Antipsoriatic Activity of gel containing Methylsulphonylmethane Powder and seed oil of Pongamia pinnata Linn.

 

Ms. Salunke Pradnya Balasaheb1*, Dr. Patil Sandeep Balvant2, Ms. Kadam A.T.1

1Assistant Professor, LNBC Institute of Pharmacy, Raigaon Satara, Maharashtra, India 415020

2Associate Professor, Department of Pharmacology, Adarsh College of Pharmacy Vita Dist Sangli

*Corresponding Author E-mail: pradnyasalunke89@gmail.com

 

ABSTRACT:

To develop gel using Methylsulphonylmethane powder and seed oil of Pongamia pinnata Linn. to minimize side effects of current therapy. All prepared formulations were evaluated for pH, Viscosity, spradability, Extrudability. The release of optimized batch was carried out using Cellophane membrane into Phosphate buffer 6.8 at 37°C. The Characterization was carried out by using FTIR, DSC, SEM analysis. In-vtro & In-vivo activities were performed by using HaCaT Cell line and Mouse tail model respectively.

 

KEYWORDS: Psoriasis, Orthokeratosis, Pongamia pinnata, Methylsulphonylmethane.

 

 


1. INTRODUCTION:

Psoriasis is ancient common, chronic, inflammatory, autoimmune dermatitis. Psoriasis is a common disorder in which environmental factors contribute to the development of sharply demarcated erythematous scaly plaques in genetically predisposed. It is characterized by epidermal keratinocytes hyper proliferation, abnormal keratinocytes differentiation, erythematous, sharply demarcated papules and rounded plaques, covered by silvery micaceous scale). Psoriasis is one of the most common non infectious skin disease affecting upto 2.5 % of world population. It is Uncommon under the age of 10 years, it apperears between age 15-30 years affecting men and women. Pongamia pinnata Linn. and Methylsulphonymethane (Sulphur powder) used in many dermatological disorders like Eczema, Scabies, Psoriasis from ancient time. A gel has good elasticity, satisfactory bioadhesion and is without irritation or sensitizing action. Natural or synthetic polymers that are Biodegradable may offer advantage over Non-Biodegradable polymers for topical drug delivery applications.1,2.

 

2. MATERIAL AND METHODS:

2.1. Materials:

Methylsulphonylmetane and Pongamia pinnata oil purchased from Loba Chemie Pvt. Ltd. Mumbai and Local area of Sangli respectively. All the materials, solvents and Chemicals were of analytical grade.

 

2.2 Preparation of formulations:

Accurately weighed quantity of carbapol 934 was taken in a beaker and dispersed in 80 ml of distilled water. Kept the beaker aside to swell carbapol 934 for 24 hours and then stirring should be done using mechanical stirrer at 1200 rpm for 30 min. Take 5 ml of propylene glycol and required quantity of methyl paraben and propyl paraben to it and kept in distilled water aside for 20 minutes and then stirred properly. In small beaker take drug and seed oil of Pongamia pinnata Linn and kept on sonicator for sonication. Finally after all carbapol dispersed solution in beaker was mixed and kept for stirring at 800 rpm for 20 minutes and then drug was added. Eventually Triethanolamine was added drop wise to formulation for adjustment of required skin pH (6.8-7) and to obtain the gel at required consistency.3

 

 


Formulation and of Gel:

The 5 different batches were developed as below.

 

Table no.1: Different batches of gel formulation.

Composition

B1

B2

B3

B4

B5

Methylsulphonylmethane (mg)

1.3gm

1.3gm

1.3gm

1.3gm

1.3gm

Pongamia pinnata Linn oil (ml)

5ml

5ml

5ml

5ml

5ml

Carbapol 934 (gm)

0.5

1.0

1.25

1.50

1.75

Propylene Glycol (ml)

5

5

5

5

5

Methyl paraben (mg)

0.2

0.2

0.2

0.2

0.2

Propyl paraben (mg)

0.02

0.02

0.02

0.02

0.02

Distilled water (ml)

100

100

100

100

100

Triethanolamine (ml)

q.s.

q.s.

q.s.

q.s.

q.s.

 


2.3 Evaluation of prepared gel:

2.3.1.Physical appearance and Homogeneity :

The prepared gel formulations were inspected visually for their colour, Homogeneity

 

2.3.2. pH:

To determined pH, 1 gm of each gel formulation were transferred into 100ml beaker with distilled water and ph measured by using digital pH meter.

 

2.3.3. Viscosity:

A Brookfield Viscometer RVT with spindle no.64.The spindle was rotated at 10r/min and samples were allowed to settle over 30 min at temperature [25±1]°C before measurements were taken.

 

2.3.4. Spreadability :

It was determined by using spredability apparatus.

 

2.3.4. Extruidability study:

The gel formulations were filled in standard capped Collapsible Aluminum tubes and sealed by crimping to the end. Weights of the tubes were recorded. The tubes were placed between two glass slides and were clamped. 50 gm was placed over the slide and then the cap was removed.

 

2.4.Optimization of batch:

The batches were optimized by checking, and by studying physical evaluation of their pH, Viscosity, Spradability and Extrudability of all formulations batches. By studying the evaluations parameters of all batches, from gel formulation the optimized batch was decided and was further studied.

 

2.5.Evaluated parameter of optimized batch:

2.5.1.In vitro Diffusion Study:

The diffusion studies of the prepared gel can be carrying out in Franz diffusion cell for studying the dissolution release of gel through a synthetic Cellophane membrane was used as diffusion membrane. Gel sample (0.5 gm) was taken in cellophane membrane and the diffusion studies were carried out at 37±0.5°C using 250 ml of phosphate buffer (pH 7.4) as the dissolution medium.5 ml of each sample was withdrawn periodically at 1,2,3,4,5,6.7 and 8 hours. And each sample was replaced with equal volume of fresh dissolution medium. Then the samples were analyzed for the drug content by using phosphate buffer as blank. The absorbance was taken by UV spectroscopy

 

2. 5.2.Skin Irritation Study:

The Wistar rats of either sex of weight 150-200 gm were used for this test. The intact skin was used. The hairs were removed from the back of rat 3 days before experiment. Formulated gel was applied on test animal. Gel base was applied on the back of animal as control. The animals were treated daily up to seven days and finally treated skin was examined visually for erythema and edema.

 

2.5.2. Stability Study:

Stability testing of drug product being as a part of drug discovery and ends with the commercial product, to assess the drug and formulation, stability study were done. Optimized gel formulation (50gm) was packed in the wide mouth plastic container and subjected to Accelerated stability study at 40±10oC and 75% RH for a period of 3 month as per ICH guidelines. The gels were withdrawn and evaluated for physical changes in colour, odour, consistency, pH, Viscosity and spread ability study.

 

2.5.4. FTIR:

It is the powerful tool for the determination of functional group.IR of formulated was done by using FTIR Agilent technology model Cart 630

 

2.5.5. Scanning Electron Microscopy (SEM):

The Surface topography, particle size, morphology of the gels were investigated with a scanning electron microscope. SEM is one of the common methods used owing to the simplicity of sample preparation and ease of operation

 

The analysis was performed by placing samples on a brass stub using a double-sided adhesive tape and was made electrically conductive by coating in vaccum (6pas)m with platinum using ah Ion Sputter at 15mA. The microphotographs were taken at an excitation voltage of 10KV.

2.5.6. Methods

2.5.6.1. In vitro methods:

2.5.6.1.1.HaCaT cell line (Psoriatic skin cell line):

2.5.6.2. In vivo (Pery scientific mouse tail method)

 

2.5.6.1.1.HaCaT cell line (Psoriatic skin cell line):

In vitro antipsoriatic study was performed using SRB assay. HaCaT human keratinocyte cell (procured from NCCS Pune) lines were used. Culturing of cell lines was done in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The monolayer cell culture was trypsinized and the cell count adjusted to 1.0x105 cells/ml using growth medium in a 96 well microtitre plate, 0.1 ml of the diluted cell suspension (approximately 10,000 cells/well) was added. After 24 hours, the monolayer was washed once, when a partial monolayer was formed, the supernatant was taken, and 100 μl of drug dilution prepared with above media was added per well in microtitre plates. The plates were microscopic examination was carried out and observations were recorded for every 24 hours. After 72 hours, 25 μl of 50% trichloro-acetic acid (TCA) was added to the wells such that it forms a thin layer over the drug dilutions to form a overall concentration of 10%.The plates were then incubated at 4oC for one hour. The plates were flicked; culture was washed five times with tap water to remove traces of medium, drug and serum, and was then air dried. The air-dried plates were stained with SRB for 30 minutes. The unbound dye was then removed by rapidly washing four times with 1% acetic acid. The plates were then air-dried.100μl of 10 mM tris buffer.4

 

Percent Cytotoxicity =

Reading of control - Reading of treated cells X 100

              Reading of control

 

2.5.6.2. In-vivo method (Perry scientific Mouse Tail Method for Psoriasis.)

The mouse Tail Test first described by Jarrett and Spearman (1964) with certain modifications reported by authors is a morphometry-based, sensitive and well reproducible method. It allows the quantitative evaluation of the effects of antipsoriatic drugs on epidermal differentiation crucially disturbed in psoriasis. This model is based on the induction of orthokeratosis in those parts of the adult mouse tail which have normally a Parakeratosis differentiation.

 

Albino Mice of weight 25-27 gm were divided into three groups of six each. Proximal parts Tails were treated locally with 1 gm of drug The first group was control which was untreated and second group was standard treated with 0.05% Retinol A cream. The Third group was treated with prepared gel. For the topical application gel is applied by the cotton topically to the proximal part of the tail about 2.5cms and allowed to remain in contact for 2 hrs a plastic cylinder is sliped over tail and fixed with adhesive tape. Then tail were washed with water. Treatment was given once daily for 14 days. The animals were sacrified using deep ether anesthesia by cervical dislocation, two hours after the last treatment and the proximal parts of their tails were cut and each group tails were stored in treatment and the proximal parts of their tails were cut and each group tails were stored in separate containers containing 10 % Formaline in buffer solution.

 

3. RESULTS:

3.1. Physicochemical evaluation of gel formulation:

3.1.1. Physical appearance and Homogeneity

Table no. 2 Physical appearance and Homogenecity of different batches of gel formulations

Batches

Appearance

Homogeneity

B1

White

Good

B2

White

Good

B3

White

Good

B4

White

Good

B5

White

Good

Standard drug (Retino A 0.05% cream)

White

Good

 

3.1.2. Measurement of pH

Table no. 3: pH of different batches of gel formulations of pH:

Batches

pH

B1

6.6

B2

6.8

B3

6.5

B4

6.6

B5

6.6

Standard drug (Retino A 0.05% cream)

6.9

 

Fig.No.1. pH of different batches of formulated gel

 

3.1.3. Viscosity study:

Table no 4. Viscosity of different batches of gel formulations

Batches

Viscosity (cps)

B1

4098

B2

4476

B3

4235

B4

5015

B5

5094

Standard drug (Retino A 0.05% cream)

4376

 

Fig.No.2.Viscosity (cps) of different batches of formulated gel

 

3.1.4. Spreadability study:

Table no.5: Spreadability of different batches of formulated gel

Batches

Spreadability (gm.cm/sec)

B1

19.35

B2

22.50

B3

20.56

B4

16.67

B5

18.36

Standard drug (Retino A 0.05% cream)

23.00

 

Fig.No.3.Spreadability of different formulated batches

 

6.2. Optimization of batch:

After analysis of all batches of formulations by their evaluation parameters like appearance and homogeneity, pH, viscosity, spreadability and Extrudability, the formulation batch B2 showed.

 

Good results than other batches. Hence the optimized batch was used for further study like in vitro diffusion study. In- Vitro and In and vivo study.

 

The study of optimized batch was as follows

 

Table no.6: Evaluation parameters of optimized batch B2

Optimized batch

pH

Viscosity (cps)

Spreadability (gm.cm/sec)

Extrudability (%)

B2

6.8

4476±0.5

25.02±0.001

92.45

 

Table no 7. Drug content of prepared formulations

Formulations

Absorbance (y)

% drug content

B1

0.5826

91.01

B2

0.5871

91.76

B3

0.5856

92.01

B4

0.5995

93.89

B5

0.6079

95.26

 

3.2.3. In vitro diffusion study:

In vitro diffusion study was performed for optimized batch B2. The results were depicted in table. Batch B2 showed 76.50% drug release through Cellophane membrane after 6 hours.

 

Table no.8 Percentage drug release of optimized batch B2

Time (Hrs)

% Drug release

0

0

1

19.50

2

32.05

3

43.65

4

57.09

5

63.04

6

77.6

 

Fig.No4.Percentage drug release of optimized batch B2

 

3.2.4. Skin Irritation study:

Absence of skin irritation of gel formulation is acceptable by patient. Skin irritation test performed In vivo test on swiss albino rats.. Gel formulation of batch B2 was found to be free from irritation. Observations indicates acceptability of this gel for topical use

 

Table no. 09: Skin Irritation study of optimized batch B2

Batch

No. of group

Erythema

Edema

B2

Control

0

0

B2

Test

0

0

 

6.2.5. Stability study

Accelerated stability study of batch B2 indicated that the physical appearance, pH, viscosity, Extrudability, Spreadability in the prepared gel remained unchanged upon storage for 3 month.


Table no. 10: Stability profile of optimized batch B2 gel formulation

Evaluation

 Initial

 After1 month

After 2 months

After 3 Months

pH

6.7

6.7

6.8

6.8

Viscosity (cps)

3612

3622

3624

3625

Extrudability(gm.cm/sec)

92.45

92.37

92.30

92.20

Spreadability

19.50

18.90

18.77

18.69

 

8.2.6. Infra Red (IR ) analysis: Fig. No. FTIR analysis Of Formulated Gel

 

Fig.No.5. IR spectra of formulated gel

 

Table No. 11. IR spectra wavenumber and functional groups

Sr. No.

Observed Peak (cm-1)

Peak reported (cm-1)

Bond

Functional group

1

1437

1400-1600

C=C stretch

Alkene

2

1724

1670-1820

C=O stretch

Carbonyl

3

2956

3000-3100

-OH stretch

Carboxylic acid

4

1149

1225-980

S=O stretch

Sulphonyl

5

1232

1320-1000

C-O-C

Ethylene oxide

 

 

8.2.7. Differential Scanning Colorimetry (DSC):

 

Fig no.6. Diffrential Scanning Colorimetry (DSC) curve

 


The DSC thermogram shows the endothermic peak at its melting point i.e. 92.48oc.This indicates the possible change in melting point, release kinetics.

Surface topography, particle size, morphology of the gels were investigated with a scanning electron microscope. SEM is one of the common methods used owing to the simplicity of sample


 


 

Fig. no.7. Scanning Electron Microscopy(SEM)of formulated gel

 

Fig. no. 8.SEM at 30.0kV 8.3mm×65

 


The analysis was performed by placing samples on a brass stub using a double-sided adhesive tape and was made electrically conductive by coating in vaccum (6pas) with platinum using ah Ion Sputter at 15mA. The microphotographs were taken at an excitation voltage of 10KV.

 

The scanning electron microscopic (SEM) image shown the relatively spherical.

 

 

6.3. Results

In-vitro method.

Table no. 12.Invitro activity of Formulation gel against HaCaT Cell line.

Compounds

Reading

% Inhibition

IC 50

Negative Control

0.390

 

 

Postive Control (5 FU)

0.095

75.64

 

 10 µg/ml

0.066

83.07

 

 50 µg/ml

0.050

87.17

43.63µg/ml

 100 µg/ml

0.029

92.56

 


Fig. No. 9 Percentage inhibition of samples using HaCaT cell line

 

RESULTS:

100µg/ml shows 92.56% inhibition with good antiproliferation activity compared to Positive control.

 

3.4.1. perry Scientific Mouse tail Method

 

Fig. No.10 The length of granular layer in control group.(Under 10x)

[L1= 0.00166432 µm and L2=0.003042155µm and mean=0.002353µm]

 

Fig. no.11 The length of granular layer in Standard (Retino A 0.005% cream) group. under 10X magnification [L1=0.004876474µm and L2= 0.007889234µm Mean=0.006312µm]

 

Fig. no. 12The length of granular layer in Test group (Formulated gel) [L1=0.002059126µm and L2= 0.002846050µm Mean=0.002452µm]

 


 

DISCUSSION:

The present study was aimed to investigate the Antipsoriatic activity of gel containing Methylsulphonylmethane Powder and seed oil of Pongamia pinnata Linn.

 

The physicochemical properties of gel formulation in which different batches (B1-B5) showed good appearance and homogeneity. The physical appearance of the gel formulation was white in nature.

 

The pH of the gel formulations was determined by pH meter in which different batches (B1-B5) showed pH in range of 6.5-6.8 and the pH of optimized batch B2 was found to be 6.8. It lies in the normal pH range of skin and with time no skin irriatation was observed.

 

The viscosity of formulated gel was determined by using Brookfield Viscometer of different batches (B1-B5) and were ranging between 3782 to 5094 CPS. The Viscosity of optimized batch B2 was found to be 4476 cps Gels with high viscosity may not extrude from tubes whereas low viscous gels may flow quickly. Hence suitable viscosity is required. Gel formulation majorly depends upon its viscosity. Viscosity of the formulation affects the drug release from the gel. If a gel consists of more viscosity the drug release from the formulation is decreased and if the same gel possess less viscosity the drug diffuses immediately into the diffusion medium. Hence for the gel formulation optimum viscosity is necessary to get the maximum drug release.

 

The values of optimized batch B2(25.02(gm.cm/sec)) in spreadability indicates that gel was easily spreadable by small amount of shear as compared with other batches. It denotes the extent of area on which gel readily spreads on application to skin. Therapeutic efficacy of formulation depends upon spreadability of formulation.

 

The extrusion of formulated gel of different batches (B1-B5) was determined and were showed in percentage in ranging between 82.65-95% The extrusion of optimized batch B2 was found to be 92.45%. The extrusion of gel from tube is important during its application and in patient acceptance. Gels with high consistency may not extrude from tubes whereas low viscous gels may flow quickly. Hence suitable viscosity is required.

 

The Calibration curve of MSM:

The different concentrations were prepared and their absorbance was taken at 470nm their absorbance. The percent drug content of all formulations was found to be in the range of 91.01 to 95.26 % w/v

The difference in drug content may be due to human error during dilution or may be due to production loss during formulation.

In-vitro diffusion study:

It was performed by using cellophane membrane of pore size 40µm using Franz diffusion cell. Batch B2 shows 77.60 % drug release. Generally it is said that cross-linking of polymers in formulation such as increases drug diffusion rate decreases. From observation it can be concluded that concentration of polymer increases drug diffusion rate was reduced. Thus diffusion is polymer concentration dependent phenomenon.

 

Skin irritation study:

The skin Irritancy study indicated that formulated gel were free from dermatological action like signs of erythema and edema.

 

Stability study:

Stability of pharmaceutical product is capability of a particular formulation in a specific container closure system, to remain within its physical, chemical, microbiological, therapeutic and toxicological specifications. Accelerated stability study was done for optimized Batch B2 for three months at 40oC and 75% relative humidity as per ICH guidelines. It indicates that that the physical appearance, pH, viscosity, Extrudability, Spreadability in the prepared gel remained unchanged upon storage for 3 month The results of stability study indicates that formulation was stable at accelarated condition of temperature and relative humidity.

 

The IR spectrum was carried out for the determination of functional groups in the sample. The optimized batch B2 of gel was characterized, the observation of principle peak was found at 1724 which indicates carbonyl group while Peak at 1149 indicates the presence of sulphonyl group.

 

DSC analysis of formulated gel:

DSC curves of optimized Batch (B2) showed at 92.61oC which indicates possible change in melting point, release kinetics and bioavailability of drug.

 

SEM was used for determination of Surface topography, particle size, morphology of the gels.

The scanning electron microscopic (SEM) image shown the relatively spherical and uniform particles of diameter 7.8mm

 

Pharmacological activity:

In vitro activity of formulated gel of optimized batch B2 against HaCaT Cell line:

The present study aimed to investigate the anti-proliferative properties of formulated gel. Antiprolifearive effects against keratinocytes using cultured HaCaT cells as a psoriasis relevant experimental model.IC50 value was found to be 43.63µg/ml.

Antipsoriatic activity by Perry’s scientific mouse tail model:

In vitro antipsoriatic activity of batch B2 showed the significant orthokeratosis in the mouse tail when compared to control thus indicating that the formulation is effective in treating psoriasis.

 

Haematoxylin-eosin staining act as an indicator of orthokeratosis. The granular layer is present in epidermis. It is greatly reduced or almost absent in epidermis of psoriatic lesions. This Condition is known as Parakeratosis, one of the most important characteristic features of psoriasis. The granular layer formation around the epidermis is known as Orthokeratosis condition. The main principle behind the mouse tail test is conversion of parakeratosis to orthokeratosis.

 

Morphometric measurement by motic microscope shows the thickness of granular layer around the epidermis is less in control group while moderate in standard group and highest in test group. Many herbs used in the treatment of psoriasis have been evaluated by this method, and were found to have significant effects.Psoriasis is a disease resulted from the hyperproliferation and abnormal differentiation of Keratinocytes. A successful antipsoriatic drug that targets the epidermis is a compound that ideally shows low toxicity and restores skin homeostasis by suppressing Keratinocytes hyper proliferation, abnormal differentiation, or both.

 

Substances like dithranol and vitamin D analogues that form keratinocytes differentiation are effective in bringing homeostasis of the epidermis in psoriasis conditions. The granular layer is greatly reduced or almost absent in epidermis of psoriatic lesions.5,6

 

The results of the histopathological study indicates that in control group, the nucleus was present in the keratinocytes of epidermis as well as the length of granular layer was found 0.002353 µm around epidermis. While In Standard group and Test group the nucleus was absent and the length of granular layer around the epidermis were 0.006312µm and 0.002452 µm respectively.

 

CONCLUSION:

Since psoriasis is a recurrent skin disease of multiple etiologies, we selected the drug of herbal origin to minimize the related side effects associated with synthetic drugs. The external application is be beneficial in the management of psoriasis. Topical formulations are widely accepted because it is having effective and easy administer. Our findings reveals that MSM and seed oil containing gel formulations is good in appearance, homogeneity, viscosity. Our study provides evidences that gel containing of seed oil of Pongamia pinnata Linn and methylsulphonylmethane combination shows antipsoriatic activity. In vitro antipsoriatic activity of batch B2 show the significant inhibition of proliferation of keratinocyte cell (HaCaT cell line).

 

However there is need for further investigations of antipsoriatic drugs to establish the clinical utility.

 

ACKNOWLEDGEMENT:

Firstly, I would like to express my sincere gratitude to my guide Dr. Sandeep. B. Patil, Assistant Professor of Pharmacology, for His expert guidance and scholarly supervision, endless motivation immense knowledge, encouragements and freedom to carry research work. A sincere gratitude Dr. Mrs. N. S. Naikwade Head of Pharmacology department for Her expertise guidance and moral support every time I needed during my research work.

 

REFRENCES:

1.       Misal G., Dixit. Gulkari V, Formulation and evaluation of herbal gel, Indian journal of Natural Products and Resources, 2012; 3(4): 501-506.

2.       Vogel H.G, 2002; Drug discovery and evaluation, pharmacological assays, 2:1323-1325.

3.       Vijayalakshmi A, Ravichandiran V, Malarkodi Velraj, Nirmala S, Jayakumari S 2012, Screening of flavonoid quercetin from the rhizome of Smilax china Linn. For antipsoriatic activity, Asian Pacific Journal of Tropical Biomedicine 2012; 2(4): 269-275.

4.       Dhanabal S, Priyanka Shamugam, Antipsoriatic activity and cytotoxicity of ethanolic extract of Psoralia coryfoli seeds; Hygeia Journal of Drugs and Medicines.2012; 4(2):41-48.

5.       Bosman B, Matthiesen T, Quantitative method for measuring antipsoriatic activity by drug by mouse tail test. Skin pharmacology, 1992:41-48.

6.       Misal G, Dixit, Gulkari V, 2012, Formulation and evaluation of herbal gel, Indian journal of Natural Products and Resources, 2012; 3(4):501-506.

 

 

 

 

 

 

 

Received on 24.04.2017       Modified on 28.08.2017

Accepted on 12.09.2017      ©A&V Publications All right reserved

Res. J. Pharmacology & Pharmacodynamics.2017; 9(3): 122-130.

DOI: 10.5958/2321-5836.2017.00021.0