INTRODUCTION:

Skin diseases are due to poor hygiene, overcrowding, malnourishment, non-availability of potable water, high temperature and humidity. Further, drugs used to treat them are antibiotics, steroids and sulfonamides, which are not only out of reach of local population in remote areas but also associated with adverse effects like atrophy, telangiectasia, hirsutism and sensitizing dermatitis which are far more troublesome. Indigenous medicinal plants have been a readily available source of drugs since ancient times and even today almost 50% new drugs have been patterned after phytochemicals. Majority of the population in developing countries and approximately 25% people in developed regions use herbal medicine for prevention and treatment of diseases. Recognizing the medicinal significance of indigenous plants, World Health Organization (WHO) in its 1997-guideline states that ‘‘effective locally available plants be used as substitutes for drugs. Research work on medicinal plants and exchange of informations obtained will go a long way in scientific exploration of medicinal plants for the benefit of man and is likely to decrease dependence on imported drugs’’^[1].

Staphylococcus aureus is the main pathogens that cause skin infections. Development of microbial resistance to antibacterial is a global concern.

Plants are important sources of potentially useful constituents for the development of new therapeutic agents because most of them are safe with little side effects ^[2].

The Kalanchoe pinnata is a divine herb, which contains a wide range of active compounds, including alkaloids, triterpenes, glycosides, flavonoids, steroids, bufadienolides, lipids and organic acids. Due to these constituents it has many pharmacological activity. The antimicrobial activity may be due to presence of triterpenes, bufadienolides and phenolic compound^[3].Several reports are available on the antimicrobial activity of plant extracts on human pathogenic bacteria ^[4,5]. The presence of phenolic compounds indicate that the plant possess anti-microbial activity. Plant is effective in the treatment of typhoid fever and other bacterial infections, particularly those caused by S. aureus, E. coli, B. subtilis, P. aeruginosa, K. aerogenes, K. pneumoniae and S. typhi ^[3].The antibacterial potential of 60% methanolic extract of leaves of Kalanchoe pinnata was evaluated and MIC and MBC against Stapyloccocus aureus was found out as 30mg ^[6].

Creams are semisolid dosage forms intended mainly for external use and commonly consist of two immiscible phases, an oily internal phase and an aqueous external phase. Due to emulsified nature of skin surface, drugs formulated as cream more effectively interact with skin and more readily penetrate through biological membranes^[2]. Herbal products are popular due to their minimum risk of side-effects with maximum efficacy.

MATERIAL AND METHODS:

Preparation of plant extract:

60 % Methanolic extract - Fresh leaves of Kalanchoe pinnata were coarsely grounded and was macerated with 60% methanol for not less than seven days in a close vessel and thereafter concentrated by evaporation at room temperature ^{[7, 8]}.

Total phenolic assay:

The total phenolic content of extract is determined by using folin ciocalteu assay. An aliquot (1ml) of extract or standard solution of gallic acid was added to 25ml volumetric flask containing 9ml of distilled water. A reagent blank using distilled water was prepared, 1ml of folin ciocalteu phenol reagent was added to the mixture and shaken. After 5 min.10ml of 7% Na₂Co₃ solution was added to the mixture. The solution was diluted to volume 25 ml with distilled water and mixed. After incubation of 90 min at RT, the absorbance against the prepared reagent bank was determined at 750nm with an UV-Vis spectrophotometer. Total phenolic content of extract was expressed as mg gallic acid equivalents (GAE)/100gm weight ^[9].

Antibacterial activity:

The antibacterial activity of extract was studied against Stapyloccocus aureus ^[6].

Formulation and physico-chemical evaluation:

The compositions and amounts of the formulation ingredients are as shown in table 1, 2.

Table 1: Formulation no. – 1^[2]

Sr.No	Ingredients	Qty in gm
1	Stearic acid	1
2	Spermaceti	0.5
3	Cetyl alcohol	0.5
4	Glycerin	0.5
5	Triethanolamine	0.2
6	Water q.s.	10

Table 2: Formulation no. – 2^[10]

Sr.No	Ingredients	Qty in gm
1	Stearic acid	2.4
2	KOH	0.135
3	Glycerin	1.05
4	Water q.s.	10

Base cream contains water and oil phases. Both phases were mixed at same temperature 65^oC. In order to prepare the cream, different amount of ingredients were incorporated together, and then 0.3 gms of herbal extract was added.

Antimicrobial Activity of Cream Formulations:

The antibacterial activity of different formulations was performed by standard agar well diffusion method against Staphylococcus aureus. Nutrient broth/agar was used to cultivate bacteria. In order to recover the lyophilized culture, the desire amount of culture was aseptically transferred in nutrient broth and maintained in an incubator at 37^o C for 3 hrs to form inoculums. The media was poured in Petri plates aseptically and kept for 30 minutes for solidification. After 30 minutes, the fresh inoculums of different culture were spread on to solidified nutrient agar plates. Wells were made using sterile small test tubes of 5mm diameter on petri plates at proper positions. The cream were added in to agar wells, aseptically. Then the agar plates were incubated at 37⁰C for 24hrs. After 24 hrs of incubation, the zone of inhibition was investigated ^[6,
11,12].

Evaluation of Cream:

General appearance: The general appearance of cream was observed visually and by applying it on human skin.

pH :The pH of a cream was determined by using digital pH meter . Accurately weighed 5 g of the cream was dispersed in 45 ml of water to determined the pH of the suspension at 27°C using digital pH meter ^[13,16,18].

Spreadability:

Spreadability is a term expressed to denote the extent of area to which the topical application spreads on application to skin on the affected parts. The therapeutic efficiency of the formulation also depends upon its spreading value. Hence, determination of spreadability is very important in evaluating topical application characteristics. For the determination of spreadability, excess of sample (3g) was applied in between two glass slides and was compressed to uniform thickness by placing 1000 g weight for 5 minute. Thereafter weight (50g) was added to the pan and the top plate was subjected to pull with the help of string attached to the hook. The time in which the upper glass slide moves the lower plate to cover a distance of 10 cm is noted. A shorter interval indicates better spreadability. The spreadability (S) was calculated using the formula

S = m.l/t,

Where, S – spreadability, m- weight tied to upper glass slide. l-length moved on glass slide t- time ^[14].

Tube extrudability:

The method for evaluating cream formulation for extrudability was based upon the quantity in percentage of cream extruded from tube on application of finger pressure. More quantity extruded better was extrudability. The formulation under study was fill in a clean, aluminum collapsible tube with a nasal tip of 5mm opening and applied the pressure on the tube with the help of finger. Tube extrudability was then determined by measuring the amount of cream extruded through the tip when a pressure was applied on the tube ^{[13, 16, 17]}.

Viscosity:

The viscosity of cream was measured by Brookfield viscometer at 25⁰ C ^[13].

Drug release:

The in vitro diffusion study of the cream was performed by using cellophane membrane. The membrane soaked in phosphate buffer pH 7.4 for 2-3 hrs was clamped carefully to one end of the hollow glass tube of dialysis cell (2cm in diameter and 3.14 cm² area). 14 ml of phosphate buffer was taken in a beaker, which was used as receptor compartment for the study. 1 gm of cream was spread uniformly on the membrane.

Fig. 1: Calibration curve

The donor compartment was kept in contact with the receptor compartment and the temperature was maintained at 37⁰ ± 0.5⁰ C. The solution in the receptor compartment was continuously stirred with magnetic stirrer. At pre-determined time intervals, 0.5 ml of solution from the receptor compartment was pippeted out and immediately replaced with 0.5 ml fresh phosphate buffer solution. Pippeted 0.5ml added to 25ml volumetric flask containing 9ml of distilled water.A reagent blank using distilled water was prepared,1ml of folin ciocalteu phenol reagent was added to the mixture and shaken. After 5 min, 10ml of 7% Na₂Co₃ solution was added to the mixture. The solution was diluted to volume 25 ml with distilled water and mixed. After incubation of 90 min at RT, the absorbance against the prepared reagent bank was determined at 750nm.The drug concentration of the receptor fluid was determined spectrophotometrically at 750 nm ^{[9, 15]}.

Skin irritation test: Test was performed on healthy human volunteer. For each formulation five volunteers were selected and 1 gm of weighed formulation was applied on area of 2 sq. inch to the back of the hand and covered with cotton. Volunteers were asked to report after 24hrs to observe for any reaction or irritation ^[17].

Accelerated stability testing: Since period of of stability testing can be as long as two years, it is time consuming and expensive. Therefore it is essential to devise a method that will help rapid prediction or long-term stability of drug. The accelerated stability testing is defined as the validated method by which the product stability may be predicted by storage of the product under condition that accelerate the change in defined and predictable manner. The stability studies of formulated cream were carried out at 40/75(0C/RH) ^{[18, 19]}.

RESULT AND DISCUSSION:

Antibacterial activity: The antibacterial activity of extract was studied against Stapyloccocus aureus. The antibacterial potential of 60% methanolic extract of leaves of Kalanchoe pinnata was evaluated and MIC and MBC against Stapyloccocus aureus was found out as 30mg ^[6].

Total phenolic assay:

Total phenolic content of 60% methanolic extract was found to be 1.49% in terms of gallic acid equivalents.

Table 3: Calibration Curve

Sr. No.	Concentration(ppm)	Absorbance
1	0	0.00
2	2	0.07
3	4	0.15
4	6	0.22
5	8	0.29
6	10	0.35
7	Test extract	0.53

Antimicrobial Activity of Cream Formulations: Antimicrobial activity of various cream (Diameter of zone of inhibition in mm) was as shown in Table 4.

Table 4: Antimicrobial activity of various cream.

Sr.No	Pathogen	Formulation-1	Formulation-2	Marketed formulation
1.	Staphylococcus aureus	22	16	21

From above, formulation-1 was found to be best formulation as compare to formulation -2 and marketed formulation. It shows best antibacterial activity so this formulation-1 is taken for further evaluation test for cream.

General Appearance:

The cream was creamish white in colour, having glossy and smooth feel on application. The consistency was found to be excellent.

pH:

The pH of cream was found to be 7.0, which is neutral. The cream was non-irritant upon application on to the skin. Thus, the cream formulation fulfills the acceptance criteria for pH required for skin formulations.

Spreadability:

The average spreadability of cream was found to be 16.66 g.cm/ sec. Thus, spreadability was good.

Tube Extrudability:

The average percent extrudability was found to be 80.67%. So, tube extrudability was good.

Viscosity:

It was found that the viscosity at 50 rpm was 23130 cps. It is near to the viscosity of standard creams.

Skin irritation test:

No skin irritation was observed.

Drug release:

The release profile of extract at different time intervals was as shown in Table 5.

Table 5: Percentage release of extract from cream.

Sr. No.	Time (min)	% Release
1	0	0.000
2	5	53.897
3	15	55.822
4	30	57.880
5	60	59.944
6	90	62.816
7	120	65.185
8	150	67.569
9	180	69.164
10	210	69.941
11	240	74.425

The % release of drug from cream after 240 min was found to be 74.425% in terms of gallic acid equivalents.

CONCLUSION:

Total phenolic content of 60% methanolic extract was found to be 1.49% in terms of gallic acid equivalents. The % release of drug from cream after 240 min was found to be 74.425% in terms of gallic acid equivalents. The prepared cream shows better antibacterial activity than marketed cream. The prepared cream was herbal , easily washable thereby increases the patient compliance. This cream could be used topically in order to protect skin against damage caused by Staphylococcus aureus pathogens. Staphylococcus aureus is a bacterium that is a member of the Firmicutes, and is frequently found in the human respiratory tract and on the skin. Although S. aureus is not always pathogenic, it is a common cause of skin infections (e.g. boils), respiratory disease (e.g. sinusitis), and food poisoning. Disease-associated strains often promote infections by producing potent protein toxins, and expressing cell-surface proteins that bind and inactivate antibodies. Staphylococcus aureus causes cellulitis, folliculitis, or impetigo. If these bacteria can reach the bloodstream and end up in many different body sites, causes wound infections, abscesses, osteomyelitis, endocarditis, pneumonia that may severely harm or kill the infected person. So the prepared formulation can act against such diseases, thus can save the life of person.

AKNOWLEDGEMENT:

The authors are grateful to Management of Sanjivani Rural Education Society’s, Sanjivani Institute of Pharmacy and Research, Kopargaon for their valuable support for this project.

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Received on 04.03.2014 Accepted on 22.04.2014

Research J. Topical and Cosmetic Sci. 5(1):Jan.–June 2014 page 1-4