GC-MS Profiling of Reproductive Stage Withania somnifera for Antimicrobial and Anticancer Phytochemicals

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. 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). 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. In this study, phytochemicals with antimicrobial and anticancer properties were identified in leaf, root and stem parts of W. somnifera at reproductive stage.

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. 13 W. 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 in India. 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 yield 24 . 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.
The study mentioned the rationale for selecting the root at plant's reproductive stage. Why do they compare the same with leaf and stem? This needs some clarification or explanation.
Rationale for selecting only methanol why not other solvents?

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.

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 costefficient 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-[2,4bis(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-2trimethylsilyloxy-, 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 properties 69 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 â-nitrostyrene derivatives compounds has been reported to be antibacterial. 36 Some steroids compounds identified in this study which include á-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 properties 80 and its derivatives like methyl-á-D-glucopyranoside in Tulbghia violacea, 70 and á-D-glucopyranoside,Oá-D-glucopyranosyl-(1.fwdarw.3)-ß-D-fructo in Foeniculum vulgare 81 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-one 84 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.
The authors should have been compared the GC-MS analysis between the three parts during activity growing stage and reproductive stage in discussion part, so that a wider knowledge have been developed. Usually actively growing plant will not produce secondary metabolites unless it undergoes a variety of stress.
The work is under progress, we will report in our next publication.