Development and evaluation of  Rubia cordifolia  gel for treatment of Acne

 

Archana Gorle1 *, Swati  Patil2

1NCRD’S Sterling Institute of Pharmacy, plot No 93, Sector -19 Seawoods, Nerul (E) , Navi Mumbai- 400706, Maharashtra, India.

2Principal K. M. Kundnani College of Pharmacy, Plot NO. 23, Jote Joy Bldg, Cuffe Parade, Colaba, Mumbai 400 005, India.

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

 

ABSTRACT:

Acne is disorder of pilosebaceous unit, and generally characterized by formation of seborrhea, comedones, inflammatory lesions and presence of bacteria Propionibacterium acnes in the follicular canal and sebum production. Each of these factors provides a potential target for treatment. Propionibacterium acnes are the pharmacological target site of anti-acne drugs. methanolic extract was prepared and evaluated for antioxidant and antibacterial activity The aim of present study was to prepare herbal gel formulation containing methanolic extract of Rubia cordifolia on acne.  Topical gel formulation was designed by using methanolic extract of roots and stems of Rubia cordifolia in varied concentrations. The gel was prepared by using carbopol 940(1%w/v), Rubia cordifolia Extract, ethanol, propylene glycol, methyl paraben, propyl paraben, EDTA disodium, tri-ethanolamine and required amount of distilled water The prepared gels were evaluated for physical appearance, pH, drug content, diffusion study, viscosity, antibacterial activity. Thus, it may be concluded that gel formulations were good antibacterial and antioxidant activity can be used in antiacne activity.

 

KEYWORDS: Rubia cordifolia, carbopol, Herbal gel, Acne.

 


INTRODUCTION:

Acne is disorder of pilosebaceous unit, and generally characterized by formation of seborrhea, comedones, inflammatory lesions and presence of bacteria Propionibacterium acnes [1,2] in the follicular canal and sebum production[3].Each of these factors provides a potential target for treatment. Propionibacterium acnes are the pharmacological target site of anti-acne drugs [4]. Propionibacterium acnes, an anaerobic pathogen, plays important role in the pathogenesis of acne inflammation by inducing polymorphonulcear leukocytes (PMNL), monocyte and macrophages to produce pro-inflammatory mediators. These organisms produce neutrophil chemotactic factors, which attract neutrophils to release inflammatory mediators such as reactive oxygen species (ROS)to create an oxidative stress which is responsible for acne inflammation [5].

Herbal medicines are now being developed in modern dosage forms using modern manufacturing and processing condition compared to past. If concentrated extracts of herbal plants are employed, the dose can be reduced.

 

Herbal cosmetics are safe, effective and potent formula for not only curing the disorders it also beautifying. Topical gel formulations were selected as these are easy to prepare and also economic with respect to cost factor.

In  present study Rubia cordifolia roots and stems were  standardized [6] and methanolic extract was prepared and  evaluated for  antioxidant and antibacterial activity ( P. acne)

 

Gels are transparent to opaque semisolids containing a high ratio of solvent to gelling agent. A characteristic of gel is presence of some continuous structure which provides solid like properties. The system may be clear or opaque because the gelling agent does not fully dissolve or because it forms aggregates which disperse the light. Typical polymers include natural gums – tragacanth, carrageenan, pectin, agar, semisynthetic materials such as methyl cellulose, hydroxy ethyl cellulose, HPMC and CMC, and synthetic polymers carbopol.

 

Among different gelling agents, carbopol is used widely. The carbopols (carbomers) are a group of carboxy vinyl polymers are hydrophilic colloidal materials which thicken better than the natural gums. They disperse in water to form cloudy solutions which are neutralized by strong bases such as sodium hydroxide, triethanolamine or by weak inorganic bases like ammonium hydroxide which increase consistency and decrease turbidity. Different grades of carbopol thicken over slightly different pH ranges; carbopol 934, pH 5.5-11.0, carbopol 940, pH 4.5-11.0., carbopol 941, pH 3.5-11.0, etc. Carbomer gel possesses good thermal stability at different gel viscosity. As a topical product carbomer gel possesses optimum rheological properties. Carbopol gel provides good freeze thaw stability and they can withstand autoclaving without serious loss of consistency. Dispersions of carbomers should contain a suitable antimicrobial preservative [7,8].

 

MATERIALS AND METHODS:

Plant Material and preparation of the Extracts

The root and stem of Rubia cordifolia were collected from the Pydhonie, Mumbai Market and authenticated at the Agharkar Institute (No.: Auth. 07-40) Poona. The dried powdered of Rubia cordifolia was extracted with methanol using soxhlet apparatus at temperature 500. The concentrated extract was evaporated to dryness under reduced pressure at450.

 

The culture of Propionibacterium acnes (MTCC 1951) was obtained from IMTECH (Institute of Microbial Technology) Chandigarh. The microorganism was grown using Brain Heart Infusion Broth which was procured from (Hi media Laboratories limited, Mumbai). All solvents used were of LR  grade.

 

Formulation of Topical Gels

The Rubia cordifolia has demonstrated a good antibacterial activity; hence development of a topical formulation was tried. Among different bases reviewed like carbopol gel base, vanishing cream base, ointment base, the carbopol gel base was found to be suitable for developing an antiacne gel formulation because it is non oily, non greasy base, greasy bases cannot be used as in acne skin oil secretion is large.       

 

For topical gel, the grade of carbopol used was 940. Carbopol 940 is widely used in topical carbomer gel formulations. Base like triethanolamine was used as a neutralizing agent for carbopol, the quantity was estimated for pH of the gel.  The following formula used for preparing for the topical gel.

 

Preparation of the Gel

Carbopol 940 was soaked in sufficient quantity of water for a 12 hrs and then stirred to disperse uniformly with help of a magnetic stirrer and heated if necessary. To this carbopol solution, glycerine was mixed and then parabens were added and stirred till dissolved. Extract was dissolved in sufficient water solution. Propylene glycol was dissolved in this extract solution. To carbopol solution this extract solution was mixed homogenously. Triethanolamine (TEA) base was added to carbopol solution. The formed gel was mixed homogenously. Air entrapment should be avoided during stirring.  The composition of herbal gel prepared from methanolic extract of Rubia cordifolia tabulated in Table 1.

 

Table 1: Composition of gel formulation

Sr. No.

Ingredients      

Quantity (% wt/wt)

1

Carbopol 940

2

2

R. cordifolia extract

**

3

Propylene glycol      

10

4

Glycerin

5

5

Methyl paraben        

0.18

6

Propyl paraben        

0.02

7

Water

q.s

** Various concentrations of the R. cordifolia extract (1%, 2% and 4%) were subsequently incorporated into the separate base.

 

Evaluation of pH of Topical Gel

pH

The pH of gel formulations were determined by using digital pH meter. 2.5gm of gel was accurately weighed and dispersed in 25ml of distilled water and stored for two hours. The measurement of pH of formulation was carried out in triplicate (Table 2).pH of all gel formulations was found to be in the range of 6.5-7.0.

Viscosity

Viscosities of gels were determined using Brookfield viscometer. Gels were tested for their rheological characteristics at 250C using Brookfield viscometer. The measurement was made over the whole range of speed settings from 10 rpm- 100 rpm with 30 seconds between 2 successive speeds and then in a descending orders (Table 2).

 

Spreadability

Spreadability is a term expressed to denote the extent of area to which the gel readily spreads on application to skin or affected part.

 

Spreadability (S) is calculated by using the

formula:

S=ml/t

Where, m = weight tide to upper slide

l = length moved on the glass slide

t = time taken to separate the slides

completely from each other

 

Extrudability

Extrudability is the force required to exude material out of tube; determining the consistency of preparation. The extrudability was calculated using the following formula

 

Extrudability = Applied weight to extrude gel

from tube (gm) / Area (cm2)

 

 Diffusion Study of Topical Gels

In vitro permeation studies through dialysis membrane: [9]

The in vitro diffusion study of the gels was performed using dialysis membrane (Sigma Inc, MO, USA; Cat. No.: 250-7U; dry unwashed, pre-cut and open ended; flat width: 35 mm; inflated diameter: 21 mm; Length: 30 mm). The membrane soaked in phosphate buffer, pH 7.4 (PB) for 6-8 h and then was clamped carefully to one end of the hollow glass tube of dialysis cell (2.3 cm diameter; 4.16 cm2 area). 100 ml of phosphate buffer was taken in a beaker, which was used as receptor compartment. Then 50 mg gel was spread uniformly on the membrane. The donor compartment was kept in a contact with the receptor compartment and the temperature was maintained at 37±0.5.  The solutions on the receptor side were stirred by externally driven Teflon-coated magnetic bars. At predetermined time intervals, pipetted out 5 ml phosphate buffer was pipette out. The drug concentration of the receptor fluid was determined spectrophotometrically (Shimadzu, Tokyo, Japan) against appropriate blank. The experiment was carried out in triplicate for each gel containing 1%, 2% and 4% R. cordifolia methanolic extract.

 

 

 

Antibacterial Activity

The gels were evaluated for their antibacterial activity against Propionibacterium acne using cup plate method.

 

METHODOLOGY

Standardization of culture

Culture of the test organism P. acnes was standardized to obtain a suspension of 1x106 to 5x106 cells/ml using McFarland turbidity standard method [10].

 

b) Preparation of test materials

The various gels containing different concentrations of extract (1% w/w, 2% w/w and 4%w/w) were used for testing. Clindamycin gel 1% w/w (Wallace Pharmaceuticals Pvt. Ltd) was used as positive control.

 

c) Procedure for evaluation of activity using cup plate method

The antibacterial activity of gel was evaluated using cup plate method as mention earlier. The wells were filled with gels containing different concentrations of extract, incubated and zone of inhibition was calculated [11].

 

RESULTS AND DISCUSSION:

The prepared gel formulations were evaluated for various pharmaceutical parameters and results were mentioned in Table 2. From the results it is clearly evident that all the gel formulations showed good gelling property and homogeneity. The pH of all the formulations was in the range compatible with normal pH range of the skin. The drug content released was also found to be moderate releases drug with in optimum range of time period (Figure 2). The rheological behaviors of the gel formulations were studied with Brookfield viscometer. The results indicated the viscosity of gel formulations was consistent. A comparative study of viscosity and Spreadability showed that with increase in viscosity of the formulation, the Spreadability decreased and vice versa.

 

The average reading of three determinations for the zone of inhibition values of methanolic extract of Rubia cordifolia in various formulations are as given in table 4The results of antibacterial activity of topical gels were shown in fig. 2 to fig. 5.

 

Table 2: Results of evaluation parameters of various gel formulations

Sr No.

Formulation

Parameters

Ph

Viscosity

Spredability

Excrudability

1

1% Gel

6.5

1652

28.60

76.8

2

2% Gel

6.6

1544

28.80

79.5

3

4% Gel

6.7

1530

30.92

81.5

 

 

 

Fig: 1. In vitro Diffusion profile of R. cordifolia Gels

 

Table:3. Drug release of Rubia cordifolia topical gel formulation

Sr. No.

Various conc. Gels

Time in

min.

% Cumulative drug release

1.

1% Gel

20

14.26

40

33.34

60

54.02

80

75.66

100

98.42

2.

2% Gel

20

16.82

40

34.74

60

55.49

80

77.28

100

99.70

3.

4% Gel

20

16.99

40

35.06

60

55.86

80

77.58

100

99.98

1% Gel containing 1% R. cordifolia extract

2% Gel containing 2% R. cordifolia extract

4% Gel containing 4% R. cordifolia extract

 

Table: 4.  Antibacterial activity of topical gel formulation

Sr. No.

Concentration of R. cordifolia extract in the formulation

Diameter of zone of inhibition* (mm)

1.

Gel containing  1% of R. Cordifolia extract

14

  2.

Gel containing  2% of R. Cordifolia extract

18

3.

Gel containing  4% of R. Cordifolia extract

23

4.

Clindamycin Gel (1%wt/wt)

29

*Average of three readings

Fig :2. Gel containing 1% wt/wt of R. cordifolia extract

 

Fig :3. Gel containing 2% wt/wt of R. cordifolia extract

 

 

Fig: 4. Gel containing 4 % wt/wt of R. cordifolia extract

 

 

Fig: 5. Positive control (Clindamycin Gel 1%wt/wt)

 

 

CONCLUSIONS:

The extract of R. cordifolia exhibited good antiacne activity, in earlier experiments, it was proved by evaluating the antibacterial activity and antioxidant activity that are potential for antiacne therapy. This may be attributed to presence of anthraquinone in R. cordifolia. For easy application of this extract, it was formulated in form of a topical gel by using carbopol 940, the formulation was then evaluated for pH which was found in range between 6.5-6.7, which is suited for topical application without any discomfort.

 

Drug release was evaluated by using in vitro diffusion study results show that 1% gel, 2% gel and 4% gel release more than 15% of the drug within the first 20 minutes and further releases 98% of the drug after 100 minutes.

 

These gels were also evaluated for antibacterial activity by comparing with positive control clindamycin and it showed significant results compared with it.

 

Thus, it may be concluded that R.cordifolia with good antibacterial and antioxidant activity can be used in antiacne activity. Since the activity is comparable to that of clindamycin, the constituents of R.cordifolia may be exhibiting antibacterial activity by inhibiting protein synthesis like clindamycin.

 

ACKNOWLEDGEMENTS:

The authors thank Agharkar Research Institute, Pune, for identifying and authenticating Rubia cordifolia (Linn.) family Rubiaceae and IMTECH (Institute of Microbial Technology) Chandigarh, for sending the culture of Propionibacterium acnes (MTCC 1951) for research work

 

REFERENCES:

1.      Gopal M.G, Farahana B, Dr. Kala Suhas Kulkarni, Effectiveness of Herbal Medication in treatment of Acne vulgaris- A pilot Study, The Indian Practioner; 2001, 54, 10, 723.

2.      Jappe Utta, Pathological Mechanism of Acne with special emphasis on P. acnes and related Therapy, Acta Derm Venerol, 2003,83,241-8

3.      Leyden, J. J., Journal of American Acad. Dermatol., 1995, 32, S15.

4.      Lever, L and Marks, R., Drugs. 1990, 29, 681.

5.      Jain A, Basal E, “Inhibition of Propionibacterium acnes-induced mediators of inflammation by Indian herbs” Phytomedicine, 2003, 10, 34-38.

6.      Indian Council of Medicinal Research “Quality Standards of Indian medicinal plants” New Delhi 2005, 3 307-315.

7.      Brian W. Barry, In “Dermatological formulations Percutaneous Absorption”, Drugs, and Pharmaceutical Sciences, Marcell Dekker Inc., New York, 1983, 18.

8.      David B. Osborne, Anton H. Amann, In “Topical Drug Delivery Formulations”, Drugs and Pharmaceutical Sciences, 42, Marcell Dekker Inc., New York, 1990.

9.      Reddy M. S; Mutalik S; Rao G. V; “Preparation and evaluation of minoxidil gels for topical application in alopecia” Indian Journal of Pharmaceutical Sciences, 68(4),432-436, 2006.

10.   Baueg A.W. et al, American Journal of Clinical Pathology, 45(4), 493-496, 1996.

11.   Barry A.L., In: ‘The antimicrobial susceptibility test principles and Practices’ Lea and Febiger, Philadelphia, 1976, 4164.

 

 

 

 

 

Received on 18.06.2018                    Accepted on 29.06.2018

©A&V Publications all right reserved

Research J. Topical and Cosmetic Sci. 9(2): July- Dec. 2018 page 39-43.

DOI: 10.5958/2321-5844.2018.00008.0