{"id":47777,"date":"2023-03-21T11:22:06","date_gmt":"2023-03-21T11:22:06","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=47777"},"modified":"2023-04-01T06:06:44","modified_gmt":"2023-04-01T06:06:44","slug":"gc-ms-profiling-of-reproductive-stage-withania-somnifera-for-antimicrobial-and-anticancer-phytochemicals","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no1\/gc-ms-profiling-of-reproductive-stage-withania-somnifera-for-antimicrobial-and-anticancer-phytochemicals\/","title":{"rendered":"GC-MS Profiling of Reproductive Stage Withania somnifera for Antimicrobial and Anticancer Phytochemicals"},"content":{"rendered":"

Introduction <\/strong><\/p>\n

Human civilization has been in an intimate relationship with plants since time immemorial.1<\/sup> They rely on plants and other natural sources for their survival and well-being.2<\/sup> Herbal medicines have become increasingly popular in recent years due to their therapeutic properties, minimal side effects, and cost efficiency.3,4<\/sup> 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<\/sup> The data collected on phytochemicals helps in the discovery of new therapeutic prospects.6<\/sup> However, the therapeutic potential of various plants and their parts that are available in nature has yet to be explored.7<\/sup><\/p>\n

Withania somnifera <\/em>(L.<\/a>)\u00a0Dunal 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<\/sup> 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<\/sup> In Latin, the term \u201csomnifera\u201d means sleep-inducing.10<\/sup> The name \u201cashwagandha\u201d is derived from the Sanskrit words \u201cashva\u201d (horse) and \u201cgandha\u201d (smell), indicating that the root has a strong horse-like scent.11<\/sup> 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<\/sup><\/p>\n

W. somnifera<\/em> is used in over 100 formulations in Indian traditional medicine, including Ayurveda, Unani, and Siddha, and is therapeutically equivalent to ginseng.13<\/sup>W. somnifera<\/em> leaf extract has been shown to be effective against Staphylococcus aureus<\/em> and Enterococcus spp<\/em>.14 <\/sup>Other health benefits of W. somnifera<\/em> 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<\/sup> The medicinal use of ashwagandha for cognitive and neurological diseases, such as anxiety, Parkinson\u2019s disease, and inflammation, is also backed by clinical trials and animal research.16<\/sup> Furthermore, Ashwagandha’s chemopreventive properties make it a potentially effective adjuvant for radiation and chemotherapy patients.17<\/sup> 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 <\/sup>The major phytoconstituents of ashwagandha root are withanolides, which include steroidal alkaloids and steroidal lactones.19<\/sup><\/p>\n

The selection of different plant parts roots, stem and leaf of W. somnifera<\/em> could provide a biological and biochemical basis for identifying new pharmacologically important phytochemicals of therapeutic value.20<\/sup> Extraction and characterization of bioactive compounds from W. somnifera<\/em> have given birth to various phytochemicals with therapeutic importance\u00a0 like\u00a0 anaferine, anahygrine and isopelletierine etc., \u00a0belonging to alkaloids, withaferins as well as withanolides belonging to steroidal lactone compounds, and saponins.21 <\/sup>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<\/sup><\/p>\n

However, there is a lacuna in comparative profiling of\u00a0 phytochemicalsin W. somnifera<\/em> leaf, stem, and root qualitatively and quantitatively.23 <\/sup>Moreover, W. somnifera<\/em>\u00a0 is harvested at reproductive stage for the optimum dry root yield24<\/sup>. 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\u00a0 Gas chromatography\u2013mass spectrometry (GC-MS).25 <\/sup>The GC-MS technique has the highest sensitivity and specificity to detect the presence of phytochemical constituents.26<\/sup> GC-MS analysis has long been the method of choice for determining steroid levels in clinical samples.27 <\/sup>\u00a0Moreover, GC-MS allows effective chromatographic separation, quantification, and identification of sample constituents by using mass spectral libraries.28 <\/sup>Hence, in this study, GC-MS analysis was chosen as a standard approach for phytochemical profiling of leaf, stem and root\u00a0 at reproductive stage in W. sominfera<\/em>.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Preparation of plant extract<\/strong><\/p>\n

W. sominifera <\/em>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\u00b0C using a rotary evaporator to get a viscous semi solid mass.<\/p>\n

GC\u2011MS analysis<\/strong><\/p>\n

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\u00b0C and a temperature of 250\u00b0C 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 \u00b5m. The injector’s temperature was set to 250\u00b0C, and it had a split injection mode. The initial temperature was set at 80\u00b0C for 3 minutes, then the temperature was steadily increased to 280\u00b0C at a rate of 10\u00b0C\/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.<\/p>\n

Identification of phytochemicals<\/strong><\/p>\n

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.<\/p>\n

Results<\/strong><\/p>\n

GC-MS analysis of reproductive stage Leaf<\/strong><\/p>\n

As shown in Figure 1, a total of 16 phytochemicals were exclusively identified in the methanolic leaf extracts viz.<\/em> 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-.<\/p>\n\n\n\n
\"Vol16No1_GC-MS_Sri_fig1\"<\/a><\/td>\nFigure 1:\u00a0GC-MS chromatogram of leaf methanolic extract in W. somnifera<\/em><\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

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%.<\/p>\n

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%.<\/p>\n

Table 1:\u00a0Phytochemicals identified for antimicrobial and anticancer properties in the methanolic leaf extracts of W. somnifera<\/em>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Sl. No.<\/strong><\/td>\nPeak<\/strong><\/td>\nRT<\/strong><\/td>\nName of the compound<\/strong><\/td>\nMolecular formula<\/strong><\/td>\nM.W<\/strong><\/td>\nPeak area (%)<\/strong><\/td>\nTherapeutic Activity<\/strong><\/td>\n<\/tr>\n
1<\/td>\n2<\/td>\n1.115<\/td>\n2-Pentanone, 5-chloro-<\/td>\nC5<\/sub>H9<\/sub>ClO<\/td>\n120<\/td>\n3.29<\/td>\nAntibacterial29<\/sup><\/td>\n<\/tr>\n
2<\/td>\n6<\/td>\n12.820<\/td>\n3-Butoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tris(trimethylsiloxy)tetrasiloxane<\/td>\nC19<\/sub>H54<\/sub>O7<\/sub>Si7<\/sub><\/td>\n590<\/td>\n1.66<\/td>\nAntibacterial30<\/sup><\/td>\n<\/tr>\n
3<\/td>\n8<\/td>\n28.405<\/td>\nBenzaldehyde, 3-methoxy-4-[(trimethylsilyl)oxy]-, O-methyloxime<\/td>\nC12<\/sub>H19<\/sub>NO3<\/sub>Si<\/td>\n253<\/td>\n1.12<\/td>\nAntibacterial31<\/sup><\/td>\n<\/tr>\n
4<\/td>\n16<\/td>\n29.560<\/td>\n2-(7-Methoxymethylphenanthren-3-yl)propan-2-ol<\/td>\nC19<\/sub>H20<\/sub>O2<\/sub><\/td>\n280<\/td>\n1.15<\/td>\nAnticancer and Antibacterial32<\/sup><\/td>\n<\/tr>\n
5<\/td>\n17<\/td>\n29.975<\/td>\nCyclopropanecarbonyl chloride, 1-fluoro-2,2-diphenyl-<\/td>\nC16<\/sub>H12<\/sub>ClFO<\/td>\n274<\/td>\n1.12<\/td>\nAnticancer33<\/sup><\/td>\n<\/tr>\n
6<\/td>\n22<\/td>\n30.960<\/td>\n4′,6-Dimethoxyaurone<\/td>\nC17<\/sub>H14<\/sub>O4<\/sub><\/td>\n282<\/td>\n1.78<\/td>\nAntifungal34<\/sup><\/td>\n<\/tr>\n
7<\/td>\n25<\/td>\n32.630<\/td>\nDibenzo[a,e]cyclooctene, 5,6,11,12-tetrahydro-<\/td>\nC16<\/sub>H16<\/sub><\/td>\n208<\/td>\n1.08<\/td>\nAnticancer35<\/sup><\/td>\n<\/tr>\n
8<\/td>\n29<\/td>\n34.020<\/td>\n2,5-Dimethoxy-4-propoxy-.beta.-methyl-.beta.-nitrostyrene<\/td>\nC14<\/sub>H19<\/sub>NO5<\/sub><\/td>\n281<\/td>\n1.08<\/td>\nAntibacterial36<\/sup><\/td>\n<\/tr>\n
9<\/td>\n31<\/td>\n35.250<\/td>\n1,3-Dihydroxy-2,4,5-trifluoro-6-nitrobenzene<\/td>\nC6<\/sub>H2<\/sub>F3<\/sub>NO4<\/sub><\/td>\n209<\/td>\n1.24<\/td>\nAntibacterial37<\/sup><\/td>\n<\/tr>\n
10<\/td>\n37<\/td>\n36.980<\/td>\n1-Phenazinecarboxylic acid, 6-(1-methoxyethyl)-, methyl ester<\/td>\nC17<\/sub>H16<\/sub>N2<\/sub>O3<\/sub><\/td>\n296<\/td>\n1.23<\/td>\nAntibacterial38<\/sup><\/td>\n<\/tr>\n
11<\/td>\n40<\/td>\n39.066<\/td>\ncis,syn,cis-Perhydrophenanthrene<\/td>\nC14<\/sub>H24<\/sub><\/td>\n192<\/td>\n1.40<\/td>\nAnticancer39<\/sup><\/td>\n<\/tr>\n
12<\/td>\n47<\/td>\n42.525<\/td>\nBenzene, 1,1′-(1,2-ethenediyl)bis[2-methyl-<\/td>\nC16<\/sub>H16<\/sub><\/td>\n208<\/td>\n1.41<\/td>\nAntibacterial and Anticancer40<\/sup><\/td>\n<\/tr>\n
13<\/td>\n21<\/td>\n30.740<\/td>\nBenzene, 1,2,3-trimethoxy-5-(2-propenyl)-<\/td>\nC12<\/sub>H16<\/sub>O3<\/sub><\/td>\n208<\/td>\n2.77<\/td>\nAntifungal41<\/sup><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

GC-MS analysis of reproductive stage root:<\/strong><\/p>\n

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.<\/p>\n\n\n\n
\"Vol16No1_GC-MS_Sri_fig2\"<\/a><\/td>\nFigure 2: GC-MS chromatogram of root methanolic extract \u00a0in Withania somnifera<\/em>.<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

The phytochemicals identified with antimicrobial properties in the methanolic root extracts is given in Table 2. The phytochemicals\u00a0 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. \u00a0Phytochemicals 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%.<\/p>\n

Table 2:\u00a0\u00a0Phytochemicals identified for antimicrobial and anticancer activity in methanolic root extracts of W. somnifera<\/em>.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
Sl.<\/strong><\/p>\n

No.<\/strong><\/td>\n

Peak<\/strong><\/td>\nRT<\/strong><\/td>\nName of the compound<\/strong><\/td>\nMolecular formula<\/strong><\/td>\nMW<\/strong><\/td>\nPeak area (%)<\/strong><\/td>\nTherapeutic Activity<\/strong><\/td>\n<\/tr>\n
1<\/td>\n10<\/td>\n28.290<\/td>\nTris(trimethylsilyl)hydroxylamine<\/td>\nC9<\/sub>H27<\/sub>NOSi3<\/sub><\/td>\n249<\/td>\n1.18<\/td>\nAntibacterial28<\/sup><\/td>\n<\/tr>\n
2<\/td>\n15<\/td>\n29.531<\/p>\n

 <\/td>\n

4-(2,6,6-Trimethylcyclohexa-1,3-dienyl)pent-3-en-2-ol<\/td>\nC14<\/sub>H22<\/sub>O<\/td>\n206<\/td>\n2.36<\/td>\nAntibacterial42<\/sup><\/td>\n<\/tr>\n
3<\/td>\n16<\/td>\n29.765<\/td>\nStannane, 1,3-dithian-2-ylidenebis[trimethyl-<\/td>\nC10<\/sub>H24<\/sub>S2<\/sub>Sn2<\/sub><\/td>\n448<\/td>\n1.57<\/td>\nAntibacterial43<\/sup><\/td>\n<\/tr>\n
4<\/td>\n27<\/td>\n34.230<\/td>\n2-{4-[2-(4-Methoxymethylphenyl)vinyl]phenyl}propan-2-ol<\/td>\nC19<\/sub>H22<\/sub>O2<\/sub><\/td>\n282<\/td>\n1.23<\/td>\nAnticancer and Antimicrobial32<\/sup><\/td>\n<\/tr>\n
5<\/td>\n40<\/td>\n38.505<\/td>\nBenzenepropanoic acid, 4-benzoyl-, methyl ester<\/td>\nC17<\/sub>H16<\/sub>O3<\/sub><\/td>\n268<\/td>\n1.75<\/td>\nAntibacterial44<\/sup><\/td>\n<\/tr>\n
6<\/td>\n44<\/td>\n39.580<\/td>\nUleine<\/td>\nC18<\/sub>H22<\/sub>N2<\/sub><\/td>\n266<\/td>\n1.90<\/td>\nAnticancer and Antibacterial45<\/sup><\/td>\n<\/tr>\n
7<\/td>\n34<\/td>\n36.850<\/td>\nBenzoic acid, 3-methyl-2-trimethylsilyloxy-, trimethylsilyl ester<\/td>\nC14<\/sub>H24<\/sub>O3<\/sub>Si2<\/sub><\/td>\n296<\/td>\n3.53<\/td>\nAntibacterial46<\/sup><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

GC-MS analysis of reproductive stage stem<\/strong><\/p>\n

As shown in Figure 3, a total of 18 phytochemicals were exclusively identified in the methanolic stem extracts. The phytochemicals identified included \u00a0viz. 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-.<\/p>\n\n\n\n
\"Vol16No1_GC-MS_Sri_fig3\"<\/a><\/td>\nFigure 3:\u00a0GC-MS chromatogram ofmethanolic stem extract in W.somnifera<\/em>.<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

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.<\/p>\n

Table 3: Phytochemicals identified for antimicrobial and anticancer in reproductive stage stem extracts of W.somnifera<\/em>.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Sl. No<\/strong><\/td>\nPeak<\/strong><\/td>\nRT<\/strong><\/td>\nName of the compound<\/strong><\/td>\nMolecular formula<\/strong><\/td>\nMolecular weight<\/strong><\/td>\nPeak area (%)<\/strong><\/td>\nTherapeutic Activity<\/strong><\/td>\n<\/tr>\n
1<\/td>\n4<\/td>\n1.170<\/td>\nAcetohydroxamic Acid<\/td>\nC2<\/sub>H5<\/sub>NO2<\/sub><\/td>\n75<\/td>\n3.12<\/td>\nAntibacterial5<\/sup><\/td>\n<\/tr>\n
2<\/td>\n9<\/td>\n27.030<\/td>\nPentasiloxane, dodecamethyl-<\/td>\nC12<\/sub>H36<\/sub>O4<\/sub>Si5<\/sub><\/td>\n384<\/td>\n1.12<\/td>\nAntibacterial47<\/sup><\/td>\n<\/tr>\n
3<\/td>\n12<\/td>\n27.800<\/td>\n1,4-Dibromo-2,3-butanediol<\/td>\nC4<\/sub>H8<\/sub>Br2<\/sub>O2<\/sub><\/td>\n246<\/td>\n2.01<\/td>\nAntileishmanial48<\/sup><\/td>\n<\/tr>\n
4<\/td>\n13<\/td>\n28.510<\/td>\ntrans-2,3,6-Trimethoxy-b-methyl-b-nitrostyrene<\/td>\nC12<\/sub>H15<\/sub>NO5<\/sub><\/td>\n253<\/td>\n1.06<\/td>\nAntibacterial36<\/sup><\/td>\n<\/tr>\n
5<\/td>\n20<\/td>\n29.395<\/td>\nBenzylamine, 2-hydroxy-N,N-di-[2-aminoethyl]-<\/td>\nC11<\/sub>H19<\/sub>N3<\/sub>O<\/td>\n209<\/td>\n1.43<\/td>\nAntibacterial49<\/sup><\/td>\n<\/tr>\n
6<\/td>\n22<\/td>\n29.804<\/td>\nAndrost-9(11)-en-17-one, 3-[(trimethylsilyl)oxy]-, O-methyloxime<\/td>\nC23<\/sub>H39<\/sub>NO2<\/sub>Si<\/td>\n389<\/td>\n1.21<\/td>\nAntibacterial50<\/sup><\/td>\n<\/tr>\n
7<\/td>\n25<\/td>\n30.970<\/td>\n3-Acetoxy-2,3′-bibenzo[b]thiophene<\/td>\nC18<\/sub>H12<\/sub>O2<\/sub>S2<\/sub><\/td>\n324<\/td>\n1.11<\/td>\nAnticancer and Antibacterial47,51<\/sup><\/td>\n<\/tr>\n
8<\/td>\n26<\/td>\n32.579<\/td>\n1,3,5,7-Tetraethyl-1-oxycyclotetrasiloxane<\/td>\nC8<\/sub>H24<\/sub>O5<\/sub>Si4<\/sub><\/td>\n312<\/td>\n1.51<\/td>\nAntibacterial52<\/sup><\/td>\n<\/tr>\n
9<\/td>\n27<\/td>\n33.825<\/td>\n1,3-Methylene-d-arabitol<\/td>\nC6<\/sub>H12<\/sub>O5<\/sub><\/td>\n164<\/td>\n1.13<\/td>\nAntibacterial53<\/sup><\/td>\n<\/tr>\n
10<\/td>\n32<\/td>\n36.255<\/td>\n3.alpha.,4.alpha.,9.beta.,11-Diepoxymuurolan-10-ol<\/td>\n <\/p>\n

C15<\/sub>H24<\/sub>O3<\/sub><\/td>\n

252<\/td>\n1.74<\/td>\nAntibacterial54<\/sup><\/td>\n<\/tr>\n
11<\/td>\n37<\/td>\n40.350<\/td>\nBenzenamine, N-(3,4,5,6-tetraethyl-1-phenyl-2(1H)-pyridinylidene<\/td>\nC25<\/sub>H30<\/sub>N2<\/sub><\/td>\n358<\/td>\n1.15<\/td>\nAntibacterial55<\/sup><\/td>\n<\/tr>\n
12<\/td>\n41<\/td>\n41.310<\/td>\n3-(3-Ethoxy-4-hydroxyphenyl)-2-isothiocyanatopropionic acid, ethyl<\/td>\nC17<\/sub>H25<\/sub>NO4<\/sub>SSi<\/td>\n367<\/td>\n1.15<\/td>\nAntibacterial56<\/sup><\/td>\n<\/tr>\n
13<\/td>\n43<\/td>\n41.619<\/td>\nBenzeneacetic acid, .alpha.,3,4-tris[(trimethylsilyl)oxy]-, trimethylsilyl<\/td>\nC20<\/sub>H40<\/sub>O5<\/sub>Si4<\/sub><\/td>\n472<\/td>\n1.18<\/td>\nAntibacterial57<\/sup><\/td>\n<\/tr>\n
14<\/td>\n46<\/td>\n44.074<\/td>\nSilanamine, N-[(4-methoxyphenyl)methyl]-1,1,1-trimethyl-<\/td>\nC11<\/sub>H19<\/sub>NOSi<\/td>\n209<\/td>\n1.92<\/td>\nAntibacterial58<\/sup><\/td>\n<\/tr>\n
15<\/td>\n10<\/td>\n27.480<\/td>\nCyclotetrasiloxane, octamethyl-<\/td>\nC8<\/sub>H24<\/sub>O4<\/sub>Si4<\/sub><\/td>\n296<\/td>\n1.33<\/td>\nAntibacterial47<\/sup><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

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. <\/em>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\u00a0 identified at RT. 35.215.<\/p>\n\n\n\n
\"Vol16No1_GC-MS_Sri_fig4\"<\/a><\/td>\nFigure 4: (a-e). Phytochemicals with steroid structures identified in the W. sominifera <\/em>methanolic root extracts.<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Discussion<\/strong><\/p>\n

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

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 <\/sup>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<\/em>. In the methanolic leaf extracts, the identified phenolic compounds 1,2-bis(trimethylsilyl)benzene have previously been reported for antibacterial and anticancer activities,65 <\/sup>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 <\/sup>and phenolic compound 1,3-Dihydroxy-2,4,5-trifluoro-6-nitrobenzene \u00a0is \u00a0a nitrobenzene derivative shown to have antitumor and antibacterial properties.67, 41 <\/sup>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 <\/sup>Phenolic stilbenes1-Methyl-1,2,2-triphenylindan has been reported to be antibacterial in property,69 <\/sup>silyl ethers 1,3,5,7-tetraethyl-1-butoxycyclotetrasiloxane has been reported to be antibacterial properties,70<\/sup> 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 <\/sup>and phenolic compounds 2-(7-methoxymethylphenanthren-3-yl)propan-2-ol has been reported to be both anticancer and antimicrobial in properties.71<\/sup><\/p>\n

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 <\/sup>Amines compounds tris(trimethylsilyl)hydroxylamine a hydroxylamine derivative, has been reported to be antibacterial properties.28 <\/sup>Ester compound\u00a0 boric acid, trimethyl ester has been reported to be anticancer properties69<\/sup> and dextroamphetamine compound was reported to be brain stimulant and antibiotic.72 <\/sup>Steroid compound morphinan found in the Opium poppy (Papaver somniferum<\/em>),73 <\/sup>has been identified in this study in the root extracts \u00a0of W. somnigfera<\/em> which was found to have antibacterial properties.74 <\/sup>Somephytochemicals identified in the root and stem methanolic extracts alkanes like ethane, 1,2-dichloro-1-ethoxy which is antimicrobial,75 <\/sup>steroid amines like dextroamphetamine, acids like boric acid, trimethyl which is anticancer,76 <\/sup>ketones like tartronic acid, 4-(dimethylethylsilyl)phenyl-, dimethyl ester, which is antibacterial,77<\/sup> and aromatic acid like 3,5-dichloro-4-hydroxybenzoic acid which is antimicrobial in properties.78<\/sup><\/p>\n

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 <\/sup>The identified terpenoid phytoalexins such as acetohydroxamic acid has been reported to be antibacterial in the previous study.5 <\/sup>Identified alkanes such as pentasiloxane, dodecamethyl- has previously been reported to be anticancer,79<\/sup> and aromatic nitroalkene such as trans-2,3,6-trimethoxy-b-methyl-b-nitrostyrene which is \u03b2-nitrostyrene derivatives compounds has been reported to be antibacterial.36<\/sup><\/p>\n

Some steroids compounds identified in this study which include \u03b1-D-glucopyranoside, methyl 2-(acetylamino)-2-deoxy-3-O-(trimethylsilyl)-, cyclic methylboronate were reported to be found in the flowers of Jacaranda mimosifolia, <\/em>that poses antibacterial properties80 <\/sup>and its derivatives like methyl-\u03b1-D-glucopyranoside in Tulbghia violacea,<\/em>70 <\/sup>and \u03b1-D-glucopyranoside,O-\u03b1-D-glucopyranosyl- (1.fwdarw.3)-\u00df-D-fructo in Foeniculum vulgare<\/em>81 <\/sup>were reported to be anticancer in properties. Similarly, carbromal is found in Decalepis hamiltonii,<\/em>82<\/sup> has been used to treat mild insomnia.83 <\/sup>Derivatives of novel steroids 19-norpregn-5(10)-en-20-yn-3-one84<\/sup> 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.<\/em>85<\/sup> Cucurbitacin b is an effective anticancer and antibacterial agent,57 <\/sup>found in the Cucurbitaceae plant families.87<\/sup><\/p>\n

This study agrees with previous researchers of W. somnifera <\/em>to be antimicrobial and anticancer in properties.88,89<\/sup> Moreover, this GC-MS investigation rationally evaluated and identified the phytochemicals that attributes the antimicrobial and anticancer properties of W. somnifera<\/em>. Further research is needed to isolate and purify the identified phytochemicals for bioactivities in order to develop W. somnifera<\/em> herbal based products for \u00a0applications in the antimicrobial and anticancer therapies.<\/p>\n

Conclusion<\/strong><\/p>\n

The present study describes comparative GC-MS analysis of methanol extract of W. sominfera <\/em>phytochemicals distribution in the leaf, stem, and root.\u00a0 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<\/em>, 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.).<\/em>This information can be utilized further to develop W. somnifera <\/em>based traditional herbal medicines that are playing an important role in healthcare system.<\/p>\n

Acknowledgement<\/strong><\/p>\n

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.<\/p>\n

Conflicts of Interest<\/strong><\/p>\n

The authors declare no conflicts of interest<\/p>\n

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