Phytochemical analysis of Withania somnifera leaf extracts by GC-MS and evaluating antioxidants and antibacterial activities

ABSTRACT Many people in developing countries mainly depend on medicinal plants like Withania somnifera and their products for healthcare. In this study, reflux and maceration extraction methods were applied to investigate total phenolic (TPC) and flavonoid contents (TFC); and antioxidant properties of Withania somnifera extracts toward DPPH and hydrogen peroxide (H2O2) were studied. Highest TPC was detected with methanol extracts with value of 198.24 ± 1.16 mg GAE/g and TFC of 31.52 ± 0.91 mg QE/g. The minimum amount of TPC was observed in aqueous extract (101.41 ± 1.07 mg GAE/g) and the TFC in ethyl acetate extract 9.72 ± 1.32 mg QE/g. The maximum value of DPPH radical scavenging was estimated in maceration methanol extract 81.98 ± 0.49% and value of H2O2 was obtained in maceration acetone extract 76.18 ± 1.06%. The major identified molecules were Heptasiloxane,1,1,3,3,5,5,7,7,9,9,11,11,13,13-tetradecamethyl-(50.04%), and trans-2,4 Dimethylthiane, S, S- dioxide (4.09%) The broad spectrum of antibacterial activity was observed in Escherichia coli reflux methanol extract. In both extraction techniques methanol was the best extracting solvent for phenolic and flavonoid contents as antioxidants as well as antibacterial activities.


Introduction
Plants are not only a primary source of food and fuel, but also used for folk medication due to the presence of active chemical compounds or secondary metabolites. [1,2]Due to inadequate health centers, shortage of medicines and low-income community, almost 70% people in developing countries mainly dependent on less cost plants. [3]For the last few decades, phytomolecules such as alkaloids, tannins and flavonoids extracted from plant are eco-friendly with negligible toxicity, and are non-immunogenic in nature. [4]Among these plants, Withania somnifera, in the family of Solanaceae, is a known medicinal plant grown throughout the world. [5]n most part of Ethiopia w. somnifera is mainly used as traditional medicine to treat snake bite; cancer and chest pain. [6,7]The roots the leaves, seeds, fruits and flowers of w. somnifera are used for various purposes, the leaves for ointment and hemorrhoids and rheumatism treatments; and berries is also applied to treat wounds, cuts, abscesses and inflammation. [8]iterature reports showed that W. somnifera consists of more than 35 identified, extracted and isolated phytochemicals such as alkaloid, flavonoids, glycosides, saponins and tannins. [9]Due to these chemicals consumption of the plant in general reduces swelling, stress, and hair loss, and regulates cholesterol, improves cardiovascular health, antiepileptic, anxiety, anti-arthritic, CNS depressant and antimicrobial and strengthens heart muscles. [2,10]Since phenols are found either in free or bound forms through ether, ester or acetal bonds, phenolic in plant foods, to increase the antioxidant capacity some processing methods were required to extract them with appreciable amount. [11]xtraction techniques such as maceration, infusion, decoction, percolation, digestion and soxhlet and microwave-assisted extraction have been used extraction of medicinal plants.The selection of an appropriate extraction technique depends on the nature and type of the plant material, solvent type and ratio, part of plant to be extracted, nature of the compounds and temperature. [12]Polar solvents like ethanol, water and methanol are used to extract polar components, whereas less polar solvents like dichloromethane and hexane can extract nonpolar molecules. [13]Due to simplicity and low economic outlay, conventional extractions such as maceration and reflux techniques were used for isolating the chemical constituents present in w. somnifera leaves. [14]lthough W. somnifera is widely used as a traditional medicine for treating various diseases, in Ethiopia, scarce information is available on total phenolic content, flavonoid content and antioxidant activity. [15]To the best of our knowledge, there are no reports on the bio-constituents and bacterial activity of W. somnifera have been documented, and therefore, the current study was aimed to: (i) determine total phenolic content and flavonoid content, (ii) evaluate the antioxidant capacity and antibacterial activity, and (iii) investigate the chemical components responsible for antioxidant activity of w. somnifera leaves using GC-MS.

Sample collection
Plants were collected from various areas of Arba Minch town of southern region, Ethiopia from May to September 2021.After the representative plant samples were collected the leaves of plants were preserved in the chemistry laboratory of Chemistry department, Arba Minch University.Then after, the leaves were washed with distilled water to remove dust particles and debris and then shadedried.

Extractions
Dried leaves of W. somnifera were ground into powder followed by extraction using acetone, methanol, ethyl acetate and water.Forty gram (40 g) of the W. somnifera leaves were soaked separately in 400 mL of acetone, ethyl acetate, methanol and distilled water in a conical flask and kept at room temperature in laboratory and shacked with electrical shaker at speed of 800 rpm for 24 h.
For reflux extraction technique, the same solvents were refluxed for 2:30 h at 50°C, then allowed to cool, and filtered through using Whatman No. 42 filter paper.The supernatant was processed for evaporation at 50°C in a rotary evaporator to remove excess solvent until dryness.Concentrated extracts were kept at 4°C for future experiment and analysis.

Phenolic content
The total polyphenol contents (TPC) of leaves extracts were determined based on Mahendran et al., with some modifications. [16]A 0.3 mL (1 mg/mL) of the solvent extracts sample was mixed with 1.75 mL of Folin-ciocalteu reagents.After 6 min the mixture 2 mL of 7.5% of Na 2 CO 3 was added.Then, the mixture was incubated at 25°C for 1 h, and the absorbance was measured at 765 nm using spectrophotometer at 765 nm for generating calibration curve.

Flavonoid content
The total flavonoid content (TFC) in W. somnifera leave extracts evaluated according to method explained Oinam et al. with some modifications. [17]A 0.5 mL of solution from each fractionated components was combined with 0.160 mL of NaNO 2 (5%), 0.16 mL of AlCl 3 (10%) and 1 mL of 1 M NaOH.The absorbance of was measured at 510 nm by spectrophotometer.Total flavonoid content of all fractionated solvent extracts was estimated using the calibration curve of quercetin standard (mg QE) equivalents per g of dry mass.

DPPH radical scavenging activity
The antioxidant capacity of extracts was evaluated by using the 1-1-Diphenyl-2-picryl-hydrazyl (DPPH) based on the method developed by Mondal et al (2021). [18]Briefly, in different test tubes, 1.5 mL each of methanol, ethyl acetate, acetone and aqueous extracts were allowed to react with 3 mL DPPH solution (0.004%) to make the final volume of 4.5 mL and incubated the mixture in room temperature for 45 min in a dark place until pink color was noted.The scavenging of DPPH was evaluated in terms of its absorbance using UV-Vis spectrophotometer at 517 nm.The radical scavenging capacities of all extracts were calculated with Eqn.1: where, A o is the absorbance of control and A s is the absorbance of the sample.

Hydrogen peroxide radical scavenging activity
Free radical scavenging activity of W. somnifera extracts was evaluated by hydrogen peroxide based on the method described by Ratan et al. [19] To 100 μg/mL of all extract solutions were mixed with 0.5 mL hydrogen peroxide (50 mM) prepared using phosphate buffer (pH 7.4).The mixtures of all components were vigorously shaken using vortex mixer and incubated well for 25 min.Then, the absorbance of the reaction mixture and blank solution of phosphate buffer was measured using double beam UVvis spectrophotometer at 230 nm.Hydrogen peroxide inhibition capacities of extracts were calculated using Eqn.2: where A o is the absorbance of the control, A s is the absorbance of the sample.

Antibacterial activities
The antibacterial activities of all extracts were evaluated based on agar well method as described by Khan et al. with minor modifications. [20]The bacterial strains incubated for 24 h to obtain turbidity with reference to McFarland turbidity standard having 0.5 McFarland units.Each Mueller-Hinton agar plate was inoculated with a sterile wash and spread evenly on the surface of the agar plate.With the use of sterile cork borer (5 mm diameter) wells were made in agar medium.Briefly, 0.1 mL of sample was added into the wells in the plate.A 5% of DMSO added to the well as a control.The plates were incubated at 37°C for 24 h.Extracts of antibacterial activities were estimated by detecting the diameters of zone of inhibition for each method and extracting solvent.The antibacterial activities of samples were evaluated in the cleared zone of inhibition.

Gas chromatography mass spectrometry analysis of Withania somnifera extract
The molecules in methanol extract of W. somnifera leaves were identified based on gas chromatogram coupled with mass spectrometer (GC-MS).The GC-MS of Agilent Technologies 8890 GC system equipped with an Agilent Technologies GC-MS capillary column HP-5 MS measured with Column Elite-1 fused silica capillary column (30 m x 0.25 mm 1D x 25 μL) contained 100% dimethyl polysiloxane, 70 eV ionization voltage was for GC-MS operation.The carrier gas was helium gas (99.99%) with flow rate of 1 mL/min and 2 μL an injection volume was employed at an injector and ion source temperature of 250°C and 280°C, respectively.The identified compounds were based on the comparisons of their mass spectra with NIST library.

Statistical analysis
The analysis were made in triplicates, and the results were reported as mean � SD (n = 3).The calibration curve of total polyphenol content, flavonoid content and antioxidants activities was determined using MS excel.The TPC, TFC and antioxidant activities were analyzed using analysis of variance (ANOVA) followed by Tukey test at a significance level of α = 0.05.

Total phenolic content
The antioxidant activities of plant extracts like W. somnifera are due to phenols, flavonoids, anthocyanin, alkaloids, tannin and steroidal lactones. [21]Effective extraction methods and solvent types have an influence on the nature and amount of extracts with minimal changes of physical and chemical nature of the required extracts. [22,23]As a result, it is important to choose appropriate extraction solvents and methods based on sample matrix properties, chemical properties of the analytes, analytematrix interaction, efficiency and desired properties. [24]he TPC of the leaves extracts prepared by two extraction techniques (i.e., reflux and maceration) using methanol, acetone, water and ethyl acetate extraction solvents is presented in Table 1.The TPC in reflux method with methanol extract (198.24 ± 1.16 mg GAE/g) was the highest, followed by aqueous (140.59� 1.82 mg GAE/g), acetone (123.83 ± 1.07 mg GAE/g) and ethyl acetate (109.48 ± 1.24 mg GAE/g), which in line with literature reports [25] and the differences were highly significant (p < .05).
However, the TPC values in maceration with different extraction solvents were significantly different and the highest TPC was found in methanol extract, which was 187.74 ± 1.02 mg GAE/g, followed by ethyl acetate extract with the value 144.06 ± 2.01 mg GAE/g, acetone extract with value of 110.69 ± 1.19 mg GAE/g and water with value of 101.41 ± 1.07; the differences are highly significant.The highest TPC values with methanol extract compared with other solvents attributed to its better polarity and its better solubilization of phenolic components in W. somnifera. [26,27]There are significance differences (p < .05) on the value of TPC for all solvents in each extraction methods.The TPC values between the two extraction methods from leaves of W. somnifera were found to be higher in reflux methods except ethyl acetate extracts, which is in agreement with previous data. [28,29]owever, The TPC values in this study are higher than literature reports. [30]

Total flavonoid content
The TFC was calculated based on the calibration curve of quercetin standards.As shown in Table 1, the TFC in reflux extracts varied from 11.89 ± 1.21 mg QE/g to 31.52 ± 0.91 mg QE/g.The highest values were investigated in methanol extract (31.52 ± 0.91 mg QE/g), followed by aqueous (26.19 ± 0.62 mg QE/g), acetone extract (17.21 ± 1.22 mg QE/g) and ethyl acetate extract (12.89 ± 1.29 mg QE/g) of dry matter, which were in line with TPC values.While in maceration extraction the highest TFC was noted in methanol extract (24.10 ± 0.97 mg QE/g), followed by acetone extract (18.25 ± 1.48 mg QE/g), aqueous extract (12.06 ± 1.68 mg QE/g) and ethyl acetate (9.72 ± 1.37 mg QE/g).Extracting capacity of polar solvent in reflux was in the order: methanol > aqueous > acetone > ethyl acetate while in maceration: methanol > acetone > aqueous > ethyl acetate.The TFC values in both solvents within the two extraction methods showed significant differences (p < .05), in agreement with results reported by. [28,30,31]Our results are entirely below the findings of other authors, [32] who demonstrated that the flavonoid content of ethyl acetate extract was 65.6 mg of QE/g, but our results of methanol extracts are greater compared with the study of [33] whose value is 5.06 ± 1.16 (mg QE/100 g dw).

DPPH Radical scavenging capacity:
The capacity of W. somnifera leaves to scavenge DPPH free radical in both extraction methods are displayed in Table 2. DPPH is a stable organic free radical, which is widely used to assess the antioxidant capacities of compounds in plants using spectrophotometer by quenching of stable purple-colored DPPH into yellow. [34]It can be seen that with the exception of aqueous extracts, stronger scavenging capacities were observed in maceration method than reflux method with percentage of inhibition values ranging from 50.67 ± 0.90 to 81.98 ± 0.49 and differed significantly (p < .05).A value in the same column for each solvent extracts followed by different letters (a-g &h-n) mean significant differences between extraction techniques and solvents estimated by Tukey's multiple range tests (p < 0.05).
Hydrogen peroxide radical scavenging activity: The accumulation of H 2 O 2 is responsible for oxidative stress and inflammation reactions, which are correlated with pathological conditions like cancer, diabetes, and cardiovascular diseases. [38]This is due to the decomposition of H 2 O 2 and generation of the OH radical that initiates lipid peroxidation and damaging cellular components. [39]lant antioxidants were the promising sources for regulating the damaging effects of those hydroxyl free radicals.
As shown in Table 2, all solvents and both extraction techniques showed remarkable antiradical trapping abilities using hydrogen peroxide.The order of antioxidant capacity of leaf extracts using maceration on H 2 O 2 toward was acetone (76.18 ± 1.06) > methanol (68.01 ± 0.39)> aqueous (57.19 ± 1.28) > ethyl acetate 946.09 ± 1.94).In reflux extraction, the antioxidant of the plant extracts is in the order: aqueous > methanol > acetone > ethyl acetate, with percentage inhibition of 62.01 ± 2.17, 60.94 ± 2.01, 51.82 ± 1.33 and 38.07 ± 1.02, respectively.In comparison with extraction methods, except aqueous extracts of reflux methods have relatively lower than that of maceration extraction.The one-way ANOVA statistical evaluation revealed that the type of extraction method, solvent type and the interaction of both factors had a significant effect on the polyphenol content, flavonoids content and antioxidant activity.

Antibacterial activity
The antibacterial susceptibility test of W. somnifera leaves extracts indicated varying degrees of antibacterial activities for the selected bacterial species.Among the different solvent extracts in both extraction methods, reflux extracts showed the highest and widest spectrum of activity (Table 3) to inhibit bacterial growth in the given zone.As shown in the table, except ethyl acetate and acetone maceration extracts, reflux extracts via methanol, acetone and ethyl acetate crude extracts effectively inhibited the growth of all gram positive (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli).In comparative analysis using gram-positive and gram-negative bacteria, methanol extracts were more effective to inhibit the growth of S. aureus and E. coli than other solvents, with estimated trapping zone ranging from 39.75 � 0.08 mm in reflux method to 18.16 ± 0.18 mm in maceration extracts.In all solvent extracts, the highest inhibition zone was observed toward gramnegative E. coli, which may be attributed to the structural difference between gram-positive and gramnegative bacteria. [40]imilarly, acetone extract gives an inhibitory zone in the range between 30.09 ± 0.17 mm and 14.51 ± 0.91 mm and minimum inhibition zone was recorded in ethyl acetate extract and varied  [41] on the antibacterial activity of the methanolic extract of W. somnifera leaves against E. coli 16 ± 0.57 mm and S. aureus 15 ± 0.33 mm of zones of inhibition.The findings of our study indicate that reflux of all solvent extracts was efficient to inhibit the activity of pathogenic bacteria.The bioactive molecules in plants were found to be source of various phytochemicals that could be directly used as intermediates for the production of new drugs. [42]It was concluded that methanol is the best solvent to extract secondary metabolites to inhibit different types of disease caused by pathogenic bacteria.

Phytocomponent identification by GC MS
The GC-MS method was help to identify different substances within a test sample.

Table 1 .
Total phenolic content and flavonoid content of W. somnifera leaves extract.

Table 2 .
Radical scavenging activities of leaves extract in different solvents and extraction methods..05mm to 13.07 ± 0.38 mm.In this study no remarkable zone of inhibition was found in maceration extracts with ethyl acetate and acetone extracts.Our study values were greater than the reported values of Mahesh et al.

Table 3 .
Antibacterial activity of W. somnifera leaves in different solvent and extraction techniques.

Table 4 .
Identified compounds in W. somnifera methanol leaves extract.ConclusionThe evaluation of antioxidant activities in our study indicates that W. somnifera leaves is the main source of phenolic compounds and flavonoids and it is regarded as sources of natural antioxidants.Extraction methods are the key factors to analyze the level of antioxidant properties of phenolic compounds and flavonoids which are found in the selected plant parts.In this study, the maximum radical scavenging abilities were observed in maceration than that of reflux extraction technique.As it can be clearly seen, extraction of W. somnifera leaves using methanol has depicted the highest phenolic content (198.24 ± 1.16 mg GAE/g) and flavonoids (31.52 ± 0.91 mg QE/g), while the least values of TPC (109.48 ± 1.24 mg GAE/g) and TFC (12.89 ± 1.29 mg QE/g) were recorded in ethyl acetate.Similarly, the highest DPPH (81.98 ± 0.49%) of W. somnifera leaves extracted with methanol and hydrogen peroxide scavenging activities (76.18 ± 1.06) of W. somnifera leaves in acetone were recorded.In both extraction techniques, W. somnifera leaves extract showed strong antibacterial activities against all the S. aureus and E. coli bacterial species.Except maceration with acetone and ethyl acetate extracts, all extracts clearly indicate inhibitory activities against gram-positive and gramnegative bacterial strains.Therefore, W. somnifera leaves act as defensive parts against bacterial infections, and it is important to investigate active constituents on it.The investigation clearly indicates that the stronger extraction capacity of methanol could have produced a number of active constituents responsible for many biological activities.Those obtained compounds might be utilized for the development of traditional medicines, and further investigation needs to elute novel active compounds which may be created a new way to treat many incurable diseases.