Chemicals
Porcine pancreatic α-amylase (10 units/mg), starch, (DPPH) 2,2'-diphenyl-1-picrylhydrayl, sodium carbonate, p-Nitrophenyl-α-D-glucopyranoside, ascorbic acid, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide, and bovine serum albumin (BSA), were all purchased from HiMedia chemicals, India. HPLC grade acetonitrile and methanol, gallic acid, quercetin, tannic acid, α-glucosidase (1U/mL), trichloroacetic acid, metformin hydrochloride, diclofenac sodium, ABTS (2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, metformin, penicillin-streptomycin and Dulbecco’s Modified Eagle Medium (DMEM) from Sigma-Aldrich.
Collection and identification of the plant
C. cuspidatus was collected in November month from Yelagiri mountain in the Tirupathur district of Tamil Nadu, India. The plant sample was taxonomically identified by Prof. P. Jayaraman, Director of Plant Anatomy Research Centre (PARC), West Tambaram, Chennai. The identification was conducted under the authorization number PARC/2022/4736.
Preparation of extract
The leaf of C. cuspidatus was separated into parts and all possible impurities were meticulously removed using deionized water. The cleaned leaf parts were then dried in a shaded area at a temperature of 38oC. In a processor, the chopped leaf components were then ground into a powder. The fine leaf powder was stored in a dry and sealed container, protected from sunlight and moisture. To the maceration, course-dried leaf powder was mixed with different polarity solvents (distilled water, methanol, chloroform, ethyl acetate, hexane). Following that, the extract performed filtration using a muslin cloth. The extracts were concentrated through a rotary evaporator and refrigerated for further application [8].
Qualitative Evaluation of C. cuspidatus Leaf
The extraction required primary phytochemical analysis to determine the major bioactive components contained in the different polarity solvent extract [9]. Standard chemical procedures were employed for evaluating a plant extract [10].
Fourier Transform Infrared Spectroscopy
The concentrated extracts were subjected to analysis using FTIR (IR-affinity-1, SHIMAZDU, Japan 2011) to identify predominant functional groups and stretching and bending vibrations in different solvent samples within the range of 400–4000 cm− 1. Further, screen the effective extract based on the preliminary and IR spectrum.
Gas Chromatography-Mass Spectrometry Analysis
The selected methanolic leaf extract of C. cuspidatus was analyzed using GC-MS on the Perkin Elmer Clarus 600 C model from the USA. Phytochemical identification was achieved by determining a retention time of 30 minutes and comparing the MS of the identified substance.
Nuclear Magnetic Resonance Spectroscopy
The methanolic extract of C. cuspidatus was dissolved (20 mg/mL) in deuterated DMSO and subjected to sonicated for 15 minutes and centrifuge at 2500 rpm for 5 minutes. 800 µL of supernatant was transferred to the NMR tube [11]. The sample was analyzed using NMR Spectroscopy specifically FT-NMR at a frequency of 400 MHz using a Bruker instrument.
Determination of Total Phenolic Content
The Folin-Ciocalteu method was used to determine the total phenolic content, as explained by Ramasubramaniyan et al. (2015) with minor changes. A gallic acid curve was calculated by diluting a standard gallic acid (1 mg/mL stock) solution to varying concentrations (5, 25, 50, 75, and 100 µL) in methanol [13]. 2.5 mL of Folin-Ciocalteu reagent was mixed with the unknown sample, and the mixture was incubated for 5 to 8 minutes at 38oC. Two milliliters of a 75% sodium carbonate solution were added and the mixture was incubated at a temperature of 50oC for a duration of 10 to 15 minutes. At OD765 nm, the reaction mixture’s absorbance was measured. Using a calibration curve based on gallic acid and reported in milligrams of gallic acid equivalent per gram (mg GAE/g), the phenolic content of the unknown sample was determined.
Determination of Total Flavonoid Content
The total flavonoid content was estimated using an aluminum chloride colorimetric assay, following the method described by Nikzad and Parastar (2021) with slight modifications. 1 mg/mL of quercetin standard with various concentrations (5, 25, 50, 75, and 100 µL) and 1 mL unknown sample. The resulting mixture was mixed with 200 µL of 10% aluminum chloride. 1 M of 200 µL potassium acetate was mixed and 5.6 mL of milli-Q water was incubated for 3 to 5 mins [15]. UV spectrophotometer was used to evaluate the absorbance of the standard and sample solution at 415 nm in comparison to the reagent blank. The flavonoid content of the unknown sample was quantified using a quercetin calibration curve, reported in milligrams of quercetin equivalent per gram (mg QE/g).
Determination of Total Tannin Content
The Folin-Ciocalteu method was used to calculate the total tannin concentration [16]. A tannin acid standard with a concentration of 1 mg/mL of tannic acid standard with various concentrations (5, 25, 50, 75, and 100 µL) and 0.5 ml of Folin-Ciocalteu reagent in a final volume of 10 mL milli-Q water was mixed in the reaction. The reaction mixture was incubated for 30 mins and the absorbance was measured at 700 nm. Using a tannic acid equivalent curve, the tannin content of the unknown sample was determined and reported in milligrams of tannin acid equivalent per gram (mg TAE/g).
Evaluation of Antioxidants by Invitro Techniques
Activated oxygen species which include single molecule oxygen and (H2O2) hydrogen peroxide, can cause cell damage by reacting with free radicals [17] [18]. We performed the FRAP, ABTS, and DPPH assays to assess the reducing potency and radical scavenging properties. Various aliquots (5, 25, 50, 75, and 100 mg/mL) of the sample, along with ascorbic acid as a positive control. A blank for antioxidant activities was created using a phosphate buffer and methanol. To assess the reduction of Fe3+ to Fe2+, we conducted the FRAP assay. A mixture of 1 mL methanol solution (sample and standard) and phosphate buffer (pH 6.6) was prepared. Subsequently, the reaction mixture was treated with 1% of 2.5 mL potassium ferricyanide solution [19]. The reactant was vigorously and subjected to incubation at a temperature of 50oC for a duration of 20 to 30 minutes. Following this, 10% of 2.5 mL trichloroacetic acid was added and the solution was centrifuged at 3000 rpm for 3 minutes. The supernatant was diluted with an equal volume of mill-Q water. Then, 0.5 mL of ferric chloride (20 mM) was added and incubated for 2 to 3 minutes. Using a UV spectrophotometer, the absorbance of the reaction mixture was determined at 700 nm. The 2 mL of extract was mixed with a DPPH (0.1 mM) and incubated in darkness for a duration of 30 minutes. The absorbance of the reaction was measured at wavelength of 517 nm. The ABTS activity was determined by mixing a reaction mixture of 7 mM ABTS solution with 2.45 mM potassium persulfate to generate radical cations. 1 mL volume of both sample and standard solution were mixed with 2 mL of ABTS radical cation reactant solution. The resulting mixture was then incubated for duration of 25 to 30 minutes. The reaction was quantified at wavelength of 734 nm [20]. The inhibition potential percentage was determined through the employing of an equation.
$$\% Inhibition=\frac{Acontrol-Asample}{Acontrol}*100$$
Evaluation of Antidiabetic by In-vitro Techniques
The evaluation of α-amylase and α-glucosidase enzyme inhibition has gained considerable interest in the analysis of blood hemostasis. Subsequently, the α-amylase and α-glucosidase enzyme inhibition of the samples (10 to 100 mg/mL) was evaluated using 0.1 mL of each sample [21]. Metformin was employed as a positive correlation with the samples [22] [23]. The α-amylase inhibitory assay was assessed using the standard DNS method. Difference sample and standard concentration solutions were treated with 0.2 M of 500 µL phosphate buffer (pH 6.9) and 1 U/mL amylase enzyme solution. The reaction was incubated for 20 to 30 minutes at 38oC. A 250 µL aliquot of 1% starch solution was incubated for 15 minutes. To terminate the reaction, 1 mL of DNS reagent was added and incubated in a boiling water bath for 10 to 15 minutes. The results were measured at a wavelength of 540 nm. The α-glucosidase inhibitory assay was assessed using the para nitrophenyl α-D-glucopyranoside substrate method. The experiment involved using 0.1 M 150 µL phosphate buffer (pH 6.9) as the sample and standard concentrations. Additionally, 1 U/mL of glucosidase enzyme was added to initiate the enzyme reaction. The reaction was incubated for 10 to 12 minutes. The enzyme reaction was terminated by adding 50 µL of 2mM PNP substrate and incubating the reaction mixture for a duration of 15 to 20 minutes. The enzyme reaction was terminated using 50 µL of 0.1 M sodium carbonate, and the resulting measurement was taken at 405 nm.
$$\% Inhibition=\frac{Acontrol-Asample}{Acontrol}*100$$
Invitro Bovine Serum Albumin Assay
Bovine serum albumin (BSA), when subjected to heat, undergoes denaturation and initiates the expression of the antigen-associated response. The anti-denaturation assay is evaluated as a significant method for stabilizing compounds against allergies [24]. Various concentrations of (2.5, 5, 25, 50, 75, and 100 mg/mL) were evaluated and compared to a positive control to diclofenac [25]. A 0.2% solution of BSA was prepared using phosphate buffer at pH 6.5. A 1 mL sample and standard were subjected to treatment with 500 µL of BSA solution. The resulting reaction mixture was heated at 60oC for a duration of 2 to 4 minutes. After cooling, the reaction was incubated for a period of 15 to 20 minutes [26]. The UV-visible spectrophotometer was used to measure the absorbance of the solution at a wavelength of 520 nm [27].
$$\% Inhibition=\frac{Acontrol-Asample}{Acontrol}*100$$
Antibacterial activity
The antibacterial activity of three different bacterial strains including Escherichia coli, Klebsiella pneumonia, and Staphylococcus aureus, was examined against the methanolic leaf extract of C. cuspidatus methanolic leaf extract. Muller Hinton Agar served as the substrate for the agar diffusion procedure, and plates were then utilized using the standardized microbe culture method performed in Muller Hinton Agar (MHA), plates were lawn cultured with standardized microbial culture broth. Plant extract showed indeed effectiveness so, the concentration of the sample was fixed with a microgram. 20 µL of the plant extract was poured into each well after it had been dissolved in 1% DMSO using distilled water. The plates were incubated at 37oC for an extended period. Following incubation, the emergence of a clear zone around the well proved. The size of the zone of inhibition was measured and evaluated. For positive control, an ampicillin antibiotic disc was utilized.
Cell culture - MTT assay
The absorption phase of a drug is crucial for its effectiveness, as it determines the degree to which the drug is taken up by liver cells. Therefore, it is important to perform cytotoxic analysis to assess the effect of the drug on the HepG2 cell line. The HepG2 cell line, provided by the National Centre for Cell Science, Pune (NCCS), was utilized to evaluate the cytotoxicity of a leaf sample from C. cuspidatus. The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM media) supplemented with 10% fetal calf serum and penicillin (100 U/mL) in a humidified environment. The cells were cultured at 37oC in a temperature-controlled atmosphere containing 5% CO2 and 95% atmosphere [28]. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) test was conducted using varying concentrations of the sample to determine cell viability [25]. The absorbance at 540 nm was calculated using a UV spectrophotometer [29].
$$\% of Cell viability=HepG2 of\frac{Treated cells}{Control cells}*100$$
In silico study
Druglikeness rule and ADMET analysis
The importance of pharmacokinetic and pharmacodynamic studies in determining standard drug design through the process of drug-likeness is expanding. Limited studies have examined the screening of compound parameters in the overall framework of target drug design. All compounds were evaluated and screened using Lipinski’s rule of five [30], Pfizer rule, GSK, and the golden triangle, and further selected screened compounds investigated the ADMET properties for molecular docking. To objective is to assess the pharmacokinetics and toxicity of C. cuspidatus screened lead compounds utilization of the SwissADME (www.swissadme.ch) online tool. In addition to its conformation, ADMETlab 2.0 used canonical smiles to forecast ligand characteristics and determine their pharmacokinetics, potentially lowering the hazards involved in upcoming research studies.
Molecular docking
The crystal structure of α-amylase (1B2Y) and α-glucosidase (5NN8) was retrieved from the RCSB protein data bank with the co-crystallized inhibitor of acarbose. Molecular docking analysis is employed to forecast the binding between ligands and proteins in biological activities, facilitating recognition of molecular biology and computer-aided design. According to the literature survey, 1B2Y protein (X = 18.909389; Y = 5.79070; Z = 47.006148) and 5NN8 protein (X = 12.10300, Y= -16.549000, Z = 106.548000) exhibit the active sites which allowing the ligand to identification of high-affinity binding compounds. Various software tools, including Autodock, Autodock vina package v1.2.3, MGL software, and Discovery Studio are utilized for virtual molecular docking. Protein Data Bank was used to obtain 3D crystallographic structures of proteins (www.rcsb.org). the protein complex was selected for analysis after removing water molecules, heteroatoms, and inhibitors. Subsequently, the PDBQT format was generated [31].
The screened ligands of nor-diazepam, akuammilan-17-ol, 1,2-benzene dicarboxylic acid, and standard metformin were used in the molecular docking study, ligands were obtained from the PubChem database (pubchem.ncbi.nlm.nih.gov) and prepared for analysis in PDBQT format using CADD group Chemoinformatics tools (cactus.nci.nih.gov) and Autodock vina software. Proteins and ligands possessed a total of 10 degrees of freedom. Among the 10 binding poses generated, the initial negative binding pose had a lower root mean square deviation (RMSD) value of atomic alignment. This result suggests that the initial negative binding pose is highly valid and indicates a stronger binding efficacy. The binding affinity was determined, and the ligand’s predicted pose was analyzed. The interactions of the 2D results were visualized using Discovery Studio.
Statistical Analysis
The experiments were conducted in triplicate, and the quantitative analysis data were subjected to regression analysis in Microsoft Excel. The in vitro results were analyzed using OriginPro software while the IC50 value was determined in Microsoft Excel. The in vitro anticancer activity results were analyzed using GraphPad Prism 8 and Dunnett’s test. A significance level of P < 0.005 was employed.