Phytochemical and In-SilicoAnalysis of the PotentAntidiabetic Compound from Cactus (Opuntia ficus–indica) cladode

The Cactaceae family includes Opuntia ficus-indica, also calledNopal or Prickly pear (Beatriz N. Guedes et al, 2023).This plant has long been used traditionally to treat various illnesses, including diabetes, especially in Mexico and Central America(Sáenz et al., 2004).Scientific studies have validated these conventional beliefs, showing that Opuntia ficus-indica has hypoglycemic effects in human and experimental animals. Studies have shown that the ingestion of stems can lead to a decrease in blood glucose levels in individuals with non-insulin-dependent diabetes mellitus (NIDDM). Research involving patients with NIDDM found that ingesting 500g of O. ficus indica in various preparations (entire broiled, blended broiled, blended crude, or blended crude and heated) resulted in a statistically significant decrease in glycemia after 120 and 180 minutes. The hypoglycemic effect appears independent of heating or blending during preparation(Frati et al., 1990). Several potential mechanisms have been proposed to explain the anti-diabetic properties of Opuntia ficus-indica. These include the role of dietary fiber in reducing glucose absorption in the intestine and inhibiting α-glucosidase activity, which can postpone the release of glucose. Water extracts of fresh nopal stems have shown significant decreases in blood glucose levels and exhibit α-glucosidase inhibitory activity(Hwang et al.,2017). Opuntia ficus-indica contains a complex array of chemical constituents, including polysaccharides, flavonoids, sterols, and various other bioactive compounds (Alqudah, S.M., et al., 2024). Given the intricate chemical profile of Opuntia ficus-indica and the demonstrated anti-diabetic effects, metabolomic mining represents a valuable approach to comprehensively identify and characterize the specific metabolites responsible for its therapeutic actions (Beatriz N. Guedes et al, 2023). Understanding the complete metabolome and its interaction with biological systems can lead to developing novel and effective anti-diabetic treatments with potentially fewer side effects than existing pharmacological agents. The increasing global prevalence of diabetes underscores the importance of exploring natural resources like Opuntia ficus-indicato discover new therapeutic strategies(Hwang et al.,2017). Furthermore, a study on Wistar rats with induced hyperglycemia demonstrated the normoglycemic activity of Opuntia ficus indica stem extracts (Alqudah, S.M., et al., 2024).  The acetone extract of OFI showed a high efficiency in controlling blood glucose levels, comparable to Metformin(Frati et al.,1990). Considering the growing demand for the development of economical, eco-friendly plant compounds with unique antidiabetic activity, the current study was performed with the objective of untapping the bioactive compounds and in silico docking against target potential for the treatment of diabetes.

The Cactaceae family includes Opuntia ficus-indica, also calledNopal or Prickly pear (Beatriz N. Guedes et al, 2023).This plant has long been used traditionally to treat various illnesses, including diabetes, especially in Mexico and Central America(Sáenz et al., 2004).Scientific studies have validated these conventional beliefs, showing that Opuntia ficus-indica has hypoglycemic effects in human and experimental animals. Studies have shown that the ingestion of stems can lead to a decrease in blood glucose levels in individuals with non-insulin-dependent diabetes mellitus (NIDDM). Research involving patients with NIDDM found that ingesting 500g of O. ficus indica in various preparations (entire broiled, blended broiled, blended crude, or blended crude and heated) resulted in a statistically significant decrease in glycemia after 120 and 180 minutes. The hypoglycemic effect appears independent of heating or blending during preparation(Frati et al., 1990). Several potential mechanisms have been proposed to explain the anti-diabetic properties of Opuntia ficus-indica. These include the role of dietary fiber in reducing glucose absorption in the intestine and inhibiting α-glucosidase activity, which can postpone the release of glucose. Water extracts of fresh nopal stems have shown significant decreases in blood glucose levels and exhibit α-glucosidase inhibitory activity(Hwang et al.,2017). Opuntia ficus-indica contains a complex array of chemical constituents, including polysaccharides, flavonoids, sterols, and various other bioactive compounds (Alqudah, S.M., et al., 2024). Given the intricate chemical profile of Opuntia ficus-indica and the demonstrated anti-diabetic effects, metabolomic mining represents a valuable approach to comprehensively identify and characterize the specific metabolites responsible for its therapeutic actions (Beatriz N. Guedes et al, 2023). Understanding the complete metabolome and its interaction with biological systems can lead to developing novel and effective anti-diabetic treatments with potentially fewer side effects than existing pharmacological agents. The increasing global prevalence of diabetes underscores the importance of exploring natural resources like Opuntia ficus-indicato discover new therapeutic strategies(Hwang et al.,2017). Furthermore, a study on Wistar rats with induced hyperglycemia demonstrated the normoglycemic activity of Opuntia ficus indica stem extracts (Alqudah, S.M., et al., 2024).  The acetone extract of OFI showed a high efficiency in controlling blood glucose levels, comparable to Metformin(Frati et al.,1990). Considering the growing demand for the development of economical, eco-friendly plant compounds with unique antidiabetic activity, the current study was performed with the objective of untapping the bioactive compounds and in silico docking against target potential for the treatment of diabetes.

Determination of total phenolic contents

The Folin Ciocalteu method (Elizabeth Rojas-Ocampo  et al., 2021) was used with minor adjustments to determine the total phenolic contents in the in various solvent extracts of cladode and fruit, viz., hexane, ethyl acetate, 40% ethanol, and water. To the aliquot of 0.25 mL, 1.25 mL Folin-Ciocalteu reagent was added, diluted ten times. Then, 1 mL of sodium carbonate (7.5%) was added. The mixture was incubated in the dark for 30 min. The absorbance was measured at 765 nm against a blank. The total phenolic contents were expressed as gallic acid equivalents (GAE) in milligrams per 100 g dry material.

Determination of flavonoid contents

The flavonoid compounds content wasascertained in various solvent extracts ofcladode and fruit, viz., hexane, ethyl acetate, 40% ethanol, and water,as described by Chouguiet al., 2013. In summary, 1.5 mL of extract was added to 1.5 mL of AlCl₃ reagent (2%). After 30 min of incubation in the dark, the absorbance was read at 430 nm against a blank. Quercetin was used as a standard for the calibration curve. The results are expressed as mg equivalent of quercetin (QE) per 100 g of dry matter.

Gas Chromatography – Mass Spectrometry Analysis (GC-MS)

            The extract was prepared according to the protocol Rahul et. al. (2012) described with appropriate modification. Five (5) g of the powdered cladode was soaked in 25 mL of methanol for 48 hours. The extract was centrifuged at 5000 rpm for 10 min, then filtered through Whatman filter paper No. 41 along with 0.5 g sodium sulphate to remove any sediments and traces of water in the filtrate, and finally passed through a polyvinylidenedifluoride membrane filter of 0.22 micron (PDVF, Agilent Technologies, India).

Analysis of the extract through GC-MS

            GC-MS analysis, the methanol cladode extract of Opuntia ficus – indica was performed using a Shimadzu QP2020 (Shimadzu, Japan) gas chromatographconnected to a mass spectrometer. GC was equipped with Rx Sil 5MS capillary column (30 m x 0.25 mm i.d. x 0.25 μm film thickness) consisting of a stationary phase 5% phenyl and 95% methyl polysiloxane. The injection was carried out in splitless mode at an injector temperature of 260℃. Helium gas was a carrier gas with a 1.0 mL/min flow rate. The oven temperature programming was as follows: the initial oven temperature was held at 70℃ for 2.0 min, and then increased to 210℃ at a rate of 20℃/min, and then increased to 290℃at a rate of 10 ℃/min held for 5 min. The ion source and transfer temperature were 230℃ and 280℃, respectively.

Identification of the compounds

            GC-MS, which detected the various compounds present in the methanol extract of the cladode. Identification and interpretation of each compound’s mass spectrum was carried out using National Institute Standard and Technology (NIST) database available in the instrument. The spectrum of the unknown components was compared with those of the known components stored in the NIST library.

In silico analysis of the biomolecules present in Cactus cladode methanolic extract

The names, molecular formula, molecular weight, and structure of the compounds in the methanol cladode extract were identified using GC-MS analysis, and the NIST library was ascertained via PubChem database and Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp.kegg).

Screening of ligands for pharmacological activity

The three-dimensional (3D) structures of the Biomolecules with enzymatic inhibitory activity were obtained from PubChem compound-specific database. The pharmacological activity for the selected structures was screened using the QikProp module from the Schrodinger 9.3 and Prediction of Activity Spectra for Substances (PASS).

Ligand–based docking using GLIDE (Grid-based Ligand Docking with Energetics)

To carry out glide analysis, 3D conformation structures of the ligands for antidiabetic propertywere  obtained from Pubchem in .sdf format. The receptors were prepared, grid generation was done, the ligands were prepared, and finally, docking analysis was carried out.

Analysis of the docking result

            GLIDE XP visualization of Schrodinger software was used in viewing the docking results and incorporated them into the project table as an XP descriptor file in .xpdes format. The analyzed results were saved in .csv format.

Screening of the identified compound for pharmacological activity

From the list of compounds  in table 2, the highest peak area (%) compound was screened for various pharmacologicalactivities. The respective 3D conformation structure and biological activity were obtained from PDB and PASS database, respectively (Table 3).

Statistical analysis

Data are expressed as mean ± standard deviation from three separate observations. For total phenol compounds assays (phenol and flavonoid) a way ANOVA test was used to analyze the significance of the difference between various extracts studied (P < 0.05).

Phytochemical analysis of Cactus cladode

            The Opuntia indicaficus cladode was subjected to preliminary Phytochemical screening using different qualitative tests. The results indicated the presence of alkaloids, tannins, flavonoids, phenols, proteins, terpenoids, reducing sugars, glycosides, carbohydrates, steroids, and saponins as shown in Table 1.

 Table 1. Phytochemical analysis of Cactus cladode methanol extract

Total phenolic estimation using Folin-Ciocalteu assay (TPC)

Figure 2. Total phenolic estimation by the Folin-Ciocalteu method in Opuntia plant fruits and

Cladodes. A. The standard graph uses gallic acid as a standard polyphenol. B. Cactus fruit; C. Cactus

cladode.

Total Flavonoid content analysis (TFC)

Total flavonoid analysis refers to determining the total amount of flavonoids present in

a sample. This analysis is essential because flavonoids are known for their antioxidant and

other health-promoting properties. TFC is typically determined, often involving aluminum chloride,

and results are expressed as equivalents of a standard flavonoid, like catechin or quercetin.

Figure 3. Total flavonoid estimation by Aluminium chloride method in Opuntia plant fruit

and cladode. A. Standard graph using Quercetin as a standard flavonoid. B. Cactus fruit; C.

Cactus cladode.

Cactus cladode and fruit possess higher health-promoting properties in terms of high antioxidant potential in ethyl acetate fraction thanwith other solvent fractions.

GC-MS analysis of the cladode of Cactus

            The methanol cladode extract of cactus was subjected to GC-MS analysis using Shimadzu QP2020 gas chromatography mass spectrometry. The compounds identified are recorded in Table 2.

Methanol extract of the cladode:

Table 2. Compounds identified in the methanol extract of cactus cladode by GC-MS

Medicinal plants are the source of potent drugs, and the use of plants as a medicine has been inherited as an important component of the health care system from the ancient period. India is also regarded as the largest producer of medicinal herbs (Sathyaprabha, 2010).  Opuntia ficus-indica is also used traditionally to treat various illnesses, including diabetes, especially in Mexico and Central America (Saenz et al, 2004).

            Successful isolation of the bioactive principle from plant materials largely depends on the type of solvent used in the extraction procedure. The solvents can also contribute to the variation since it has been proven that the extraction of biologically active compounds from plants depends upon the polarity of the solvent used (Ghosh et al., 2012).

            In the present study, the cladode was subjected to qualitative phytochemical screening. Seven different solvents: Hexane, chloroform, diethyl ether, ethyl acetate, methanol, ethanol and water were used. The presence of alkaloids, tannins, flavonoids, proteins, terpenoids, reducing sugars, glycosides, carbohydrates, steroids and saponins was documented and these results were similar to the results of Shimaa Ali et al., 2022.

Docking analysis

            Docking analysis was carried out for the compound 2-Thophene carboxylic acid-5methyl (2-carboxy-5-methylthiophene) obtained from the methanolic extract of the cactus cladode for four human protein targets namely, Human Estrogen receptor (PDB ID: 3ERT) (anti-cancer), HIV-1 envelope glycoprotein (PDB ID: 4CC8) (anti-HIV),  Human C-reactive protein (PDB ID: 1GNH) (anti-inflammation) and Human 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1) (PDB ID: 1XU7) (anti-diabetes) respectively via Schrodinger version 9.3.

The bioactive compounds obtained from plants, include alkaloids, flavonoids, tannins, phenolic compounds, etc.,are the main drivers of the pharmacological actions of medicinal plants (Hussein and El-Anssary, 2018). The chemical constituents of the plants are the economic source for the synthesizing complex chemical substances (Moniruzzaman Sohag Howlader  et al., 2016).

A total of 25 compounds were identified from the GC-MS result, the concentration of 2-Thophene carboxylic acid-5methyl (2-carboxy-5-methylthiophene) was found to higher in terms of area %. So far, this compound is not reported from the methanolic extract of the cactus cladode and was found to be a first report. Other therapeutic compounds such as heneicosane, behenic alcohol, pentatriscontene, and docosane were also found.

Gas chromatography-mass spectrometry has been firmly established as a key technological platform for identifying secondary metabolites in both plant and non-plant species. Its ability to separate complex organic molecules with high efficiency (Zhang et al., 2012), the mass fragmentation pattern based on m/z values, provide a clue to the molecular structure. Additionally, the data library along with the molecular ion peaksignificantly improves the compound identification.

Table: Information of binding affinity and the 2D interaction diagram

We conducted the in-silico analysis using the biologically active secondary metabolites identified from the GC-MS screening.  Among the chosen targets, the highest binding affinity of -5.25 kcal/mol wasobserved withdiabetes target Human 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1), which showed two hydrogen bond interactions (GLN 21 and ARG 252 residues) with 2-Thiophenecarboxylic acid, 5-Methyl-,2-(Methylamino)-2-Oxoethylester.  The second highest binding affinity was shown against the anti-HIV target, HIV-1 envelope glycoprotein (-5.14 kcal/mol), with PRO 167 and SER 176 residues involved in hydrogen bond interactions at the active site. Further, 2-Thiophenecarboxylic acid,5-Methyl,2-(Methylamino)-2-Oxoethyl estershowed binding value of -4.55 kcal/mol and -3.46 kcal/mol against cancer target and inflammation target respectively. Further, this compound has a bioavailability score of 0.55 and follows the Lipinski rule of five, which is an important property for the drug's likelihood.

Based on the above docking results, Thiophenecarboxylic acid,5-Methyl,2-(Methylamino)-2-Oxoethyl ester is predicted as the best anti-diabetic compound and may be considered as a drug-like molecule for further investigation to develop as a drug.

Phytochemical analysis of Cactus cladode

            The Opuntia indicaficus cladode was subjected to preliminary Phytochemical screening using different qualitative tests. The results indicated the presence of alkaloids, tannins, flavonoids, phenols, proteins, terpenoids, reducing sugars, glycosides, carbohydrates, steroids, and saponins as shown in Table 1.

Table 1. Phytochemical analysis of Cactus cladode methanol extract

Total phenolic content estimation using Folin-Ciocalteu assay (TPC)

Figure 2. Total phenolic estimation by the Folin-Ciocalteu method in Opuntia plant fruits and

Cladodes. A. The standard graph uses gallic acid as a standard polyphenol. B. Cactus fruit; C. Cactus

cladode.

Total Flavonoid content analysis (TFC)

Total flavonoid analysis refers to determining the total amount of flavonoids present in

a sample. This analysis is essential because flavonoids are known for their antioxidant and

other health-promoting properties. TFC is typically determined, often involving aluminum chloride,

and results are expressed as equivalents of a standard flavonoid, like catechin or quercetin.

Figure 3. Total flavonoid estimation by Aluminium chloride method in Opuntia plant fruit

and cladode. A. Standard graph using Quercetin as a standard flavonoid. B. Cactus fruit; C.

Cactus cladode.

Cactus cladode and fruit possess higher health-promoting properties in terms of high antioxidant potential in ethyl acetate fraction thanwith other solvent fractions.

GC-MS analysis of the cladode of Cactus

            The methanol cladode extract of cactus was subjected to GC-MS analysis using Shimadzu QP2020 gas chromatography mass spectrometry. The compounds identified are recorded in Table 2.

Methanol extract of the cladode:

Table 2. Compounds identified in the methanol extract of cactus cladode by GC-MS

Medicinal plants are the source of potent drugs, and the use of plants as a medicine has been inherited as an important component of the health care system from the ancient period. India is also regarded as the largest producer of medicinal herbs (Sathyaprabha, 2010).  Opuntia ficus-indica is also used traditionally to treat various illnesses, including diabetes, especially in Mexico and Central America (Saenz et al, 2004).

            Successful isolation of the bioactive principle from plant materials largely depends on the type of solvent used in the extraction procedure. The solvents can also contribute to the variation since it has been proven that the extraction of biologically active compounds from plants depends upon the polarity of the solvent used (Ghosh et al., 2012).

            In the present study, the cladode was subjected to qualitative phytochemical screening. Seven different solvents: Hexane, chloroform, diethyl ether, ethyl acetate, methanol, ethanol and water were used. The presence of alkaloids, tannins, flavonoids, proteins, terpenoids, reducing sugars, glycosides, carbohydrates, steroids and saponins was documented and these results were similar to the results of Shimaa Ali et al., 2022.

Docking analysis

            Docking analysis was carried out for the compound 2-Thophene carboxylic acid-5methyl (2-carboxy-5-methylthiophene) obtained from the methanolic extract of the cactus cladode for four human protein targets namely, Human Estrogen receptor (PDB ID: 3ERT) (anti-cancer), HIV-1 envelope glycoprotein (PDB ID: 4CC8) (anti-HIV),  Human C-reactive protein (PDB ID: 1GNH) (anti-inflammation) and Human 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1) (PDB ID: 1XU7) (anti-diabetes) respectively via Schrodinger version 9.3.

The bioactive compounds obtained from plants, include alkaloids, flavonoids, tannins, phenolic compounds, etc.,are the main drivers of the pharmacological actions of medicinal plants (Hussein and El-Anssary, 2018). The chemical constituents of the plants are the economic source for the synthesizing complex chemical substances (Moniruzzaman Sohag Howlader  et al., 2016).

A total of 25 compounds were identified from the GC-MS result, the concentration of 2-Thophene carboxylic acid-5methyl (2-carboxy-5-methylthiophene) was found to higher in terms of area %. So far, this compound is not reported from the methanolic extract of the cactus cladode and was found to be a first report. Other therapeutic compounds such as heneicosane, behenic alcohol, pentatriscontene, and docosane were also found.

Gas chromatography-mass spectrometry has been firmly established as a key technological platform for identifying secondary metabolites in both plant and non-plant species. Its ability to separate complex organic molecules with high efficiency (Zhang et al., 2012), the mass fragmentation pattern based on m/z values, provide a clue to the molecular structure. Additionally, the data library along with the molecular ion peaksignificantly improves the compound identification.

Table: Information of binding affinity and the 2D interaction diagram

We conducted the in-silico analysis using the biologically active secondary metabolites identified from the GC-MS screening.  Among the chosen targets, the highest binding affinity of -5.25 kcal/mol wasobserved withdiabetes target Human 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1), which showed two hydrogen bond interactions (GLN 21 and ARG 252 residues) with 2-Thiophenecarboxylic acid, 5-Methyl-,2-(Methylamino)-2-Oxoethylester.  The second highest binding affinity was shown against the anti-HIV target, HIV-1 envelope glycoprotein (-5.14 kcal/mol), with PRO 167 and SER 176 residues involved in hydrogen bond interactions at the active site. Further, 2-Thiophenecarboxylic acid,5-Methyl,2-(Methylamino)-2-Oxoethyl estershowed binding value of -4.55 kcal/mol and -3.46 kcal/mol against cancer target and inflammation target respectively. Further, this compound has a bioavailability score of 0.55 and follows the Lipinski rule of five, which is an important property for the drug's likelihood.

Based on the above docking results, Thiophenecarboxylic acid,5-Methyl,2-(Methylamino)-2-Oxoethyl ester is predicted as the best anti-diabetic compound and may be considered as a drug-like molecule for further investigation to develop as a drug.

The medicinal/pharmacological property of the cactus cladode is attributed to the presence of various bioactive compounds. The findings recorded in the present research will be helpful in designing an anti-diabetic drug. Therefore, this study supports the management of type-2 diabetes with the plant-based compound as they are practical, easily accessible, and have no known adverse effects.

Acknowledgement:The authors are grateful to the Tamil Nadu Agricultural University for sanctioning the University research project and the Centre for Plant Molecular Biology and Biotechnology (CPMB&B), TNAU, Coimbatore, for their support throughout this research.

Conflict of Interest :The authors hereby declare no conflict of interest.

Ethics statement:No specific permits were required for the described field studies because no human or animal subjects were involved in this research.

Consent for publication :All the authors agreed to publish the content.

Competing interests

There were no conflicts of interest in the publication of this content.

Author contributions

Idea conceptualization, writing original draft – Vellaikumar, Experiments – Vellaikumar, Saranya, reviewing and editing – Saranya andPreethieswari.