Plant collection and processing
Desmodium gangeticum (L.) DC. was collected from the Idukki District of Kerala, India, during March–April 2019 (the atmospheric temperature found to be 25–33 °C with a humidity of 74%). The plant was identified by Dr. S. Soosairaj, Department of Botany, St. Joseph's College, Tiruchirappalli, Tamil Nadu, India (voucher number: 3006). Freshly collected plant material was cleaned to remove adhering dust, divided into different parts, and then dried under shade. The dried samples were powdered and stored at room temperature (25 °C) for further studies.
Reagents and chemicals
All the reagents and chemicals used in this research work were procured from M/s Merck India, Ltd., Godrej One, 8th floor, Pirojsha Nagar, Eastern Express Highway, Vikroli East, Mumbai, Maharashtra 400079.
DNA Barcoding
DNA extraction
Genomic DNA was extracted from young leaves of D. gangeticum using a modified cetyl trimethyl ammonium bromide (CTAB) method [21]. The quantity of DNA was measured using a NanoDrop Spectrophotometer (ND-2000, Thermo Scientific, Wilmington, USA), and the quality of DNA was checked via a 0.8% (w/v) agarose gel electrophoresis. The isolated DNA was stored at − 20 °C.
DNA amplification
The PCR was set with 50 μl reaction mixture containing 300 ng genomic DNA, 25 mM MgCl2, 1 mM dNTPs, 10 pmol rbcL primer (rbcL [R]: GTAAAATCAAGTCCACCRCG; rbcL [F]: ATGTCACCACAAACAGAAACTAAAGC), and 1 U Taq DNA polymerase (GeNet Bio, Daejeon, Korea). The PCR was performed using a master cycler (Eppendorf, Germany) with an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 30 s denaturation at 94 °C, 45 s annealing at 54 °C, and 45 s extension at 72 °C, with a final extension at 72 °C for 5 min. The PCR products were resolved on a 1% (w/v) agarose gel at 70 V and stained with ethidium bromide. The agarose gel was photographed using the ChemiDoc XRS imaging system (Bio-Rad, Hercules, California, USA) to check the presence or absence of bands.
Sequencing and plant identification
The amplified products were used for DNA sequencing (sequencing PCR was performed in a final volume of 10 μl containing BigDye sequencing buffer (1X), template 40 ng (for 1000–2000 bp amplicon sequencing), primers (3.2 pM), and BigDye (0.5 μl)), and the sequences were processed with BioEdit software version 7.1.11 [22]. The chimeric artifacts were removed using the online tool Decipher [23]. The sequences were compared by using BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with the non-redundant database of sequences deposited at the NCBI GenBank database [24].
Soil analysis
Soil samples were collected from the location of plant population grown (Idukki District of Kerala, India). The soil profile of different depths (0–10, 10–20, and 20–30 cm) was taken using the soil auger. Immediately after collection, soil samples were air-dried at room temperature (22 ± 1 °C), sieved (2 mm), and analyzed for different soil parameters. The dried sample (10 g) was used to evaluate the soil pH [25], electrical conductivity [26], total organic carbon (TOC) by titration method [27], and determination of total NPK and nitrogen [28]. Other elements (S, Fe, Mn, Cu, Zn, and B) are evaluated by spectrophotometric method [29] and flame photometer [30].
Organoleptic and macroscopic evaluation
To carry out organoleptic evaluation, various sensory parameters of the plant material, such as color, odor, size, shape, and taste, were studied [31].
Powder microscopy
The dried powdered aerial parts of D. gangeticum were studied by placing small amounts of powder on a slide and observing under the microscope. Samples were mounted on 5% glycerin solution or stained with reagents such as N/50 iodine for the observation of starches or 0.1% w/v phloroglucinol plus a drop of concentrated hydrochloric for the observation of lignified cells [32]. Characteristic structures and cell contents such as fibers, vessels, cork cells, calcium oxalate crystals, and plant cells were observed at various magnifications, and photomicrographs were taken.
Physicochemical analysis
The dried powdered aerial parts and root were used for determination of physicochemical contents, viz. total ash, acid-soluble ash, water-soluble ash, and alcohol-soluble as hand extractive values using different solvents, viz. ethanol, chloroform, ethyl acetate, hexane, toluene, petroleum ether, and water [19, 33].
Proximate analysis
The dried powdered aerial parts and root were used for evaluation of proximate contents through total ash, crude fiber, crude protein, carbohydrate, crude fat, dry matter, and moisture content [34].
Preparation of extracts
The dried aerial/root (100 g) was powdered in a plant sample grinder at a controlled temperature and used for extraction using suitable solvents in a Soxhlet extraction apparatus attached with a rotary vacuum evaporator (Buchi, Switzerland). Solvents were removed using a rotary vacuum evaporator at 175 mbar at a controlled temperature.
Phytochemical analysis
Preliminary phytochemical screening was done as per the standard procedure [35] for various phyto-constituents such as steroids (Liebermann–Burchard test), terpenoids (Salkowaski test), alkaloids (Mayer’s test), tannins (ferric chloride test), flavonoids (alkaline reagent test), carbohydrates (Benedict’s test), and amino acids (Biuret test).
UV and FTIR profiling
The UV spectra of the ethanol extracts of the aerial and root were recorded on a Jasco UV spectrophotometer at a wavelength range of 200–600 nm with a scan speed of 400 nm/min. The spectra used in obtaining the structural properties of the selected plant extract were obtained from the Fourier-transform infrared spectrometer equipped with an attenuated total reflectance (ATR-FTIR), model PerkinElmer Spectrum 400. In the ATR-FTIR method, the sample to be analyzed is placed directly into the sample cell, where a good and reproducible contact between the sample and the crystal of reflection is obtained nondestructively, producing good-quality infrared spectra. The FTIR spectra were recorded in the range of 4000–700 cm−1.
ADME properties
Pharmacokinetics and drug-likeness prediction for the compounds 1a-d were performed by online tool SwissADME [36] of the Swiss Institute of Bioinformatics (http://www.sib.swiss) [37]. 2D structural models were drawn in ChemBioDraw Ultra version 15.0 (Cambridge Software), and SMILES of 1a-d was translated into molfile by online SMILES translator and structure file generator found in online tool SwissADME. The analysis task was done to check whether the compound was an inhibitor of isoforms of the Cytochrome P450 (CYP) family, such as CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4. Also, pharmacokinetics (such as gastrointestinal absorption, P-glycoprotein, and blood–brain barrier) and drug-likeness prediction was done, such as Lipinski, Ghose, and Veber rules and bioavailability score [38,39,40]. The Lipinski, Ghose, Egan, Mugges, and Veber rules were applied to assess drug-likeness to predict whether a compound is likely to be bioactive according to some important parameters such as molecular weight, Log P, number of HPA, and HBD. The SwissADME tool used a vector machine algorithm (SVM) [41] with fastidiously cleaned large datasets of known inhibitors/non-inhibitors as well as substrates/non-substrates.