Thirty-Two Selected Cissus quadrangularis Phytoconstituents as Modulating Agents of Human Intestinal-Type Fatty Acid Binding Protein and Carnosinase 2: An In silico Study

INTRODUCTION

Cissus quadrangularis (Pirandai) is a perennial climber plant, which belongs to Vitaceae (grape) family (Kumar et al., 2020). This plant has been traditionally used in Africa, Bangladesh, Cameroon, India, Sri Lanka and Thailand to treat various diseases (Sawangjit et al., 2017). Till date 350 Cissus species have been reported (Rahmawati et al., 2021) which includes C. aralioides, C. assamica, C. bathyrakodes, C. cactiformis, C. cornifolia, C. hastata, C. hypoglauca, C. ibuensis, C. incisa, C. javana, C. latifolia, C. oliveri, C. quadrangularis, C. rhombifolia, C. repanda, C. repens, C. rotundifolia, C. rubiginosa, C. sicyoides, C. subtetragona, C. trifoliata, C. verticillata and C. vitiginea.

Among above mentioned Cissus species Cissus quadrangularis is one of the popularly known species used for more 100 years in the Indian traditional medicine that to particularly in Ayurvedic medicine (Brahmkshatriya et al., 2015). The vernacular names for Cissus quadrangularis are “Adamant Creeper” in English, “Chodhari” in Gujarati, “Hadjod” in Hindi, “Peranta” in Malayalam, “Mangarahalli” in Kanada, “Hadavhanga” in Oriya, “Pirandai” in Tamil, “Nalleru” in Telugu, “Phet sang-Khaat” in Thai, “Asthisanghata” in Sanskrit, “Heeressa” in Sinhala, “Harjora” in Urdu (Rex and Ravi, 2020). Different plant parts of Cissus quadrangularis (Pirandai) are traditional used as follows i) whole plant is used to treat osteoarthritis, osteoporosis and rheumatoid; ii) both roots and stems extracts are used for repairing fractured bones and torn ligaments; iii) stem extract is used to cure epistaxis, menstrual irregularities and scurvy; iv) fresh leaves and stems are used to treat dysmenorrhea, dyspepsia, hemorrhoids and scurvy (Sawangjit et al., 2017; Aarthi et al., 2024).

Cissus quadrangularis (Pirandai) has been reported to possess various pharmacological activities such as anti-bacterial, anti-diabetic, anti-fungal, anti-inflammatory, anti-microbial, anti-obesity, anti-osteoporotic, anti-oxidant, anti-pyretic, anti-ulcer, anxiolytic, bone-healing, gastro-protective (Vinoth and Kumar, 2025).

The earlier reports motivated us to perform the current investigation on thirty two chosen constituents which includes i) Aglacin I, ii) Agropinic acid, iii) Alpha-amyrin, iv) Apigenin 6-C-glucoside 8-C-arabinoside, v) Apiin, vi) Atractylodin, vii) Cis-P-coumaric acid, viii) Cosmosiine, ix) 3,4-dimethoxyphenethyl alcohol, x) Dyphylline, xi) 5,8,11-Eicosatriynoic acid, xii) 2- Furancarboxaldehyde, 5-(hydroxymethyl), xiii) Gallocatechin gallate, xiv) Ginkolide B, xv) Hexadecanedioic acid, xvi) n-Hexadecanoic acid, xvii) Isoquercitrin, xviii) Kaempferol-3-o-galactoside, xix) Kaempferol 3-rutinoside, xx) Ligustrosidic acid, xxi) Linarin, xxii) Malic acid, xxiii) 3-O-Methylgallic acid, xxiv) 9Z,12Z,15Z-Octadecatrienoic acid, xxv) 2-Oxo-4-Methylthiobutanoic acid, xxvi) Phytol, xxvii) Preskimmianine, xxviii) 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl, xxix) Quercetin-3-o-xyloside, xxx) Quercitrin, xxxi) Secologanin and xxxii) Tetradecanoic acid.

These above-mentioned C. quadrangularis (Pirandai) phytochemicals were aimed to investigate on the molecular docking analysis of human intestinal-type fatty acid binding protein (hI-FABP) and human Carnosinase 2 (hCN 2) by using the swissdock method, which aids in developing anti-obesity agents for managing obesity related disorders.

MATERIALS AND METHODS

Ligand preparation

The chemical structures of thirty-two selected C. quadrangularis (Pirandai) ligands were chosen for the current study based on earlier reports (Mehta et al., 2001; Dinesh Kumar et al., 2020; Kannaa et al., 2022, Aarthi et al., 2024; Mondal et al., 2025), that includes 1) Aglacin I (CID 21578048); 2) Agropinic acid (CID 173285); 3) Alpha-amyrin (CID 73170; 4) Apigenin 6-C-glucoside 8-C-arabinoside (CID 131750832); 5) Apiin (CID 5280746); 6) Atractylodin (CID 442004); 7) Cis-P-coumaric acid (CID 1549106); 8) Cosmosiine (CID 5280704); 9) 3,4-dimethoxyphenethyl alcohol (CID 81911); 10) Dyphylline (CID 3182); 11) 5,8,11-Eicosatriynoic acid (CID 1781); 12) 2- Furancarboxaldehyde, 5-(hydroxymethyl)- (CID 237332); 13) Gallocatechin gallate (CID 5276890); 14) Ginkolide B (CID 65243); 15) Hexadecanedioic acid (CID 10459); 16) n-Hexadecanoic acid (CID 985); 17) Isoquercitrin (CID 5484006); 18) Kaempferol-3-o-galactoside (CID 5282149); 19) Kaempferol 3-rutinoside (CID 5318767); 20) Ligustrosidic acid (CID 146014676); 21) Linarin (CID 5317025); 22) Malic acid (CID 525); 23) 3-O-Methylgallic acid (CID 19829); 24) 9Z,12Z,15Z-Octadecatrienoic acid (CID 5280934); 25) 2-Oxo-4-Methylthiobutanoic acid (CID 473); 26) Phytol (CID 5280435); 27) Preskimmianine (CID 12305721); 28) 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- (CID 119838); 29) Quercetin-3-o-xyloside (CID 5321278); 30) Quercitrin (CID 5280459); 31) Secologanin (CID 161276); 32) Tetradecanoic acid (CID 11005) and 33) Standard drug (Orlistat) (CID 3034010) were downloaded from PubChem compound database. These thirty-two selected C. quadrangularis (Pirandai) structures were drawn and prepared by using ChemDraw 2D and 3D software tools. Thus, these prepared three-dimensional structures were used for further (swissdock) studies (Mohan et al., 2023).

Preparation of target proteins

The 3-D [three-dimensional] structure of human intestinal-type fatty acid binding protein [hI-FABP] (PDB^◊◊ ID: 3AKM with a resolution of 1.90 A^ᵒ) and human carnosinase 2 [hCN 2] (PDB^◊◊ ID: 4RUH with a resolution of 2.25 A^ᵒ) was downloaded from C. quadrangularis (Pirandai) ^◊◊Protein Data Bank (PDB). “A” chain of these two proteins was prepared separately by deleting other chains, ligands, and even the crystallographically observed “water” (H₂O) molecules by using UCSF Chimera software tool (Mohan et al., 2023).

Toxicity analysis

Pro-Tox 3 online server was used to predict the toxicity effect of 32 selected C. quadrangularis (Pirandai) ligands (Mohan et al., 2022).

Docking study

A docking study was performed for thirty-two selected phytoconstituents of C. quadrangularis (Pirandai) and one standard drug (Orlistat) with two target proteins (hI-FABP and hCN 2) using the Swissdock free web server (Prakash et al., 2023). Finally, “PLIP” [Protein-Ligand Interaction Profiler] free online server was utilized to determine the binding site of best-docked pose for each ligand. Docking protocol was validated using orlistat as standard drug. And the Root Mean Square Deviation (RMSD^◘) analysis of all the docked complexes [32 ligands] was separately compared with that of orlistat [standard drug] docked complex for each chosen target protein by using the “align” command in ‘PyMOL’ software (Ramsbottom et al., 2018; Narayanaswamy et al., 2024).

In present investigation, no experimental animals (or) human subjects were used, thus ethical approval was not needed.

With regard to statistical analysis, in the present study orlistat (standard drug) was used for the comparison purpose.

RESULTS

In the present investigation, Table 1 represents the toxicity analysis of thirty-two chosen C. quadrangularis (Pirandai) ligands, in which no hepatotoxicity was predicted by any of the ligands. Two ligands (3-O-Methylgallic acid and 9Z,12Z,15Z-Octadecatrienoic acid) of C. quadrangularis (Pirandai) were predicted to have cytochrome P450 1A2 inhibitory effect (as shown in Table 1).

In the present molecular docking analysis showed that Ligustrosidic acid has the Highest Binding Energy (HBE) (-9.50 kcal/mol) with the human Intestinal-Type Fatty Acid Binding Protein (hI-FABP). In contrast, Alpha -amyrin had the lowest binding energy (LBE) (-5.70 kcal/mol) with the human Intestinal-type Fatty Acid Binding Protein (hI-FABP) (as shown in Table 2).

Twelve ligands (Agropinic acid, Cis-p-coumaric acid, Cosmosiin, 5,8,11-Eicosatriynoic acid, Gallocatechin gallate, n-Hexadecanoic acid, Isoquercitrin, Malic acid, 3-O-Methylgallic acid, Quercetin-3-o-xyloside, Quercitrin and Standard drug-Orlistat) have shown interactions with ASP 34 amino acid residue of human Intestinal-type Fatty Acid Binding Protein (hI-FABP). Similarly, twelve ligands (Agropinic acid, Cosmosiin, 5,8,11-Eicosatriynoic acid, Gallocatechin gallate, Isoquercitrin, Kaempferol-3-rutinoside, Ligustrosidic acid, Malic acid, 2-oxo-4- methylthiobutanoic acid, Quercetin-3-o-xyloside, Quercitrin and Orlistat) have shown interactions with SER 53 amino acid residue of human Intestinal-type Fatty Acid Binding Protein (hI-FABP) (as shown in Table 2). However, five ligands (Aglancin I, Alpha -amyrin, Atractylodin, Apiin and 9Z,12Z,15Z-Octadecatrienoic acid) does not shown any interaction with the amino acid residue of human Intestinal-type Fatty Acid Binding Protein (hI-FABP).

The current docking results showed that Linarin has the Maximum Binding Energy (MBE) (-10.01 kcal/mol) with the human Carnosinase 2 (hCN 2) enzyme. On the other hand, 4H-pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl had the Least Binding Energy (LBE) (-5.92 kcal/mol) with the human Carnosinase 2 (hCN 2) enzyme (as shown in Table 3).

Seven ligands (Aglancin I, Apiin, Hexadecanedioic acid, Isoquercitrin, Kaempferol-3-rutinoside, Secologanin and Orlistat) have shown interactions with ARG 211 amino acid residue of human Carnosinase 2 (hCN 2) enzyme. Interestingly, four ligands (Apigenin- 6-c-glucoside 8-c-arabinoside, 5,8,11-Eicosatriynoic acid, Ginkgolide B and Secologanin) have shown interactions with GLU 414 amino acid residue of human Carnosinase 2 (hCN 2) enzyme (as shown in Table 3). However, three ligands (Alpha-amyrin, Atractylodin and Quercetin-3-o-xyloside) does not shown any interaction with the amino acid residue of human Carnosinase 2 (hCN 2) enzyme.

DISCUSSION

Sharp and coworkers (2007) had demonstrated that Cissus quadrangularis (Pirandai- stem and leaf extracts) shown to inhibit human lipase, porcine pancreatic amylase and Sacccharomyces cerevisiae alpha glucosidase activities. Interestingly, Oben and colleagues (2015) had clinical demonstrated the effectiveness of using C. quadrangularis (CQR-300 and CORE) product in decreasing i) Body Weight (BW), ii) Body Fat (BF), iii) Total Cholesterol (T-CHO), iv) LDL-Cholesterol (LDL-C), v) Triglycerides (TG) and vi) fasting blood glucose (FBG) levels in humans. Similarly, Kuate and colleagues (2015) had clinical demonstrated the effectiveness of using C. quadrangularis (CQR-300) product in decreasing Body Weight (BW), along with enhancing blood parameters associated Metabolic Syndrome (MS) and obese in humans. Further, Lee and colleagues (2016) had demonstrated that C. quadrangularis (CQR-300 extracts) decrease Body Fat (BF) via by regulating the fatty acid biosynthesis in high fat diet induced obese mice. Furthermore, Lee and coworkers (2018) had reported that C. quadrangularis (CQR-300 extracts) suppress lipid accumulation via by down-regulating adipogenesis and lipogenesis in 3T3-L1 (mouse pre-adipocytes) cells.

Before performing docking, toxicity analysis was carried out in the present study, where no hepatotoxicity effect was predicted for thirty-two selected C. quadrangularis (Pirandai) ligands (as shown in Table ). However, Orlistat (reference drug) has been predicated to have hepatotoxicity effect. This finding was on par with earlier report, where Orlistat showed fulminant hepatic failure (Sall et al., 2014). Similarly, two ligands (3-O-Methylgallic acid and 9Z,12Z,15Z-Octadecatrienoic acid) of C. quadrangularis (Pirandai) were predicted to have Cytochrome P450 1A2 inhibitory activity. This result was in excellent correlation with previous reports, where gallic acid (parent compound of methyl gallic acid) and 9Z,12Z,15Z-Octadecatrienoic acid (from Moringa oleifera) showed Cytochrome P450 1A2 inhibitory activity (Jumpa-Ngern, 2022; Parvathi et al., 2022).

In the present docking analysis, eleven ligands of Pirandai and standard drug (Orlistat) have exhibited interaction with ASP 34 amino acid residue of Human Intestinal-type Fatty Acid Binding Protein (hI-FABP). This result was in excellent agreement with earlier report, where nitrazepam (lipophilic drug) showed interaction with ASP 34 amino acid residue of hI-FABP (Velkov et al., 2005). Similarly, eleven ligands of Pirandai and standard drug (Orlistat) have shown interactions with SER 53 amino acid residue of human intestinal-type fatty acid binding protein (hI-FABP). This result was in excellent correlation with previous report, where nitrazepam (lipophilic drug) showed interaction with Ser 53 amino acid residue of hI-FABP (Velkov et al., 2005). Further, eight ligands of Pirandai (Cosmosiin, 3,4-dimethoxyphenethyl alcohol, Gallocatechin gallate, Kaempferol-3-o-galactoside, Kaempferol-3-rutinoside, Quercetin-3-o-xyloside, Quercitrin and Secologanin) have exhibited interaction with TYR 14 amino acid residue of human Intestinal-type Fatty Acid Binding Protein (hI-FABP). This result was in excellent agreement with earlier report, where nitrazepam (lipophilic drug) showed interaction with TYR 14 amino acid residue of hI-FABP (Velkov et al., 2005).

In the current docking analysis, four ligands of Pirandai (3,4-dimethoxyphenethyl alcohol, Kaempferol-3-rutinoside, 2-oxo-4-methylthiobutanoic acid and Secologanin) have exhibited interaction with ARG 343 amino acid residue of human Carnosinase 2 (hCN 2) enzyme. This result was in excellent agreement with earlier reports, where Bestatin (BES) and KKL-35 showed interaction with ARG 343 amino acid residue of hCN 2 (Toviwek et al., 2024, Homma et al., 2025). Similarly, three ligands of Pirandai and standard drug (Orlistat) have shown interactions with GLU 166 amino acid residue of human Carnosinase 2 (hCN 2) enzyme. This result was in excellent correlation with previous report, where Bestatin (BES) and KKL-35 showed interaction with GLU 166 amino acid residue of hCN 2 (Homma et al., 2025). Further, two ligands of Pirandai (Dyphylline and n-Hexadecanoic acid) have shown interactions with HIS 380 amino acid residue of human Carnosinase 2 (hCN 2) enzyme. This result was in excellent agreement with earlier report, where Bestatin (BES) and KKL-35 showed interaction with HIS 380 amino acid residue of hCN 2 (Homma et al., 2025).

The present finding is only based on in silico (molecular docking) method which gives new understanding about the 32 chosen C. quadrangularis (Pirandai) ligands and their interactions with 2 selected target proteins. However, further in vitro [8-anilino-1-naphthalene sulfonate (ANS) binding assay] and in vivo studies are required to confirm 32 chosen ligands as modulating agents of two human proteins (hI-FABP and hCN 2).

CONCLUSION

In the current study, the thirty-two chosen Cissus quadrangularis (Pirandai) phytochemicals have shown the potential to dock with two target human proteins (hI-FABP and hCN 2). Moreover, two ligands of Pirandai (Alpha-amyrin, Atractylodin) do not exhibit any interaction with amino acid residues of both hI-FABP and hCN 2 respectively. Thus, the current finding give new knowledge about the thirty-two selected phytochemicals of C. quadrangularis (Pirandai) as potent modulating agents of hI-FABP and hCN 2, which will aid in managing good health and well-being especially obesity related disorders.