Isolation, characterization, and cytotoxic studies of secondary metabolites from the leaves of Bauhinia foveolata Dalzell: An endemic tree from the Western Ghats, India

Prasanna V. Habbu1*, Narayan Miskin1, Venkatrao H. Kulkarni2, Pradeep Bhat3, Arun Joshi4, Anant Bhandarkar4, Satish Kulkarni5,Sheshagiri R. Dixit6, Jaishree Vaijanathappa6 1Postgraduate Department of Pharmacognosy and Phytochemistry, SET’s College of Pharmacy, Dharwad 580 002, India. 2Postgraduate Department of Pharmacology, SET’s College of Pharmacy, Dharwad 580 002, India. 3Indian Council of Medical Research, National Institute of Traditional Medicine, Belagavi 590 010, India. 4Department of Pharmacognosy, Goa College of Pharmacy, Panaji, India. 5B N Degree College, Dandeli 581 325, India. 6Department of Pharmaceutical Chemistry, JSS College of Pharmacy, S S Nagar, Mysuru 570015, India.


INTRODUCTION
One of the most ancient sources of traditional medicines are raw materials extracted from the plants, and the heritage knowledge on preventive and curative medicines is based on the works of Atharva Veda, Charaka, and Sushruta. Globally, around 13,000 plant species are known to have been used as drugs, as they possess a wide variety of biologically active compounds that are essential to protect human health. Phytopharmaceuticals are one of the important parts of modern therapy for the alleviation of diseases of the cardiac and vascular system (Baharvand-Ahmadi et al., 2016), nervous system (Srivastava and Yadav, 2016), and immune system (Ford et al., 2016). Besides their therapeutic use, a large number of herbal drugs have been known for their prophylactic effect (Amirghofran et al., 2009;Kim et al., 2009). To develop new anticancer drugs in an estimated 7-10 years, we require a cost over $5,000,000, and it is an inclusive cost for the initial collection of plant material; evaluation of crude fractions; purification, identification, and laboratory synthesis of the bioactive compounds; and preclinical and clinical studies. Many medicinal plants available in the market might contain chemical substances with potential mutagenic (Rattanachaikunsopon et al., 2009;Yao et al., 2007) as well as antitumor properties (Orfali et al., 2016;Wang et al., 2016). Such constituents act by destroying or blocking the DNA-damaging mutagens, thereby acting as protective agents by preventing cell mutation. Hence, herbs have a vital role in the prevention and treatment of cancer.
Bauhinia foveolata Dalzell (Leguminosae) is an endemic tree, native to Southwest India (the Western Ghats), naturally occurring only in Uttara Kannada district. Leaves are suborbicular, deeply cordate at base, glabrous above, and pubescent beneath, 15-17 nerved with minutely pitted beneath. Flowers are small, unisexual, in dense much-branched panicles, 5-lobed calyx tomentose, not much exerted and white-to-light cream corolla, alternatively long and short stamens, densely hairy ovary, linear-oblong pods, and red tomentose. The scientific name "foveolata" is derived from the minute pores covered on the underside of the leaves (foveoli). Recent literature reveals the antibacterial activity of B. foveolata Dalzell acetone fraction of bark against Streptococcus pyogenes (Gamit et al. 2018). No pharmacological/phytochemical reports are available for B. foveolata leaves until now. However, the phytochemical and pharmacological literatures are available on the other species of Bauhinia.
A wide range of investigations on the cytotoxicity of flavonoids, such as apigenin, eriodictyol, 3-hydroxyflavone, kaempferol, luteolin, naringenin, taxifolin, quercetin, and rutin, toward cultured and tumor cells has been reported (Akbas et al., 2005;Matsuo et al., 2005). Although several bioactive constituents including flavonoids have been isolated from Bauhinia species, this is the first report on cytotoxic secondary metabolites from the leaves of B. foveolata Dalzell. The reported constituents were also isolated from several other medicinal plants, and some biological activities are reported (Dos et al., 2019;Rukshana, et al., 2017;).

Collection and authentication of plant material
The leaves of B. foveolata Dalzell were collected from Dandeli reserve forest, Karnataka, India, and were authenticated by Dr. Harsha Hegde, Scientist, Regional Medical Research Centre, Belagavi, India. A voucher specimen has been kept in the herbarium of pharmacognosy department (SETCPD/Ph.cog/ herb/04/09/2016) for future reference.

Extraction and preparation of crude fractions
The authenticated leaves of B. foveolata Dalzell were dried and reduced to coarse powdered material. It was passed through 120-mesh sieves to remove any fine dust or powder, and the coarse powder was used for extraction. The powder was extracted exhaustively with 95% ethanol in a Soxhlet extractor. The total ethanolic extract was further fractionated with ethyl acetate and n-butanol by solvent-solvent extraction technique.

Flash chromatography
About 1.5 g of the EtOAc:MeOH (80:20 and 70:30) fraction was adsorbed on 4 g of flash grade silica (230-400 mesh) using motor and pestle. Prepacked silica column RediSep (12 g) was used. The sample was loaded and placed in flash chromatography instrument. All the parameters were set and monitored using CombiFlash software. The column was eluted with gradient elution system using EtOAc:MeOH and MeOH, with concentration ranging from 0% to 50%. The eluents were monitored on TLC and visualized under UV 254 and 366 nm. Detection was performed using ferric chloride as spraying reagent. Identical elutes were collected, combined (TLC monitored), concentrated, and kept aside. A prominent peak was obtained with 10% of methanol in ethyl acetate. Elutions which were collected separately in test tubes and TLC studies were performed using EtOAc:MeOH (8:2) as a mobile phase with the R f of 0.52. After concentration of the solvent, a dark brown residue was obtained, which was designated as DN 1 (70 mg).

Conditions used for liquid chromatography-mass spectrometry (LC-MS) analysis
The mobile phase consisted of water containing 0.05% phosphoric acid (A) and acetonitrile (B) at a flow rate of 0.8 ml minute −1 using the following gradients: 0.1-23 minutes, 10%-40% of solvent B in A, and 23.01-40 minutes, 10% solvent B and 90% solvent A. The detection was done on a Diode-Array Detection (DAD) detector set at 340 nm. The mobile phase was prepared daily, filtered through a 0.45-mm membrane filter (Millipore), and sonicated before use. The LC/Electrospray ionization (ESI)-MS was conducted in positive-ion mode and operated according to the defined conditions: nitrogen gas temperature-320°C, drying gas flow rate-7 l minute −1 , capillary voltage-4,000 V, and nebulizing pressure -27 psi. Mass spectra were recorded using the full scan mode in the range of 200-800 Daltons.
Mass spectra (MS) were recorded using a JEOL GCMATE II GC-Mass spectrometer and Shimadzu QP 20105 GC-Mass spectrometer. Analytical TLC was performed on precoated TLC sheets of silica gel 60 F254 (Merck, Darmstadt, Germany) visualized by long-and short-wavelength UV lamps. Chromatographic purifications were performed on Merck aluminum oxide (70-230 mesh) and Merck silica gel (70-230 mesh).

Cell cultures
HT-29 and HCT-15 (Human colon cancer) cells were procured from the National Centre for Cell Science, Pune, India. The stock cells of all cell lines were cultured in DMEM supplemented with 10% inactivated FBS, penicillin (100 IU/ml), and streptomycin (100 µg/ml) in a humidified atmosphere of 5% CO 2 at 37°C until confluent. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, and 0.05% glucose in PBS). The stock cultures were grown in 25-ml flat bottles, and all the experiments were carried out in either 96-well microtiter plates.

Cell proliferation assessment-MTT assay
The monolayer of cell culture was trypsinized and the cell count was adjusted to 3.0 × 10 4 cells/ml using DMEM medium containing 10% FBS. 0.1 ml of the diluted cell suspension was added to each well of the 96-well microtiter plate. After 24 hours, the supernatant of monolayer formed was removed and washed with medium, and 100 µl of different concentrations of phytoconstituents were added on to the partial monolayer in microtiter plates. Microscopic examination of the samples was carried out, and the observations were noted every 24 hours interval after incubating the plates at 37°C for 3 days in a 5% CO 2 atmosphere. After 72 hours, 50 µl of MTT in PBS was added to each well after discarding the drug solutions. The plates were gently shaken and incubated for 3 hours at 37°C in 5% CO 2 atmosphere. The supernatant was removed and 100 µl of propanol was added, and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader (ELISA reader, BioTek) at a wavelength of 570 nm. The growth inhibition percentage was calculated, and the concentration of test compound required to inhibit 50% cell growth by CTC 50 values is generated from the dose-response curves for each cell line (Alley et al., 1988;Langner et al., 2019).

CONCLUSION
The research work successfully explores the cytotoxic principles from the leaves of B. foveolata Dalzell, an endemic tree of the Western Ghats, India. It is evident that these metabolites belong to the class polyphenolic compounds. Among the compounds studied against HT-29 and HCT-15 colon cancer cell lines of human, odoratin-7-glucoside and quercetin have proved to be highly cytotoxic by inducing apoptosis of cancer cells. Further research on the in vivo anticancer activity of potential fractions and to study the chemical diversity of this plant for other pharmacological activities is under progress in the laboratory.