INTRODUCTION:

In developing countries, herbal medicine is strength of about 75–80% of the whole population for primary health care because of better compatibility with the human body and less side effects. Herbal medicines usage is increased globally¹. Numerous medicinal plants are used in the management of a number of disease conditions including pain and inflammatory conditions^2,3,4. Dioscorea bulbifera is a member of yam family also called as Aerial yam. Diosgenin and dihydroradioscorine are the important phyto constituent present in aerial yam with potent anti-inflammatory and Anti-tumor properties^5,6. There are number of flavonoids, saponin and isoflavanoids have been spotted by the researchers with the oestrogenic, heart protective, antioxidant, anti-cancer properties and free radical scavenging activity.

The authentication of the folkloric claims of these medicinal plants will afford scientific basis for the conservation of tropical medicinal resources. The phyto constituents are the prime health care and the increase of potential bioactive constituents as novel lead compounds against the life threatening diseases. In the present study, the anti-inflammatory activity of saponin which is one of the phyto constituent of the fruit of Dioscorea bulbifera using the in vitro and in silico methods.

MATERIAL AND METHODS:

Collection of Plant material:

Dioscorea bulbifera fruit were collected from Medicinal Plant Garden at Sri Sairam Siddha Medical College and Research Centre, West Tambaram, Chennai 600 044, a recognized institution of Government of Tamil Nadu and the Department of AYUSH, Government of India.

Phytochemical analysis of the Plant extract:

The aqueous methanol Dioscorea bulbifera fruit extract is freshly prepared and divided into different test tubes and phyto constituents was analysed according to methods described^7,8

Test for saponin:

About 2g of the powdered sample was boiled in 20ml of distilled water in a water bath and filtered. 10ml of the filtrate was mixed with 5ml of distilled water and shaken vigorously for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously, then observed for the formation of emulsion.

The Partial characterization of Thin Layer Chromatography in Dioscorea bulbifera fruit extract:

The Methanol extract of Dioscorea bulbifera fruit were loaded on to pre coated TLC (60 F₂54) and it was developed using solvent system in the ratio of 1:0.5:0.1 (Hexane, Chloroform and Methanol) visible and the non-visible spot given and it is fluorescent with UV light at 360nm.

Determination of total saponins:

Saponin content of the sample was determined by double solvent extraction gravimetric method. 2g of the powdered sample was mixed with 50mls of 20% aqueous ethanol solution. The mixture was heated with periodic agitation in water bath for 90 minutes at 55^◦C. It was filtered through filter paper. The residue was extracted with 50ml of the 30% ethanol and both extracts were collected together. The combined extract was reduced to about 40ml at 90^◦C and transferred to a separating funnel where 40ml of diethyl ether was added and shaken vigorously. Separation was done by partition during which the ethyl ether layer was discarded and aqueous layer reserved. Re-extraction by partition was done repeatedly until the aqueous layer become clear in color.

The saponins were extracted with 60ml of normal butanol. The combined extracts were washed twice with 10ml of 5% aq. NaCl solution and evaporated to dryness in a pre-weighed evaporating dish. It was dried at 60^◦C in the oven and re-weighed. The experiment was repeated two more times to get an average.

% Saponins = (W₂-W₁/ Weight of sample) * 100/1

Whereas, W₁= weight of evaporating dish, W₂= weight of dish + sample

Cyclooxygenase inhibition assay:

The cyclooxygenase inhibition assay was performed according to a modified method⁹.The oxidation of leuco-dichlorofluorescein (1-DCF) in the presence of phenol by the hydroperoxide formed in the cyclooxygenase reaction can be used as a sensitive spectrophotometric assay for PGH synthaseactivity. Leuco-2, 7-dichlorofluoresceindiacetate (5 mg) was hydrolysed at room temperature in 1 M NaOH (50 μl) for 10 min, then 1 M HCl (30 μl) was added to neutralize excess NaOH before the resulting 1-DCF was diluted in 0.1 M Tris-buffer, pH 8. Cyclooxygenase enzyme (COX-1 or COX-2) was diluted in 0.1 M Tris-buffer, pH 8, so that a known aliquot gave an absorbance change of 0.05/min in the test reaction.

Test samples (or the equivalent volume of methanol, 20 μl) were pre-incubated with enzyme at room temperature for 5 min in the presence of hematin. Premixedphenol, 1-DCF and arachidonic acid were added to the enzyme mixture to begin the reaction, and to give a final reaction mixture of arachidonic acid (50 μm), phenol (500μm), 1-DCF (20μm) and hematin (1 μM) in 1ml final volume of0.1 M Tris-buffer, pH 8. The reaction was recorded spectrophotometrically over 1 min at 502nm. A blank reaction mixture was analysed in the spectrophotometer reference cell against each test reaction to account for any non-enzymatic activity attributed to the test sample. This blank consisted of the reaction mixture without the addition of enzyme.

Lipoxygenase inhibition assay:

A spectrophotometric assay for determination of LOX activity was used as reported with slight modification¹⁰. Soybean 15-lipoxygenase (15-LOX) was used for the assay. Inhibition experiments were run by measuring the loss of soybean 15-LOX activity (5μg) with 0.2μM linoleic acid (Sigma) as the substrate prepared in solubilized state in 0.2M borate buffer (pH 9.0). Inhibition studies in presence of various concentrations of extracts (5, 10, 15, 20μg/ml) and reference compound viz., quercetin was recorded at 234nm using UV-Vis spectrophotometer (Beckman Coulter, DU 730 Life Sciences). The inhibitory effect of the extracts was also expressed as percentage of enzyme activity inhibition.

IC₅₀ indicating the concentration required to inhibit 50 % LOX activity was also calculated. Values of hydroperoxide content and lipoxygenase activity were calculated from equation,

Specific activity (LOX) =ΔA. V/ε.l.c

Where, ΔA is the value of absorbance increase per min, V is the volume of incubation mixture, ε is the extinction coefficient for linoleic acid (25 x 10-3 mol/l/cm), l is the length of the cuvette (1cm) and c is the concentration of enzyme in mg (0.005). The values are mean of three independent experiments.

Inhibition of protein denaturation activity of Dioscorea bulbifera fruits saponin extract:

In vitro anti-arthritic activity of Dioscorea bulbifera fruit, the method used was “inhibition of protein denaturation”^11,12,13,14 using diclofenac sodium a standard. The test solution (0.5 ml) consists of 0.45 ml of bovine serum albumin (5% w/v aqueous solution) and 0.05 ml of test solution (Dioscorea bulbifera fruit).

The test control solution (0.5ml) consists of 0.45ml of bovine serum albumin (5% w/v aqueous solution) and 0.05ml of distilled water. Product control (0.5ml) consists of 0.45ml of distilled water and 0.05ml of test solution. Standard solution (0.5ml) consists of 0.45ml of bovine serum albumin (5% w/v aqueous solution) and 0.05ml of diclofenac sodium. Various concentrations (5, 10, 15, 20μg/ml) of Dioscorea bulbifera fruit and diclofenac sodium (standard) were taken, respectively.

All the solutions were adjusted to pH 6.3 using 1 N HCl. The samples were incubated at 37°C for 20 min and the temperature was increased to keep the samples at 57°C for 3 min. After cooling, 2.5 ml of phosphate buffer was added to the previous solutions. The absorbance was measured using UV-Visible spectrophotometer at 416 nm.

Docking Studies:

iGEMDOCK¹⁵ was the automated docking tool utilised for the present study. The binding of chemical molecules were predicted by docking tags. To begin the docking process, the target COX-2 was modelled using Mus musculus COX-2 receptor as the template using homology modelling. The ligand and the receptor were fed as input in PDB format respectively using Open babel online server¹⁶. The list of energy of the poses and the amino acids interacted along with the different bonding energies involved were displayed for analysis respectively.

RESULTS:

The phytochemical screening of the Dioscorea bulbifera fruit extract were studied presently showed the presence of saponins. Total saponin contents of Dioscorea bulbifera extracted was 30µg/g.

The Partial characterization of Dioscorea bulbifera by TLC:

The saponin extract of Dioscorea bulbifera loaded on Pre-coated TLC plates and developed with a solvent system of Toluene, tetrahydrofuran and acetic acid in the ratio of 1:0.5:0.1 were and it is used for further studies. The developed plate was viewed under UV 240nm and 360nm (Fig-1).

a b

Fig 1: a) TLC viewed under UV 360nm b) TLC viewed under UV 240nm

Cycloxygenase 2 and Lipoxygenase inhibition activity of Dioscorea bulbifera and saponin extract:

The inhibitory effects of COX-mediated TMPD oxidation activity were examined using purified COX as enzyme sources. COX-2 activity was strongly inhibited by saponin extract from Dioscorea bulbifera fruit 84% at the concentration 20µl (Fig 2). The anti-inflammatory activity of the was evaluated by measuring the inhibition of LOX using linoleic acid as substrate. The LOX inhibition assay showed the specific activity of 4.7, 4.72, 4.75 and 4.8μg at the concentration of 5µl, 10µl, 15µl and 20µl/ml respectively.

Fig 2: Cycloxygenase 2 inhibition activity of Dioscorea bulbifera and saponin extract

Inhibition of protein denaturation of Saponin extract from Dioscorea bulbifera fruit:

Examination of Dioscorea bulbifera fruit saponin of momentous activity on inhibition of protein denaturation and its effect was compared with the standard drug Diclofenac sodium. The production of auto antigen in certain arthritic disease may be due to denaturation of protein. The maximum percentage inhibition of protein denaturation was observed as 71.51% at 20μg/ml which was close to the percentage of inhibition of diclofenac sodium (76.90%).

Antihemolytic activity of Saponin extract from Dioscorea bulbifera fruit:

The Saponin extract of Dioscorea bulbifera fruit showed higher antihemolytic activity with the increase in concentration (Fig 3).

Fig 3: Antihemolytic activity of Saponin extract from Dioscorea bulbifera fruit

Molecular docking analysis to predict the lead molecule possessing Anti-inflammatory activity:

The lead-likeliness property and its binding efficacy with COX-2 receptor is studied using in-silico studies. Literature survey enabled in enumerating the possible saponin moieties present in D.bulbifera possessing anti-inflammatory activity. Amidst the various moieties present, Dioscin, Diosgenin, Spiraconazole A and Floribundasaponin D were chosen to predict the binding efficacy through in-silico studies respectively. The docking results revealed that dioscin was predicted to have the highest binding efficacy with COX-2 receptor with a binding energy of -662.07 kcal/mol (Table 1, Fig 4).

Table 1: Binding efficacy prediction of COX-2 receptor with D.bulbifera saponins

Compound	Energy (kcal/mol)	Binding site amino acids
Dioscin	-662.07	Arg-424, Asn-425, Leu-167, Leu-168, Pro-375, Val-420, Gly-422, Gly-423, Arg-424, Arg-424, Asn-425, Val-426, Leu-494, Val-495
diosgenin	-323.5	Leu-167, Leu-168, Pro-375, Arg-419, Val-420, Ala-421, Gly-422, Gly-423, Arg-424, Asn-425, Val-426, Val-426, Leu-494, Val-495
Spiraconazole A	-326.9	Ala-118, Asp-119, Asp-119, Tyr-120, Gly-121, Pro-140, Val-141, Asp-143, Asp-143
Floribundasaponin D	-346.1	Val -102, Arg-106, Val-335, Ser-339, Tyr-341, Leu-345, Ala-513, Leu-517

(a) (b)

(c) (d)

Fig 4: Docked pose of ligand and human COX-2 receptor

a) Dioscin b) Diosgenin c ) Floribundasaponin D d) Spiraconazole A

DISCUSSION:

Various parts of plants have been used as herbal medicine to treat illness, to promote health, to restore the body's ability to protect, regulate and heal on its own. Several preparations are now available in market also as successful products. About 40% of the anticancer drugs made from plants and some herbal remedies are reported to have adverse effects also and some of them have a tendency to interact with the synthetic preparations^17,18. Several clinical trials of herbal drugs have been done, representing them as a better aid in the treatment of anti-inflammatory diseases.

Lipoxygenases (LOXs) (LOX; EC 1.13.11.12) are a family of non-heme iron-containing dioxygenases catalyzing the biosynthesis of leukotrienes. Leukotrienes function as initiators of inflammation and their inhibition is considered to be partly responsible for the anti-inflammatory activity¹⁹. In the present study saponin extracts of Dioscorea bulbifera showed good anti-LOX activity with an IC₅₀ value of 29.87μg/ml. LOX inhibition was used to evaluate anti-inflammatory activity of a few medicinal plants used in Limousin country. Filipendula ulmaria (Meadowsweet) recorded LOX inhibition with IC₅₀ of 60 μg/ml and Urtica dioica (Nettle) methanolic extract inhibited LOX with IC₅₀ of 348μg/ml²⁰. In another study, Bidens pilosa extract exhibited IC₅₀ of 21.8μg/ml and Emex australis extract recorded IC₅₀ of 81.4μg/ml for LOX inhibition²¹^. Another hypothesis proposed indicated that inhibition by antioxidant could be attained via chelation of its non-heme bound iron or by reduction of its ferric form suggesting a competitive kind of inhibition as reported for Mahonia aquifolium²². The present work would like to speculate that LOX inhibition could be due to antioxidant properties of the anthocynin extract with the mechanism of action to be elucidated.

Some studies have demonstrated that Saponin anti-inflammatory activities by inhibition of cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-activated RAW 264 cells or inhibiting inducible nitric oxide (iNOS) protein and mRNA expression in LPS-activated murine J774 macrophages^23,²⁴. Some antioxidants inhibit the expression of COX-2 by interfering with the signalling mechanisms that regulate the COX-2 gene²⁵^,26. The late phase is sustained and mediated by the release of Prostoglandins (PGs), the products of cycloxygenase and lipoxygenase enzymes²^7,28. It has been clearly reported that caragennan induced paw oedema for the administered dose (500mg/kg, p.o.) is comparable with reference standard indomethacine, which is a cycloxygenase inhibitor^29,30. Similar studies have been conducted by many researchers on the Anti-inflammatory effect of plant extracts^31,32,33. Thus the present study results strongly suggest that the extracts possess Anti-inflammatory activity and their mechanisms of action may be mediated through inhibition of cyclooxygenases.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 21.03.2021 Modified on 25.11.2021

Research J. Pharm. and Tech 2022; 15(10):4434-4438.

DOI: 10.52711/0974-360X.2022.00743