INTRODUCTION:

Plant tissue culture technique is an important tool used in both basic and applied sciences. Tissue culture techniques that ensure genetic stability are mostly useful for in vitro mutation induction and mutant plant regeneration¹. The chemical mutagen, ethyl methanesulfonate (EMS) has been widely used in mutagenesis of many plant species². Mutagenic treatment to the tissues under in vitro condition enhances the frequency rate of spontaneous mutations with the high spectrum³. Plant cell culture is an effective system to study the biological significance of bioactive metabolites under in vitro conditions.

The present work deals with the studies on quantitative changes in bioactive constituents due to EMS-induced mutagenesis in cultured cells of E. scaber.

Elephantopus saber Linn is a member of Asteraceae family and is an erect herb 15-38cm in height. Triterpenoids, sterols and caffeic acids were reported by Liang et al.⁴ Wang et al.⁵. Phytoconstituents like de-oxyelephantopin and iso-deoxyelephantopin in this plant are tumor inhibitors^6,7,8. The plant part (root, leaf, and bark) has medicinal value, because of the presence of epifriedelinol, lupeol, and Stigmasterol^9,10. Previous phytochemical investigations on Elephantopus have also resulted in the isolation of flavonoids and Diosgenin^11,12. Triterpene among these compounds, sesquiterpene lactone is a chemotaxonomic marker for the genus Elephantopus¹³. It is reported that this species is also used for antitumor, hepatoprotective, wound healing treatments. E. scaber is reported to contain a large number of bioactive compounds such as lipids, phytochemicals, pharmaceutics, and pigments. For example, ethyl hexadecanoate, ethyl-9, 12-octadecadienoate, ethyl-(Z)-9-octadecenoate, ethyl octadecanoate, lupeol, stigmasterol, stigmasterol glucoside, de-oxyelephantopin) and two new germacranolide sesquiterpene lactones named 17, 19-dihydrodeoxyelephantopin (2) and iso-17, 19-dihydrodeoxyelephantopin¹⁴. This plant is reported for the tumor suppression effect of deoxyelephantopin and it is investigated to show effect on mammary adenocarcinoma, the obtained results also provide evidence of the anti-tumor activity of the compound. Plants produce a significant amount of antioxidants to prevent the oxidative stress caused by reactive oxygen species and are thought to be safer and healthier than synthetic antioxidants¹⁵. The impact of various growth regulators, growth media and induction of callus biomass was studied in Elephantopus scaber L.¹⁶.

There are no previously reported studies of EMS induced mutations in E. scaber. The goal of the present investigation was to induce mutations in suspension cell culture for the enhancement of some secondary metabolites and to evaluate its effect on treated suspension culture using the HPLC and HPTLC analytical techniques and its antioxidant potentials.

MATERIAL AND METHODS:

Chemical reagents:

The standard Elephantopin was procured from Hi-hang Industry Co, LTD. Jinan City, China, Ethyl methanesulfonate procured from Himedia, 1,1-Diphenyl-2-picrylhydrazyl (DPPH), Quercetin, Rutin and Stigmasterol from Sigma Aldrich, L-Ascorbic acid, 2,4-D, BAP, Agar-agar (Himedia), Methanol, n-Hexane, Acetic acid, Formic acid, Ethyl acetate (SD-Fine), HPLC grade solvents and water was used in the present analysis.

Plant materials, Callus induction, Cell cultures and Mutagen preparation:

The leaves of E. scaber were used as explants for callus induction. In vitro grown 8 weeks older leaf was cut into small pieces and inoculated aseptically in 50mL MS basal medium supplemented with different combinations of 2,4-D and BAP (1:1 ratio; Fig. 1). The explants produce yellowish green calli. After 3 successive trials of callus induction, 2,4-D and BAP fortified medium (1:1 ratio) was the media combination selected for the further work as a best suitable and favorable. All experiments in this study were performed in suspension culture of E. scaber cells in a liquid medium of the same composition excluding agar. The cell suspension culture was maintained in 250mL Erlenmeyer flasks on a gyratory shaker operated at 110–120rpm, at 25°C in the dark for 30 days. Each of the flasks was filled with 50 mL medium and inoculated with 3.0g of fresh callus cells from 18–21day old culture. Before the EMS treatment the sub culturing of callus was done for the next experiment.

Preparation of chemical mutagen doses:

The stock solution of ethyl methyl sulfonate (EMS) was prepared to dissolve the liquid EMS solution (1mL) in 100mL distilled water. From this stock, the appropriate dilutions of 0.1% 0.2% and 0.5% EMS (v/v) was prepared in aqueous media and used for the treatment.

Treatment administration:

The mutagenic solution was added to the Dippy’s Jar containing the E. scaber cell suspension culture. The cell suspension culture was subjected to the EMS treatment for 1, 2 and 3 h duration. After each treatment each sample was washed four times with HPLC grade water using whatmann filter paper no.1 to remove the traces of EMS in treated suspension cultures. Then it was kept in oven for drying. The experiment was conducted in triplicate with a control set for every treatment duration.

Measurement of cell growth:

The cell viability was determined using Guava cell count assay with Guava Easy CD4 System (Guava Technology). The 10µl cell suspension culture was taken in Eppendorf tube and 190µl of via count solution was added into the tube (viability counting solution) and the mixture was then vortex for 1 min and incubated for 5 min at the room temperature. The results were obtained in the form of viable cell count, total cell count (both in the number of cells/mL) and percent viability. The cell growth phase was determined using the colorimetric method. The density of cell suspension was determined daily by observing optical density at 430nm for every day up to 30 days duration. The spectrum of the samples was recorded using the UV-Spectrophotometer (Shimadzu-1800) for cell growth index. The MS-medium without agar was used as a blank reference.

Extraction of Sesquiterpene, Flavonoids and Stigmasterol samples for analysis:

The cell suspension samples were filtered and washed four times with the HPLC water after mutagen treatment. The fresh weight of the collected cells was noted and then it was dried in the oven at 50° to obtained constant dry weight. The fine powder was prepared using mortar and pestle and 150mg of powdered sample was extracted in 2mL of methanol by sonication using Sonics Vibra Cell (VCX 130). The extracts were used for the determination of bioactive compounds. The extracted solutions were analyzed for the content of Deoxyelephantopin, Quercetin, Rutin, and Stigmasterol. The cell suspension samples (EMS treated) were subjected for the quantitative analysis of secondary metabolites using HPTLC technique.

HPTLC assay:

High-Performance Thin-Layer Chromatography (HPTLC) is emerging as a versatile and cost-effective technology that is uniquely suitable to assess the identity and quality of plant secondary metabolites. Plant drug quantification using HPTLC is the most ideal analytical method.

Preparation of standard stock solutions for HPTLC:

Standard stock solutions for Deoxyelephantopin, Quercetin, Rutin, and Stigmasterol was prepared by dissolving 5mg of powder in 5mL of Methanol and sonicated for 10minutes. From this solution, 10µl of each of these sample solutions was applied using sample applicator. The control and treated samples of methanolic extract of E. scaber L. with 10µl quantity were used for the HPTLC chemo-profiling. Only HPLC grade solvents were used for the present analysis.

Chromatographic conditions for HPTLC:

Chromatography was performed on pre-activated (at 1100^°C) silica gel 60 F₂₅₄ HPTLC plates. Samples and standard compound each in 10µl quantities were applied to the layer as 6.0mm wide bands, positioned at 8.0mm from the bottom of the plate, using an automated CAMAG LINOMAT-5, TLC applicator instrument with nitrogen flow providing the delivery by 100 micro-litres Hamilton syringe.

Detection of compounds by HPTLC:

Chromatography was developed at room temperature (24±10°C) in glass twin-though chambers (10mm × 10 mm, with metal lids), previously saturated with mobile phase vapors for 30min. The development distance was 86mm. The ascending mode was used for the development of thin layer chromatography. The plate was placed in the photo-documentation chamber (CAMAG Reprostar-3) and the images were captured at 254nm and 366nm. The densitogram was obtained under absorbance reflection mode using CAMAG-TLC Scanner-3 equipment. The compounds were investigated according to their R_F values with the corresponding spot of standards. Calculations for percentage were performed considering standard and sample R_F, AUC and dilution factor. For validation of the method, the calibration curve was obtained by plotting the peak area against the concentration of Elephantopin, Quercetin, Rutin, and Stigmasterol. The spectra obtained from the samples were correlated to the standard compound used.

Validation method for studied compounds:

The validation methods used for the four standard compounds (Elephantopin, Quercetin, Rutin, and Stigmasterol) are initially standardized with the help of calibration parameters as specified below.

Calibration curve and Quantification of Elephantopin, Flavonoids and Stigmasterol using HPTLC:

The results of HPTLC quantification of Elephantopin compound are presented in Figure 2. The standard solution (10µL) of studied phytoconstituent was used in different concentrations in triplicate mode and these concentrations were applied on TLC plate using CAMAG Linomat-5 automatic sample spotter. On the development of TLC plate using specific mobile phase, the plate was air dried and scanned for spectral analysis. The spectrum was recorded at the start point, middle point and end point to check the purity of the bands. The reading was taken at 366 nm using CAMAG TLC Scanner-3 equipment and winCATS software. The calibration curve for the respective standard was plotted using peak area vs. concentration of the applied standard solution. The R_Fvalue obtained was correlated to the spot of standard compound. The method was validated using the calibration curve obtained for a respective standard. The calculation was performed considering the standard R_Fvalue and area against concentration (AUC) for the respective standard.

Fig.1: Callus induction from leaf explant of E. scaber using 2,4-D:BAP hormonal combination.

The concentrations for Elephantopin standard solution were 50, 100, 200, 300, 400, 500, 600 and 700 ng spot^-1. The n-hexane: ethyl acetate: [7.2:2.8 v/v] was used as a mobile phase. The HPTLC method was validated in terms of specificity, accuracy, sensitivity (LOD and LOQ).

FREE RADICAL SCAVENGING ACTIVITY:

DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging assay)

The DPPH is a stable - free radical and is widely used to assess the radical scavenging activity of the antioxidant component. DPPH scavenging activity was measured using the method described by Brand-Williams et al., (1995)¹⁷ with slight modifications. 1,1-diphenyl-2-picrylhydrazyl (DPPH) methanolic solution (0.002%) was used. DPPH concentration is reduced by the existence of an antioxidant at 515nm and the absorption gradually disappears with the time. This method is based on the reduction of DPPH in methanol solution in the presence of a hydrogen donating antioxidant due to the formation of the non-radical form of DPPH. Briefly, 0.002% solution of DPPH in methanol was prepared and 180μl of this solution was added to 20μl of the solution of all control and treated stock samples at different concentrations. The aliquot of stock solutions was prepared (50, 100, 150, 200 and 250μg/mL). The diluted working solutions of the test samples were prepared in methanol (sonicated for 15min.) The mixtures were kept at room temperature for 30 minutes. The analysis was performed using a Synergy H1-Multi-plate reader (Biotek) instrument. Ascorbic acid was used as a positive control (standard) and methanol as a blank source. The experiments were performed in triplicate and percentage scavenging activity was calculated using the following equation - Absorbance _control - Absorbance _sample / Absorbance _control x 100. All experiments were repeated in triplicate and means with standard deviation were calculated. The radical scavenging activity result (radical scavenging % and IC₅₀ value) summarized in the results and discussion. For evaluation of data, standard statistical methods were used.

Statistical Analysis:

The observation data obtained on different parameters as studied in the present work was subjected for statistical analysis using the Microsoft Excel and Graph pad prism-5 software. The analysis was performed in triplicate mode and the data represented as the mean ± standard deviation.

RESULTS AND DISCUSSION:

In the present work, in vitro chemical mutagenesis was carried out to understand the impact of this technique on the quantitative status of four bioactive compounds present in E. scaber cell suspension culture. Research information on this particular aspect has not yet surfaced out in the selected experimental system, E. scaber. This experimental system is widely available in the forest. This plant survives in all type of environmental conditions and is disease resistant. It is medicinally useful and its medicinal potential needs to be explored using various techniques and methodologies. In context to this, the present investigation was conducted in E. scaber cell suspension culture by administration of in vitro mutagenic treatment using a potent chemical mutagen, Ethyl methanesulfonate (EMS). The results were assessed using HPTLC technique for Deoxyelephantopin, flavonoids (Quercetin and Rutin) and Stigmasterol compound.

In vitro mutagenesis is commonly used for the quantitative improvement of plant bioactive metabolites. Plant tissue culture offers a wide choice of plant material (axillary buds, leaf, shoot, root, organs, tissues, cells or any other part as an explants) for the treatment and the induction of mutation. Ethyl methanesulfonate is an alkylating agent and capable to induce point mutations in the DNA molecule. Chemical mutagenesis under in vitro culture increases the mutation efficiency. Our objective was to explore the possibility of enhancement in the synthesis of targeted metabolites using EMS-induced mutagenesis in cell suspension culture of E. scaber.

Cell viability:

The 25 days old cell suspension culture samples were evaluated for cell viability using Guava cell counter. The analysis was performed in the triplicate set. The results were obtained in the form of viable cells count/mL, total cells/mL count and the percent viability. All the treated samples of Ethyl methanesulfonate treatment show the decline in the cell count at higher doses and treatment durations.

Cell viability assay for Ethyl methanesulfonate (EMS) treated samples:

The cell viability was drastically affected in EMS-induced mutation treatment. The higher doses and longer duration treatment appeared to be prominently responsible for retardation of cell viability count. The lowest concentration of EMS (0.1%) revealed 72.16% cell viability count against 96.10% in control and it was highest within and between all the studied concentrations and treatment durations. It is evident that 0.1% EMS treatment for 2 and 3 h treatment duration exhibits 54.60% and 40.30% viability count, respectively. The decline in the percentage cell viability was more prominent at higher doses and treatments durations. The cell viability was retarded up to 39.20% in EMS 1.0% concentration for 3 h treatment and it was least amongst all the studied treatments. It was evident that average cell viability (92.60%) was marginally more than EMS treated samples and it was remarkably declined (54.03%).

The results of HPTLC analysis is presented in Figure 2 for Elephantopin. The control sample exhibit low Elephantopin content (0.00795±0.05µg/mL) (Fig. 2c) compared to EMS 0.1% concentration for 1 h treatment content (0.02998±0.04µg/mL) (Fig. 2d). The effect of the treatment on the quantitative status of the compound was not dose and time-dependent. EMS treatment with 0.2% revealed slight decrease in the content of Elephantopin (0.02017±0.08µg/mL) (Fig. 2e). The higher concentration of 0.5% EMS, the estimate was very low (0.01593±0.02µg/mL) (Fig. 2f) in the same treatment duration. In the EMS treatment of 0.1% concentration for 2h, it was observed that the Elephantopin content declined (0.01559±0.05µg/mL) compared to control (0.00818±0.07µg/mL) and other doses.

Fig.2a. HPTLC densitogram for Elephantopin standard

Fig.2b. Calibration curve for standard Elephantopin

Fig.2c. HPTLC densitogram for Elephantopin in control cell suspension culture of E. scaber L.

Fig.2d. HPTLC densitogram for Elephantopin in EMS treated 0.1% cell suspension culture of E. scaber L.

Fig.2e. HPTLC densitogram for Elephantopin in EMS treated 0.2% cell suspension culture of E. scaber L.

Fig.2f. HPTLC densitogram for Elephantopin in EMS treated 0.5% cell suspension culture of E. scaber L.

The result obtained from HPTLC assay indicates that the higher EMS concentration and treatment duration has a negative impact on the production or synthesis of a target compound. The higher dose with longer treatment duration might have induced toxicity or interferes in the metabolic pathway by retarding the functional potentiality of metabolic factors or enzymes involved in the synthesis pathway of the compound. This is very possible since EMS is known to cause variations in the expression of genes by inducing genetic changes.

In vitro culture mediated mutation breeding techniques used in vegetatively propagated crops. In vitro mutagenesis is mostly used to exploit genetic variability in economically important plants using other chemical mutagens. Induced mutagenesis is used to generate a wide range of plants with improved resistance to abiotic and biotic stresses as reported by Jain³. The induced in vitro mutation technique is a powerful tool using physical and chemical mutagens on horticultural crops due to their ease of application and high mutational frequency¹⁸. Ali et al.¹⁹ reported the effect of sodium azide on embryonic calli and found that 4.0 mg/L -NaN3 induces the largest number of genetic variants, but the regenerated plant did not survive. One other researcher²⁰ has tried EMS chemical mutagen in sugarcane calli. The average LD-50 for a 2.5 – 4 h exposure was recorded for 32.2 mM EMS when expressed in terms of callus production and plantlet regeneration. Ghani et al.²¹ reported that chemically-mutated plants showed the highest acclimatization (72.89 %) when exposed to the lowest dose of EMS (0.1% for 10 min.) and the lowest acclimatization percentage (43%) for same concentration but for 20 min. treatment duration in Gerbera jamesonii Hook. It is evident that higher treatment duration affects the plants and survival percentage drastically.

Fig.3a. HPTLC densitogram for Quercetin compound

Fig.3b. Calibration curve for standard Quercetin

Fig.3c. HPTLC densitogram for Rutin compound

Fig.3d. Calibration curve for standard Rutin

Fig.3e. HPTLC densitogram for Quercetin and Rutin in control cell suspension culture of E. scaber L.

Fig.3 f. HPTLC densitogram for Quercetin and Rutin in EMS treated 0.1% cell suspension culture of E. scaber L.

Fig.3 g. HPTLC densitogram for Quercetin in EMS treated 0.2% cell suspension culture of E. scaber L.

Fig.3 h. HPTLC densitogram for Quercetin in EMS treated 0.5% cell suspension culture of E. scaber L.

The results of HPTLC analysis is presented in Figure 3 for Quercetin and Rutin. The cell suspension culture of control sample for Quercetin content reveals lower values (0.06991±0.05µg/mL) (Fig. 3e) compared to treated with EMS 0.1% concentration for 1 h treatment duration (0.08151±0.04µg/mL) (Fig. 3f) and other doses. Whereas, the treatment of 2 h EMS for 0.1% concentration exhibits slightly high Quercetin content (0.07195±0.05µg/mL) when compared to control (0.07019±0.07µg/mL). But, 0.2% and 0.5% EMS treatment for same treatment duration (2 h) indicates decline in the content of compound. From the results obtained, it is evident that 0.1% EMS treatment for 1 h duration revealed optimized impact of EMS treatment with respect to the enhancement of Quercetin compound (Fig. 3g, 3h). The increase in the treatment duration and doses showed unremarkable quantitative variations in the content.

The Quercetin is known to generate the mutagenic quinone type^22,23,24. This compound is reported to have mutagenic properties. Quercetin is composed of flavonoid ring structure with a free hydroxyl group at 3-position, a double bond between 2, 3-position and a keto-group at 4-positon allowing the proton of the 3-hydroxyl group to tautomerize to a 3-keto moiety. Hamid et al.²⁵ reported that in-vitro mutagenesis is the best technique to elicit the synthesis of secondary metabolites. Juan et al.²⁶ have reported high amounts of esters using gradient reverse phase high-performance liquid chromatography (HPLC) method in EMS treated lines of Haematococcus pluvialis. EMS was used for inducing mutations in banana by treating shoot tips and then regenerating adventitious buds ²⁷.

The other flavonoid compound, Rutin was treated by EMS mutagen and the quantification was done using HPTLC assay. Rutin content was quantitatively assessed in control and EMS treated samples. The level of the release of Rutin compound was lower than Quercetin compound in the studied plant, E. scaber. Rutin content could not be traced out in the samples of 1 and 2 h EMS treatments but it was observed in control. However, the optimized Rutin content was estimated in the cell suspension culture treated with 0.1%, 0.2% and 0.5% EMS treatment for 3 h duration. The Rutin content in 0.1% EMS for 3 h treatments was 0.01353 ± 0.02 µg/mL (Fig. 3e, 3f) and it was insignificantly high compared to 2 and 3 h EMS treatments and respective control.

Induced mutations through tissue cultures are desirable for developing new cultivars to be used in plant breeding, and floriculture industry. The low profile occurrence of Rutin in treated samples attracts our attention. It is reported that Rutin and rosmarinic acid encircle the nucleotides and fill-up the EMS binding space around the nucleus thereby acting as impermeable barriers for EMS molecule to interact with DNA and to trigger alkylation²⁸. It is possible that the mutagen might have interfered the synthesis pathway undesirably and as a consequence, the compound reflect low or untraceable quantum of its occurrence in some EMS concentrations and treatment durations.

The results of HPTLC analysis is presented in Figure 4 for Stigmasterol. The EMS treatment of 0.1%, 0.2% and 0.5% concentrations for 1h duration does not reveal Stigmasterol content up to quantifying range but it was traceable in control. In the control sample Stigmasterol indicated slightly lower content (0.06820 ± 0.05 µg/mL) (Fig. 4c) compared to the EMS treatment for 2 h duration with 0.1% concentration (0.07713 ± 0.02 µg/mL) (Fig. 4d). The 0.2% (Fig. 4e) and 0.5% (Fig. 4f) EMS treatment for the same duration indicated the appearance of the compound but it was less to the quantity obtained in 0.1% EMS treatment.

Fig.4a. HPTLC densitogram for Stigmasterol standard

Fig.4b. Calibration curve for standard Stigmasterol

Fig.4c. HPTLC densitogram for Stigmasterol in control cell suspension culture of E. scaber L.

Fig.4d. HPTLC densitogram for Stigmasterol in EMS treated 0.1% cell suspension culture of E. scaber

Fig.4e. HPTLC densitogram for Stigmasterol in EMS treated 0.2% cell suspension culture of E. scaber L.

Fig.4f. HPTLC densitogram for Stigmasterol in EMS treated 0.5% cell suspension culture of E. scaber L.

It is evident that for 0.1% EMS for 2 h treatment showed the positive but unremarkable impact on the quantitative release of Stigmasterol compared to control and other studied doses.

In vitro EMS mutagenesis in cell suspension culture of oryza sativa is earlier reported by Chen, et al.²⁹ these coworkers have observed that EMS treated cultured cells regenerated self-pollinated plant with high mutation rates and stated that EMS treatment of 0.4% for 18 to 22 h is most favorable to induce mutations. Serrat et al.³⁰ worked on a rice mutant and reported that 0.2% EMS for 2 h is the most effective treatment to generate a mutant population with high mutation frequency. An increase in the concentration of EMS results in reducing the mutation rate and causes proportionally greater seedling damage accompanied by a decrease in the rate of survival. However, it is known that the lower or moderate mutagenic dose with longer treatment durations enhances mutation frequency with minimum chromosomal damage and survival rate. The estimation of flavonoids by HPTLC method was already reported in Syzygium cumini³¹. Muslek et al.³² reported HPTLC finger print analysis and in vitro cytotoxicity study in Erythrina stricta. Recently we reported HPTLC quantification and estimation of Rutin and Kaempferol using aerial parts of Ixora javanica and Ixora barbata³³.

The optimized impact of EMS treatment for Elephantopin, and Quercetin was observed in 0.1% concentration for 1h treatment and for Rutin 0.1% for 3 h duration in all treated samples compared to control and other doses. Stigmasterol compound was observed in 0.1% EMS concentration for 2h treatment. It also revealed that the HPTLC technique showed the quantity, occurrence, and release of Elephantopin, Quercetin, Rutin and Stigmasterol content.

Free radical scavenging activity in control and Chemical mutagen (EMS) treated cell suspension culture samples of E. scaber:

The result of DPPH free radical scavenging activity is presented in Figure 5 and Table 1. The 1 h EMS treated sample for 0.5% concentration exhibits high percentage of free radical scavenging (58.18±0.02µg/mL) with low IC₅₀ value (0.40±0.02μg/mL) for respective treatment duration compared to high IC₅₀ value of 0.46±0.05 μg/mL in control sample. The decrease in the scavenging percentage was observed for all other treated doses. For 2 h treatment with 0.5% EMS, the radical scavenging percentage was (65.73%) with IC₅₀ value of 0.33±0.04μg/mL for respective treatment duration compared to control (IC₅₀ 0.40±0.08μg/mL). However, 3 h treatment 0.5% EMS showed 71.56% scavenging activity with IC₅₀ value of 0.27±0.05μg/mL for respective duration against control with IC₅₀ value of 0.31±0.03μg/mL. It is evident that EMS treated samples showed potential scavenging activity compared to control samples IC₅₀ value. Ascorbic acid used as reference standard in the range of 20 to 140μg/mL and it indicates 52.61% scavenging activity with the IC₅₀ value, 42.62±0.05μg/mL.

Fig.5: DPPH radical scavenging ability of EMS treated cell suspension culture [in %] of E. scaber.

Ganie et al.³⁴ reported DPPH antioxidant radical scavenging activity of methanolic extract in Arnebia benthamii. These workers have recorded free radical (DPPH) scavenging activity in terms of percent inhibition and suggested that the methanolic extract of A. benthamii exhibits the radical scavenging activity for the plant extract concentrations in the range of 100-700 μg/mL.. Gulcin et al.³⁵ compared antioxidant activity of tannic acid against other antioxidant compounds like Butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT), α-tocopherol and Trolox and opinioned that the tannic acid is the most effective natural antioxidant. From the results obtained it is revealed that chemical mutagen treated cell suspension culture samples of E. scaber indicated high potential of free radical scavenging activity.

It is reported that, in DPPH assay, the non-radical form of DPPH is formed due to its reduction by donating hydrogen atom. DPPH contains free radicals and has a purple color. However, when DPPH reacts with antioxidant, it undergoes reduction mechanism due to hydrogen radical from the antioxidant molecule and then it appears yellow in color. This change in the color indicates the decaying of DPPH due to the scavenging of free radicals in the cells^36,37. The change in the color is evaluated by spectrophotometric analysis at the wavelength 515nm. The antioxidant activity of silver nanoparticles synthesized from the leaf extract of E. scaber was already reported by us³⁸. In vitro antioxidant activity was done in medicinal plant Schleichera oleosa³⁹. One researcher has reported the in vitro antioxidant and anti-inflammatory activity of the aqueous and ethanol leaf extracts of Clerodendrum phlomidi⁴⁰. HPTLC and antioxidant analysis was done in Ipomoea obscura (L.) using ethanolic extract⁴¹. Number of researchers reported antioxidant activity in plant extracts like, Murraya koiengii and Ficus palmate⁴², Cannabis sativa, Triticum aestivum and Dascus carota L.⁴³, Ocimum sanctum and Terminalia arjuna⁴⁴, Cucurbita maxima, Artemisia vulgaris and Clitoria ternatea⁴⁵.

The role of chemical mutagen EMS in genetic alterations is well documented. The conspicuous variation in antioxidant properties of treated samples may, therefore, be correlated to their interaction at molecular and enzymatic level. As a consequence, the scavenging potential of DPPH added in sample extract probably elevated thereby revealing enhanced scavenging activities.

CONCLUSION:

The concept of in vitro enhancement of important bioactive constituents by in vitro mutagenesis is preferably aimed in the present work. E. scaber responded very well for callus induction in 2,4-D : BAP hormonal combination. The work was focused on four bioactive metabolites i.e. Elephantopin (sesquiterpene compound), Quercetin and Rutin (flavonoid compounds) and Stigmasterol (a steroidal compound) in E. scaber for in vitro mutation studies. The most undesirable impact was evident in EMS treated samples with retardation in percent cell viability up to 39.86% against control (i.e., 93.60%). EMS induced in vitro mutagenesis in cell suspension culture of E. scaber provides an insight to understand the possible role of EMS in the biosynthesis pathway of studied metabolites. The higher treatment durations and concentrations have negative impact on the synthesis of targeted compounds as revealed in HPTLC assay for Deoxyelephantopin. The results obtained revealed that the concentrations and treatment durations of EMS was effective in enhancement of the content of studied metabolites. The EMS 1 h treatment duration was suitable for Deoxyelephantopin and Quercetin enhancement while for Rutin it was 3 h and for Stigmasterol compound it was 2 h the content of all studied constituents was elevated in EMS concentration of 0.1% compared to 0.2% and 0.5%. The response of compounds to the EMS induced in vitro mutagenesis was in the order of Elephantopin < Rutin < Quercetin < Stigmasterol. The extracts of E. scaber have a good radical scavenging activity. Exploring the importance of active constituents in medicinal plants and investigations on understanding the role of different techniques to enhance their quantity, is a need of hour. It has a social as well as pharmaceutical relevance in context to the present threat of non-availability of useful flora in adequate quantity for the extraction of life saving natural drug constituents because of the rare and extinct occurrence of some medicinal plants.

CONFLICT OF INTEREST:

We declare that we have no conflict of interest.

ACKNOWLEDGEMENT:

The authors are thankful to Director, The Institute of Science, Mumbai, Dr. Rajesh Raut, Dr. Umesh kakde and Dr. Atul Babar for their kind help during sample preparation and instrumental analysis.

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Received on 22.01.2023 Modified on 19.06.2023

Research J. Pharm. and Tech 2023; 16(12):5834-5843.

DOI: 10.52711/0974-360X.2023.00945