Int J App Pharm, Vol 17, Issue 1, 2025, 190-198Original Article
GC-MS ANALYSIS AND MOLECULAR DOCKING STUDIES OF LASERPITIUM LATIFOLIUM L EXTRACT FOR ANTI-DIABETIC ACTIVITY
DIVYA MUKKA, KRISHNA PRASAD DEVARAKONDA*
School of Pharmacy, Department of Pharmacology, Anurag University, Venkatapur (M), Ghatkesar, Medchal (D), Hyderabad-500088, Telangana, India
*Corresponding author: Krishna Prasad Devarakonda; *Email: krishnaprasadpharmacy@anurag.edu.in
Received: 13 Aug 2024, Revised and Accepted: 02 Dec 2024
ABSTRACT
Objective: To assess the anti-diabetic activity of Laserpitium latifolium l extract through computational docking studies.
Methods: Crude extract of Laserpitium latifolium l whole plant was obtained using a rotary evaporator. The extract was analyzed for bioactive compounds using Gas chromatography/Mass spectrometry (GC-MS). The extract was examined for their anti-diabetic effects through computational docking studies and in vitro. For docking studies, proteins of diabetes mellitus, α-amylase and α-glucosidase, were selected from the literature.
Results: Binding affinity assessed by score function identified 2,4-Thiazolidinedione, 5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z)-(highest docking score of-7.8 kcal/mol) and cycloartenol acetate (docking score of-7.5kcal/mol) are the most promising compounds that showed strong affinity to target proteins of diabetes mellitus. These compounds have the highest docking scores, suggesting that they are potential candidates for anti-diabetic drug development. Extract of L. latifolium exhibited 68.1% potent activity at 250 μg/ml.
Conclusion: In the anti-diabetic potential of crude extracts, multiple pancreatic and extra-pancreatic mechanisms may occur synergistically to achieve a strong anti-diabetic effect.
Keywords: Laserpitium latifolium L, Alpha-amylase and alpha-glucosidase, 2,4-Thiazolidinedione, 5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z)-, Cycloartenol acetate
© 2025 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2025v17i1.52343 Journal homepage: https://innovareacademics.in/journals/index.php/ijap
INTRODUCTION
In many recent years, the effect and affection that Diabetes Mellitus (DM) has generated inside the global community has been alarming. In 2019, the range of people with diabetes is 463 million and is projected to obtain 700 million by 2045 [1]. In Mexico, it is taken into consideration the second biggest reason of mortality [2]. DM is a persistent degenerative disease associated with accelerated blood sugar ranges, which results in damage to cells, organs, and structures [3]. Greater glucose inside the blood results in the increment of reactive oxygen species [4], inducing the proliferation of oxidative strain [5], which induces the formation of advanced glycation end products [6], the development of dyslipidemia [7], as well as a chain of metabolic troubles associated with the sickness. There are notable forms of diabetes, which consist of Type 1 diabetes, that's as a result of insulin dependence. There may be additionally Type II DM related to insulin resistance or sensitivity [8]. DM is complicated and tough to treat. These days, new alternatives that are able to control diabetes is still in research. Medicinal vegetation has traditionally been used as therapeutic systems [9] and ethnobotanical extracts were analysed for richness in phytochemical compounds [10] due to their useful biological activities [11]. Those bioactive compounds had been isolated from exclusive elements of flowers (bark, lumen, branches, leaves, flora, fruits, roots, and seeds) [12].
Laserpitium latifolium L. (L. latifolium), belonging to family Apiaceae has long been used as a diuretic, purgative, and tonic in belly sicknesses, pulmonary tuberculosis, and fever; in gynecology; for treating coronary heart and liver and in rheumatism; and externally in toothache and puritic dermatomycoses; and also in brewing and veterinary medicine. It is widely recognized that quercetin has been isolated from L. latifolium. Some research has pronounced the biologically active additives of L. latifolium, in particular sesquiterpene lactones.
Chemical investigations have discovered the presence of daucane derivatives such as laserpitin, laserpitinol, isolaserpitine, and deoxodehydrolasepitine. Tests on essential oils obtained from leaves and fruits of L. latifolium confirmed the presence of monoterpenoids like sabinene, α-pinene, and limonene. From the leaves of L. latifolium, phenolic compounds were isolated and recognized with the assistance of a paper chromatograph and showed with High-Performance Liquid Chromatography (HPLC). The consequences confirmed the presence of quercetin 3-zero-arabinoside (avicularin); quercetin 3-O-glucoside (isoquercitrin); quercetin O-rhamnoside (quercitrin); and quercetin 3-O-rutinoside (rutin). The aim of the study was to assess the anti-diabetic activity of L. latifolium l extracts through computational docking and in vitro anti-diabetic studies.
MATERIALS AND METHODS
Materials0
Ethanol, sodium phosphate, NaCl, starch, dinitro salicylic acid, acarbose, yeast, and glucose were purchased from SD. Fine Chemicals, Hyderabad. α-amylase solution was acquired from Signa and Metronidazole was acquired from M. S. N. Labs, Hyderabad, India.
Plant collection and authentication
L. latifolium L. whole plant had been acquired from the neighborhood places of Tirupati, AP. L. latifolium L. Plant (Voucher No. 0211) was authenticated by Dr. Madhava Shetty, Asst. Professor, Department of Botany, Sri Venkateshwara University, Thirupathi, Andhra Pradesh, India.
Extraction by maceration
The whole plant of L. latifolium L. washed with water to get rid of contaminants like dirt and other impurities and was shade-dried. These dried wholes were ground and sieved to get uniform, coarse powder. Powdered plant material was weighed (1 kg) and immersed in ethanol and kept for maceration for a period of 7 d with occasional stirring. On the eighth day, the solvent was filtered by pressing with a muslin cloth and was evaporated in a rotary evaporator at 40 °C. The resultant extract was put in a desiccator to remove any solvent left in it. The dried ethanolic extract of L. latifolium (EELL) was packed in an airtight bottle and put in a dry place for further studies [13].
Qualitative evaluation of phytoconstituents
The extract was screened for the presence of various phytoconstituents like carbohydrates, amino acids, proteins, alkaloids, cardiac glycosides, triterpenoids, saponins, flavonoids, phenolic compounds, tannins, and steroids [14].
Gas chromatography-mass spectrometry (GC-MS) analysis
GC-MS evaluation is completed in an Agilent 8890 gas chromatograph device and mass spectrophotometer [15], equipped with a HP-5 MS fused silica column (5% phenyl methyl siloxane, 30.0 m × 250 μm, film thickness 0.25 μm), interfaced with a mass selective detector. Helium fuel was used as carrier gas and adjusted to column pace with the flow of 1.2 ml/min. Different GC-MS specifications are ion-source temperature, 250 °C; interface temperature, 300 °C; pressure, 11.367 psi; holdup time, 1.2376 min; and split ratio 15:1 with injection temperature of 250 °C. The column temperature began at 75 °C for 5 min and changed to 150 °C at a rate of 4 °C/min. The temperature was raised to 250 °C at a rate of 20 °C/min and held for 5 min. The entire elution became 53.5 min.
Identification of compounds
Identification of components was achieved based on their retention indices and interpretation of the mass spectrum was conducted using the database of the National Institute of Standards and Technology (NIST). The database consists of more than 62,000 patterns of known compounds. The spectra of the unknown components of L. latifolium fraction obtained were compared with the standard mass spectra of known components stored in NIST library.
Molecular docking
As a result of structural molecular biology and structure-based drug discovery, the area of molecular docking has arisen during the last three decades. Automated molecular docking software attempts to comprehend and anticipate molecular recognition by estimating binding affinity and detecting probable binding patterns. The most common molecular docking partners are a small molecule and a target macromolecule. Although ligand-protein docking is more widely used, protein-protein docking is gaining prominence. In this study, virtual screening tool like AutoDock Vina was used for docking program, and Biovia Discovery studio 2024 was used for visualization [16].
Selection of target protein
The target protein of DM was found from literature are Alpha-Amylase and Alpha-Glucosidase. The 3D structure of this target protein having Protein Data Bank (PBD) ID: 2QV4 for alpha-amylase and PBD ID: 2QMJ for alpha-glucosidase were retrieved from PDB database in. pdb format.
Ligand selection
The initial 3D structures of the selected ligands were retrieved in. sdf from Pubchem. PubChem and ChemSpider are repositories for chemical substances and their related biological activities. The optimized structures were then converted into. pdb format using Open Babel software.
Docking technique
Protein preparation
After opening the protein molecule in AutoDock Vina, the water molecules must be deleted and polar hydrogens added.
The PDB files of both ligands and proteins were converted in an extended PDB format, termed PDBQT, to perform molecular docking analysis using AutoDock Vina.
Data of target protein, ligand, and size of grid were saved in the. txt file and named as config. Grid box size was set up at 40×40×40 points for XYZ with a grid spacing of 0.5 Å.
Alpha-glucosidase
The conversion of oligosaccharides and disaccharides into glucose by the enzyme alpha-glucosidase is needed for carbohydrate hydrolysis. The resultant monosaccharides may be absorbed by the small intestine and raise blood glucose levels [17]. This enzyme may originate from a bacterial plant, fungus or mammal [18]. Acarbose, miglitol, nojirimycin, and other compounds should be assessed for their capacity to avoid or treat Type-II DM (fig. 1).
Fig. 1: 3D structure of alpha-glucosidase (2QMJ)
Alpha-amylase
The alpha-1-4 glycosidic linkages seen in the endo-region of amylase or amylopectin chains can be broken down by endo-amylase. A well-known endo-amylase is alpha-amylase, and it may be found in a wide variety of microbes, including bacteria and archaea. Because it’s a calcium metalloenzyme, a metal cofactor must be present for it to function [18, 19]. Alpha configuration and alpha-limit dextrin are the end products of alpha-amylase activity, which results in branched oligosaccharides. Depending on how much the substrate has been hydrolyzed, alpha-amylase has 2 types. Endo-and exo-hydrolases, where endo-hydrolases work on the terminal non-reducing ends while endo-hydrolases function inside the substrate molecule (fig. 2).
Fig. 2: 3D Structure of Alpha-amylase (2QV4)
In vitro anti-diabetic assay
α-Amylase inhibition assay
Around 0.5 ml of extract was mixed with 0.5 ml of α-amylase solution (0.5 mg/ml) with 0.02 M of sodium phosphate buffer (pH-6.9) with 0.006 M NaCl. The mixture was incubated at room temperature for 10 min and 0.5 ml of starch solution (1%) in 0.02 M sodium buffer was added. Resultant mixture was incubated at room temperature for 10 min and the reaction was terminated using 1 ml of dinitro salicylic acid color agent. At this time, the test was placed in water bath (100 °C for 5 min) and cooled until room temperature was obtained. The mixture was then diluted with 10 ml of deionized water and absorbance was determined at 540 nm. The adsorbent of blank (buffer instead of extract and amylase solution) and control (buffer instead of extract) sample was also determined. Acarbose was used as standard drug. The inhibition of α-amylase was calculated using the following equation.
% Inhibition of α-amylase = x 100
Where Abs controls correspond to the absorbance of the solution without extract (buffer instead of extract) and with α-amylase solutions and abs sample corresponds to the solution with extract and α-amylase solution [19, 20].
Table 1: Bioactive compounds found in Ethanolic extract of Laserpitium Latifolium L
| S. No. | Compound name | Retention time | Molecular formula | Molecular structure |
| Benzeneacetonitrile, α-hydroxy- | 3.475 min | C8H7N0 | ||
| Tyr-Ala | 5.694 min | C12H16N2O4 | ||
2(4H)-Benzofuranone , 5, 6, 7, 7a-tetrahydro-4,4,7a-trimethyl- |
15.821 min | C11H16O2 | ||
| Caryophyllene | 17.084 min | C15H 24 | ||
| Cycloartenol acetate | 17.084 min | C32H52O2 | ||
| Cyclopentanecarboxamide,N-(4-fluorophenyl)- | 15.821 min | C12H14FNO | ||
| 3-O-Methyl-d-glucose | 18.421 min | C7H14O6 | ||
| Bicyclo[3.1.1]heptane, 2,6,6-trimethyl-, (1α,2β,5α)- | 22.603 min | C10H18 | ||
| 4-Methoxy-6-methyl-6,7-dihydro-4H-furo [3,2-c] pyran | 22.747 min | C9H12O3 | ||
| Hexadecanoic acid, ethyl ester | 26.835 min | C18H36O2 | ||
| Phytol | 29.630 min | C20H40O | ||
| Neophytadiene | 29.630 min | C20H38 | ||
| Ipriflavone | 37.000 min | C18H16O3 | ||
| 2,4-Thiazolidinedione, 5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z)- | 38.481 min | C18H11N3O2S |
Glucose uptake by yeast cells
The yeast suspended in distilled water was subjected to repeated centrifugation (, 5 min) until clear supernatant fluid was obtained and 10% (v/v) of the suspension was prepared in distilled water. Various concentrations of plant extract (50 to 250 μg/ml) were added to 1 ml of glucose solution () and incubated together for 10 min at . Reaction was started by adding 100 μl of yeast suspension, followed by vertexing and further incubating at for 60 min. After 60 min the tubes were centrifuged (, 5 min) and amount of glucose was estimated in supernatant. Metronidazole was used as standard drug. The percentage increase in glucose uptake by yeast cells was calculated using following formula.
Increase in glucose uptake = x 100
Where Abs sample is the absorbance of test samples and abs control is the absorbance of control reaction (containing all regions except the test sample). All the experiments were carried out in triplicates [21].
Statistical analysis
Data were analysed and compared by one-way analysis of variance followed by post-hoc Tukey’s test. P<0.05 was considered to be statistically significant. Data are presented as a mean±SEM
RESULTS AND DISCUSSION
In phytochemical screening of ethanolic extraction of L. Latifolium l carbohydrates, amino acids, proteins, alkaloids, cardiac glycosides, triterpenoids, saponins, flavonoids, phenolic compounds, tannins, and steroids showed positive results, whereas gums showed negative results.
GC-MS evaluation carried on the ethanolic fraction of L. Latifolium l confirmed bioactive compounds like Glycerin, Benzeneacetonitrile, α-hydroxy-, Tyr-Ala, 2(4H)-Benzofuranone,5,6,7,7a-tetrahydro-4,4,7a-trimethyl-, cycloartenol acetate, 4-Methoxy-6-methyl-6,7-dihydro-4H-furo[3,2-c] pyran, Phytol, Neophytadiene, Ipriflavone, 2,4-Thiazolidinedione, 5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z)-.(table 1).
Molecular docking study is done to identify the significant binding interactions at the active pocket of targeted protein alpha-amylase with PDB ID: 2QV4. A docking study of standard drug acarbose was conducted along with 10 selected molecules for comparing the interactions. The standard drug showed a binding affinity of-8.5 kcal/mol and was found stabilized by hydrogen bonding with ARG-A: 252 and vander Waals bond with GLY-A: 334, ASP-A: 402, PHE-A: 335, PRO-A: 4, THR-A: 11 (fig. 3).
Among 10 compounds, 2,4-Thiazolidinedione,5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z)-was found with highest docking score of-7.8 kcal/mol and found stabilized by hydrogen bonding with ARG-A: 421, ARG-A: 398 and vander Waals bonds GLY-A: 403, PRO-A: 332, ASP-A: 402, THR-A: 11 (fig. 4).
The compound has formed similar interactions in comparison with standard interactions with GLY-A: 334, ASP-A: 402, PHE-A: 335, PRO-A: 4, THR-A: 11, ARG-A: 252, which might be significant and contribute to the docking score.
Similarly, cycloartenol acetate has demonstrated a docking score of 7.5kcal/mol. Interaction with PHE-A: 335, THR-A: 6, THR-A: 11, ASP-A: 402, GYL-A: 334, ARG-A: 252, ARG-A: 10, PRO-A: 4 was found common for both the compounds (fig. 5).
Further experimental investigation might help us to understand significant interactions and correlate docking results (table 2 and table 3).
Table 2: Docking scores of compounds with alpha-amylase (PDB: 2QV4)
| S. No. | Name of the compound | Docking score (K. Cal/mol) |
| Tyr-Ala | -6.4 | |
| 2(4H)-Benzofuranone,5,6,7,7a-tetrahydro-4,4,7a-trimethyl- | -6.1 | |
| Caryophyllene | -6.1 | |
| Cycloartenol acetate | -7.5 | |
| Cyclopentanecarboxamide,N-(4-fluorophenyl)- | -5.9 | |
| 3-O-Methyl-d-glucose | -5.4 | |
| 4-Methoxy-6-methyl-6,7-dihydro-4H-furo [3,2-c] pyran | -5.2 | |
| Hexadecanoic acid, ethyl ester | -4.0 | |
| Neophytadiene | -4.8 | |
| 2,4-Thiazolidinedione, 5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z)- | -7.8 | |
| Acarbose | -8.5 |
Fig. 3: Standard-acarbose (-8.5)
Docking with Alpha-Amylase (PDB: 2QV4)
A molecular docking study is also done to identify the significant binding interactions at the active pocket of targeted protein alpha-amylase with PDB ID 2QMJ. The standard drug showed a binding affinity of-8.5 kcal/mol and was found stabilized by hydrogen bonding with LYS-A: 765, TYR-A: 605, and vander waals bond with GLU-A: 767, PRO-A: 676, LEU-A: 640, TYR-A: 636, TYR-A: 733, GLY-A: 766, ILE-A: 734 (fig. 6).
Among 10 compounds, 2,4-Thiazolidinedione,5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z)-was found with the highest docking score of-8.1 kcal/mol and found stabilized by hydrogen bonding with GLU-A: 767, PRO-A: 676, GLU-A: 788, vander Waals bond with TYR-A: 733, TYR-A: 636, ARG-A: 643, LEU-A: 640, ARG-A: 647, ARG-A: 653, ASP-A: 649. The compound has formed similar interactions in comparison with standard interactions with TYR-A: 733, TYR-A: 636, LEU-A: 640, PRO-A: 676, and GLU-A: 767, which might be significant and contribute to the docking score (fig. 7).
Similarly, cycloartenol acetate has demonstrated a docking score of-7.6 kcal/mol. Interactions with GLU-A: 767, LYS-A: 765, PRO-A: 676, LEU-A: 640, TYR-A: 636, TYR-A: 733, and GLY-A: 766 were found common for both compounds (fig. 8).
Further experimental investigation might help us to understand significant interactions and correlate docking results (table 4 and table 5).
Fig. 4: 2, 4-Thiazolidinedione, 5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z) (-8.1)
Table 3: Types of interactions obtained with the molecules and the target proteins
| Name of the compound | Type of interaction | Interacting residues |
| Tyr-Ala | Van der waals | ILE-A: 312, ASP-A: 317, TRP-A: 316, TRP-A: 539, PHE-A: 348 |
| Conventional-Hydrogen bond | ARG-A: 346, THR-A: 314 | |
| 2(4H)-Benzofuranone,5,6,7,7a-tetrahydro-4,4,7a-trimethyl- | Van der waals | ASN-A: 352, ALA-A: 310, GLY-A: 304, GLY-A: 351, GLY-A: 309, ASP-A: 353, ARG-A: 303, TRP-A: 344, GLN-A: 302 |
| Conventional-Hydrogen bond | ARG-A: 346 | |
| Pi-Alkyl | PHE-A: 348 | |
| Cyclopentanecarboxamide, N-(4-fluorophenyl)- | Van der waals | ASP-A: 212, LEU-A: 211 |
| Conventional-Hydrogen bond | ASN-A: 216, HIS-A: 215, LEU-A: 214 | |
| Pi-Alkyl | PRO-A: 228, LYS-A: 227 | |
| 3-O-Methyl-d-glucose | Van der waals | PHE-A: 348 |
| Carbon-Hydrogen bond | ARG-A: 346 | |
| 4-Methoxy-6-methyl-6,7-dihydro-4H-furo [3,2-c] pyran | Van der waals | ASP-A: 353, ARG-A: 303, GLY-A: 304 |
| Pi-Alkyl | TRP-A: 344 | |
| Conventional-Hydrogen bond | ARG-A: 346 | |
| Alkyl | ALA-A: 310, PHE-A: 348 | |
| Hexadecanoic acid, ethyl ester | Van der waals | TRP-A: 406, GLN-A: 603, PHE-A: 575, GLY-A: 602, ALA-A: 576, ASP-A: 542, MET-A: 444, ASP-A: 203, PHE-A: 450, LYS-A: 480 |
| Pi-Alkyl | TYR-A: 299 | |
| Conventional-Hydrogen bond | TYR-A: 605 | |
| Caryophyllene | Van der waals | ARG-A: 303, ASN-A: 301, GLY-A: 309, GLY-A: 304, ALA-A: 310, GLN-A: 302, ILE-A: 312, ARG-A: 346, ARG-A: 267, THR-A: 314 |
| Conventional-Hydrogen bond | PHE-A: 450 | |
| Alkyl | PHE-A: 348 | |
| Cycloartenol acetate | Van der waals | PHE-A: 335, THR-A: 6, THR-A: 11, ASP-A: 290, ASP-A: 402, GYL-A: 334, ARG-A: 252, ARG-A10, GLY-A: 403, SER-A: 289 |
| Alkyl | TYR-A: 333, HIS-A: 331, PRO-A: 332, PRO-A: 4 | |
| Neophytadiene | Van der waals | GLY-A: 249, ASN-A: 250, ASP-A: 212, PCA-A: 1 |
| Alkyl | LYS-A: 208, LYS-A: 227, LEU-A: 211, LEU-A: 214, ILE-A: 230, PRO-A: 228 | |
| 2,4-Thiazolidinedione, 5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z)- | Van der waals | GLY-A: 403, PRO-A: 332, ASP-A: 402, THR-A: 11 |
| Conventional-Hydrogen bond | ARG-A: 421, ARG-A: 398 | |
| Acarbose | Van der waals | GLY-A: 334, ASP-A: 402, PHE-A: 335, PRO-A: 4, THR-A: 11 |
| Conventional-Hydrogen bond | ARG-A: 252 | |
| Carbon-Hydrogen bond | TYR-A: 605 |
Table 4: Docking scores of compounds with alpha-glucosidase (PDB: 2QMJ)
| S. No. | Name of the compound | Docking score (KCal/mol) |
| Tyr-Ala | -7.5 | |
| 2(4H)-Benzofuranone,5,6,7,7a-tetrahydro-4,4,7a-trimethyl- | -5.8 | |
| caryophyllene | -7.3 | |
| Cycloartenol acetate | -7.6 | |
| Cyclopentanecarboxamide,N-(4-fluorophenyl)- | -6.5 | |
| 3-O-Methyl-d-glucose | -5.2 | |
| 4-Methoxy-6-methyl-6,7-dihydro-4H-furo [3,2-c] pyran | -5.8 | |
| Hexadecanoic acid, ethyl ester | -4.6 | |
| Neophytadiene | -5.0 | |
| 2,4-Thiazolidinedione, 5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z)- | -8.1 | |
| Acarbose | -8.5 |
Fig. 5: Cycloartenol acetate docking score(-7.6)
Table 5: Types of interactions obtained with the molecules and the target proteins
| Name of the compound | Type of interaction | Interacting residues |
| Tyr-Ala | Van der waals | ASP-A: 649, ARG-A: 647 |
| Carbon hydrogen bond | PRO-A: 676 | |
| Conventional-Hydrogen bond | ARG-A: 653 | |
2(4H)-Benzofuranone ,5,6,7,7a-tetrahydro-4,4,7a-trimethyl- |
Van der waals | GLY-A: 766, GLU-A: 767, TYR-A: 636 TYR-A: 733, ASP-A: 649, ARG-A: 643, ARG-A: 653, LEU-A: 640 |
| Conventional-Hydrogen bond | ARG-A: 647 | |
| Alkyl | PRO-A: 676 | |
| Cyclopentanecarboxamide,N-(4-fluorophenyl)- | Van der waals | GLU-A: 767, GLU-A: 788, GLY-A: 766, TYR-A: 733 |
| Pi-Alkyl | LYS-A: 765 | |
| 3-O-Methyl-d-glucose | van der waals | ARG-A: 643, THR-A: 639, LEU-A: 640, TYR-A: 636 |
| Pi-Alkyl | PRO-A: 676 | |
| Conventional Hydrogen bond | GLU-A: 767 | |
| 4-Methoxy-6-methyl-6,7-dihydro-4H-furo [3,2-c] pyran | Van der waals | LYS-A: 534, ALA-A: 509 |
| Pi-Alkyl | ARG-A: 520 | |
| Carbon Hydrogen bond | PHE-A: 535, LEU-A: 286 | |
| Pi-Carbon | LYS-A: 776 | |
| Pi-Sigma | ALA-A: 285 | |
| Hexadecanoic acid, ethyl ester | van der waals | GLU-A: 767, THR-A: 639 |
| Pi-Alkyl | TYR-A: 636 | |
| Alkyl | LEU-A: 605 | |
| Caryophyllene | Van der waals | TYR-A: 733, TYR-A: 636, LYS-A: 765, GLY-A: 766, GLU-A: 767, ILE-A: 734, ARG-A: 643, ARG-A: 647, LEU-A: 640 |
| Alkyl | PRO-A: 676 | |
| Cycloartenol acetate | Van der waals | LYS-A: 765, ARG-A: 647, TYR-A: 733, ARG-A: 653, GLN-A: 272, THR-A: 269, TYR-A: 660, GLU-A: 767, GLY-A: 766, |
| Pi-Alkyl | PRO-A: 676 | |
| Alkyl | LEU-A: 640, TYR-A: 636 | |
| Neophytadiene | Van der waals | ARG-A: 643, ARG-A: 647, ARG-A: 653, ASP-A: 649, THR-A: 639, TYR-A: 636, GLU-A: 767, GLY-A: 766, TYR-A: 733 |
| Alkyl | LEU-A: 640, PRO-A: 676 | |
| 2,4-Thiazolidinedione, 5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z)- | Van der waals | TYR-A: 733, TYR-A: 636, ARG-A: 643, LEU-A: 640, ARG-A: 647, ARG-A: 653, ASP-A: 649 |
| Carbon Hydrogen bond | GLU-A: 788 | |
| Pi-Alkyl | PRO-A: 676 | |
| Pi-Anion | GLU-A: 767 | |
| Acarbose | Van der waals | GLU-A: 767, PRO-A: 676, LEU-A: 640, TYR-A: 636, TYR-A: 733, GLY-A: 766, ILE-A: 734 |
| Conventional-Hydrogen bond | LYS-A: 765 | |
| Carbon-Hydrogen bond | TYR-A: 605 | |
| Unfavorable donar-donar | ASP-A: 542, ARG-A: 526 | |
| Unfavorable aceptor-aceptor | ASP-A: 542, ARG-A: 526 |
Fig. 6: Standard-acarbose (-8.5)
Fig. 7: 2, 4-Thiazolidinedione, 5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z)-docking score(-8.9)
Fig. 8: Cycloartenol acetate docking score(-8.9)
In vitro anti-diabetic assay
L. latifolium extract of different concentrations was assessed for in vitro anti-diabetic activity by glucose uptake assay using a yeast model. EELL exhibited potent activity (68.1% at 250 μg/ml). A dose-dependent rise in the % of glucose uptake with increasing concentration (50–250 µg/ml) of L. latifolium extract was observed (table 6 and table 7).
Table 6: Glucose uptake by yeast cells
| Sample | Concentration (µg/ml) | Increase in glucose uptake% |
| Extract (EELL) | 50 | 25.1±3.3 |
| 100 | 30±4.6 | |
| 150 | 51.9±5.7 | |
| 200 | 66.5±8.9 | |
| 250 | 68.1±10.1 | |
| Standard (Metronidazole) | 50 | 89.0±13.7 |
| 100 | 89.9±12.9 | |
| 150 | 89.5±12.7 | |
| 200 | 89.5±14.3 | |
| 250 | 89.5±20.1 |
All the values were expressed in (n=3) Mean±SD
Table 7: Alpha amylase inhibition assay
| Sample | Concentration | IC50 (µg/ml) |
| Ethanol (Extract) | 0.5 mg/ml | 745±45.8 |
| Standard (Acarbose) | 0.5 mg/ml | 458±58.2 |
All the values were expressed in (n=3) mean±SD
DISCUSSION
Molecular docking studies are frequently employed in drug layout to predict interactions between ligands and proteins. This is performed with the aid of calculating the binding affinity and visualizing the amino acid interactions contributing to it. Docking permits the prediction of anti-diabetic pastime with the aid of assessing the binding affinity of isolated compounds for proteins involved in glucose metabolism. Anti-diabetic treatment plans are commonly advanced to target numerous mechanisms of glucose metabolism related to more than one pathway. In this study, molecular docking was done against proteins α-amylase α-glucosidase to decide the efficacy of the extract. The binding conformation of the compounds is assessed totally on the scoring characteristic and predicting the power of the compound-receptor interaction. Ten docking interactions had been selected due to the fact they'd the great (lower ratings) free power of binding (ΔG kcal/mol). All the compounds interacted with the receptors to various degrees. The binding affinities are evaluated through scoring functions. The compounds 2,4-Thiazolidinedione, 5-[[4-(4-pyridinyl)-6-quinolinyl] methylene]-(5Z), cycloartenol acetate exhibited advanced binding to more than one receptor, suggesting their capacity as applicants for anti-diabetic drug development. In diabetes, high postprandial blood glucose leads to microvascular complications, including retinopathy, nephropathy, and neuropathy, and macrovascular complications refer to increased atherosclerosis-related events such as myocardial infarction and stroke [22, 23]. One of the therapeutic approaches for controlling postprandial hyperglycemia in diabetic patients is to prevent or decrease absorption of carbohydrate after food intake. Complex starches, oligosaccharides, and disaccharides must be broken down into monosaccharides by α-amylase and α-glucosidases before they are absorbed in the duodenum and upper jejunum [24]. Recent advances in understanding the activity of intestinal enzymes helped in the development of newer pharmacological agents [25]. α-glucosidase inhibitors reduce intestinal absorption of starch, dextrin, and disaccharides by inhibiting the action of α-glucosidase in the intestinal brush border. Inhibition of this enzyme slows the absorption of carbohydrates from the GI tract and decreases the rate of rise of postprandial glucose (PP hyperglycemia). This delay in digestion and breakdown of starch may have beneficial effects on insulin resistance and glycemic index control in people with diabetes [26]. Acarbose is an α-glucosidase inhibitor that reduces digestion of complex carbohydrates and slows their absorption from the gut. These drugs also increase the release of the glucoregulatory hormone glucagon-like peptide-1 into the circulation, which may contribute to their glucose-lowering effects [27–29]. However, they may cause side effects such as malabsorption, abdominal pain, flatulence, and diarrhea, which lead to a high discontinuation rate [30, 31]. Acarbose and miglitol should not be prescribed in individuals with renal impairment. Acarbose should be used with caution in patients with hepatic diseases because it may cause reversible elevation of hepatic enzymes. Experimental results showed that EELL inhibited the α-glucosidase and α-amylase enzymes.
CONCLUSION
Although there are many treatments for diabetes, the disease is inevitable, and its epidemiological impact is rapidly worsening. The identification of new anti-diabetic drugs has been greatly accelerated by protein-ligand docking and simulation techniques. This study provided evidence that L. latifolium L. a plant rich in important terpenoids, flavonoids and phenolic compounds, could be used as an anti-diabetic drug. The in vitro and docking studies are in fairly good agreement. The docking calculations revealed the importance of binding energy, electrostatic energy and van der Waals energy. Several aspects need to be considered when developing new amylase and 𝛼-glucosidase inhibitors. Further studies are needed to elucidate the exact pharmacodynamic effects and demonstrate efficacy in the treatment of diabetes.
FUNDING
This research received no external funding.
AUTHORS CONTRIBUTIONS
MD completed the research work, execution, and writing. DKP did the work plan, review, and corrections of the publication. Both authors agree with the submission and publication. Both authors have read and agreed to the published version of the manuscript.
CONFLICTS OF INTERESTS
No conflict of interest
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