α-Amylase, α-glucosidase and aldose reductase inhibitory and molecular docking studies on Tinospora cordifolia (Guduchi) leaf extract

Background Type II diabetes mellitus is posing a severe health threat throughout the globe due to its associated pathophysiological risks and high mortality rate. Carbohydrate catabolic enzymes, including α-amylase, α-glucosidase and aldose reductase, play an important role in the development of diabetes. The natural or synthetic inhibitors of these enzymes are crucial in reducing diabetes and its related complications. Tinospora cordifolia is a plant of great significance in Ayurveda due to its unique biological activities, including anti-diabetic properties. The present study aims to identify the active constituents of T . cordifolia leaves and evaluate the in vitro inhibitory potential of its ethanol extract constituents against α-amylase, α-glucosidase and aldose reductase activities. Results The ethanolic leaf extract of T . cordifolia inhibited the activities of α-amylase, α-glucosidase and aldose reductase in a dose-dependent manner. It was on par with the standard inhibitors acarbose and quercetin. At 5 mg/ ml, the noted % inhibition values of extract were 69.27 ± 0.17, 67.8 ± 0.26 and 62.55 ± 0.24, respectively, for α-amylase, α-glucosidase and aldose reductase. Using GC-MS analysis, neophytadiene, γ-sitosterol, phytol, phytyl palmitate, and phytyl acetate were identified as prominent constituents of the ethanolic extract. Based on molecular docking and ADME analysis, γ-sitosterol was found as the major reactive phytoconstituent, which showed the highest inhibitory potential against α-amylase, α-glucosidase and aldose reductase activities. Conclusions The present study identified γ-sitosterol as triplet inhibitor of α-amylase, α-glucosidase and aldose reductase and affirmed the ethno-medicinal significance of T . cordifolia leaves in the development of new anti-diabetic leads.

by diabetes globally, and the frequentness is supposed to rise to 643 million by 2030 and 783 million by 2045 [2].
Diabetes is recognized as one of the significant listed ten causes of deaths occurring worldwide.It also increases 2-3 folds risk of all-cause-mortality in affected individuals [3].The lifestyle modifications, including a healthy and balanced diet, physical exercise and weight management, are vital parameters in the managing diabetes [4].Although these lifestyle changes are helpful in reducing blood glucose levels, controlling weight and lowering the risk of cardiovascular complications, their regular and long-term follow-up may not be possible for most of the patients [5], and hence, they are primarily dependent on diabetic medications for treatment.The exogenous insulin application and many glucose-lowering compounds are commercially available and routinely used to manage diabetes.However, the pharmacologic management of diabetes through available anti-diabetic drugs is complicated and requires a combination therapy of two oral drugs with or without insulin for high-risk patients [6].
The three carbohydrate digestive enzymes, α-amylase (AML), α-glucosidase (AGA) and aldose reductase (AR), play an essential role in the development of diabetes and associated complications.The AML and AGA act on complex dietary carbohydrates and convert them into simple, absorbable sugars (Fig. 1a), [7].The postprandial hyperglycemia resulting from diabetes can be lowered by preventing the absorption of simple sugars resulting from the metabolism of complex sugars in the intestine.The pancreatic AGA and AML enzymes are attached to the brush border of intestinal cells and involved in digesting complex carbohydrates [8].The AR is an additional enzyme involved in glucose metabolism and produces sorbitol via the polyol pathway in the eye lens (Fig. 1b), [9].It acts as a connecting link between diabetic retinopathy and elevated blood sugar levels.Diabetic retinopathy is a common microvascular complication arising due to the prolonged effects of diabetes and may lead to severe vision problems and even blindness in affected individuals [10].Inhibition or suppression of these principal carbohydrate metabolic enzymes using natural sources is considered one of the centered approaches in managing diabetes.Medicinal plants rich in phytochemicals of therapeutic significance have been used to cure many ailments for a long time.The use of herbal remedies in the management of diabetes is also recommended by the World Health Organization [11].
Tinospora cordifolia (Guduchi, Giloy, Amrita or Indian bitter) is mainly used in the Indian traditional medicinal system to combat complications of diabetes.The antidiabetic utility of guduchi is also documented in Ayurvedic Pharmacopoeia of India [12].In the Melghat region of Maharashtra state, the tribals of Korkus make use of this herb to relieve polyuria, diabetes and fever [13].Recently, Ahsan et al. [14] reviewed and summarized the therapeutic potential of various phytochemical constituents of Guduchi in terms of anti-cancer, immuno-modulatory, anti-viral, anti-oxidant, anti-microbial and anti-diabetic activities.However, well-established scientific or experimental evidence on the inhibitory effects of Guduchi leaf extracts on the activities of AML, AGA and AR enzymes is less documented.In-depth analysis and understanding of the inhibition properties of various leaf phytoconstituents by molecular docking will be promising in revealing additional knowledge on its therapeutic significance.The present research work aimed to investigate the inhibitory action of Guduchi leaf extract on principal carbohydrate metabolic enzymes (AML, AGA and AR) and to study the nature of interactions of various chemical constituents of the extract with these target enzymes.Further, the physiological and pharmacological properties of these constituents are studied to assess their drug likeliness and to find their suitability for the development of potential inhibitors.

Materials and Methods
Porcine pancreatic AML and AGA from Saccharomyces, NADPH, DL-glyceraldehyde, and quercetin were procured from Sigma Aldrich., India.Ethanol and dimethyl sulfoxide (DMSO) were purchased from Thermo Fisher Scientific, India.

Collection and identification of plant material
The fresh green leaves of Tinospora cordifolia (Fig. 2) were collected from in and nearby regions of Vishnupuri, Nanded district (19°06′43″N 77°17′20″E), of Maharashtra during November-December 2022.The plant material was identified in the School of Life Sciences, SRTM University, Nanded, and a voucher specimen (SLS21) was deposited at the School.

Processing of plant material and preparation of extracts
The freshly collected leaves of T. cordifolia were washed thoroughly under running tap water, blotted on filter paper and dried in shade for 10-15 days.The completely dried leaves were ground to obtain a fine powder using a mechanical grinder.The powdered material was stored in sealed containers at 4°C for further use.About 10 gm of dried leaf sample was dissolved in 100 ml ethanol in a 500-ml Erlenmeyer flask and macerated completely under shaking conditions of 120 rpm for 24 h in an orbital shaker (Remi-396LAG).The extract was filtered through Whatman filter paper (No. 1), and the filtrate was evaporated to dryness using rotavapor.The dried extract was weighed and dissolved in 10% dimethyl sulfoxide (DMSO) at a final concentration of 5 mg/ml for further studies.The yield of the extract (1.09 g, 10.9%) was determined as explained by Tomlinson et al. [15].

Enzyme inhibitory activities of ethanolic leaf extract (ELE) AML and AGA inhibition assay
Porcine pancreatic AML and AGA from Saccharomyces (Sigma-Aldrich) were used for inhibition studies.Acarbose was used as a standard AML and AGA inhibitor.The method described by Kwon et al. [16] and modified by Poovitha and Parani [17] was used to determine inhibition of porcine α-amylase activity.To determine the AGA inhibition activity of ELE, the method followed by Kim et al. [18] was used.The extract was used at varying concentrations of 0.5-5 mg/ml, and acarbose as a reference drug was used at a 1 mg/ml concentration.The extract concentrations and acarbose were prepared in water.

AR inhibition assay
The method described by Kinoshita [19] with modifications followed by Ranjan et al. [20] was used to study the AR inhibition activities of leaf extract.To determine enzyme inhibition activity, spectrophotometric measurements of the decrease in absorption of NADPH at 340 nm over a time interval of 5 min and the use of DLglyceraldehyde as a substrate were studied in the presence of different concentrations of leaf extract (0.5-5 mg/ ml).Quercetin (100 µg/ml) was used as a control in this method.
Percentage inhibition in AML, AGA or AR activities in the presence of ELE was calculated using the following formula:

GC-MS analysis of ELE
Gas chromatography-mass spectrometry (GC-MS) analysis of the ethanolic extract of T. cordifolia leaves was performed using a GC-MS (Model: TQ80 50) equipped with a DB5MS silica capillary column (30 m × 0.25 mm × 0.25 µm length), and helium was used as the carrier gas at a constant flow rate of 1.01 ml/min.Initial injection temperature was set to 250 °C and oven temperature increased from 50 to 180 °C at 10 °C / min rise, and finally set to 250 °C for 5 min.The sample (1 µl) was injected in split mode with a scanning range of 20 to 700 m/z.The complete run time of GC-MS was 46 min.The relative percentages indicating the amount of each component were calculated from peak areas and identified by using the National Institute of Standards and Technology (NIST) library, 2014 [21].

Molecular docking studies
The GC-MS analysis of the ethanol extract of T. cordifolia leaves identified five principal phytocompounds: gamma sitosterol, 3,7,11,15-tetramethyl 2-hexadecen-1-ol, neophytadiene, phytyl palmitate and phytyl acetate.In order to define the potency correlation of results observed in vitro with the structure of phytocompounds, an attempt was made to investigate our findings by using an in silico molecular docking approach.The 3D structures of all selected phytocompounds and standards (acarbose and quercetin) used in this study were obtained from the PubChem database (https:// pubch em.ncbi.nlm.nih.gov/) (accessed on 21 December 2023) [22].The 3D structures of three target diabetic-relevant proteins, AML, AGA and AR, were retrieved from the protein databank database (PDB) (http:// www.rcsb.org) (accessed on 21 December 2023) [23].Following energy minimization, the ligands were converted to pdbqt format for further investigations by Auto Dock.By using the Uniprot database (http:// www.unipr ot.org/) (accessed on 22 December 2023), the Uniprot IDs of these target proteins were taken as 1U2Y (AML), 5KZW (AGA) and 3S3G (AR).Docking studies for all three target proteins, α-amylase, α-glucosidase and aldose reductase, and five phytoconstituents of T. cordifolia leaf extract were carried out using Auto Dock 4.2.The results were visualized using PyMol V 0.99 (http:// www.pymol.org) (accessed on 22 December 2023).

Physiological and pharmacokinetic properties of phytocompounds
The Swiss web server www.swiss adme.ch (accessed on 23 December 2023) was used for computing the adsorption, % Inhibition = Enzyme activity in absence of ELE − Enzyme activity in presence of ELE Enzyme activity in absence of ELE × 100 distribution, metabolism and excretion (ADME) properties of each phytocompound [24].The parameters considered for analysis were molecular weight, flexibility (number of rotational bonds), topological polar surface area (TPSA, A 2 ), aqueous solubility (Logs by ESOL method), polarity (Log P water: octanol), gastrointestinal tract (GI) absorption, blood-brain barrier (BBB) permeant, substrate for Pg-P and inhibition of CYP450 isoforms, skin permeation coefficient (KP), Lipinski's rule of 5, Veber's rule and pan assay interference (PAINS) alert.The SMILE format of each phytocompound was uploaded to the web software, and the obtained results were recorded manually.

Statistical analysis
All the experiments related to AML, AGA and AR inhibition assays were performed in triplicate, and the results were presented in terms of the mean ± standard deviation.Linear regression analysis of dose-response curves was performed to calculate the IC 50 values.Regression coefficients, mean, standard deviations, and P values were computed using Graph Pad Prison 5.0 software (Graph Pad, San Diego, CA, USA).

Results
The results of in vitro enzyme inhibitory activities of ELE are shown in Table 1.ELE of T. cordifolia inhibited AML and AGA activities at all studied concentrations, the highest being recorded at 5 mg/ml.A maximum of 69.27% and 67.8% inhibition of AML and AGA activities were observed at 5 mg/ml concentration of T. cordifolia ELE with IC 50 values of 2.96 ± 0.59 mg/ml and 3.398± 0.16 mg/ml respectively.Acarbose, at 1 mg/ml, showed 72.15% and 81.20% inhibition in AML (IC 50 , 0.574 ± 0.39 mg/ml) and AGA (IC 50 , 0.612 ± 0.24 mg/ ml) activities, respectively.The ethanolic leaf extract of T. cordifolia showed AR inhibition at concentrations of and above 2 mg/ml in a dose-dependent manner with an IC 50 value of 3.86 ± 0.42 mg/ml.Quercetin at 100 µg/ml inhibited 73.18% of AR activity, attaining an IC 50 value of 58.76 ± 0.68 µg/ml.The GC-MS analysis of T. cordifolia leaf extract revealed the identification of 24 chemical compounds.The elemental analysis and molecular details of these       4).
Although phytyl palmitate was the first revealed best phytocompound, when analyzed for pharmacokinetic properties using a Swiss web server, it did not satisfy the ADMET criteria of drug likeliness due to its higher molecular weight, presence of more rotational bonds, aqueous insolubility, low GI absorption, poor bioavailability score and not satisfying Lipinski's rule of 5 (Table 5).Hence, the second best phytocompound, γ sitosterol, was selected further.The binding energies of − 2.8, − 16.Table 5 shows the physicochemical and pharmacokinetic properties of selected phytocompounds in the ethanolic leaf extract of T. crdifolia.The chosen compounds exhibited molecular weights ranging from 296.53 to 534.94 Da, and log P (O/W) values were ranged from 4.71-8.67.The TPSA and log values ranged between 0.00 to 26.30 and − 5.98 to − 11.38, respectively.The compounds were insoluble to moderately soluble in water, while their log Kp values were between + 1.86 and − 2.29.The number of rotational bonds was limited only for γ-sitosterol.All phytocompounds except phytyl palmitate obeyed Lipinski's rule of 5, and only γ-sitosterol satisfied both Lipinski's rule of 5 and Veber's law.All compounds were not BBB-permeant, and their bioavailability scores varied from 0.17 to 0.55.The γ-sitosterol and phytyl palmitate were not inhibitory to all isoforms of CYP 450, whereas other phytocompounds inhibited either CYP 450 enzyme.

Discussion
The chronic metabolic disorder, diabetes, is one of the leading causes of death in the 21st century and is currently affecting more than half a billion people worldwide.The pathophysiological symptoms associated with diabetes are mainly due to impairment in insulin secretion, accompanied to some extent by genetic predisposition, obesity, hypertension and cardiovascular complications [25].The urgent need for effective treatment of diabetes is a matter of great concern due to its common occurrence and associated pathophysiological complications.The worldwide search for effective anti-diabetic drugs has intensified in the past few decades to meet the challenges associated with diabetes and overcome related disorders.
Presently available treatments for diabetes are either insulin-dependent and/or make use of oral drugs containing sulfonylureas, biguanides, thiazolidinediones and glinides [26].Most of these drugs show adverse side effects, indicating the need for safer and more effective alternatives.As AMA, AGA and AR are the principal enzymes involved in carbohydrate metabolism and linked with the cause or origin of diabetic problems, a broad range of studies have been focused on inhibitors of these significant enzymes in the past few decades.
Medicinal plants rich in bioactive metabolites are the principal candidates for drug screening programs, and since the ancient era, plants have been viewed as the fountainhead of anti-diabetic molecules.Although many traditional plants are used for the treatment of diabetes, only a few of them are critically examined at the scientific or clinical level.In the present study, we have selected T. cordifolia, a member of Menispermaceae, which has an ancient history in Ayurveda, as a source for anti-cancer [27], immunomodulatory [28], hepatoprotective [29] and antidiabetic [30] molecules.A critical and systematic review by Sharma et al. [30] has also provided the pharmacological and clinical evidence indicating the hypoglycemic potential of T. cordifolia plant parts.However, these studies do not support sound knowledge regarding the inhibition of diabetic-relevant AMA, AGA and AR enzymes.
The AML, AGA and AR inhibitory activities of ELE were promising and significant, although they were slightly lower than the standards acarbose and quercetin.GC-MS analysis of ELE carried out to identify active compounds showed five major constituents: neophytadiene, γ-sitosterol, 3,7,11,15-tetramethyl 2-hexadecen-1-ol, phytyl palmitate and phytyl acetate.The neophytadiene is a terpene identified with great anti-diabetic potential in many plants, including Achillea ligustica [31] and Eryngium caeruleum [32].In addition to this, the anti-inflammatory and antibacterial activities of neophytadiene have been reported by Bharadwaj et al. [33].Similarly, the anti-diabetic activity of γ-sitosterol, a steroidal compound isolated from Lippla nodiflora, has been reported by Balamurgan et al. [34].The study found a significant decrease in blood glucose and glycosylated hemoglobin in streptozotocin-induced rats in the presence of γ-sitosterol.However, the reports on its interaction with AML, AGA and AR are the least found.Phytol (3,7,11,15-tetramethyl 2-hexadecen-1-ol), an acyclic diterpene alcohol and its derivative phytol acetate, the other two major constituents of ELE, were reported earlier for their anti-tuberculosis activity against Mycobacterium tuberculosis and anti-oxidant, anti-inflammatory and anti-convulsion activities [35,36,37].Recently, Mariyammal et al. [38] found phytyl palmitate by GC-MS analysis of Aristolochia tagala cham leaf extracts and indicated the antibacterial potential of extracts against Proteus vulgaris and Pseudomonas aeruginosa.However, its anti-diabetic relevance has been indicated rarely in earlier studies.
By considering the promising results of AML, AGA and AR inhibition and exploring further opportunities with ELE to appear as a suitable target of an anti-diabetic molecule, we employed a molecular docking approach to evaluate their receptor binding affinities and mechanisms of inhibition activity.Our docking results showed that active constituents from the ELE of T. cordifolia are reactive with all three enzyme targets.However, the overall binding affinities of phytyl palmitate and γ-sitosterol for AML, AGA and AR were higher than those of neophytadiene, phytyl acetate and phytol.The role of AGA and AML [8,16,39] and AR [40,41] inhibitors in diabetes has been authorized by many researchers.Although we found γ-sitosterol and phytyl palmitate as the bestsuited antidiabetic molecules, based on ADMET analysis, the pharmacokinetic properties of phytyl palmitate did not satisfy the selection criteria of drug likeliness, and we came up with γ-sitosterol as the most suitable anti-diabetic compound from the ELE of T. cordifolia.
All five selected phytocompounds from the ethanolic leaf extract of T. cordifolia showed varying binding affinities with 3 target proteins.Molecular docking analysis of AML and AGA shows that among all phytocompounds, only γ-sitosterol and phytyl palmitate have binding energies greater than those of the standard drug acarbose.The amino acid residues of target proteins involved in binding interactions that are common to both acarbose and γ-sitosterol are Gln349, Asn352, Gly351, and Ser 251, Trp273, Tyr133 and Pro85, respectively, for AML and AGA.The presence of common binding residues of acarbose and γ-sitosterol indicated that γ-sitosterol binds to AML and AGA in a space nearer to where the acarbose binds to the respective protein.As per the previous studies, acarbose is a known competitive inhibitor of AML and AGA [42].This shows that γ-sitosterol might also act as a competitive inhibitor of AML and AGA.Molecular docking analysis of AR also indicates that the binding energies of γ-sitosterol and phytyl palmitate are more than the standard inhibitor quercetin used in this study.However, the amino acid residues of involved in binding interactions of γ-sitosterol are different from quercetin, indicating the possibility of sharing dissimilar inhibition modes.

Fig. 1
Fig. 1 Role of AML, AGA and AR in diabetic complications.a: Action of AML and AGA on dietary carbohydrates containing starch, b: polyol pathway for conversion of glucose to sorbitol and fructose by AR and sorbitol dehydrogenase

Fig. 2 T
Fig. 2 T. cordifolia plant growing as a climbing shrub (a) and enlarged view of leaves (b)

Fig.
Fig. GC-MS chromatogram of T. cordifolia leaf extract

phytocompounds are shown in Table 2 .
The five prominent chemical structures selected on the basis of their peak area values (%) showed excellent affinity with chosen target proteins.The GC-MS chromatogram with identified prominent peaks is displayed in Fig.3.The fragmentation patterns and mass spectrums of selected principle phytocompounds are shown in Figs. 4, 5, 6, 7, 8.The 2D structures of selected phytocompounds derived from NIST Chemistry Web-Book are shown in Table3.The binding affinities of phytocompounds revealed in terms of binding energies varied from − 1.6 to − 3.0 kcal/ mol, − 10.9 to − 20.2 kcal/mol and − 10.8 to − 20.4 kcal/ mol for AML, AGA and AR, respectively.Phytyl palmitate showed very good binding affinities with all three

Fig. 9
Fig. 9 3D (a) and 2D (b) interactions of γ-sitosterol with amino acid residues of AML 5 and − 15.0 kcal/mol were observed during the docking analysis of γ sitosterol with AML, AGA and AR, respectively.It showed very good affinity with Asp 352, Gln 347, Val 354, Asn 350, Gly 351, Asp 353 and Phe 348 amino acid residues of AML, Arg 154, Thr 156, Ser 135, Pro 238, Tyr 133 and Ser 132 residues of AGA, and Leu 6, Asn 7, Leu 5, Asn 8 and Ala 10 residues of AR.The interaction details of gamma sitosterol with the AML, AGA and AR active sites are depicted in Figs. 9, 10 and 11.

Table 1
In vitro enzyme inhibitory activities of ethanolic leaf extract from T. cordifolia a Each value of percent inhibition of AML, AGA, and AR activity is presented as mean ± standard deviation (n = 3).IC 50 : concentration at which half-maximal activity is inhibited.A significant difference was observed among mean percent inhibition values (P < 0.05)Concentration

Table 2
Structural details of phytocompounds of ethanolic extract of T. cordifolia leaves

Table 3
2D structures of selected phytocompounds from ELE of T. cordifolia leaves

Table 4
Details of interaction of selected phytocompounds with AAL, AGA and AR

Table 5
Physicochemical and pharmacokinetic properties of selected phytocompounds