.01 mg/mL seeds, 0.065 mg/mL ufp and 0.39 mg/mL leaves for the HLE gene.  The gene expression levels of the mixed extracts, comparing the three Mc parts, i.e., ufp, rawfs and seeds (containing α-EG and pinoresinol), and Morus alba (Ma) leaves containing protein were tested. The result showed that there were various levels of expression depending on the kind and concentration of Mc parts and genes. Additionally, both higher and lower expression were observed when Ma was added; for example, percentage expressions were 3200% and 2450% from 5.06 mg/mL rawfs and 6.50 mg/mL ufp for the COL1A1 gene, i.e., higher than without Ma. However, with1.04 mg/mL seeds, higher expression, i.e., 2930%, was observed without Ma for the COL1A2 gene. All results are shown in Table 3 and Figure 5. The results for the inhibitory genes showed that all studied mixtures, i.e., Mc leaves, rawfs and seeds with and without Ma, affected expression to a lower extent than with and without Ma, as shown in Table 4 and Figure 6. Rawfs at a concentration of 5.06 mg/mL and leaves at a concentration of 3.90 mg/mL without Ma showed lower activity, i.e., 0.10% and 0.20% for TYR and HLE, respectively, but showed higher activity, i.e., 0.19% and 1.40%, at a concentration of 5.06 mg/mL rawfs and 3.90 mg/mL leaves with added Ma for α-MSH, NRF2. With various activity found from the Mc parts, leaves and rawfs, the mixtures of Mc leaves and Ma leaves and rawfs were further tested for toxicity at a working concentration, as described in Table 5, Figure 7.

Cytotoxicity and Genotoxicity Testing via MTT and Comet Assays
The results of a MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) showed IC 50 values at 3.00 mg/mL, 2.90 mg/mL and 3.60 mg/mL in Mc parts and leaves, and in the mixture of Mc and Ma leaves. Following depth toxicity testing on the DNA level by comet assay, the samples showed significant DNA damage (Table 5, Figure 8). With the LD 50 value calculated using IC 50 values, the toxicity was revealed as Class II, i.e., slightly hazardous, corresponding to over 2000 mg/kg body weight for rats by oral or dermal application.

Cytotoxicity and Genotoxicity Testing via MTT and Comet Assays
The results of a MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) showed IC50 values at 3.00 mg/mL, 2.90 mg/mL and 3.60 mg/mL in Mc parts and leaves, and in the mixture of Mc and Ma leaves. Following depth toxicity testing on the DNA level by comet assay, the samples showed significant DNA damage (Table 5, Figure 8). With the LD50 value calculated using IC50 values, the toxicity was revealed as Class II, i.e., slightly hazardous, corresponding to over 2000 mg/kg body weight for rats by oral or dermal application.

Discussion
When analyzing substances using the GC-MS method, α-EG was not found in Ma. Therefore, the quantity and concentration of this substance were not further studied.
The first finding regarding α-EG in Mc (grown in Thailand) leaves ufp, rfp, rawfs, and seeds provided very important information following the discovery of pinoresinol [9]. The research results can be applied for the creation of natural products including traditional forms, modified forms like supplements, nutraceuticals, functional foods, and cosmetics. α-EG acts to stimulate collagen production; collagen is an extracellular matrix protein, i.e., the major component of the extracellular matrix of the dermis. It accounts for 90% of the fiber component. Thus, change in dermal collagen content greatly affects dermal homeostasis [15]. Collagen produced by the body is synthesized mainly by fibroblasts, i.e., the main structural protein found in skin, tendon and bone. Additionally, collagen has been found to have many pharmaceutical, medicinal, nutritional and cosmetic applications [16,17]. Pinoresinol, which is present in Mc fruit, has several functions. It was tested and shown to have tyrosinase inhibitory activity in melanogenesis, an inhibitory effect on matrix metalloproteinase-1 (MMP-1) secretion (MMP-1 is a major collagenolytic enzyme which is responsible for collagen damage) and an inhibitory effect on wrinkle formation by HLE creation, which degrades collagen I and elastic fiber in human skin. Thus, HLE inhibition may be useful for the prevention of wrinkle formation [9].
On this basis, human normal fibroblast was selected in this study for experiments to show the expression of angiogenesis and fibroblast proliferation genes FGF1 and FGF7, type I collagen genes COL1A1 and COL1A2, and type III collagen gene COL3A1. Fibroblasts produce substances that constitute the basic structure of skin; a decrease in their proliferation and activity causes the deterioration of the skin structure [18]. The activities of inhibiting α-MSH-stimulated melanogenesis and an inhibitory effect on MMP-1 secretion (HLE activates MMP-1) mean that it is expected that these activities and the two substances, α-EG and pinoresinol, will be useful for the prevention of photoaging and wrinkle formation, as well as collagen creation and skin whitening. However, the amount and concentrations in each studied Mc part should be clarified. From the studied parts, leaves ufp, rfp, rawfs and seeds, and differences (concentrations in mg/mL and amounts in mg/g sample) contained 0.30/1.51, 0.11/1.75, 0.15/2.48, 0.28/3.77, and 0.07/0.94 α-EG and 0.09/0.20, 0.13/0.34, 0.14/0.38, 0.11/0.28 and 0.08/0.20 pinoresinol. These values do not reveal, however, whether the concentrations and quantities are sufficient to stimulate and inhibit the effects of the aforementioned genes. Therefore, mRNA gene expression was measured by qRT-PCR from the total RNA extracted from extracts treated with NHDF. The result showed strong effects compared to the control, with expression stimulation levels of up to 3480-4160%, 2930% and 2870% from ufp and seeds for type I and III collagen COL1A1, COL1A2, COL3A1, and 2700-4870% from seeds and ufp for angiogenesis and fibroblast proliferation genes FGF1 and FGF7 ( Table 4). The inhibitory expression showed low levels but high efficacy in rawfs at 0.10% for the TYR gene, seeds at 1.29% α-MSH, and rawfs and seeds at 0.20-0.30% HLE. Additionally, leaves also showed low percentages but high efficacy for inhibition of TRY, i.e., 0.20%, and of NRF2, i.e., 3.80%. The NRF2 gene regulates oxidative stress and toxicity [19]. In line with expectations, if the protein substrate from Ma leaves was added, gene expression in the stimulatory (COL1A1, COL1A2, COL3A1, FGF1 and FGF7) and inhibitory groups showed various higher or lower expressed values for different genes. Similarly, with and without Ma leaves, TYR and HLE showed higher or lower expression. This indicated that leaves and ufp, as well as rawfs, have the potential to stimulate and inhibit the expression of all of the studied genes. However, the other studied Mc parts seemed to have advantages, depending on the objective, gene type and plant part used. Given this, the applications of Mc parts including leaves, rawfs and these two parts combined with Ma leaves at a ratio 6:4 passed toxicity tests, both in cell and DNA by MTT and comet assays. The overall results showed no toxicity in rawfs.
As such, the raw fruit with seeds can be safely applied in natural products, i.e., both foods and cosmetics. However, IC 50 values indicated that these three parts should be used within limits, because of the observed Class II toxicity [20].

Plant Materials and Extract Preparation
The plant species were collected from a field in Muangkhonkaen District, Khon Kaen Province, Thailand and were identified by a proficient botanist, prof. Arunrat Chaveerach, Ph.D. Permission was not required to collect the Morus alba and M. citrifolia materials used in this study, because they are common species which grow widely in gardens, fields and forests for domestic consumption, i.e., eating the leaves and fruits by drinking and cooking. Specimen numbers will be granted 2 years after the deposition. Mature leaves of Morus alba and mature leaves and fruits of M. citrifolia were collected, washed and air-dried, and then ground into a powder. The powder was combined with hexane or ethanol, separately at a rate of 1:5, and soaked for 72 h. Each solution was filtered through a Whatman no.1 filter paper. The filtrates were kept at −20 • C until used in experiments, including phytochemical component analysis by GC-MS and for GC comparisons.

Proximate Analysis of Nutritional Content
The air-dried Morus alba leaf samples were ground to a fine powder. The powder was employed for proximate analyses at the Central Laboratory (Thailand) Co., Ltd., Muangkhonkaen, Thailand following the standard protocols of the Association of Analytical Chemists (AOAC) [21].

Gas Chromatography-Mass Spectrometry (GC-MS)
Phytochemical analyses of M. alba and M. citrifolia were performed using an Agilent Technologies GC 6890 N/5973 (Santa Clara, CA, USA) inert mass spectrometer fused with a capillary column (30.0 m × 250 µm × 0.25 µm). Helium was used as the carrier at a constant flow rate of 1 mL/min. The injection and mass-transferred line temperature was set at 280 • C. The oven temperature was programmed to increase from 70 • C to 120 • C at 3 • C/min. It was then maintained isothermally for 2 min, and subsequently raised to 270 • C at 5 • C/min. A 1 µL aliquot of the extract was injected in split mode. The relative percentages of the extract constituents were expressed as percentages using peak area normalization. Components were identified by comparing the obtained mass spectra with reference compounds in the Wiley 7N.1 library.2.4.

Ethyl-α-d-Glucoside (α-EG) and 3,3 -Bisdemethylpinoresinol (Pinoresinol) Detection by GC Compared to the Standard
Ten-gram samples of M. citrifolia including leaves, ufp, rfp, rawfs and seeds were soaked in 50 mL ethanol (at a ratio of 1 g:5 mL), and the solution was kept in a dark room for 3 days. The mixture was filtered through Whatman no.1 filter paper (125 mm diameter) and fractioned by adding a mixture of ethyl acetate and dichloromethane (at a ratio of 1:2). The separated clear mixture was precipitated by adding a solution mixture of petroleum ether:diethyl ether (ratio 2:1) for 400 µL per 1 µL sample, until the solution precipitated. The clear solution was filtered by syringe filter, with pore sizes of 0.45 µm, and each final extract was used for α-EG and pinoresinol detection.
The chromatographic conditions for pinoresinol detection were as follows: the GC was an Agilent Technologies GC7890B (Santa Clara, CA, USA) equipped with a flame ionization detector (FID) and HP-5 capillary column (30.0 m × 320 µm i.d. × 0.25 µm film thickness) (Agilent, Santa Clara, CA, USA). Helium was used as the carrier gas at a flow rate of 1 mL/min. The injector and detector temperatures were 260 • C and 320 • C, respectively. The oven temperature was programed at an initial temperature of 120 • C and a ramp rate of 10 • C/min for a final temperature at 300 • C, which was maintained for 10 min. Then, 1 µL of each sample was injected into a column. and positive controls, respectively. After this, the plates were centrifuged at 1500 rpm for 10 min and the medium was removed. MTT (Sigma, St. Louis, MO, USA) was added to a final concentration of 0.5 mg/mL and the plates were wrapped with aluminum foil and incubated for 4 h at 37 • C. Formazan crystals were solubilized by adding 100 µL DMSO to each well. The plates were kept in the dark for 2-4 h. The absorbance was read at 570 nm with a microtiter plate spectrophotometer (Multifunction microplate reader; Molecular devices, San Jose, CA, USA). Wells containing medium and MTT without cells were used as blanks. Each concentration treatment was performed in triplicate. All values were expressed as the mean ± SD. The cellular reduction of MTT formed violet crystal formazan through mitochondrial succinate dehydrogenase activity of the viable cells. The violet crystal formazan was quantified following the methods of Freshney et al. [29]. Percentages of cell viability were calculated using the equation cell viability (%) = average viable treated cells/average viable negative control cells × 100 to reveal the cytotoxicity of the plant extracts. Doses inducing 50% inhibition of cell viability (IC 50 value) were determined by plotting a graph of the extract concentration against the cell viability. The IC 50 value was used for the LD 50 calculation [30] to infer hazardous levels, according to the World Health Organization guidelines [20]. 4.
Comet assay. The concentration at IC 50 value or the maximum-treated concentration in the case of no IC 50 value was used in the comet assay to assess the genotoxicity of plant extracts, following the method described by Singh et al. [31]. Briefly, 500 µL cells in media were added with 50 µL extracts in a 1.5 mL microtube and incubated for 24 h in a humidified CO 2 incubator at 37 • C and 5% CO 2 . Then, the DNA was checked by electrophoresis. The electrophoresis buffer consisted of 0.3 M NaOH and 1 mM EDTA (pH = 10). Power was supplied at a constant of 3.4 v/cm, with an adjustment to 300 mA, for 25 min. To quantify the level of DNA damage, the extent of DNA migration was defined using the Olive Tail Moment (OTM), which is the relative amount of DNA in the tail of the comet multiplied by the median migration distance. The comets were observed at 200× magnifications and images were obtained using an image analysis system (Zeiss, Jena, Germany) attached to a fluorescence microscope (Nikon, Minato-ku, Japan), equipped with a 560 nm excitation filter, 590 nm barrier filter, and a CCD video camera PCO (Kelheim, Germany). At least 150 cells (50 cells for each of the triplicate slides) were examined for each experiment. The CASP software version 1.2.3 (CASPlab, Wroclaw, Poland) was used to analyze the OTM. The negative and positive controls were untreated cells and UV-treated cells, respectively. All experiments were performed in triplicate. The cultures were scored for the experiment. All values were expressed as the median ± S.D. The nonparametric Mann-Whitney U test was used for statistical analyses of the comet assay results; statistical significance was set at p < 0.05.

Conclusions
Mc rawfs contain a new substance, identified as α-EG, along with pinoresinol, which was previously found in sufficient concentrations and quantities to stimulate collagen growth and promote whitening and minimize dull skin, photoaging and wrinkle formation. These two substances are useful for natural product creation in natural and modified forms. They were found to be without toxicity at both cellular and DNA levels. Additionally, the leaves of the plant can be consumed but in limited amounts, i.e., not more than 2000 mg/kg body weight.