Phytochemical Profiling, Biological Activities, and In Silico Molecular Docking Studies of Causonis trifolia (L.) Mabb. & J.Wen Shoot

Abstract

Causonis trifolia (L.) Mabb. & J.Wen, commonly known as "fox grape", is an ethnomedicinally important twining herb of the Vitaceae family, and it is used by ethnic communities for its wide range of therapeutic properties. Our research aims to investigate the chemical composition; antioxidant, anti-inflammatory, and antidiabetic activities; and mechanisms of interaction between the identified selective chemical compounds and the target proteins associated with antioxidant, anti-inflammatory, and antidiabetic effects of the optimised phenolic extract of Causonis trifolia (L.) Mabb. & J.Wen, shoot (PECTS) to endorse the plant as a potential drug candidate for a future bioprospecting programme. Here, we employed the response surface methodology (RSM) with a Box-Behnken design to enrich the methanolic extract of C. trifolia shoot with phenolic ingredients by optimising three key parameters: solvent concentration (% v/v, methanol:water), extraction temperature (°C), and extraction duration (hours). From the quantitative phytochemical estimation, it was evident that the PECTS contained good amounts of phenolics, flavonoids, tannins, and alkaloids. During the HPLC analysis, we identified a total of eight phenolic and flavonoid compounds (gallic acid, catechin hydrate, chlorogenic acid, caffeic acid, p-coumaric acid, sinapic acid, coumarin, and kaempferol) and quantified their respective contents from the PECTS. The GC-MS analysis of the PECTS highlighted the presence of 19 phytochemicals. In addition, the bioactivity study of the PECTS showed remarkable potentiality as antioxidant, anti-inflammatory, and antidiabetic agents. In silico molecular docking and computational molecular modelling were employed to investigate the anti-inflammatory, antioxidant, and antidiabetic properties of the putative bioactive compounds derived from the PECTS using the GC-MS technique to understand the drug-receptor interactions, including their binding pattern. Out of the 19 phytocompounds identified by the GC-MS analysis, one compound, ergosta-5,22-dien-3-ol, acetate, (3β,22E), exhibited the best binding conformations with the target proteins involved in anti-inflammatory (e.g., Tnf-α and Cox-2), antioxidant (SOD), and antidiabetic (e.g., α-amylase and aldo reductase) activities. The nontoxic nature of this optimised extract was also evident during the in vitro cell toxicity assay against the Vero cell line and the in vivo acute toxicity study on BALB/c mice. We believe the results of the present study will pave the way for the invention of novel drugs efficacious for several ailments using the C. trifolia plant.

Keywords: Causonis trifolia; acute toxicity; molecular docking; phytochemical profiling; response surface methodology.

Figure 1

Statistical optimisation of the extraction parameters by the response surface methodology. The response surface plots demonstrate the combined effect of the (A) temperature (°C) and solvent (%); (B) extraction time (hour) and temperature (°C); (C) extraction time (hour) and solvent (%) on the yield of TPC. (D) Perturbation plot showing the relative influence of each parameter on the extraction of TPC. (E) Normal probability plot showing scatters along the standard curve supporting the prediction of the ANOVA analysis. (F) Comparison of the experimental and predicted results, showing a greater degree of agreement between them.

Figure 2

Predictive extraction model designed for the effective extraction of the highest amount of bioactive phenols. The model highlights the most suitable values of the three parameters, namely, methanol concentration (67.6754%), extraction temperature (49.7753 °C), and extraction time, (12.5837 h) predicted for the highest yield of bioactive phenols (152.774 mg GAE/g dry tissue).

Figure 3

HPLC chromatogram obtained from the optimised phenolic extract of Causonis trifolia shoot (PECTS).

Figure 4

Phenolic and flavonoid profiles of the optimised phenolic extract of Causonis trifolia shoot (PECTS).

Figure 5

GC-MS chromatogram obtained from optimised phenolic extract of Causonis trifolia shoot.

Figure 6

Chemical structure and retention time of the compounds identified from PECTS.

Figure 7

In vitro antioxidant activities exhibited by the PECTS in different radical scavenging assays and a comparison with ascorbic acid: (A) DPPH radical scavenging activity of PECTS (Y = 0.5907x + 14.557; IC₅₀ = 59.96 μg/mL) and ascorbic acid (Y = 0.4368x + 37.336; IC₅₀ = 28.99 μg/mL); (B) hydrogen peroxide scavenging activity of the PECTS (Y = 0.5288x + 13.969; IC₅₀ = 68.13 μg/mL) and ascorbic acid (Y = 0.4526x + 33.847; IC₅₀ = 35.68 μg/mL); (C) ABTS radical scavenging activity of the PECTS (Y = 0.58x − 2.0551; IC₅₀ = 47.94 μg/mL) and ascorbic acid (Y = 0.4406x + 35.233; IC₅₀ = 33.51 μg/mL).

Figure 8

In vitro anti-inflammatory activities of the optimised phenolic extract of Causonis trifolia shoot and a comparison with the anti-inflammatory activity of the standard drug diclofenac sodium: (A) inhibition of the BSA denaturation activity of th ePECTS (Y = 0.1833x − 6.133; IC₅₀ = 306.78 μg/mL) and diclofenac sodium (Y = 0.1852x − 3.66; IC₅₀ = 288.74 μg/mL); (B) antiprotease activity of the PECTS (Y = 0.1541x + 5.161; IC₅₀ = 290.97 μg/mL) and diclofenac sodium (Y = 0.1764x + 2.337; IC₅₀ = 270.19 μg/mL); (C) inhibition of the heat-induced haemolysis activity of the PECTS (Y = 0.1547x − 0.594; IC₅₀ = 327.04 μg/mL) and diclofenac sodium (Y = 0.177x + 5.131; IC₅₀ = 253.48 μg/mL).

Figure 9

In vitro antidiabetic activity of the optimised phenolic extract of Causonis trifolia (PECTS) and a comparison with acarbose’s activity: (A) inhibition of the α-amylase activity of the PECTS (Y = 0.3021x − 14.55; IC₅₀ = 117.38 μg/mL) and acarbose (Y = 0.2541x − 28.76; IC₅₀ = 84.48 μg/mL); (B) yeast cell glucose uptake activity of the PECTS (Y = 0.1126x + 32.385; IC₅₀ = 156.42 μg/mL) and acarbose (Y = 0.1284x + 43.2; IC₅₀ = 52.95 μg/mL).

Figure 10

Binding energy (−Kcal/mol) of Ergosta-5,22-dien-3-ol, acetate, (3β,22E) with five target proteins.

Figure 11

(a) Surface view of the docked complex of ergosta-5,22-dien-3-ol, acetate, (3β,22E), and an anti-inflammatory target protein (TNF-α); (b) binding pose of ergosta-5,22-dien-3-ol, acetate, (3β,22E); (c) ligand–protein docked complex showing the binding interaction of ergosta-5,22-dien-3-ol, acetate, (3β,22E), with TNF-α. Two hydrogen bonds (yellow, dotted line) formed between the ligand ergosta-5,22-dien-3-ol, acetate, (3β,22E), and two amino acid residues (Leu-93 (3.0 Å) and Ser-95 (2.6 Å)) of the target protein TNF-α.

Figure 12

(a) Surface view of the docked complex of ergosta-5,22-dien-3-ol, acetate, (3β,22E), and the anti-inflammatory target protein (Cox-2); (b) binding pose of ergosta-5,22-dien-3-ol, acetate, (3β,22E); (c) ligand–protein docked complex showing the binding interaction of ergosta-5,22-dien-3-ol, acetate, (3β,22E), with Cox-2. Four hydrogen bonds (yellow, dotted line) formed between the ligand ergosta-5,22-dien-3-ol, acetate, (3β,22E), and two amino acid residues (Ser-120 and Arg-121) of the target protein Cox-2.

Figure 13

(a) Surface view of the docked complex of ergosta-5,22-dien-3-ol, acetate, (3β,22E), and the antioxidant target protein (SOD); (b) binding pose of ergosta-5,22-dien-3-ol, acetate, (3β,22E); (c) ligand–protein docked complex showing the binding interaction of ergosta-5,22-dien-3-ol, acetate, (3β,22E), with SOD. One hydrogen bond (yellow, dotted line) formed between the ligand ergosta-5,22-dien-3-ol, acetate, (3β,22E), and one amino acid residue (Lys-23 (2.2 Å)) of the target protein SOD.

Figure 14

(a) Surface view of the docked complex of ergosta-5,22-dien-3-ol, acetate, (3β,22E), and the antidiabetic target protein (α-amylase); (b) binding pose of ergosta-5,22-dien-3-ol, acetate, (3β,22E); (c) ligand–protein docked complex showing the binding interaction of ergosta-5,22-dien-3-ol, acetate, (3β,22E), with α-amylase. One hydrogen bond (yellow, dotted line) formed between the ligand ergosta-5,22-dien-3-ol, acetate, (3β,22E), and one amino acid residue (Gln-63 (2.2 Å)) of the target protein α-amylase.

Figure 15

(a) Surface view of the docked complex of ergosta-5,22-dien-3-ol, acetate, (3β,22E), and the antidiabetic target protein (aldo reductase); (b) binding pose of ergosta-5,22-dien-3-ol, acetate, (3β,22E); (c) ligand–protein docked complex showing the binding interaction of ergosta-5,22-dien-3-ol, acetate, (3β,22E), with aldo reductase. Four hydrogen bonds (yellow, dotted line) formed between the ligand ergosta-5,22-dien-3-ol, acetate, (3β,22E), and four amino acid residues (Thr-19 (2.2 Å), Lys-21 (2.0 Å), Ser-22 (3.4 Å), and Tyr-48 (3.2 Å)) of the target protein aldo reductase.

Figure 16

Cell viability (%) at different concentrations (50–250 mg/L) of the PECTS.

Figure 17

Changes in body weight of (A) female BALB/c and (B) male BALB/c mice over 14 days of acute exposure (oral administration) of the PECTS.