Phytochemical and chromatographic characterization ofMimosa diplotricha Wright ex. Sauvalle ethanolic leaf extract and assessment of antioxidant potential and anti-proliferative effect on colorectal cancer (HCT-116) cell lines

Present study aims to evaluate phytochemical and chromatographic pro ile, along with antioxidant and anti proliferative properties ofMimosa diplotricha ethanolic leaf extract. Qualitative screening of phytoconstituents by consecutive solvent extraction in increased polarity basis and standardization of potential extract based on phytochemical elution pro ile was done. Selected secondary metabolites like phenols, lavanoids, tannins and alkaloids were quanti ied in ethanolic extract. Chromatographic pro ile was determined by HPTLC and GC-MS analysis. In vitro antioxidant potential was assessed by DPPH, superoxide, nitric oxide, hydrogen peroxide and hydroxyl radical scavenging assay. Ferric-reducing antioxidant power assay (FRAP) and reducing potential of the respective extract were also determined. Anticancer potential was con irmed by cytotoxic screening in colorectal (HCT-116) cancer cell lines by MTT assay. Qualitative phytochemical analysis and chromatographic pro ile reveal a phytoconstituent rich pro ile for the ethanolic leaf extract. The amount of, phenols (56 ± 0.57 mg/g), lavanoids (27 ± 0.76 mg/g), tannins (33± 0.15 mg/g) were quanti ied as equivalent of gallic acid, quercetin and tannic acid standards respectively and alkaloids (2.51 ± 0.47 mg/g of extracted plant material) were expressed based on respective analysis. Results also reveal convincing antioxidant potential for respective extract. In vitro cytotoxicity con irmed byMTT assay represents an IC50 value of 97.82 μg/ml. From the above results it can be concluded that M.diplotricha has got pharmacologically signi icant phytoconstituents and therapeutic active ingredients as evident in HPTLC and GC-MS analysis. This is further supported by considerable antioxidant and anti proliferative properties observed in respective assays.


INTRODUCTION
Plant kingdom with its medicinally signi icant herbs and pharmacologically active phytochemical principles constitute the foundation basis of therapeutics historically, traditionally and also in folk lore remedies. Literature reference of plants as cure to pathogenesis dates back to vedic times (Rigveda, 4500-1600 BC); Egyptian papyrus, 1550 BC and Hippocrates holistic system which sets the beginning of modern system of medicine (Agrawal and Paridhavi, 2007;Atanasov et al., 2015). Secondary metabolites of vegetable kingdom constituting phenols, alkaloids, tannins, lavanoids, terpenoids, glycosides, steroids etc. manifest experimentally validated reports on antioxidant, antimicrobial, antiin lammatory, antifungal, anticancer and antidiabetic potential and also secondary metabolites represent major raw material for drug development (Nasseri et al., 2019;Natarajan and Yadav, 2018). These phytoconstituents represent the fundamental ingredients for developing and designing potent drugs from plants. According to World Health Organisation (WHO) herbal drugs serves as major source of primary curative medicine for about 80% of population of developing countries because of low cost and easy availability (Calixto, 2005). National Centre for Complementary and Integrative Health of National Institute of Health (NIH) maintains records and funds research based on phytotherapeutics (Burstein et al., 1999).
Cancer is a serious menace globally in the present scenario and numerous phytochemicals are screened to develop potential anticancer drug. Abnormal proliferation of cells, ability to overcome apoptosis and metastasis is the main characteristic feature of cancer cells leading to tumorogenesis mediated by a cascade of molecular signaling pathways. Phytoconstituents especially polyphenols and isolated compounds like curcumin, resveratrol, catechin, genistein etc. have clinically proven to be bene icial in cancer therapy (Elvin-Lewis, 2001;Li, 2015). As per FDA (Food and Drug Administration) 60-75% phytochemicals and isolated active compounds are approved as valid chemo preventive agents (Jacobs, 2018). Phytochemicals currently practiced as anticancer chemo drugs include; taxol, vinblastine, vincristine, camptothecin derivatives, topotecan, etoposide and irinotecan. Phytochemicals are potentially rich in antioxidant bioactive principles and oxidative stress is directly correlated to cancer onset and progression (Narmadha and Devaki, 2012;Perumal et al., 2012). Damage to biomolecules like DNA, lipids and proteins as a result of oxidative stress contributes to the onset of various types of malignancies.
Mimosa diplotricha, a species which occupy subfamily Mimosoideae of legume family (Fabaceae), comprises 40 genera and more than 2000 species. Distribution is pantropical with few species occupying temperate region (Aguiar et al., 2012). It is a perennial shrub, highly invasive and characterized by scrambling or straggling habit. Economically M.diplotricha is signi icant as green manure and frequently used in crop rotation due to its nitrogen ixing potential. Information regarding phytochmecial composition and medicinal uses of M.diplotricha is scanty. In Formozan folk remedies roots of the plant is used as an antidote, analgesic, tranquilizer, anticancer supplement and hemostatic agent (Chirumbolo, 2012). Plant is rich in polyphenols as peculiar to other Fabaceae members and polyphenols are reported to have antioxidant, antimicrobial, anticancer and anti-in lammatory properties (Lin et al., 2011). There are reports of few isolated compounds like pinoresinol, salicifoliol, quercetin, deoxy lavones, chalcones and diterpenoids from aerial parts and roots of M.diplotricha (Chiou et al., 2016;Trease and Evans, 1996). Present study is focused to evaluate phytochemical pro ile by qualitative and quantitative means, chromatographic evaluation by HPTLC & GC-MS analysis, antioxidant and in vitro antiproliferative properties of M.diplotricha ethanolic leaf extract on colorectal cancer (HCT-116) cell lines.

Preparatory phase collection of sample, purchase of chemicals and cell line
Samples of M. diplotricha were procured from Palakkad District, Kerala, South India in the month of July-August.
The specimens were identi ied, authenticated (Botanical Survey of India) and the respective voucher number is BSI/SRC/5/23/2018/Tech./ 2040. All chemicals required for study were obtained from Sigma-Aldrich (St. Louis, MO) and HiMedia laboratories (India) and of analytical grade. Colon cancer cell lines (HCT-116) were obtained from National Centre for Cell Science (NCCS), Pune.

Qualitative Phytochemical studies
Collected plant specimens were washed in distilled water and shade dried. The powdered sample is then subjected to extraction in organic solvents sequentially in ascending order of polarity; petroleum ether, chloroform, ethyl acetate, ethanol and water respectively. Each consecutive extract is then analysed for characteristic phytochemical pro ile including, alkaloids, lavanoids, phenols, tannins, steroids, cardioglycosides, carbohydrates, aminoacids/proteins, saponins, oils/fats and terpenoids according to (Harborne, 1989;Trease and Evans, 1996).

Estimation of secondary metabolites
50g of powdered leaf sample was extracted with 250ml of ethanol by mild maceration for 72 hours. Extract concentrated by evaporation of solvent and stored in air tight bottle for further phytochemical and in vitro free radical scavenging and cytotoxic   Phenolics was quanti ied according to the standard protocol, (Singleton and Rossi, 1965). Experimental analysis is based on the principle that phenolics in alkaline medium react with phosphomolybdic acid of Folin-Ciocalteu reagent resulting in intense blue colour which is estimated colorimetrically at 650nm. Gallic acid was used as the reference standard. Phenolic concentration is calculated from calibration graph and represented as gallic acid equivalents in milligrams per gram of dry sample weight (mg/g).

Flavanoids
Total lavanoids were determined according to (Soong and Barlow, 2004). To sample and standard (quercetin) aliquots, 0.5ml of 2% aluminium chloride was added and kept for 1 hour at room temperature. Flavanoids gives yellow colour with ethanolic aluminium chloride which was colorimetrically quanti ied at 420nm. From calibration curve lavanoid concentration in sample was calculated and expressed as quercetin equivalent in mg/g of dry matter.

Tannins
Tannins were determined according to the method described by (Amadi et al., 2004). Tannins give yellow colour by reduction of phosphomolybdic acid in alkaline medium which can be measured spectrophotometrically at 700nm. Tannin concentration expressed as tannic acid equivalent in mg/g of dry matter.

Alkaloids
Alkaloids were quanti ied according to the standardized protocol of (Harbone, 1973). About 50g of powdered leaf sample dissolved in 40ml of 10% acetic acid prepared in ethanol and kept aside for 4 hours. The mixture was then iltered and reduced to one quarter volume. Concentrated ammonium hydroxide is added to the extract, precipitate formed is iltered, washed in dilute ammonium hydroxide and weight recorded in a pre weighed vial. Total alkaloid content was estimated as follows; Where Wx is the weight of empty vial, Wy weight of vial having extract and W initial weight of powdered sample.

In vitro free radical scavenging assays
Extract dissolved in 95% of ethanol and concentration ixed as 1mg/1ml. From this stock solution appropriate dilution is carried out and sample concentration is standardized to 20µg, 40µg, 60µg, 80µg and 100µg. Ascorbic acid is used as standard. Antioxidant potential of the plant sample was quanti ied by executing the following free radical scavenging assays; DPPH, superoxide, nitric oxide, hydrogen peroxide, hydroxyl, reducing power and ferric reducing antioxidant potential (FRAP) by means of standard cited protocols (Blois, 1958;Liu et al., 2000;Klein et al., 1981;Ruch et al., 1989;Garrat, 1964;Oyaizu, 1986;Benzie and Strain, 1996). Percentage of inhibition or radical scavenging is directly proportional to antioxidant ability of respective extract. For superoxide, nitric oxide, DPPH, and hydrogen peroxide radical scavenging assay inhibition percentage was evaluated by the formula as represented, where C A is absorbance of control and S A is the absorbance of sample. For hydroxyl radical scavenging assay percentage inhibition was calculated as ratio of difference in absorbance of sample and control. The value obtained is then subtracted by one and expressed graphically.

MTT assay
Inhibition of tumor cell proliferation by ethanolic leaf extract of M.diplotricha was assessed by MTT (3-(4) 5-Dimethyl-thiazol-Zyl) -2, 5 biphenyl tetrazolium bromide) assay. Colorectal cancer cell lines were seeded in 96 well plate. Sample fraction in 0.1% DMSO diluted in accordance to get appropriate concentration. Cell lines treated with varying concentration of extract were incubated for 24 hours. Control group left alone having media and 0.1% DMSO. Plates were washed with PBS and MTT dye was added. Absorbance recorded at 540nm. Cell viability was calculated as follows, Where A o is the absorbance of control and A t is the absorbance of cells subjected to treatment. IC 50 value was calculated from calibration graph.

GC-MS chromatographic analysis
GC-MS is a combined chromatographic technique that facilitates separation of volatile constituents and fragmentation of separated entities on the basis of molecular mass and detection of compounds by means of associated mass spectrophotometer utilizing the principle of charge to mass ratio. Gas chromatogram and mass spectra of the components in sample were detected and recorded by Thermo GC-Trace Ultra Version: 5.0, Thermo MS DSQ II and mass with those of Wiley library and NBS (National Insitute Standard and Technology) database available in computer library.

HPTLC analysis
HPTLC (High performance thin layer chromatography) is a precise and reliable chromatographic technique for the documentation of phytochemical proile. It combines the principle of TLC and scanning densitometery. The sample loaded plate was kept in saturated TLC chamber with speci ic mobile phase for alkaloids (Ethyl acetate : Methanol : Water; 8 : 1 : 1), lavanoids (Toluene : Ethyl acetate : Methanol, 7:3:1), phenols (Toluene : Ethyl acetate : Acetic acid, 6 : 3: 1) and tannins (Toluene : Ethyl acetate : Methanol : Formic acid, 6:3:0.8:0.2) . The plate after development was air dried and scanned at 366nm using Camag Scanner with WINCATS software. The retention factor of each compound and peak area percentage was also recorded.

Statistical evaluation
All the experimental observations were performed in triplicates and data were recorded as mean ± standard deviation of experimental triplicates.

Phytochemical parameters
Qualitative phytochemical pro ile of ethanolic leaf extract revealed phyto compounds like alkaloids, lavanoids, steroids, glycosides, saponins, aminoacids, carbohydrates, terpenoid, tannins and phenols (Table 1). Data as per consecutive solvent extraction, it was evident that ethanolic extract have maximum phytochemical pro ile. Quantitative estimation of secondary metabolites indicates that the plant is comparatively rich in phenolic group of compounds. The total phenol, lavanoid, tannin and alkaloid content of the plant sample was found to be 56 ± 0.57 (mg/g gallic acid equivalent), 27 ± 0.76 (mg/g quercetin equivalent), 33 ± 0.15 (mg/g tannic acid equivalent) and 2.51 ± 0.47 (mg/g of extracted plant material) respectively.

In vitro free radical scavenging assay
Antioxidant ability of plants is mainly due to nonnutritive phytochemicals classi ied as secondary metabolites. Each plant species is characteristic in having its own unique phytochemical pro ile which contributes to speci ic pharmacological attributes of medicinal herbs. From the results of antioxidant assays it is evident that the plant is having signi icant free radical scavenging potential. IC 50 value of DPPH, superoxide, nitric oxide, hydrogen peroxide and hydroxyl radical scavenging assay are shown in (Table 2 ). FRAP and reducing power assay also exhibit increased percentage of scavenging with increase in concentration (Figures 1 and 2).

GC-MS analysis
GC-MS chromatographic pro ile of ethanolic leaf extract of M.diplotricha revealed 45 peaks corresponding to 45 volatile Phytoconstituents. GC-MS chromatogram is depicited in Figure 5. Among 45 secondary metabolites, compounds having comparatively higher peak area and having reports on pharmacological activity are depicted in Table 3.

HPTLC analysis
Based on the evaluation of phytochemical quantitative estimation data HPTLC analysis was carried out speci ically for alkaloids, lavanoids, phenols and tannins to validate a preliminary documentation of respective phytochemical pro ile. HPTLC densitogram of secondary metabolites are depicted in Figure 6, which con irms that the ethanolic leaf extract of M.diplotricha is having signi icant phytoconsituents of which polyphenols being comparatively abundant.

Statistical Analysis
The data are expressed as Mean ± Standard deviation from three independent experiments.
Recent innovative strategy in pharmacological research constitutes experimental evaluation of herbal extracts for their therapeutic potency and further isolation of bioactive ingredients and biological drug interactions. Cardiovascular diseases, cancer and diabetes can be recognized as ubiquitous global menace and all over world phytochemical or herbal products based research is steering to ind an alternative for aggressive synthetic therapeutic drugs.
Understanding of phytochemical constituents of species under concern is the preliminary step in pharmacological research. As per indings of present study M.diplotricha is having all the major class of characteristic secondary metabolites and ethanolic extract is comparatively rich in almost all phytochemicals except fats & oils. Literature reports classify M.diplotricha as a nutrient rich fodder plant abundant in nitrogen and amino acids.
The species also have reports on non protein amino acid mimosine which is reported to have inhibitory effect on Cyclin E associated kinases thereby putting a check on abnormal cell division (Jayasree, 2005). Quantitative estimation and HPTLC data con irms that the plant is rich in polyphenols a characteristic feature of Fabaceae. Chemical pro iling by GC-MS analysis elucidate 45 volatile bioactive phytochemicals comprising sugar alcohols, terpenoids, fatty acid esters and steroids. Among the identi ied compounds ledol, sesquiterpene is having reports on antimicrobial, anti-in lammatory and anti-cancer properties (Kumar et al., 2007;Matos et al., 2006). Stigmasterol a steroid component is characteristic in having antioxidant and anti-in lammatory attributes (Matos et al., 2006). Tetracohexane, gamma-tochopherol, n-hexadeconoic acid, alpha.-Dmannofuranoside, 1, 5-anhydro-d-mannitol is having reports on antioxidant and antimicrobial properties (Gomathi et al., 2015;Kumar et al., 2007).
For most of the degenerative diseases like atherosclerosis, Alzheimer's, Parkinsonism, Ischemic disease, cancer and diabetes, free radical imbalance constitute as a major risk factor (Shirwaikar et al., 2006). Natural or herbal products based antioxidants is being widely exploited in the present era. Most of the phytoconstituents and isolated compounds have valid reports on antioxidant potential. The present species under consideration is having considerable antioxidant potential. IC 50 value of DPPH, hydroxyl, nitric oxide, superoxide and hydrogen peroxide in comparison with standard ascorbic acid proved that the plant is having signi icant radical scavenging activity. FRAP and reducing power assay further con irms the result. Effective antioxidant potential of the plant is attributed to the presence of potent bioactive phytoconsituents, con irmed by phytochemical and chromatographic pro iling data.
M.diplotricha exhibit convincing results on antineoplastic activity. At 97.82 µg concentration of extract 50% of cell viability is encountered at 24 hours treatment. Natural products especially herbal formulations and drugs are ever increasing in demand in pharmaceutical market particularly in oncology therapeutics as alternative to synthetic cancer drugs which are highly toxic to biological system. Colorectal carcinoma is a typical variant of gastrointestinal cancer, ranks third in incidence and fourth in mortality rate. Due to westernized diet and changed environmental conditions colorectal cancer is being evident as a frequently encountered neoplasm in persons above 50 (Kolligs, 2016). Finding a herbal alternative for colorectal carcinoma is a demanding prerequisite of current scenario. Anti-cancer property of phytochemicals is mainly due to antiapoptotic, antiangiogenic and cytostatic due to microtubule disorganization as vinca alkaloids/taxol or cell cycle arrest (altering the expression of cell cycle checkpoint proteins) (Alam et al., 2018). Respective plant is rich in bioactive constituents like tochopherol, ledol, stigmasterol having reports on anti-in lammatory and in vitro antiproliferative activity. Stigmasterol and ledol is having anti-in lammatory and anticancer activity (Kumar et al., 2007;Matos et al., 2006). In lammatory mechanism has got a direct correlation in colorectal neoplasm by mediating NF-kβ in lammatory pathway. Anti-in lammatory activity directly contributes as a preventive measure in the onset of colorectal cancer. Tochopherol stimulates the expression of wild type tumor suppressor (p53) gene and results in down regulation of mutant (p53) gene. Tochopherol also exhibit antiangiogenic effect (Kumar et al., 2007). Quercetin a signi icant and effective antineoplastic and antiin lammatory agent is reported to be present on aerial parts of M.diplotricha. Quercetin has experimental evidence on inhibitory effect on colon, gastric, endometrial, liver, leukemia, ovary and nonsmall-cell lung cancer through mediating regulation of expression of p53 and p21 (Chirumbolo, 2012).

CONCLUSIONS
Based on the results it can be concluded that M.diplotricha ethanolic leaf extract have a rich phytochemical pro ile representing all major class of secondary components. In vitro radical scavenging and cytotoxic screening in HCT-116 cell lines indicates signi icant antioxidant and antineoplastic activity of the plant. These cumulative indings paves way to further explore the species for isolation of pharmacologically active ingredients and assessing anticancer potential by in vitro and in vivo models and consequent elaboration into molecular mechanism of action in biological system.

ACKNOWLEDGEMENT
We, the authors are sincerely thankful to our Chancellor, Chief Executive Of icer, Vice-Chancellor and Registrar of Karpagam Academy of Higher Education for providing facilities and encouragement.