Lingfa L; Ankanagari S. GC-MS Profiling of Reproductive Stage Withania somnifera for Antimicrobial and Anticancer Phytochemicals. Biomed Pharmacol J 2023;16(1).

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Manuscript received on :28-05-2022
Manuscript accepted on :05-10-2022
Published online on: 13-02-2023

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Plagiarism Check: Yes
Reviewed by: Dr. Tukaram Dudhamal
Second Review by: Dr. kiranmayee
Final Approval by: Dr. H Fai Poon

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Lali Lingfa and Srinivas Ankanagari*

Department of Genetics and Biotechnology, Osmania University, Hyderabad – 500 007 (T.S), India.

Corresponding Author E-mail:asrinivas@osmania.ac.in

DOI : https://dx.doi.org/10.13005/bpj/2601

Abstract

Background: Withanias omnifera also known as Indian ginseng is commonly found in India and other Southeast Asian countries. Various parts of this plant have been used as herbal medicine to treat a variety of diseases. However, there is a lacuna in the profiling of phytochemical constituents present in the different parts of the plant at reproductive stage. Objective: To identify phytochemicals present in the methanolic extracts of leaf, root, and stem parts of W. somnifera at reproductive stage using GC-MS analysis. Methods: The airdried parts of plant (leaf, stem and root) were extracted with methanol and concentrated under reduced pressure at 40°C using a rotary evaporator. The GCMSQP2010, Shimadzu, Kyoto, Japan with headspace sampler (AOC-20s) and autoinjector (AOC-20i), was used for sample analysis.  The phytochemicals were identified with the database provided by National Institute Standard and Technology (NIST11LIB). Results: The GC-MS analysis of leaf, root, and stem methanolic extracts of W. somnifera, revealed a total of eighty-two unique phytochemical peaks in the reproductive stage of the plant. Phytochemicals with antimicrobial and anticancer properties were identified in all the parts. In leaf, 2-pentanone, 5-chloro- was found to be most abundant and 2,5-dimethoxy-4-propoxy-.beta.-methyl-.beta.-nitrostyrene least abundant with antimicrobial nature, whereas, benzene, 1,1'-(1,2-ethenediyl)bis[2-methyl- was found to be most abundant and dibenzo[a,e]cyclooctene, 5,6,11,12-tetrahydro- least with anticancer property. In roots, the most abundant was benzoic acid, 3-methyl-2-trimethylsilyloxy-, trimethylsilyl ester and tris(trimethylsilyl)hydroxylamine the least abundant were identified to be antimicrobial, whereas high abundance uleine and low abundance 2-{4-[2-(4-methoxymethylphenyl)vinyl]phenyl}propan-2-olwere identified to be anticancer. In stem, acetohydroxamic acid was found to be most abundant and trans-2,3,6-trimethoxy-b-methyl-b-nitrostyrene least abundant for antimicrobial nature, whereas 3-acetoxy-2,3'-bibenzo[b]thiophene was found to be anticancer phytochemical. Conclusion: In this study, phytochemicals with antimicrobial and anticancer properties were identified in leaf, root and stem parts of W. somnifera at reproductive stage.

Keywords

Anticancer; Antimicrobial; GC-MS; Herbal medicine; Phytochemicals; Withania somnifera

Introduction

Human civilization has been in an intimate relationship with plants since time immemorial.¹ They rely on plants and other natural sources for their survival and well-being.² Herbal medicines have become increasingly popular in recent years due to their therapeutic properties, minimal side effects, and cost efficiency.^3,4 Phytochemical constituents in herbal plants are playing a central role in the development of herbal medicine that is critical for ensuring a healthy society.⁵ The data collected on phytochemicals helps in the discovery of new therapeutic prospects.⁶ However, the therapeutic potential of various plants and their parts that are available in nature has yet to be explored.⁷

Withania somnifera (L.) Dunal belongs to Solanaceae family is a delicate perennial shrub that grows 14-30 inches tall and grows out radial tomentose branches from a central stem.⁸ The leaves are dull green and elliptic, with a length of 3.9 – 4.7 inches and the flowers are green, small, and bell-shaped, the fruit is orange-red when fully ripe.⁹ In Latin, the term “somnifera” means sleep-inducing.¹⁰ The name “ashwagandha” is derived from the Sanskrit words “ashva” (horse) and “gandha” (smell), indicating that the root has a strong horse-like scent.¹¹ Indian ginseng, Ashwagandha, winter cherry and poison gooseberry, are some of the many names for it. This plant can be found in India, parts of Africa and in the Middle East.¹²

W. somnifera is used in over 100 formulations in Indian traditional medicine, including Ayurveda, Unani, and Siddha, and is therapeutically equivalent to ginseng.¹³W. somnifera leaf extract has been shown to be effective against Staphylococcus aureus and Enterococcus spp.¹⁴ Other health benefits of W. somnifera have been recommended for use as a liver tonic, aphrodisiac, astringent, and anti-inflammatory agent, and more recently for the treatment of insomnia, asthma, bronchitis, ulcers, senile dementia, and emaciation, among others.¹⁵ The medicinal use of ashwagandha for cognitive and neurological diseases, such as anxiety, Parkinson’s disease, and inflammation, is also backed by clinical trials and animal research.¹⁶ Furthermore, Ashwagandha’s chemopreventive properties make it a potentially effective adjuvant for radiation and chemotherapy patients.¹⁷ Ashwagandha is also used as an immune stimulant in patients with low white blood cell counts in the blood and as an adaptogen for patients with nervous exhaustion and debility related to stress.¹⁸ The major phytoconstituents of ashwagandha root are withanolides, which include steroidal alkaloids and steroidal lactones.¹⁹

The selection of different plant parts roots, stem and leaf of W. somnifera could provide a biological and biochemical basis for identifying new pharmacologically important phytochemicals of therapeutic value.²⁰ Extraction and characterization of bioactive compounds from W. somnifera have given birth to various phytochemicals with therapeutic importance  like  anaferine, anahygrine and isopelletierine etc.,  belonging to alkaloids, withaferins as well as withanolides belonging to steroidal lactone compounds, and saponins.²¹ A number of different solvent systems like chloroform, ethanol, ethyl acetate, methanol, petroleum ether and water, etc. have been reported to play important role for extraction of secondary metabolites. However, methanol is considered as an optimal solvent to obtain high variety phytochemical constituents in plant extracts.²²

However, there is a lacuna in comparative profiling of  phytochemicalsin W. somnifera leaf, stem, and root qualitatively and quantitatively.²³ Moreover, W. somnifera  is harvested at reproductive stage for the optimum dry root yield²⁴. Thus, the chemical profiling can be established for a plant extract to identify, provide quality assurance and quantitative molecular description of plant secondary metabolites using chemical analytical methods such as  Gas chromatography–mass spectrometry (GC-MS).²⁵ The GC-MS technique has the highest sensitivity and specificity to detect the presence of phytochemical constituents.²⁶ GC-MS analysis has long been the method of choice for determining steroid levels in clinical samples.²⁷  Moreover, GC-MS allows effective chromatographic separation, quantification, and identification of sample constituents by using mass spectral libraries.²⁸ Hence, in this study, GC-MS analysis was chosen as a standard approach for phytochemical profiling of leaf, stem and root  at reproductive stage in W. sominfera.

Materials and Methods

Preparation of plant extract

W. sominifera were air dried and the plant parts (leaf, stem and roots each10 g) were coarsely pulverized and extracted with methanol for 24 hours in a Soxhlet (100 ml). The extract was filtered and concentrated under reduced pressure at 40°C using a rotary evaporator to get a viscous semi solid mass.

GC‑MS analysis

The GCMSQP2010, Shimadzu, which includes the headspace sampler (AOC-20s) and autoinjector, was used for GC-MS analysis (AOC-20i). The system included a mass selective detector and an ion source with a temperature of 230°C and a temperature of 250°C at the interface. The capillary column used for MS analysis was an Rt 5ms capillary column having a length of 30 m, a diameter of 0.32 mm, and a film thickness of 0.25 µm. The injector’s temperature was set to 250°C, and it had a split injection mode. The initial temperature was set at 80°C for 3 minutes, then the temperature was steadily increased to 280°C at a rate of 10°C/min. With a linear velocity of 47.1 cm/sec, helium (>99.9%) was used as the carrier gas. A total flow of 90.0 ml/min was programmed, with a column flow of 1.71 ml/min.

Identification of phytochemicals

Components were identified based on retention time (RT) for GC and interpretation of mass spectrum was done by comparing spectral fragments obtained, to the database provided by National Institute Standard and Technology (NIST11LIB). The components of the test materials were identified by their name, molecular weight, and structure.

Results

GC-MS analysis of reproductive stage Leaf

As shown in Figure 1, a total of 16 phytochemicals were exclusively identified in the methanolic leaf extracts viz. 2-pentanone, 5-chloro-; 3-butoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tris(trimethylsiloxy)tetrasiloxane; benzaldehyde, 3-methoxy-4-[(trimethylsilyl)oxy]-, O-methyloxime; 2-(7-methoxymethylphenanthren-3-yl)propan-2-ol; cyclopropanecarbonyl chloride, 1-fluoro-2,2-diphenyl-; benzene, 1,2,3-trimethoxy-5-(2-propenyl)-; 4′,6-dimethoxyaurone; dibenzo[a,e]cyclooctene, 5,6,11,12-tetrahydro-; acetic acid, 2,3-dibromo-4-methoxymethoxy-1-methyl-pent-2-enyl ester; 2,5-dimethoxy-4-propoxy-.beta.-methyl-.beta.-nitrostyrene; 1,3-dihydroxy-2,4,5-trifluoro-6-nitrobenzene; cobalt, allyl-(pentamethylcyclopentadienyl; 1-phenazinecarboxylic acid, 6-(1-methoxyethyl)-, methyl ester; cis,syn,cis-perhydrophenanthrene; benzene, 1,1′-(1,2-ethenediyl)bis[2-methyl- and pentasiloxane, 1,1,3,3,5,5,7,7,9,9-decamethyl-.

Figure 1: GC-MS chromatogram of leaf methanolic extract in W. somnifera Click here to view Figure

The phytochemicals identified with antimicrobial properties in the methanolic leaf extracts is given in Table 1. The phytochemicals included2-pentanone, 5-chloro- at RT1.115with peak area 3.29%; 3-butoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tris(trimethylsiloxy)tetrasiloxane at RT 12.820 with peak area 1.66%; benzaldehyde, 3-methoxy-4-[(trimethylsilyl)oxy]-, O-methyloxime at RT 28.405 with peak area 1.12%; 2-(7-methoxymethylphenanthren-3-yl)propan-2-ol at RT 29.560 with peak area 1.15%; 4′,6-dimethoxyaurone at RT 30.960 with peak area 1.78%;2,5-dimethoxy-4-propoxy-.beta.-methyl-.beta.-nitrostyrene at RT 34.020 with peak area 1.08%;1,3-dihydroxy-2,4,5-trifluoro-6-nitrobenzene at RT 35.250 with peak area 1.24%;1-phenazinecarboxylic acid, 6-(1-methoxyethyl)-, methyl ester at RT 36.980 with peak area 1.23%; benzene, 1,1′-(1,2-ethenediyl)bis[2-methyl- at 42.525 with peak area 1.41%; and benzene, 1,2,3-trimethoxy-5-(2-propenyl)- at RT 30.740 with peak area 2.77%.

The phytochemicals identified with anticancer properties in the methanolic leaf extracts is given in Table 1. The phytochemicals included cyclopropanecarbonyl chloride, 1-fluoro-2,2-diphenyl- at RT 29.975 with peak area 1.12%; dibenzo[a,e]cyclooctene, 5,6,11,12-tetrahydro- at RT 32.630 with peak area 1.08%; cis,syn,cis-perhydrophenanthrene at RT 39.066 with peak area 1.40%; and some identified phytochemicals with both antimicrobial and anticancer properties were 2-(7-methoxymethylphenanthren-3-yl)propan-2-ol at RT 29.560 with peak area 1.15%and benzene, 1,1′-(1,2-ethenediyl)bis[2-methyl- at RT 42.525 with peak area 1.41%.

Table 1: Phytochemicals identified for antimicrobial and anticancer properties in the methanolic leaf extracts of W. somnifera.

GC-MS analysis of reproductive stage root:

As shown in Figure 2, a total of 19 phytochemicals were exclusively identifiedin the methanolic root extracts. The phytochemicals identified included sulfurous acid, bis(1-methylethyl) ester; 1,1′-(ethanediylidenediamino)bis(5-amino-1H-tetrazole); tris(trimethylsilyl)hydroxylamine; 1H-indole-2,3-dione, 1-(tert-butyldimethylsilyl)-5-chloro-, 3-(O-ethyloxime); N-(2-hydroxy-3,5-dimethylbenzyl)-.beta.-aminobutanoic acid; 4-(2,6,6-trimethylcyclohexa-1,3-dienyl)pent-3-en-2-ol; stannane, 1,3-dithian-2-ylidenebis[trimethyl-; S-[2-aminoethyl]-.beta.-phenyl-.alpha.-mercaptoacrylic acid; 1.alpha.-(hydroxymethyl)-7.alpha.,8.alpha.-dimethyl-7-(2-(3-furyl)ethyl)bicyclo[4.4.0]dec-2-; silane, methyltripropoxy-; 2-{4-[2-(4-methoxymethylphenyl)vinyl]phenyl}propan-2-ol; cyclohexanecarboxylic acid, 4-[[(tert-butyldimethylsilyl)amino]methyl]-, tert-butyldimethylsilyl; nickel, pentamethylcyclopentadienyl-(N,N,N’-trimethyl)-o-phenylenediamine-N’-o-; benzenepropanoic acid, 4-benzoyl-, methyl ester; silane, [[3,3-dimethyl-4-methylene-2-(trimethylsilyl)-1-cyclopenten-1-yl]methoxy]trimethyl-; uleine; benzene, dichlorodimethoxy-; 3,4,5-tris(trimethylsiloxy)-1-cyclohexene-1-carboxylic acid, trimethylsilyl ester; andbenzoic acid, 3-methyl-2-trimethylsilyloxy-, trimethylsilyl ester.

Figure 2: GC-MS chromatogram of root methanolic extract  in Withania somnifera. Click here to view Figure

The phytochemicals identified with antimicrobial properties in the methanolic root extracts is given in Table 2. The phytochemicals  included tris(trimethylsilyl)hydroxylamine with at RT 28.290 and peak area 1.18%; 4-(2,6,6-trimethylcyclohexa-1,3-dienyl)pent-3-en-2-ol at RT 29.531 with peak area 2.36%: stannane, 1,3-dithian-2-ylidenebis[trimethyl- at RT 29.765 with peak area 1.57%; benzenepropanoic acid, 4-benzoyl-, methyl ester at RT 38.505 with peak area 1.75%; and benzoic acid, 3-methyl-2-trimethylsilyloxy-, trimethylsilyl ester.  Phytochemicals identified for anticancer properties in the methanolic root extracts included 2-{4-[2-(4-methoxymethylphenyl)vinyl]phenyl}propan-2-ol at RT 34.230 with peak area 1.23 and uleine at TR 39.505 with peak area 1.90%.

Table 2:  Phytochemicals identified for antimicrobial and anticancer activity in methanolic root extracts of W. somnifera.

GC-MS analysis of reproductive stage stem

As shown in Figure 3, a total of 18 phytochemicals were exclusively identified in the methanolic stem extracts. The phytochemicals identified included  viz. acetohydroxamic acid; pentasiloxane, dodecamethyl-; 1,4-dibromo-2,3-butanediol; trans-2,3,6-trimethoxy-b-methyl-b-nitrostyrene; benzylamine, 2-hydroxy-N,N-di-[2-aminoethyl]-; androst-9(11)-en-17-one, 3-[(trimethylsilyl)oxy]-, O-methyloxime; 3-Acetoxy-2,3′-bibenzo[b]thiophene; 1,3,5,7-tetraethyl-1-oxycyclotetrasiloxane; 1,3-methylene-d-arabitol; 3.alpha.,4.alpha.,9.beta.,11-diepoxymuurolan-10-ol; benzenamine, N-(3,4,5,6-tetraethyl-1-phenyl-2(1H)-pyridinylidene; 3-(3-ethoxy-4-hydroxyphenyl)-2-isothiocyanatopropionic acid, ethyl; benzeneacetic acid, .alpha.,3,4-tris[(trimethylsilyl)oxy]-, trimethylsilyl; silanamine, N-[(4-methoxyphenyl)methyl]-1,1,1-trimethyl-; 4-Chloro-2-iodobenzoic acid; chlorotris(p-tolyl)methane; and cyclotetrasiloxane, octamethyl- and pentasiloxane, 1,1,3,3,5,5,7,7,9,9-decamethyl-.

Figure 3: GC-MS chromatogram ofmethanolic stem extract in W.somnifera. Click here to view Figure

The phytochemicals identified with antimicrobial properties in the methanolic stem extracts is given in Table 3.The phytochemicals with antimicrobial properties were identified in the methanolic stem extracts. These included acetohydroxamic acid at RT 1.170 with peak area 3.12%; pentasiloxane, dodecamethyl- at RT 27.030 with peak area 1.12%; 1,4-dibromo-2,3-butanediol at 27.800 with peak area 2.01%; trans-2,3,6-trimethoxy-b-methyl-b-nitrostyrene at RT 28.510 with peak area 1.06%; benzylamine, 2-hydroxy-N,N-di-[2-aminoethyl]- at RT 29.395 with peak area 1.43%; androst-9(11)-en-17-one, 3-[(trimethylsilyl)oxy]-, O-methyloxime at RT 29.804 with peak area 1.21%;1,3,5,7-tetraethyl-1-oxycyclotetrasiloxane at RT 32.579 with peak area 1.51%; 1,3-methylene-d-arabitol at RT 38.825 with peak area 1.13%: 3.alpha.,4.alpha.,9.beta.,11-diepoxymuurolan-10-ol at RT 36.255 with peak area 1.74%; benzenamine, N-(3,4,5,6-tetraethyl-1-phenyl-2(1H)-pyridinylidene at RT 40.350 with peak area 1.15%; 3-(3-ethoxy-4-hydroxyphenyl)-2-isothiocyanatopropionic acid, ethyl at 41.310 with peak area 1.15%; benzeneacetic acid, .alpha,3,4-tris[(trimethylsilyl)oxy]-, trimethylsilyl at RT 41.619 with peak area 1.18%; silanamine, N-[(4-methoxyphenyl)methyl]-1,1,1-trimethyl- at RT 44.074 with peak area 1.92%; and abcyclotetrasiloxane, octamethyl- at RT 27.480 with peak area 1.33%.The identified 3-acetoxy-2,3′-bibenzo[b]thiophene at RT 30.970 and peak area 1.11% has both antimicrobial and anticancer properties.

Table 3: Phytochemicals identified for antimicrobial and anticancer in reproductive stage stem extracts of W.somnifera.

As shown in the Figure 4 (a-e), some unique phytochemicals of steroid structures have been identified in the reproductive stage methanolic root extracts of W. somnifera. These include cucurbitacin b, 25-desacetoxy- identified at RT 29.205, 19-norpregn-5(10)-en-20-yn-3-one, 17-[(trimethylsilyl)oxy]-, (17.alpha.)- identified at RT 32.380 (also known as Trans-3, 5, 4 -trimethoxystilbene (TMS) derivatives), carbromal identified at RT 32.260, spirost-8-en-11-one identified at RT 32.980, alpha.-D-glucopyranoside, methyl 2-(acetylamino)-2-deoxy-3-O-(trimethylsilyl)-, cyclic methylboronate identified at RT 32.490 and morphinan  identified at RT. 35.215.

Figure 4: (a-e). Phytochemicals with steroid structures identified in the W. sominifera methanolic root extracts. Click here to view Figure

Discussion

In the plants, phytochemicals greatly vary from organ to organ.⁵⁹ It has been reported that all the parts of Withania somnifera have been used for treatment of various human illnesses.⁶⁰ W. somnifera root and leaf extracts of both aqueous and alcoholic, have previously been reported to be antimicrobial against a wide range of microorganisms.⁶¹ The roots of W. somnifera are mostly preferred for various therapeutic purposes.⁶² Moreover, methanolic extracts of various parts of W. somnifera especially roots have also been reported to be an effective against various kinds of cancers.⁶³

GC-MS analysis is a rapid and cost-efficient method as it is effective in chromatographic separation, quantification, and identification of sample constituents for assessing herbal products.⁶⁴ Based on this, we performed GC-MS analysis to profile phytochemicals in methanolic extracts of leaves, stems, and roots at the reproductive stage of W. somnifera. In the methanolic leaf extracts, the identified phenolic compounds 1,2-bis(trimethylsilyl)benzene have previously been reported for antibacterial and anticancer activities,⁶⁵ ester compound 1,2-cinnolinedicarboxylic acid, 1,2,3,5,6,7,8,8a-octahydro-4-trimethylsilyloxy-, diethyl ester have previously been reported to be antibacterial and antifungal activities,⁶⁶ and phenolic compound 1,3-Dihydroxy-2,4,5-trifluoro-6-nitrobenzene  is  a nitrobenzene derivative shown to have antitumor and antibacterial properties.^{67, 41} The identified methyl ketone1-[2,4-bis(trimethylsiloxy)phenyl]-2-[(4-trimethylsiloxy)phenyl]propan-1-one has been reported to be antibacterial in property,⁶⁸ Phenolic stilbenes1-Methyl-1,2,2-triphenylindan has been reported to be antibacterial in property,⁶⁹ silyl ethers 1,3,5,7-tetraethyl-1-butoxycyclotetrasiloxane has been reported to be antibacterial properties,⁷⁰ for the aldehyde compound cyclopropanecarbonyl chloride, 1-fluoro-2,2-diphenyl- there is no specific available reports, however its derivatives viz. cyclopropanecarbonyl chloride have been reported to be anticancer in properties,³³ and phenolic compounds 2-(7-methoxymethylphenanthren-3-yl)propan-2-ol has been reported to be both anticancer and antimicrobial in properties.⁷¹

In the root methanolic extracts, the identified esterbenzoic acid, 3-methyl-2-trimethylsilyloxy-, trimethylsilyl ester, has previously been reported to be antimicrobial in properties.⁴⁶ Amines compounds tris(trimethylsilyl)hydroxylamine a hydroxylamine derivative, has been reported to be antibacterial properties.²⁸ Ester compound  boric acid, trimethyl ester has been reported to be anticancer properties⁶⁹ and dextroamphetamine compound was reported to be brain stimulant and antibiotic.⁷² Steroid compound morphinan found in the Opium poppy (Papaver somniferum),⁷³ has been identified in this study in the root extracts  of W. somnigfera which was found to have antibacterial properties.⁷⁴ Somephytochemicals identified in the root and stem methanolic extracts alkanes like ethane, 1,2-dichloro-1-ethoxy which is antimicrobial,⁷⁵ steroid amines like dextroamphetamine, acids like boric acid, trimethyl which is anticancer,⁷⁶ ketones like tartronic acid, 4-(dimethylethylsilyl)phenyl-, dimethyl ester, which is antibacterial,⁷⁷ and aromatic acid like 3,5-dichloro-4-hydroxybenzoic acid which is antimicrobial in properties.⁷⁸

In the methanolic stem extract phenolic compound identified 3,5-dichloro-4-hydroxybenzoic; 3-acetoxy-2,3′-bibenzo[b]thiophene which is a benzo[b]thiophene derivatives has previously been reported to be anticancer and antibacterial.^{47, 52} The identified terpenoid phytoalexins such as acetohydroxamic acid has been reported to be antibacterial in the previous study.⁵ Identified alkanes such as pentasiloxane, dodecamethyl- has previously been reported to be anticancer,⁷⁹ and aromatic nitroalkene such as trans-2,3,6-trimethoxy-b-methyl-b-nitrostyrene which is β-nitrostyrene derivatives compounds has been reported to be antibacterial.³⁶

Some steroids compounds identified in this study which include α-D-glucopyranoside, methyl 2-(acetylamino)-2-deoxy-3-O-(trimethylsilyl)-, cyclic methylboronate were reported to be found in the flowers of Jacaranda mimosifolia, that poses antibacterial properties⁸⁰ and its derivatives like methyl-α-D-glucopyranoside in Tulbghia violacea,⁷⁰ and α-D-glucopyranoside,O-α-D-glucopyranosyl- (1.fwdarw.3)-ß-D-fructo in Foeniculum vulgare⁸¹ were reported to be anticancer in properties. Similarly, carbromal is found in Decalepis hamiltonii,⁸² has been used to treat mild insomnia.⁸³ Derivatives of novel steroids 19-norpregn-5(10)-en-20-yn-3-one⁸⁴ i.e., 19-norpregn-4-en-20-yn-3-one is antitumor in properties and has been reported to be found in the Curcuma aeruginosa.⁸⁵ Cucurbitacin b is an effective anticancer and antibacterial agent,⁵⁷ found in the Cucurbitaceae plant families.⁸⁷

This study agrees with previous researchers of W. somnifera to be antimicrobial and anticancer in properties.^88,89 Moreover, this GC-MS investigation rationally evaluated and identified the phytochemicals that attributes the antimicrobial and anticancer properties of W. somnifera. Further research is needed to isolate and purify the identified phytochemicals for bioactivities in order to develop W. somnifera herbal based products for  applications in the antimicrobial and anticancer therapies.

Conclusion

The present study describes comparative GC-MS analysis of methanol extract of W. sominfera phytochemicals distribution in the leaf, stem, and root.  The GC-MS analysis revealed the distribution variation of phytochemicals contributing towards antimicrobial and anticancer properties. The highest number of unique phytochemicals was identified in the root extracts and least number of unique phytochemicals were identified in the leaf. Moreover, root and stem shared the highest number of common phytochemicals. In this study, the GC-MS analysis identified the antibacterial and anticancer phytochemicals that were not previously been reported in the W. somnifera, which included steroidal compounds like cucurbitacin b, 25-desacetoxy-; spirost-8-en-11-one, 3-hydroxy-, (3.beta.,5.alpha.,14.beta.,20.beta.,22.beta.,25R)-; alpha.-D-glucopyranoside, methyl 2-(acetylamino)-2-deoxy-3-O-(trimethylsilyl)-, cyclic methylboronate and 19-norpregn-5(10)-en-20-yn-3-one, 17-[(trimethylsilyl)oxy]-, (17.alpha.).This information can be utilized further to develop W. somnifera based traditional herbal medicines that are playing an important role in healthcare system.

Acknowledgement

LL acknowledges National Fellowship for Higher Education of ST Students (NFST), Ministry of Tribal Affairs – Government of India, for fellowship. AS acknowledges Rashtriya Uchchatar Shikhsha Abhiyan (RUSA) 2.0 Programme, under Ministry of Huma Resource Development, Government of India for funding the research. AS also acknowledge funding by CAS-II, DST-PURSE II, UPE-FAR and DBT-BUILDER.

Conflicts of Interest

The authors declare no conflicts of interest

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