Isolation, Characterization and Docking Studies of Isolated Compounds as Antidiabetic Molecules from Scindapsus officinalis (Roxb)

 

Pawan Tiwari¹*, Gurdeep Singh², Ritesh Patel³, Mukesh Kr. Singh⁴

¹Department of Pharmaceutical Chemistry, Shambhunath Institute of Pharmacy, Prayagraj U.P., India.

²School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India.

³Department of Pharmaceutical Chemistry, Indore Institute of Pharmacy, Indore, M.P., India.

⁴School of Pharmaceutical Sciences, IFTM University, Moradabad, U.P., India.

 

ABSTRACT:

Natural products are essential to human life, and about half of the medications used in clinical practice today are of natural origin. The present work investigated to isolate and identify active compounds with anti-diabetic activity from Scindapsus officinalis fruits and confirm the isolated compounds' mode of action, affinity, and domain specificity relationships.  Some fractions of S. officinalis ethanolic extracts were subjected to column chromatography and preparative TLC and two compounds namely 2E,4E,6E)-5-methyl-7-(2,6,6-trimethylcyclohexa-2,4-dien-1-yl)hepta-2,4,6-trien-1-ol (CN-501A) and 9-(furan-3-yl)-4-hydroxy-1,5,6,6a,9,10,10a,10b-octahydro-3H,7H-pyrano[3,4-f]isochromene-3,7-dione (CN-501B) were isolated in pure form. The structures of the isolated compounds were confirmed by UV, IR, ¹H NMR and mass spectral data. The anti diabetic activity was measured using a molecular docking study and the three-dimensional structure of the target protein was downloaded from PDB. The Docking study recommended that CN – 501A and CN – 501B are existing photochemical from the plant of S. officinalis had the highest fitness docking score and hence could be a possible antidiabetic drug.

 

 

 

INTRODUCTION: 

Diabetes mellitus (DM) is a chronic disorder characterized by a high blood glucose level (hyperglycemia), which is caused by defects in insulin secretion, action, or both. Patients are at significant risk for long-term macro- and micro vascular problems as a result of the chronic metabolic imbalance associated with this disease, which can lead to recurrent hospitalization and consequences, including an increased risk of cardiovascular disease if not treated properly (CVDs)¹. Diabetes mellitus (DM) is a worldwide problem. It is the second most common cause of renal failure and blindness, whilst being the seventh most common cause of mortality².

 

Diabetes affects people globally, the majority of whom live in low- and middle-income countries, and diabetes is directly responsible for 1.5 million fatalities per year. Over the last few decades, both the number of cases and the prevalence of diabetes have significantly increased³. According to the International Diabetes Federation (IDF), 451 million adults worldwide have diabetes in 2017, with a projected growth to 693 million by 2045 if no effective prevention techniques are implemented⁴. Increased consumption of poor foods and sedentary lifestyles have been blamed for these developments, which have resulted in increasing BMI and fasting plasma glucose⁵. The worldwide burden of diabetes has risen dramatically in recent decades and is expected to continue to rise in the next decades. The Araceae family includes the species Scindapsus Schott, which is a medicinal plant. It is distributed all over the world and contains 115 genera and roughly 2100 species. Tropical America, India, Malaya, and South China, on the other hand, account for 90% of the species' distribution. From Northeastern India to Western Polynesia, the species Scindapsus Schott (1832) encompasses roughly 35 species. It's an epiphytic climbing shrub with bisexual flowers and ovate-oblong, oblong cordate, or oblong lanceolate leaves⁶ bare spathe. Florets lack a perianth and have four stamens as well as a one-celled ovary with a single basal ovule. The plant has about fifteen species and countless hybrids, all of which are evergreen⁷. This genus includes Scindapsus caudatus, Scindapsus decursivus Schott⁸. (Sylhet, Bangladesh), Scindapsus giganteus Schott (Penang, Singapore), Scindapsus glaucus Schott (Khasaya Paras, Nepal), Scindapsus officinalis Schott (S. officinalis) (India, Burma), Scindapsus peepla Endl (Sylhet, Bangladesh), Scindapsu spertusus Schott⁹. (Coromandel, South Konkan), Scindapsus pinnatifidus (Roxb.), Scindapsus pictus (S. pictus), Scindapsus scortechinii,¹⁰. Scindapsu shederacea, Scindapsusperakensis, Scindapsus crassipes¹¹. Scindapsus beccarii and Scindapsus cuscuaria. S. officinalis, a plant with a perennial stem, is a large epiphytic climber with adventitious aerial roots growing on trees and rocks.

 

MATERIALS AND METHOD:

Collection and identification:

During the months of August and September 2019, the plant was picked in India. Mr. S.L. Meena authenticated the taxonomic and ethnomedical identification of the plant's fruits (Deputy Director of the botanical survey of India, near Khemkakuaon , Nandan Van, Jodhpur-324008)

 

Preparation of plant material:

The plant's fruits were sun-dried, ground into a coarse powder with a grinder, and stored in an airtight container until needed¹².

 

Extraction and fractionation:

The powdered drug was extracted with petroleum ether to defat the fruit powder and filtered. The same procedure was repeated three times and it was extracted with 90% ethanol. The organic layer was combined and concentrated using a rotary vacuum evaporator and then dried to give an ethanolic extract. The extract was dispersed with warm water and n-hexane was added. The n-hexane layer was collected and dried using a rotary vacuum evaporator to obtain the n –hexane fraction. The extracts were kept in the refrigerator for testing at a later date¹³.

 

Preparation of sample:

A little amount of an adequate solvent was mixed with about 10grams of extract, 50grams of silica gel for CC (60-120mesh), and a very small amount of an appropriate solvent. This mixture was triturated in a pestle until it became free-flowing and homogeneous¹⁴.

 

Application of sample:

With the use of a hollow glass cylinder, the above-prepared combination is carefully fed into the column without disturbing the silica substrate. After that, the appropriate solvent system was loaded onto the column for component elucidation¹⁵.

 

Elution procedure:

Three main elution procedures are routinely used. Isocratic elution, stepwise elution, and gradient elution are the three methods. Isocratic elution is the process of running a solvent combination with a constant composition through a chromatographic column until separation is complete. When only one solvent is utilized, stepwise elution results in the elution of only some of the components in the mixture. As a result, a stronger eluting solvent will be required to extract the firmly bound components. Williams and Tiselius were the first to describe the gradient elution technique, which involves the use of a constantly changing eluting solution¹⁶.

 

Collection of eluting sample:

Elute was collected at a rate of 20 drops per minute, with each fraction containing approximately 100 ccs of liquid. TLC was performed on each fraction on silica gel G. To obtain pure compounds or a combination of 2-3 compounds, the fractions with the same Rf were pooled together and concentrated¹⁷.

 

Purification of elutes:

To achieve pure chemicals, elutes from column chromatography were re-chromatographed or exposed to preparative TLC (PTLC). A column was packed with the ethanolic soluble fraction derived from the ethanolic extract. The elusions in the silica gel column began with hexane, followed by 100 percent ethyl acetate, and finally 10-100 percent methanol. The fractions (100 ml) were collected from the column and blended if they had the same TLC profile. The column's mixed crude fractions were re-chromatographed using a silica gel column^18-20.

 

Isolation of compounds:

Hexane was used to begin the isolation, then ethyl acetate was used to boost the polarity. Based on  their TLC profiles, the elutions up to hexane: ethyl acetate (5:5) were combined to yield fraction (A). This fraction was concentrated and chromatographed with a hexane DCM combination containing an increasing amount of DCM. With the addition of di-ethyl ether, the elution up to hexane: DCM (7:3) was mixed and refrigerated overnight, yielding compound (A) as an accumulation of white and yellowish powders, respectively. Compounds (501A, 501B) were recrystallized as yellow and dark brown mass with acetone after further elution up to hexane: DCM (2:8) ²¹.

Molecular docking analysis:

For binding mechanism prediction with the target protein, molecular docking analysis was performed in addition to binding energy calculation. The most potent reported Crystal Structure of The Tyrosine Kinase Domain of The Human Insulin Receptor [1IRK] Crystal Structure of The Activated Insulin Receptor Tyrosine Kinase Dimer [4XLV] antagonists were selected and used as references. Ligands were prepared with Schrödinger's Ligprep tool was used to create the OPLS 2005 force field. At a target pH of 7.0 2.0, Epic was used to construct tautomers and all conceivable ionization states, in addition to  low energy ring conformations for each ligand. The Human Insulin Receptor's Tyrosine Kinase Domain Crystal Structure [1irk] Using the protein preparation wizard, the crystal structure of the activated insulin receptor tyrosine kinase dimer[4xlv] with 3.4 resolution was created by adding missing hydrogen atoms, missing atoms and loops, assigning bond order, and deleting non-binding waters. To remove steric conflicts of the atoms, the H-bond assignment was done with Propka (at pH 7.0) and restricted minimization was done with the OPLS 2005 force field. Docking was done first, then the grid (binding site) was generated using the Glide module in extra precision (XP) mode. The receptor grid box was created by eliminating the crystalline ligand from the protein's binding region. To generate conformations, the protein was set as stiff and the ligands were chosen as flexible. To create structural different poses, post docking reduction was used. The best stance was chosen, energy was reduced, and Glide Gscore was used to rank the poses.

 

Binding free energy calculation using Prime:

The relative binding energies were computed with the default parameters of the Schrödinger suite's Prime MM-GBSA program. The ligand strain and ligand binding energies of ligand-protein complexes are calculated using this tool. The binding free energy is calculated using the following equation ^22-23.

 

∆Gbind = Gcomplex – (Gprotein + Gligand)  

 

Where, ∆Gbind is the binding free energy, the Gcomplex is free energy of complex, the Gprotein is free energy of the target protein and the Gligand is Ligand-free energy.

Physiochemical properties analysis:

All compounds' physicochemical properties were calculated using Maestro's QikProp module ²⁴. In this module, all compounds were tested for drug resemblance, which is an important factor to consider when building an ideal medicine.

 

Characterization of Isolated Compounds from ethanolic extract:

Structural Elucidation of Isolated Compounds:

It was attempted to purify the chemicals obtained using column chromatography (CC) ^25-26 and re-crystallization in various solvents. The melting point and solubility of the compounds were determined after they were weighed ²⁷.  The structure of the isolated compounds was determined by analyzing spectroscopic data from UV‑VIS, FT‑IR ^28-29, ¹H‑NMR and MS ³⁰.

 

RESULTS AND DISCUSSION:

The TLC profile was evaluated using different solvent systems and R_f values were calculated. Table 1 and Table 2 shows the color and R_f values.

 

Table 1: TLC Studies of Ethanolic extract and Ethanolic fraction of Scindapsus officinalis Roxb

Fraction/ Extract	Solvent system	No of spots	TLC profile
			Rf value	Color
Ethanolic extract	Toluene:Ethyl acetate:Methanol: Water (7:6:5:2)	4	0.94; 0.90; 0.74; 0.69	Dark green, green, light yellow, light green
Ethanolic Fraction	Ethyl acetate: methanol: toluene: water (5:4:6:5)	2	0.90; 0.73;	Dark green, light yellow

 

Table 2: TLC studies of Hexane elution of ethanolic fraction of Scindapsus officinalis Roxb

Fraction/Extract	Solvent system	No of spots	TLC profile
			Rf value	Color
Hexane elution	Hexane: DCM: (7:3)	1	0.76	Dark yellow
Ethanol elution	Hexane: DCM (2:8)	1	0.58	Light green

 

Each fraction was isolated using column chromatography and resulted in two compounds (Table 3).

 

 

Table 3: IUPAC name and structure of isolated compounds from Scindapsus Officinalis Roxb

Compound Code	IUPAC Name	Structure
CN-501A	2E,4E,6E)-5-methyl-7-(2,6,6-trimethylcyclohexa-2,4-dien-1-yl)hepta-2,4,6-trien-1-ol
CN-501B	9-(furan-3-yl)-4-hydroxy-1,5,6,6a,9,10,10a,10b-octahydro-3H,7H-pyrano[3,4-f]isochromene-3,7-dione

Table 4: Spectral data of Compound -501A

IR (KBr) vmaxcm-1	1H NMR ( in ppm) δ	Melting Point range	Ultra Violet Wavelength	Mass Spectra
3683 (alcohols, phenols)  Stretch 3397 (OH stretch, H-bonded) 1635 (aromatics) 1521 (C-C stetch) 849 (C-H stretch)	0.675 s br (methyl) 1.49 s br (ethyl) 1.6 s br(ethyl) 1.9 s br (Propyl) 2.18d (J= 12.0 Hz) 5.21d (J= 4.0, 2.0 Hz) (ether)	230-232  0C	λ max= 259 Absorbance=0.813	Base Peak 207. M+ Peak 242.2

 

Table 5: Spectral data of Compound-501B

IR (KBr) vmaxcm-1	1H NMR ( in ppm) δ	Melting Point range	Ultra Violet Wavelength	Mass Spectra
3458 (alcohols, phenols)  Stretch 3487 (OH stretch, H-bonded) 2100(aldehydes) 1720 (aromatics) 1422 (C-C stetch) 1215 (alkyl halides) 1099 (carboxylic acids)	0.687 s br (methyl) 4.55 br (hydroxyl) 1.7 s S (ethyl) 2.3 s S(ethyl) 2.18d (J= 12.0 Hz) 5.21d (J= 4.0, 2.0 Hz) (ether)	287-288  0C	λ max= 220 Absorbance=0.621	Base Pea278. M+ Peak304.7

 

The substitution of a functional group resulted in the highest absorption in UV spectra (501A). The IR spectrum of compounds (501A) showed absorption bands typical of flavanol, indicating the presence of hydroxyl and tinsoporin functionalities at 3449, 1375, and 1079 cm-1, respectively, while 1689 cm-1 indicated the presence of a double bond. The peak at 1375 cm-1 represented C-H bending, while the NMR spectra was also typical of phytotaxanol, displaying the hydroxyl proton signal at 5.21 as multiplet, which helped us to characterize this compound as 2E,4E,6E)-5-methyl-7-(2,6,6-trimethylcyclohexa-2,4-dien-1-yl)hepta-2,4,6-trien-1-ol  501A, and 9-(furan-3-yl)-4-hydroxy-1,5,6,6a,9,10,10a,10b-octahydro-3H,7H-pyrano[3,4-f]isochromene-3,7-dione,501B respectively

 

Compound no (501B) tested positive for polyphenol by giving dark intense violet color with ferric chloride reagent. The IR spectrum (cm^-1) of compound (501B) showed absorption band at 3319 and 2926 (free and hydrogen-bonded OH groups), 1764 (Easter carbonyl group), 1632, 1519 and 1385 (aromatics ring), 1348 and 1193(=C-O groups) . the ¹H NMR of these compounds exhibited signals in the range of  6.89 - 7.97 and 3.4 - 6.34 ppm that belongs to phenolic hydroxyls and D- glucose molecules respectively, and signals in the region 6.23 - 7.97 ppm assigned to Gallic acid protons

 

Molecular docking studies

To further investigate the matter, we used molecular docking to compare all molecules to reference compounds. The Crystal Structure of The Human Insulin Receptor Tyrosine Kinase Domain [PDB ID: 1IRK] and Crystal Structure of The Activated Insulin Receptor Tyrosine Kinase Dimer[PDB ID: 4XLV] have been chosen for docking. Before docking, a grid was created around the protein's binding site. Table 6 shows the docking results along with the binding energy calculation.

 

Table 6: Glide docking score (PDB ID:1IRK and 4XLV] along with binding energies of compounds

PDB ID	Com. Code	Glide Gscore	dG Bind	dG Bind LovMW	dG Bind Lipo	dG Bind Hbond	dGBind vdW
1IRK	501A	-3.8	-3.8	-0.5	-0.2	-0.7	-2.0
4XLV	501B	-2.7	-2.7	-0.5	-1.6	-0.8	-1.6

 

501A and 501B (2E,4E,6E) -5-methyl-7-(2,6,6-trimethylcyclohexa-2,4-dien-1-yl)hepta-2,4,6-trien-1-ol and 9-methyl-7-(2,6,6-trimethylcyclohexa-2,4-dien-1-yl)hepta-2,4,6-trien-1-ol (furan-3-yl) -4-hydroxy-1,5,6,6a,9,10,10a,10b-octahydro-3H,7H -pyrano[3,4-f] isochromene-3,7-dione respectively have docking scores of -3.8 and -2.7, and displays 1 hydrogen bond with residue PHE1151 and no hydrogen bond with compound 501B. With Trp9, it also displays one - stacking interaction. Compound 501B has one -cation interaction with TYR1227 and one -stacking interaction with TRY. The fact that most produced compounds demonstrate hydrogen bonding with the same amino acid but with various bond distances is fascinating. Figure 1 shows the ligand interaction diagram as well as the 3D structures of docked ligands.

 

 

Figure 1: Target protein-ligand interaction diagram Compound 501A and 502B

 

A Compound  501A PDB ID:  1IRK

 

B: Compound 501B PDB ID: 4VLV

Figure 2: (A) Binding view of compound 501A Hydrogen bond interaction ofcompound. Binding view of compound 501B with π-π stacking interaction.

 

Overall, Compound 501A binds to the target protein with the highest affinity. According to the docking data, the binding residues phe1151, leu1051, and Tyr1087 are located in the protein's hydrophobic domain and are responsible for hydrophobic interactions with drugs. The binding affinity is improved by more hydrophobic contacts. As a result of the - stacking interaction, a comparison of produced compounds with reference molecules suggests that aromatic substitution at carbamate nitrogen results in excellent binding. Large bulky groups do not contribute to binding or activity in the same way that small bulky groups do. The binding pocket is made up of ten hydrophobic (leu1051), two hydrogen bonding (phe1151), and one - stacking (Tyr1087) amino acid residues, according to the ligand interaction diagram.

 

Physiochemical properties:

The physicochemical characteristics of all produced substances were evaluated using the maestro's Qikprop program. Tables 7 and 8 provide the principle and therapeutic important parameters, respectively. The lipophilic efficiency, which evaluates a drug's potency, is calculated using the QPlogPo/w value. As a result, the logP value of the Octanol-water partition coefficient is critical in rational drug design and QSAR research. The sum of all polar atoms, mostly oxygen and nitrogen, as well as connected hydrogen, is known as polar surface area (PSA). A molecule with more rotatable bonds (rotor) becomes more flexible and has a better binding affinity for the binding pocket 31.

 

Table 7: Principle pharmacokinetic parameters of selected compounds

PDB ID	Com. Code	MW	Dipole	DonorHB	accptHB	Volume	rotor	PISA
1IRK	501A	230.349	1.986	1	1.7	933.89	6	147.91
4XLV	501B	304.299	4.105	1	7.25	875.375	1	148.416

 

Table 8: Therapeutic significant parameters of all selected compounds

PDB ID	Com. Code	QPlogPo/w	QPlogS	QPPCaco	QPlogBB	QPPMDCK	Metab	QPlogKhsa	Percent HOA
1IRK	501A	4.169	-4.702	2798.701	-0.347	1504.725	3	0.569	100
4XLV	501B	0.883	-2.552	281.4	-0.806	125.642	6	-0.36	75.952

 

CONCLUSION:

Hexane was used to begin the isolation, then ethyl acetate was used to boost the polarity. Based on their TLC profiles, the elutions up to hexane: ethyl acetate (5:5) were combined  to yield fraction (A). This fraction was concentrated and chromatographed with a hexane DCM combination containing an increasing amount of DCM. With the addition of di-ethyl ether, the elution up to hexane: DCM (7:3) was mixed and refrigerated overnight, yielding compounds (A) as a white and yellowish powder, respectively. Compounds (501A, 501B) were recrystallized as yellow and dark brown mass with acetone after further elution up to hexane: DCM (2:8). The hydroxyl proton signal at 5.21 as a multiplet in the NMR spectra was also diagnostic of phytotaxanol, which enabled us to describe this molecule as 2E,4E,6E). hepta-2,4,6-trien-1-ol 501A -5-methyl-7-(2,6,6-trimethylcyclohexa-2,4-dien-1-yl)hepta-2,4,6-trien-1-ol. Phe1151, leu1051, and Tyr1087 are residues of the Human Insulin Receptor Protein's Tyrosine Kinase Domain that are involved in the binding mechanism. In comparison to compound 501A, simulation tests demonstrated that compound 501A has better binding. The compounds relative binding energy and physicochemical properties were also assessed. The majority of the drugs displayed a favorable pharmacokinetic and therapeutic profile. As a result, isolated compounds may be a potent and effective class of drugs for the treatment of diabetes.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The authors would like to thank SAIF, Punjab University, Chandigarh for their kind support during spectral investigation of isolated compounds.

 

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Accepted on 07.12.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):691-697.