Protective Effect of Mucoadhesive Gel of Phytosterol of Pongamia glabra Against Arecoline-Induced OSMF Rats

Introduction

Oral submucous fibrosis or juxta epithelial fibrosis (OSMF) is a chronic precancerous disease of mouth caused due to chewing habit of tobacco, betel nut and other related products, which contain harmful chemical ingredients such as arecoline.^1,2 The disease is commonly treated using synthetic steroids such as prednisolone, betamethasone etc., which can have fatal effects with prolonged durations of treatment. Hence there is a need to develop a herbal dosage form suitable for oral application, which serves to be a safer alternative medicine from plant source.

Pongamia glabra or Pongamia pinnata Vent. (P. glabra) is a medium sized evergreen glabrous tree found commonly distributed in alluvial and coastal regions of India. The plant commonly called as, “Karanja” is a well-known enlisted plant in Ayurveda, Siddha and Unani systems for having many medicinal claims such as bronchiolytic, antiscabies, anti-rheumatic, antimicrobial, wound healing properties.^3,4 The present study was designed to isolate the steroidal compound from the stem bark extract of the plant, characterize it spectroscopically, formulate mucoadhesive gel of the isolated phytosterol, standardize and subject to histopathological study for assessing its protective effect against arecoline induced oral submucosa fibrosis (OSMF) in rats.

Materials and Methods

Collection and authentication of stem bark of Pongamia glabra Vent.

Pieces of stem bark of Pongamia glabra Vent. were collected from surrounding areas of Kalaburagi, Karnataka, India, which were dried and authenticated (GUG/BOT/Herbarium/2008-09/09).

Extraction

The coarse powder of the shade dried bark of Pongamia glabra Vent. (Fam. – Fabaceae) was extracted with petroleum ether (60-80 o C) by soxhletion at 40 o C for 48 h to yield the petroleum ether extract of the plant.⁵

Preliminary qualitative phytochemical investigation and thin layer chromatography (TLC) studies

Preliminary qualitative phytochemical analysis of the petroleum ether extract of stem bark was carried out.⁶ The TLC studies were performed for the constituents of the extract by using precoated aluminum silica gel GF 254 plates of required size.^7,8 The details of the solvent systems and visualization methods were as follows.

TLC profile - Steroids

Solvent system: Petroleum ether: acetone (7:3).

Visulization: Anisaldehyde: sulphuric acid reagent (pink to red colored spots)

TLC profile for Saponins

Solvent system: Chloroform: glacial acetic acid: methanol: water (6.4:3.2:1.2:0.8)

Visualization: Anisaldehyde: sulphuric acid reagent (pink colored spots)

Isolation of phytosterol from petroleum ether extract of stem bark of Pongamia glabra Vent.

About 70 g of petroleum ether extract was solubilized in n-hexane and filtered. Then the filtrate (n-hexane fraction) was subjected for concentration under reduced pressure to dryness (yield of n-hexane fraction – 17 g). About 15 g of n-hexane fraction was saponified and the unsaponifiable matter (USM) was determined.

Determination of unsaponifiable matter (USM)⁹

The n-hexane fraction was subjected to determination of the unsaponifiable matter (USM) according to I.P. 1966 method.

Method

About 12 g n-hexane fraction was weighed accurately and taken into a 250 mL R.B. flask, which was fixed with reflux condenser. To this, 4% solution of potassium hydroxide in alcohol was added and heated for one hour on water bath with frequent shaking. Then the contents of flask were transferred to a 250 mL separating funnel and subjected for fractionation with 100 mL of solvent ether (diethyl ether) for 3-4 times. Combined ether extracts were distilled off to get the residue of 8 g i.e. unsaponifiable matter. This yellow residue indicated steroid composition on TLC (Rf: 0.41, 0.44, 0.57) and was further used for the isolation.

Isolation of sterols from USM portion of n-hexane fraction by column chromatography

About 8 g unsaponifiable matter (USM) residue was solubilized in 10 mL chloroform and loaded onto an activated silica gel column prepared in chloroform. The column was eluted with chloroform, 10%, 20%, 40% and 80% n-hexane in chloroform in gradient manner and then with 100% n-hexane. The elution of different fractions was verified by TLC studies (Rf: 0.41, 0.44, 0.57; Chloroform: methanol, 9.5:0.5; aluminum precoated silica gel plate; visualization: vanillin: methanolic H2SO4 reagent).

Elution was carried out with above solvent system in gradient manner and was adjusted to 10-15 drops/min. The eluates from 40% n-hexane in chloroform gave one single spot on the TLC (Rf: 0.57; Chloroform: methanol, 9.5:0.5; aluminum precoated silica gel plate; visualization: vanillin: methanolic H₂SO₄ reagent).

These eluates were concentrated, which yielded a white-colored waxy compound. On recrystallisation with n-hexane, a white crystalline powder (65 mg) was obtained, designated as compound PGS. The isolated compound responded to Libermann-Burchard and Salkowaski tests for sterols.

Acetylation of compound PGS Compound PGS (5 mg) was taken in a silica crucible, to which 0.2 mL dry pyridine and 1 mL freshly distilled AC2O (acetic anhydride) were added. The mixture was stored overnight and then ice-cold water was added, stirred, kept for 2h, and later filtered and dried.

Preparation of mucoadhesive gel of arecoline hydrobromide (OSMF induction)

The mucoadhesive gel of arecoline hydrobromide was prepared by dissolving 1% arecoline hydrobromide in 100 mL water, mixed with polymers, Viz. - 1.25% each of sodium carboxy methyl cellulose (Sodium CMC), hydroxyl propyl methyl cellulose (HPMC), and 0.01% of sodium metabisulphite (preservative) and allowed to undergo hydration for 24 h.¹⁰

Preparation of mucoadhesive gel of the isolated sterol compound (PGS)

The mucoadhesive gel of the isolated sterol (PGS) was prepared by solubilizing 0.1% of isolated phytoconstituent (PGS) in 20 mL of 1% tween 80 and the emulsified mixture was added with 80 mL glycerine. The glycerine mixture was then mixed with polymers, Viz. - 1.25% each of sodium carboxy methyl cellulose (Sodium CMC), hydroxyl propyl methyl cellulose (HPMC), and 0.01% of sodium metabisulphite (preservative) and allowed to undergo hydration for 24 h. The prepared gels were filled in empty aluminum tubes and labeled.

Evaluation of the prepared gel^11,12

Homogeneity

The prepared gel formulation PGS was allowed to set in a clean glass beaker and evaluated for proper appearance and presence of aggregates.

Grittiness

The gel formulation PGS was inspected for the presence of particulate matter using a microscope.

Spreadability

1 g of the prepared gel PGS was placed between two horizontal plates of size 20 cm × 20 cm, to which 125 g weight was placed on the upper plate. The spreading diameter of the gel was measured after 1 min of weight placement.

Extrudability

The gel-PGS was filled in one-ounce aluminum collapsible tube having a nasal opening of size 5 mm. The extrudability was determined by measuring the amount of gel extruded out through the tip when a constant load of 1 kg was applied. The amount of extruded gel collected on the pan was weighed.

pH measurement

Approximately 5 g of the gel-PGS was dissolved in 45 mL water and the pH of this suspension was measured using the pH meter at 27 °C.

Mucoadhesive study

The mucoadhesive strength of the prepared gel-PGS was calculated by the use of the assembled system constructed in our laboratory. Sections of the fresh goat buccal tissue were fixed with cyanoacrylate adhesive, enabling the buccal surface outside on two glass vials. One vial was attached to the weighing balance, while the other vial was mounted on a height-adjustable pan. Around 1 g gel was placed onto a vial's buccal tissue. The height of the other vial was subsequently adjusted such that the gel applied on one vial's mucosal surface could coincide and bind to the mucosal surface of the other vertically. The weight was increased gradually until the separation of two vials apart. The mucoadhesive strength of the gel-PGS was calculated based on the minimum weights required for the separation of two vials. At each time of measurement, the buccal tissue pieces were altered. The above study was performed in triplicates.

Drug Content Uniformity

1 g of prepared gel containing 1000 μg of PGS (Pongamia glabra sterol) was dissolved in 100 mL 6.4 pH phosphate buffer solution (concentration 10 μg/mL). The absorbance was measured at 257 nm using UV spectrophotometer against a blank solution. Blank solution was prepared by adding the respective concentration of polymers and other ingredients without the drug in a similar manner as mentioned above.

Viscosity

Viscosity of the gel-PGS was analyzed using Brookfield viscometer (spindle-01). Measurements were carried out at varying speeds i.e. from 10 to 30 rpm at 30 s between two successive speeds and then in a descending order, with a shear rate of 133 to 400 s^-1.

In vitro diffusion profile

The experiment to analyze the drug diffusion was carried out using a 10 cm open glass cylinder with an internal diameter of 3.1 cm. Covering one end of the cylinder with a dialysis membrane - 70 (Hi-Media), 1g of PGS (P. glabra sterol) was inserted within this permeation cell. The beaker with 100 mL of 6.4 pH buffer solution was used as receptor compartment. The test sample-PGS was submerged deeply such that it should be below the surface of the medium in the receptor compartment. The media in receptor compartment was stirred at 37°C at 50 rpm. UV spectroscopic assay was carried out at 257 nm for PGS gel, by removing 10 mL sample after every 10 min time interval. The removed aliquots of sample were replaced by the fresh medium of same quantity. The above experiment was performed in triplicates. The percent cumulative drug release versus time for the gel-PGS was noted.

In vivo studies for evaluation of antifibrotic effect in arecoline induced OSMF rats

Male albino rats of Wister strain (240 g to 250 g bw) (HKES/MTRIPS/IAEC/108/2019-20) were acclimatized to laboratory conditions and used for the in vivo study.¹³ Grouping of the experimental animals was as below (n=5).

Group 1: Normal control (G1)

Group 2: OSMF induced group with gel of arecoline HBr (G2)

Group 3: OSMF induced group treated with petroleum ether extract of bark of P. glabra solubilized in 1% tween 80 for application on oral mucosa (G3)

Group 4: OSMF induced group treated with gel formulation, P. glabra sterol (PGS) (G4)

Induction of OSMF in rats

Induction of OSMF was done by bilateral application of mucoadhesive gel of arecoline hydrobromide to the buccal mucosa using a cotton bud, twice daily for a period of four months. The animals were fed a standard diet after 6 h of application of the gel. On completion of 1^st, 2^nd and 4^th months’ application respectively, one animal from each group (G2, G3 and G4) having lowest weight was sacrificed and using skin biopsy punch # 3.5, the biopsy of oral mucosa was collected. The collected biopsies were preserved in formalin solution for histopathological studies.

Treatment of induced rats

P. glabra extract and the formulated gel PGS were applied every day using a cotton bud on the oral mucosa of induced rats for four months. Two rats from each group (G3 and G4) were sacrificed and biopsies of oral mucosa were collected for histopathological assessments on completion of 1^st, 2^nd and 4^th months following the continuous application of gel. The samples of oral mucosa from group 1 were collected and stored for comparison.

Histopathological study

The biopsy of buccal mucosa was stored in 10% formalin for 24 h. The specimen was placed in xylene three times (1 h each) for dehydration and later in 70, 90 and 100% alcohol for 2 h, each time, respectively. The specimen was then treated with paraffin wax twice, each time for 1 h for infiltration or impregnation. Blocks were made using “L” block field with molten wax. Sections of 3–5 μm thickness were cut with microtome and slides were made by mounting the sections on glass slides coated with egg albumin/glycerin. The sections were treated with hematoxylin and eosin for staining (HE-stain) and subjected to microscopic examination.

Measurement of body weight

Body weight measurement of arecoline induced rats was carried out on completion of 1^st, 2^nd and 4^th month of induction and was repeated during treatment period i.e. on completion of 5^th, 6^th and 8^th months of treatment.

Statistical analysis

Data were expressed as mean ± SEM and differences between the groups were statistically determined by analysis of variance (ANOVA) followed by Dunnet’s test.

Results

Preliminary phytochemical analysis and TLC-thin layer chromatography study

Preliminary phytochemical analysis of the petroleum ether (60-80o C) extract of stem bark of Pongamia glabra revealed the presence of sterols and saponins. TLC studies of petroleum ether extract showed four pink to red colored (Rf value - 0.64, 0.72, 0.78, 0.88) and two pink-colored spots (Rf value - 0.72, 0.87) for the presence of sterols and saponins, respectively.

Spectral characterization of isolated compoundPGS

The IR, ¹HNMR, ¹³CNMR and LC-MS spectral characteristics of the isolated phytoconstituent, PGS from petroleum ether extract of stem bark of P. glabra Vent. were as below.

1. Pearl white crystals.

2. Gave positive response to Libermann-Burchard test (green color) and Salkowaski test (red color) for sterols.

3. Rf value: 0.57.

4. Visualization: vanillin: methanolic H2SO4 reagent

5. Melting point: 165-168°C (Literature 170 o C)

Mass spectra

1. Molecular formula: C₂₉H₄₈O

2. Molecular weight: 412

Mass spectrum indicates the presence of molecular ion peak at 411 [M^-1], 100% which corresponded nearly to molar mass of the compound “Stigmasterol”.

From the M.P, I.R, ¹HNMR, ¹³CNMR, and mass spectral study, the isolated compound PGS has been identified as “Stigmasterol” resembling compound. The solid obtained after acetylation was crystallized from benzene as fine needles.

Evaluation of mucoadhesive gel-PGS

Homogenuity and grittiness

The prepared gel-PGS formulation was found to have no lumps and was free from grittiness. No particulate matter was noticed in the gel when observed under light microscope.

Spreadability and extrudability

The spreadability of the gel-PGS was found to be 61±2.0 after one min (Table 2). The viscosities of the gel formulation PGS prepared by mixing equal proportions of polymers (sodium CMC and HPMC) were low. Hence these formulations exhibited high spreadability and also good extrudability.

pH measurement

The pH of the gel-PGS was found to be 6.1±0.20 (Table 2), which was found to be similar to the salivary pH and hence free from irritation.

Mucoadhesive study

The strength of mucoadhesion of gel or the mucoadhesive strength was evaluated and reported to be 11.400±0.004 g (Table 2). The gel formulation, PGS exhibited good mucoadhesive strength.

Drug Content Uniformity

The uniformity of drug content of the PGS gel was evaluated and reported to be 87.48±0.174%. The gel showed uniformity in drug content.

Viscosity

The viscosities of the gel, PGS was found to be 8735 and 3876 cps at 133 s^-1 (low rate of shear) and 400 s-1 (high rate of shear), respectively (Table 2).

In vitro diffusion profile

The formulation, PGS exhibited a cumulative percent drug release evaluated and reported to be 87.48±0.174 (Table 2).

The characterization of mucoadhesive gel, PGS exhibited good physicochemical properties, viz.- homogeneity, grittiness, spreadability and extrudability, pH, mucoadhesive strength, drug content uniformity, viscosity and in vitro drug release (Table 3).

Histopathology and body weight measurement

Histopathological observations of arecoline HBr induced OSMF rats carried out by taking biopsies of buccal mucosa at end of four months of OSMF induction exhibited white patches on buccal mucosa indicating the micro changes such as atrophic epithelium with parakeratinization, dense hyalinization of submucosa due to thick collagen deposition (severe OSMF), narrowed blood vessels with perivascular fibrosis and chronic inflammation infiltrate. Biopsies of buccal mucosa of extract treated (G3) and gel PGS treated rats (G4), on completion of 1st month’s treatment showed gross detectable changes in white patch, and after 2nd month’s treatment, revealed appearance of pale mucosa. On completion of 4th month’s treatment, the following micro changes in buccal mucosa were noted.

• Thin epithelial lining noted in extract treated group and epithelium almost reorganizing to its normal thickness and appearance of rete pegs in the PGS gel treated animal group.
• Only about 20 to 30% submucosal collagen dissolution in the extract treated group and more than 70-80% collagen dissolution in PGS gel treated group.
• Chronic inflammation infiltrate present in the extract treated group and focal perivascular chronic inflammation infiltrate in the gel treated group.

Oral mucosal application of arecoline Hbr for four months produced significant (P <0.0001) reduction in body weight of animals i.e. 145±4 to 91±3 and the animals regained significant (P <0.0001) body weight (from 93±2 to 133±2) after four months of treatment involving the application of PGS gel to buccal mucosa to treat arecoline induced OSMF.

Discussion

Preliminary phytochemical analysis including TLC studies of petroleum ether (60-80^OC) extract of stem bark of the plant showed the presence of sterols and saponins. From the M.P, I.R, 1HNMR, 13CNMR and mass spectral studies, presence of molecular ion peak at 411 [M+1], 100% was noted which corresponded nearly to molar mass of the compound “Stigmasterol” and hence the isolated sterol compound PGS from petroleum ether extract of stem bark of Pongamia glabra has been identified as “Stigmasterol” resembling compound.

Mucoadhesive gels of arecoline HBr (OSMF inducing agent) and isolated phytosterol compound, PGS were prepared using equal proportions of hydrophilic synthetic mucoadhesive polymers like sodium carboxy methyl cellulose (Na CMC) and hydroxy propyl methyl cellulose (HPMC) and sodium metabisulphite (Preservative) in glycerine. The characterization of mucoadhesive gel, PGS exhibited good physicochemical properties, viz.- homogeneity, grittiness, spreadability and extrudability, pH, mucoadhesive strength, drug content uniformity, viscosity and in vitro drug release.

Histopathological observations on completion of 4th month of induction with arecoline in rats showed white patches on buccal mucosa with thick deposition of collagen, perivascular fibrosis and chronic inflammation infiltrate and significant (P <0.0001) reduction in their 37 body weight. The study on completion of 4th month treatment with the extract and the mucoadhesive gel, PGS exhibited changes such as the epithelium reorganizing to its normal thickness due to 70-80% dissolution of submucosal collagen and significant (P <0.0001) gain in body weight, especially in gel treated animal group (Group 4). However, the application of extract of the plant i.e. group 3 brought marginal weight gain as application of extract to oral mucosa could not assure proper drug release at the site compared to the application of mucoadhesive gels.

Conclusion

PGS, a steroidal compound from the petroleum ether extract of stem bark of Pongamia glabra was isolated and after characterization using FTIR, 1H and 13C NMR and LC-MS spectroscopic studies, was identified as, “stigmasterol” resembling compound. Mucoadhesive gel of the compound, PGS was developed and characterized. The formulated gel was found to be homogeneous with no grittiness, good extrudability and mucoadhesive strength, optimum pH, uniform drug content, low viscosity and 86-88% cumulative drug release property. Histopathological study of arecoline HBr induced OSMF rats, on completion of four months induction exhibited significant (P <0.0001) reduction in body weight due to difficulty in mouth opening with deposition of collagenous tissue in buccal mucosa. On completion of four months treatment with plant extract of P. glabra and the mucoadhesive gel PGS, significant (P <0.0001) gain in body weight was observed indicating normal thickened epithelium and 70-80% submucosal collagen dissolution. However, the extract treated rats showed marginal increase in body weight because of stickiness and non-uniform drug release compared to gel formulation, which was proved to be a better dosage form available for oral application.

Conflicts of interest

Nil

Source of funding

RGUHS Advance Research Fund, RGUHS, Karnataka

Acknowledgment

Authors are thankful to authorities of RGUHS Adv. Res. fund wing and HKE Society and Matoshree Taradevi Rampure Institute of Pharmaceutical Sciences, Kalaburagi, Karnataka India, for providing necessary facilities to carry out the study.