INTRODUCTION:

Acne vulgaris is the most common disorder of human skin that affects upto 80% of individuals in their lives. The area’s most vulnerable to acne are the largest oil secreting glands present in the face, back and trunk. It is a chronic inflammatory disease of multifactorial etiology affecting more than 85% of teenagers and frequently continues into adulthood.

The influencing factors of acne include excess sebum secretion, hyperkeratinisation of the hair follicle, oxidative stress and the release of inflammatory mediators ¹. Benzoyl peroxide is a potent oxidizing agent with antibacterial and keratolytic properties. The main adverse effects are bleaching of clothes, transient skin irritation, and occasional allergic contact dermatitis. Topical retinoid preparations are useful for mild to moderate acne and can be applied once or twice daily. Side effects include erythema, desquamation ². Topical antibiotics are particularly useful in mild to moderate acne and in acne which is resistant to benzoyl peroxide. The development of antibiotic resistance in P. acne may limit the prescription of topical antibiotics. Follicular keratinisation, seborrhoea, and colonisation of the pilosebaceous unit with Propionibacterium acne are central to the development of lesions. Genetic and hormonal factors also play a role, possibly by optimising the follicular environment suitable for the growth of P. acne or by influencing the inflammatory response and thus the nature of the lesions. Such understanding has led to the use of antibiotics, sebum reducing agents and anti-androgens and estrogens in conventional treatments^3-6.

Herbal plants used in folk medicine have been accepted as one of the main sources of drug discovery and their development. The pomegranate is an ancient, mystical, unique fruit used in several systems of medicine for a variety of ailments⁷. The synergistic action of the pomegranate constituents appears to be superior to that of single constituents. The potential therapeutic properties of pomegranate are wide-ranging and include treatment and prevention of cancer, cardiovascular disease, diabetes, dental conditions, erectile dysfunction, and protection from ultraviolet (UV) radiation. This research indicates the most therapeutically beneficial pomegranate constituents are ellagic acid, ellagitannins (including punicalagins), punicic acid, flavonoids, anthocyanidins, anthocyanins, and estrogenic flavonols and flavones. It has a variety of uses such as blood purifier, in various skin diseases, immunomodulant, anti-inflammatory and anti-platelet-activating factor (PAF)⁸.

Berberis aristata DC.(Berberidaceae), known locally as daruharidra in vernacular languages is an erect glabrous spinescent shrub, 3-6 m in height with obovate to elliptic, subacute to obtuse, entire or toothed leave commonly used to treat skin diseases, menorrhagia, diarrhea, cholera, jaundice, eye and ear infections, as well as urinary tract infections. The decoction of the root is used as a wash for infected wounds and ulcers, and is said to help healing and promote cicatrisation. B. aristata extracts have also been reported to cure hepatotoxicity. Despite the medicinal importance of this plant species, especially root extracts, reports on its antibacterial and antifungal activities⁹. The traditional claim on these plants for the root and fruit rind reveals the anti-acne effect which can reduces the sebum production. That is why the current research work is focused to carry out antiacne activity of the extracts as well as formulation.

The plant contains tannins, alkaloids, triterpenes and saponins and anti-microbial activities of tannins and alkaloids are well documented.The growth of many fungi, yeasts, bacteria and viruses wereinhibited by these phytomarkers.

MATERIALS AND METHODS:

Plant Collection and Extraction

The fresh fruit rind of the plant Punica and roots of Berberis were collected from the local region of Pune. It was authenticated by Department of Botany, University of Pune (Voucher No. Bot /35/11). The powder prepared was extracted by maceration with hydroalcoholic mixture (60:40) for 72 hours.

Histology

Histology was performed as to know the various structural bodies which are present in the plant such as crystals, grains and also which is a part of the authentication. The plant specimen of fruit were cut and fixed in FAA and then infiltration of specimen is carried out by paraffin wax. The specimens were sectioned with the help of microtome with thickness of 10-12 µm. The sections were stained with toluidine blue ⁹. Photographs of different magnifications were taken with Nikon lab photo 2 microscopic units ¹⁰.

Preliminary Phytochemical Screening

The extracts were then subjected to preliminary phytochemical screening to detect the presence of various phytoconstituents by various chemical tests ¹¹.

Antiacne activity of the Extracts:

The lyophilized cultures of bacteria Propionibacterium acne (MTCC No. 1951) were procured from Indian Institute of Microbial Technology (IMTECH), Chandigarh. The dilutions of extract were prepared and brain heart infusion broth was prepared. Tween 80 and 0.03ml thioglycollic acid per 100 ml was added in the prepared broth as a reducing agent¹². The 25 ml of the medium was poured in the ten test tubes followed by sterilization with autoclave at15 lb pressure and 121^oC for 30 minutes. Using sterile pipette exact amount of extract was added as indicated in the Table 1 and the final volumes were adjusted to 10ml with medium followed by inoculation of cultures and incubation at 37^oC for 48 hrs. The growth in the tubes was monitored by turbidity method and MIC of the extract was determined ^13,
14. The extracts also subjected to antiacne activity by Cup plate diffusion method using Clindamycin as internal standard (positive control) and zone of inhibition with MIC was determined ¹⁵. Both this analysis was performed thrice to confirm the efficacy of the result.

Fingerprinting and Spectroscopic analysis:

The extracts and standards then subjected to fingerprinting analysis. The calibration curves were plotted.

Free radical scavenging activity:

The activity was assayed spectrophotometrically by DPPH method. The reduction in absorbance of DPPH solution (2 mg/ml) was monitored at 516 nm after addition of various concentrations of test extracts to DPPH reagent and maintaining these solutions were maintained at room temperature for 5 m before taking the reading. The EC50 values for test material was calculated from the calibration curves of concentration of extract (μg/ml) verses % reduction in absorbance after subjecting to linear regression between 10–80 %. This activity was expressed as effective concentration at 50% (EC50) that is the concentration of the test solution required to give a 50% reduction in absorbance of the test solution as compared to that of blank solution. Ascorbic acid was used as a positive control.

Lipid peroxidation inhibitory activity:

The activity was studied by the method. The reaction mixture contained mice liver homogenate (0.2 ml, 10% w/v) in 0.15 M KCl,(0.1 ml, 150 μM), Tris buffer (0.4 ml, pH 7.5) and various concentrations of test extracts. In vitro lipid peroxidation was initiated by addition of FeSO₄.7H₂O (0.1 ml, 10 μM) and ascorbic acid (0.1 ml, 100 μM). The reaction mixture was incubated at 370 for 1 h. After the incubation period, reaction was terminated by addition of thiobarbituric acid (TBA- 2 ml, 0.8%) and by heating the contents for 15 min. for development of coloured complex. The lipid peroxides formed were measured as thiobarbituric acid reacting substances (TBARs) by method. The tubes were then centrifuged at 4000 rpm for 10 m. and cooled. The % inhibition of lipid peroxidation was determined by comparing the results of test compound with those of controls not treated with extracts by monitoring the colour intensity at 532 nm Curcumin was used as a positive control. The results were expressed as IC50 value that is the concentration of extract required for 50 % inhibition of production of lipid peroxides. ¹⁶

Formulation and evaluation of Microemulsion:

Required quantity of Surfactant and co-surfactant was mixed in oil phase under constant stirring and the aqueous phase containing extracts were poured in to the oil phase and homogenized using mechanical stirrer. The microemulsion was formed instantaneously by diffusion of the two phases leading to the formation of droplets as shown in the following table 1.¹⁷ Evaluation was done by Appearance, pH measurements, Spreadability, Viscosity, Extrudability and In-vitro drug diffusion.

Table 1: Formulation of Microemulsion gel of Punica and Berberis extract

Sr. No	Extract (in %)		Oil Phase (in %)			Distilled Water (in %)
Sr. No	A	B	Captex	Tween 80	PEG 400	Distilled Water (in %)
F1	1	4	30	20	5	40
F2	2	5	30	20	5	38
F3	3	6	30	20	5	36

F1, F2, F3: Formulation, Extract A: Punica, Extract B: Berberis

RESULT:

Pharmacognostic Study:

The quality control parameters were established and proximate analysis found to be significant. Preliminary phytochemical screening revealed the presence of tannins and alkaloids.

Histology:

The required samples of different organs were cut and removed from the plant and fixed in FAA. After 24 hrs of fixing, the specimens were dehydrated with graded series of tertiary -Butyl alcohol. The fruit or the pericarp is thick and fleshy and consist of less prominent epidermis or epicarp. Sclerides are distributed throughout in Mesocarp (Figure 1 and 2).

Figure 1: Histology showing epicarp and mesocarp region of Punica Fruit rind

Figure 2: Vascular strands in the mesocarp region of Punica Fruit rind

Figure 3: Circular section of Berberis root

Figure 4: Parenchyma region with abundant Starch grains (Berberis root)

Figure 5: Medullary rays with xylem vessels of Berberis (when treated with Thymol blue)

Figure 6: Entire transverse section of Berberis root with stratified cork

Screening of Extract for Antiacne activity

To screen the plant material for their antiacne activity in vitro experiments were carried out by using the organism P. acnes ¹⁸. The culture media was standardized using McFarland turbidity standard. The broth dilution method was used to detect the MIC of the extracts. (Table 2)

Table 2: Protocol for evaluation of MIC by broth dilution method for both the extracts

Sr. No.	Amount of Extract/ml	Amount of medium	Total Vol of Solution (ml)	Conc. of Extract in final sol (ml)
1	0.1	9.9	10	0.1
2	0.2	9.8	10	0.2
3	0.3	9.7	10	0.3
4	0.4	9.6	10	0.4
5	0.5	9.5	10	0.5
6	0.6	9.4	10	0.6
7	0.7	9.3	10	0.7
8	0.8	9.2	10	0.8
9	0.9	9.1	10	0.9
10	1.0	9.0	10	1.0

The results as shown in Table 3 depict that the MIC values of hydroalcoholic extracts of Punica was found to be 100 mg/ml and for Berberis was found to be 300 mg/ml respectively ¹⁹. The zone of inhibition was determined by cup plate diffusion method where an increase in antiacne activity was observed from zone of lysis emphasizes that the lysis may be due to the active components present in the hydroalcoholic extract of the plant. All this analysis has been carried out thrice to confirm the efficacy of the extract.

In-vitro drug diffusion

Retention content after 06 Hrs 11.86% n=3

F₁	Sr. No.	Time	% drug release
	1	15 min	2.29
	2	30 min	4.28
	3	45 min	6.77
	4	01 hour	7.11
	5	02 hour	7.48
	6	03 hour	8.99
	7	04 hour	10.16
	8	05 hour	10.78
	9	06 hour	12.80

Retention content after 06 Hrs 6.48% n=3

F₂	Sr. No.	Time	% drug release
	1	15 min	1.44
	2	30 min	3.36
	3	45 min	4.49
	4	01 hour	6.77
	5	02 hour	6.99
	6	03 hour	7.89
	7	04 hour	8.37
	8	05 hour	9.53
	9	06 hour	11.78

Retention content after 06 Hrs 4.78% n=3

F₃	Sr. No.	Time	% drug release
	1	15 min	2.34
	2	30 min	3.96
	3	45 min	4.47
	4	01 hour	5.77
	5	02 hour	6.10
	6	03 hour	6.56
	7	04 hour	8.97
	8	05 hour	9.83
	9	06 hour	11.93

DISCUSSION:

The results of the zone of inhibition for Punica granatum linn and Berberis aristata DC. hydroalcoholic extract shows good anti-acne activity when compared to standard drug clindamycin. Thus the targets in the microbial cell could be surface exposed adhesion, cell wall peptides and membrane bound enzymes. Here tannins in case of Punica and alkaloids in case of Berberis are the major phytoconstituents present in this plant which are responsible for anti-acne action due to cell lysis with leakage of cytoplasmic constituents. The probable mode of action may be the formation of irreversible complexes with nucleophilic amino acids, often leading to a loss of function of vital proteins in the microbial organism ²⁰. Both this extracts were shown significant free radical scavenging and lipid peroxidation activity. As infections being a major cause of morbidity and mortality in burn patients, the herbal extract may prevent infection that leads to high risk of sepsis. Thus the experimental findings may suggest the plant was found to be effective as to inhibit the effect caused by the P. acnes.

The investigations have revealed as a promising anti-acne agent because it inhibits the proliferation of Propionibacterium acnes and hence prevents its consequences. The result of anti-acne activity thus can be expressed in relation with antioxidant activity which controls the oxidative stress in acne inflammation.

CONCLUSION:

This study thus demonstrates the antiacne activity of hydroalcoholic extract which is effective in the treatment acne vulgaris.

ACKNOWLEDGMENT:

Authors are thankful to Indian Institute of Microbial Technology (IMTECH) Chandigarh, for providing cultures of microorganism and also to Anchrom laboratory (Mumbai) for carrying out fingerprinting analysis.

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Received on 10.09.2015 Modified on 25.09.2015

Research J. Pharm. and Tech. 8(11): Nov., 2015; Page 1548-1554

DOI: 10.5958/0974-360X.2015.00276.0

Formulations	Appearance	pH	Spreadability (g/sec)	Extrudability (g)	Viscosity (cps)
F1	**	6.9± .05	40.8± 0.5	525.1± 0.2	33.5± 0.3
F2	**	7± .08	42.1± 0.8	528.5± 0.1	34.5± 0.4
F3	***	6.8± .07	43.9± 0.4	532.4± 0.5	35.7± 0.9

Sr. No.	Amount of Extract/ml (Punica)	Zone of inhibition in mm for Punica extract (including borer size)	Amount of Extract/ml (Berberis)	Zone of inhibition in mm for Punica extract (including borer size)	Amount of Standard drug	Zone of inhibition For Standard drug
1	0.1	12	0.3	9.5	0.2	11
2	0.2	13	0.4	7.4	0.3	12
3	0.3	14	0.5	11.4	0.4	14.5