Metabolomics characterization of Senna tora (L.) Roxb. using different approaches

The present study aimed to investigate the variety of elements, chemical compounds and their corresponding functional groups in the whole plant, leaves, and seeds of Senna tora . A preliminary phytochemical analysis has revealed the presence of secondary metabolites including alkaloids, flavonoids, tannins, terpenoids, cardiac active glycosides, phenolics, etc . Gas Chromatography and Mass Spectrophotometry (GC-MS) analysis of leaves and seeds of S. tora has depicted 31 and 27 compounds, respectively. Fourier Transform Infrared (FT-IR) Spectroscopy has further unveiled the presence of different functional groups such as amines, aromatic compounds, carboxyl groups, ketones etc. associated with different metabolites . Wavelength Dispersive X-ray Fluorescence (WD-XRF) has revealed the presence of more than 20 elements (macro and micro) including Ca, Mg, Fe, K, etc . This study has highlighted the detailed account of the chemical compounds and elements present in the plant species under investigation and substantiated its medicinal importance in the traditional health care system.


INTRODUCTION
India is one of the top 12 mega biodiversity countries of the world and possesses a unique plant diversity and variety of plants in general and medicinal plants in particular. The richness of Indian floristic diversity is due to altitudinal, climatic and ecosystems variations in different parts of the country. Because of the medicinal value, plant species have their mention in Ayurveda, Unani, Siddha, etc. (Singh & Chowdhery, 2002). Medicinal plants, therefore, are being indiscriminately since by nature for a long time. Certain weeds or Neglected Underutilized Species (NUS) have not received much attention because of crops, intensive agronomic practices, social and cultural reasons. Limited cultivation and increased demand for herbal species has threatened the existence of certain species of medicinal importance. Due to this apprehension, there is an urgent need to plan effective conservation strategies to conserve plant diversity. Large numbers of NUS species contain phytoconstituents of medicinal value (Ugbaja et al., 2017).
Different species of the genus Cassia L. are being used in various health care medicinal preparations. Different species genus are occurring throughout the world out of which nearly 49 (Cassia, Senna, Chamaecrista) are growing in India (Efloraofindia, 2007;Kabila et al., 2020). Senna tora (Syn Cassia tora) is a member of the family Fabaceae and subfamily Caesalpinioideae (Singh et al., 2013). It is useful in skin diseases such as ringworm, eczema, scabies, rheumatic, asthma and also has hepatoprotective, anti-helminthic and anti-inflammatory activities. Its medicinal activity is likely due to the presence of various chemical compounds and elements (Choudhary et al., 2011;Mate et al., 2013;Vijayalakshmi et al., 2015).
Senna tora is a small foetid, herb or undershrub weed species (Figure 1), growing in tropical and subtropical regions. It is a native of southeastern Asia and grows well in Malaysia, Japan, Burma, Bangladesh, India, etc. This species has also been referred to as a destructive weed throughout the country (Kabila et al., 2017). The plant body is spreading with branches and has a glabrous stem. Leaves are long stipulate, linear, membranous or lanceolate. Leaflets are in three pairs, obovate, nearly equal on both sides, both surfaces of the leaflets are pubescent, glabrous with entire margin. The yellow colored gland is located between the lowermost pair of the leaflets. The inflorescence is an axillary raceme with bright yellow flowers, long-stalked in pairs with a short peduncle, linear bracts and acute pubescent. Sepals are 5 (3 large, 2 small) ovate, glabrous, acute apex; petals are 5 (bright yellow), subequal, oblong and upper petal is truncate; stamens are 10 with 3 staminodes and 7 antheriferous (3 large

Preparation of Extracts
The fresh plant material was washed, first with tap water followed by distilled water. The material was then allowed to dry at room temperature. The completely dried material was powdered using an electric grinder. The powdered material was stored in air tight container for further use.

GC-MS Analysis
GC-MS analysis was carried out using thermo trace 1300GC coupled with Thermo TSQ 8000 triple quadrupole MS (for GC-Thermo Trace 1300 GC; for MS-Thermo TSQ 8000) at Central Instrumentation Laboratory, Panjab University, Chandigarh. Column TG 5MS was composed of 5% Diphenyl; 95% dimethyl polysiloxane operating in electron impact mode and helium was used as carrier gas at a constant flow of 1.5 mL/min and an injection volume of 1µl was employed (Split ratio 33.3) at an injected temperature of 250 o C. The oven temperature was programmed for 60 o C with a hold time of one minute and an increase of 10 o C/min at 220 o C withhold time of four minute. Mass spectra transfer line temperature is 250 o C, Ion source temperature was 230 and Mass range from 50-700 o C. The compound identification was done by comparison of retention time and mass spectra of GC-MS. The compounds were identified using the Database of National Institute of Standards and Technology (NIST) Library 2.0.

FT-IR Spectroscopy
Fourier Transform Infrared (FTIR) Spectroscopy classify the chemical bonds/functional groups relevant to the phytochemicals. The chemical bonds in the spectrum absorb light of a particular wavelength. Thus, chemical bonds reported in the compounds can be resolved by interpreting the IR absorption spectrum (Visveshwari et al., 2017). The test has been performed using Perkin Elmer Spectrum 400 FT-IR/FT-FIR spectrometer with a scan range from 400 to 4000 cm -1 . Plant samples in the form of powder were used for analysis.

WD-XRF Analysis
WD-XRF analysis was performed to detect the elements (atoms, ions) in the studied samples. The analysis was carried out by instrument, Wavelength Dispersive X-ray fluorescence (WD-XRF), Model: S8 TIGER, Bruker, Germany at Central Instrumentation Laboratory, Panjab University, Chandigarh. X-Ray Tube: Anode material 'Rhodium', A sample pellet was made using hydraulic press and a pressure of 10 tons. Approx. the sample used for pellet was 3-4 gms, sample thickness ~ 2 mm (minimum thickness required than 1.5 mm), Sample diameter -34 mm, analysis time ~ 20 mins and software used for the analysis was SpectraPlus.

Phytochemical Screening
The phytochemical screening of Senna tora (whole plant, leaves, and seeds) was carried out using aqueous, ethanol and hexane extracts. Most of the studied phytochemicals were present in aqueous and ethanol extracts whereas, hexane extract yielded the least (Table 1). Ethanol extracts of the whole plant, seeds, and aqueous extract of seeds contained maximum number of phytoconstituents as compared to other extracts. Carbohydrates were observed in all the extracts. Similarly, cardiac active glycosides were also present in all the extracts except whole plant hexane extract. Alkaloids, anthraquinones, coumarins, diterpenoids, flavonoids, phenolics, quinones, reducing sugar, starch and tannins were present in all the extracts except hexane.
Alkaloids and tannins in methanol extract, steroid in petroleum ether and chloroform extracts of C. tora leaves were reported by Mazumder et al. (2005). Das et al. (2011) studied the phytoconstituents of Cassia tora stem bark in five different solvents and reported maximum phytochemicals including alkaloids, flavonoids, tannins, protein, steroids and glycosides in methanol extract followed by diethyl ether and ethyl acetate. Ethanol, methanol and ethyl acetate extracts of (Cassia tora) possess alkaloids, steroids, terpenoids, cardiac glycosides, carbohydrates and proteins whereas tannins and phlobatannins were reported only in methanol and ethanol extracts (Veerachari & Bopaiah, 2012). John et al. (2012) also reported the presence of tannins, anthraquinones, flavonoids, glycosides and coumarins in methanol and ethyl acetate leaves extracts of C. tora but methanol extract showed only steroids, cardiac glycosides, amino acids and saponins. The leaves of C. tora contain alkaloids, anthraquinones, flavonoids, phenolics and proteins in methanol and aqueous extracts (Khan et al., 2016). Among the leaves, bark, seeds and pods (containing seeds), ethyl acetate and methanol extracts of leaves showed all the studied phytoconstituents but hexane extract revealed the presence of only steroids (Khatak et al., 2014).
Screening of phytoconstituents in Senna tora had shown the presence of alkaloids, phenols, saponins, carbohydrates, glycosides and protein in aqueous, ethyl acetate and hexane extracts of leaf and seeds (Sabyasachi et al., 2016). Rao and Chattrerjee (2016) studied the aerial parts of S. tora and reported the presence of alkaloids, triterpenoids, steroids and tannins. Similarly, Asba and Meeta (2017) studied various phytoconstituents in roots, leaves, stem, flowers and pods of C. tora in different solvents (six solvents) and most of the phytochemicals were reported from the aqueous and methanol extract as compared to other solvent extracts. Suradkar et al. (2017) and Sahu et al. (2017) reported the presence of alkaloids, flavonoids, saponins, tannins, phenols, steroids etc. in ethanol, aqueous and methanol extracts of the leaves. These have also been reported during the present study in aqueous and ethanol extracts. According to the available literature, most of the studies have been conducted on the leaves and seeds of S. tora and these were the most preferred parts along with methanol as a solvent for phytochemical investigations. Phytochemical screening of the seed extract of Cassia tora revealed the presence of carbohydrates, glycosides, saponins and triterpenes in petroleum ether, ethanol and aqueous extracts (Roopashree et al., 2008).
It is pertinent to mention that none of the extracts contain amino acids and anthocyanin during the present investigation. However, some metabolites like anthraquinones, betaxanthin, cardiac-active glycosides, coumarins, diterpenoids, lignins, oxalate, phenolics, quinones and resin have been reported for the first time from this plant and its parts (leaves and seeds) in different solvents.

20.
Ethyl à-d-glucopyranoside C₈H₁₆O₆ 208.21 Antimicrobial, antioxidant, anti-inflammatory, hypocholesterolemic and anti-cancer, (Chidambaram et al., 2015), anticancer, anti-leukotriene, decreases epinephrine production, Dopaminergic and RNA depressant (Duke, 1992) 21. 5-Thio-D-glucose C₆H₁₂O₅S 196.22 Antispermatogenic and radioprotective (Homm et al., 1977;Schuman et al., 1982), antidote, anticancer, CNS depressant, Decongestant, diuretic and detoxicant (Duke, 1992) (Duke, 1992) Earlier, Vats and Kamal (2014) studied C. tora methanol extract and reported phenolic content, C=O carbonyl group stretching, some peaks of flavonoid compounds, OH groups and alkyl and aryl ethers. Screening of different functional groups from leaves of C. tora has revealed aromatic amines, aldehydes, amines (aliphatic and aromatic), alkanes, ethers, phenols, ketones, carboxylic acid and nitro compounds (Khyade et al., 2015). Similarly, Kumar and Roy (2018) studied the different populations of Cassia tora from different geographical regions.The FTIR spectrum has yielded functional groups like carbonyl group, C-H, C=C, C-N and C-O-C (ethers), aldehydes and carbohydrates. Polysulphides, aryl sulphides, aliphatic iodine are reported exclusively during the present study. Leaves are the most preferred part for FTIR studies and it contains more phytoconstituents in comparison to whole plant and seeds. Some functional groups such as transition metal carbonyl, alkyl carbonyl, alcohols, esters and anhydrides are present only in whole plant and seeds.

Elemental Analysis
Maximum numbers of elements have been reported from the leaves followed by whole plant and seeds ( Figure 5, Table 5). The amount of calcium was highest in leaves (48.9 mg/g), potassium in the whole plant (23.5 mg/g) and phosphorus in seeds (10.8 mg/g). The sodium, titanium and bromine were present in the whole plant and leaves, whereas molybdenum and zirconium were present in seeds and leaves respectively. Some of the reported elements are important for human health care system but their number and concentration vary in different species which may also be altered by the geographical and climatic conditions, soil nature and accumulation capacity of the plant species. Elements like sodium, potassium, calcium, magnesium etc. play a vital role in the regulation of human physiological processes (Silva et al., 2016). Kubmarawa et al. (2011) reported calcium (3.52 mg/g) and magnesium (0.86 mg/g) which is lesser than the observed 48.9 mg/g and 3.7 mg/g in the present study. Other elements like phosphorus, iron, sodium, zinc, manganese, cobalt and potassium were also present in different concentrations. The elemental details of C. tora leaves are comprised of magnesium, calcium, sulphur, iron, sodium and chlorine (Shaikh & Sayed, 2015). In addition to the previous reports, elements like silicon, aluminium, copper, lead, strontium etc have been reported in leaves of S. tora for the first time. Similarly, the number of essential minerals and trace elements, such as potassium, chlorine, phosphorus, sulphur,  c aluminium, iron, sodium, nickel, chromium and bromine were also studied in the whole plant and seeds for the first time during this study.
Calcium is good for bones, teeth and muscles whereas magnesium helps in the management of heart diseases and repair damaged cells. Similarly, iron prevents anaemia and boost immunity and chromium control sugar levels, lipid homeostasis and diabetes (Balk et al., 2007;Larsson & Wolk, 2007;Agunbiade et al., 2012). Sulphur is active against acne, eczema, psoriasis, skin irritation, microbes, involved in proteins synthesis, cell regeneration, blood purification, insolubility of keratin and provide strength to skin hair due to disulfide bonds (Afolayan & Otunola, 2014;Haque et al., 2015). Researchers have added silicon, arsenic, chromium, molybdenum and vanadium to the list of essential elements. The presence of these elements (macro and trace) in S. tora is an indicator of its medicinal potential.

CONCLUSION
Senna tora, a traditional medicinal plant, has been evaluated for the presence of phytoconstituents, elements, chemical compounds and their related functional groups. The ethanol extract has shown the presence of maximum phytochemicals followed by aqueous and hexane extracts. The WD-XRF analysis has revealed the presence of various elements including calcium, potassium, magnesium, sulphur, iron, zinc, manganese etc. GC-MS of leaves and seeds has depicted various chemical compounds of medicinal importance including anticancer, antibacterial, antitumor, antioxidant, diuretic etc. The ethanol extract of leaves and seeds had shown the presence of compounds like Silicic acid, diethyl bis(trimethylsilyl) ester, 1-Monolinoleoylglycerol trimethylsilyl ether, Hexadecen-1-ol, trans-9-, Ethyl à-d-glucopyranoside and 10-Heneicosene (c,t) etc. Some of these are precursors of the modified derivatives for the compound drug analysis. FTIR spectroscopy has shown various functional groups supporting the presence of important phytochemicals like alcohols, amines, carbonyls, alkyl carbonate, ethers and esters. Thus, the observations of the present study have explained the potentiality of Senna tora in the pharmaceutical sector. This species can provide raw materials for the preparation of new or alternate medicine. Further studies are required for the isolation and characterization of anticancer, antitumour, antidiabetic and antibacterial compounds.

ACKNOWLEDGEMENTS
I Bhavna Kabila is thankful to the Department of Science and Technology, Government of India New Delhi for financial