Int J Curr Pharm Res, Vol 7, Issue 3, 47-50Original Article


EVALUATION OFANTI-OXIDANT AND ANTI-ACNE ACTIVITIES (IN-VITRO) OF THE FORMULATED HERBAL GELS

KHUSHBU PRAVIN SHAH*, RASHMI SHUKLA SRIVASTAVA

Mumbai Education Trust’s Institute of Pharmacy, MET Complex, Bandra Reclamation, Bandra (W), Mumbai 400050, India
Email: khushbushah590@gmail.com

Received: 05 May 2015, Revised and Accepted: 06 Jun 2015

ABSTRACT

Objective: The objective of the present study was to evaluate the anti-oxidant and anti-acne activities (in-vitro) of the formulated herbal gels.

Methods: Herbal extracts and volatile oils were prepared and procured. Preliminary screenings for the anti-oxidant and anti-acne activities (in-vitro) were carried out to select the suitable candidates for the preparation of anti-acne herbal gels. Gels were further evaluated for the activities.

Results: The herbal gel (F2) containing the herbal extracts (Azadirachtaindica, Ocmium sanctum, Curcuma longa) each (1%) and volatile oils (Melaleuca alternifoliae, Salviaesclareae and Citrus sinensis) each (0.05%) showed maximum anti-oxidant activity (IC50 value 0.407 mg) amongst all four gels. Significant Anti-acne activity against P. acne and S. epidermidis was showed by F2 when compared with the marketed synthetic gel (Clindac gel).

Conclusion: The study proves that the herbal actives used in the formulation have promising anti-oxidant and anti-acne activity.

Keywords: Herbal extracts, Volatile oils, Gelling agents, Anti-oxidant activity and anti-acne activity.

INTRODUCTION

Acne is found be the universal skin disorder. The main target for the treatment of acne is to-

The conventional therapies used for acne have following problems such as dryness of skin, dermatitis, darkening of skin, recurrence after withdrawal and the excessive use of antibiotics for longer period develops resistance against P. acne and S. epidermidis, and results in adverse effects like gram-negative folliculitis [1, 2].

The herbal gel is formulated by using natural gelling agents, herbal extracts and volatile oils. This gel provides multi-therapeutic effect with negligible adverse effect and has similar rheological properties of gel. Hence they are preferred over synthetic ones [3]. The potential candidates showing anti-acne activity are Azadirachtaindica, Ocmium sanctum, Curcuma longa, Melaleuca alternifoliae, Salviaesclareae and Citrus sinensis. Hence these plants were selected along with Aloe vera mucilage powder 200 X (gelling agent) and carbopol 940 (viscosity modifier) for the formulation [4].

Table 1: List of herbs used with their specification

Herbs Part of herb Extraction method Solvent Phyto-constituents
Neem Leaves Solvent extraction Methanol Quercetin
Tulsi Leaves Maceration Methanol Eugenol
Turmeric Rhizome Solvent extraction Acetone Curcumin

Table 2: List of volatile oils used with their specification

Volatile oils Part of herb Phyto-constituents
Tea tree Leaves Terpinen-4-ol
Clary sage Flowering stem Linayl acetate, linalool, α-Terpineol
Orange Fresh peel D-limonine, linalool

Materials and Methods

Neem leaf powder, turmeric rhizome powder, tulsi leaf powder, euxyl 9010, tea tree oil, orange oil, clary sage oil, Aloe vera mucilage 200X powder was procured as gift sample. Ascorbic acid, carbopol 940, DPPH (2, 2-Diphenyl-1 picrylhydrazy) were obtained from Merck Pvt. Ltd, Mumbai. Anti-acne activity studies were conducted at Micro Bio Laboratory. All reagents used were of analytical grade.

Preparation of herbal extracts

The methanolic tulsi extract was prepared by maceration method. The methanolic neem and turmeric acetone extract were prepared by soxhlet extraction method.

Preparation of herbal gel

Step 1: Preparation of gel base

Required quantity of Aloe vera powder (200 X) was weighed and dissolved in water (100 gm) with continuous stirring using mechanical stirrer at 1000rpm (Remi motor RQT-127 HP1/8) at the temperature between 60-80 oC for 1hour in the reactor vessel (SS316). Gel forming polymers was added in various proportions to the solution (table 3). The pH of the solution was adjusted to 6.0-6.4 by addition of triethanolamine. Euxyl P. E. 9010 was used as preservative. All the gel bases were allowed to equilibrate for 12 hours at room temperature.

Table 3: Composition of herbal anti-acne gel

S. No. Formulation codes Ingredients (gm) F1 F2 F3 F4
Step1:Gelbase
Aloevera20 0Xpowder 0.4 0.4 0.5 0.5
Carbopol-94 0 0.5 0.4 0.4 0.5
Triethanolamine 0.1 0.1 0.1 0.1
EuxylP. E.9010 0.6 0.6 0.6 0.6
Distilledwaterupto 10 0 10 0 10 0 10 0
Step2:Formulationofherbalgel
Neemextract 1 1 1 1
Turmericextract 1 1 1 1
Tulsiextract 1 1 1 1
Teatreeoil 0.05 0.05 0.05 0.05
Orangeoil 0.05 0.05 0.05 0.05
Clarysageoil 0.05 0.05 0.05 0.05
Gelbase 10 0 10 0 10 0 10 0

Step 2: Preparation of the formulation

The herbal extracts (1% each) and volatile oils (0.05% each) were added with continuous stirring using mechanical stirrer at 1000rpm (Remi motor RQT-127 HP1/8)to different gel bases till the uniform dispersion of the ingredients was achieved.

All these batches were allowed to equilibrate for 24 hours at room temperature. The prepared gel was filled and stored ina wide mouth polypropylene container.

Biological evaluation

  1. Anti-oxidant activity (in vitroDPPH Method)

0.012 gm of DPPH was weighed and dissolved in 90 ml of methanol and volume was made up to 100 ml.

  1. Standard preparation

10 mg of ascorbic acid was weighed and dissolved in 100 ml of distilled water in an amber colored volumetric flask. The solution was vortexed on a cyclomixer and used as standard.

c) Sample preparation

The stock solution of 1 gm of sample in 10 ml of methanol was prepared. The solution was sonicated for 10 minsand vortexed. The clear methanolic solution obtained was used for assay.

  1. Different concentrations of test samples were prepared in methanol. 2.5 ml of each test sample was mixed with 1 ml of 0.3 mM DPPH solution. These samples were kept in dark for incubationat room temperature for 30 mins.
  2. Absorbance was measured at 516 nm using UV-visible spectrophotometer (Jasco V-630).
  3. Blank was prepared by combining 1 ml of methanol with 2.5 ml of test sample. Control sample was prepared by adding 1 ml of 0.3 mM DPPH solution to 2.5 ml of methanol. Ascorbic acid was used as reference standard. All readings were taken in triplicates.
  4. The results were expressed as % Inhibition of DPPH radical induced by both the tests and standard samples. IC50 values were calculated and compared with that of the standard.
  5. The inhibition of DPPH radicals by the samples was calculated according to the following equation:DPPH-scavenging activity (%): [1‐(A1‐A2)/A0] x 100,Where,A0=absorbance of control.A1=absorbance of the sample.A2= absorbance of blank

One month trial version of Graph Pad Prim 6.5 software was used to plot graphs and for the calculation of IC50 values by point to point curve method [5].

  1. Anti-acne activity (in vitro)-was carried out by Paper disc diffusion method.
  1. Micro-organism and media used
  1. Propionibacterium acne (MTCC1951) andbrain heart infusion media (BHI)
  2. Staphylococcus epidermidis (MTCC9041) andsoybean casein digest media (SCD)
  1. Sterile Disc: The Whatmann paper No.1 (diameter = 5.42 mm)
  2. Samples: Herbal gel formulation and Clindac A gel (Standard)
  3. Procedure:

The petri plates were washed thoroughly and sterilized in hot air oven at 160 °C for 1 ½ hours. 20 ml of sterile molten antibiotic assay medium was seeded by micro-organism; it was poured aseptically in sterile plate and allowed to solidify at room temperature. After the solidification of soft agar, sterile disc impregnated with the gel sample were placed over the solidified agar plate.

The plates were left for 10 min’s at room temperature to allow the diffusion of gel and incubated at 37 °C for 24 hours for S. epidermidis and 48 hrs for P. acne under anaerobic conditions. The plates were then observed for the zone of inhibition (mm). The zone of inhibition was calculated by measuring the diameter of the zone including the well diameter. Readings were taken in triplicates (in three different directions) and the average values were calculated

Measurement of zone of inhibition

The zone of inhibition for each sample was observed, measured and expressed in mm. From this the activity index (A. I.) and Percent Inhibition (P. I.) were calculated for all oils and extracts using the following formula.

A. I = Mean zone of inhibition of each formulation

Zone of inhibition obtained for standard

P. I. = Activity index x 100 [2, 6]

RESULTS

  1. Anti-oxidant activity (in vitro DPPH Method)
  1. Anti-oxidant activity of ascorbic acid
  2. Anti-oxidant activity of herbal anti-acne gel.From (fig. 1) the IC50 value of ascorbic acid was found to be39.563 µg/0.039563 mg.Table 4: Anti-oxidant activity of ascorbic acid
Concentration (mcg/ml) % Inhibition of DPPH radical Standard deviation
0 0 0
10 11.234 0.58845
20 23.833 0.422383
30 36.83 0.29964
40 50.16 0.541516
50 71.55 0.277978
60 81.92 0.305054
70 87.38 1.036101
80 94.69 0.925993
90 95.49 0.981949

Table 5: Anti-oxidant activity of herbal gel formulations

Concentration mg/ml) %Inhibition of F1 gel±SD % Inhibition of F2 gel±SD % Inhibition of F3 gel±SD % Inhibition of F4 gel±SD
0 0 0 0 0
0.5 60.54±1.24 61.47±2.02 61.08±1.24 61.27±1.24
1 75.33±1.19 77.48±1.02 75.87±1.19 75.87±1.19
1.5 80.61±0.87 81.68±0.94 80.61±0.76 80.61±0.76
2 82.28±0.94 82.82±0.94 82.28±0.94 82.28±0.94
2.5 83.52±0.92 84.05±0.92 83.46±0.90 83.25±0.077

Fig. 1: Plot of % inhibition of DPPH v/s concentration of ascorbic acid

From (fig. 2)

The IC50 value of F1 was found to be 0.413 mg/ml

The IC50 value of F2 was found to be 0.407 mg/ml

The IC50 value of F3 was found to be 0.409 mg/ml

The IC50 value of F4 was found to be 0.408 mg/ml

Fig. 2: Plot of % inhibition of DPPH v/s concentration of herbal gel formulations

  1. Anti-acne activity (in vitro)

The anti-acne activity of the herbal gel formulations showed significant and promising activity against P. acne and S. epidermidis in comparison with the marketed synthetic gel (Clindac gel).

Fig. 3: Anti-acne activity against P. acne


Fig. 4: Anti-acne activity against S. epidermidis


Table 6: Anti-acne activity of optimized formulations (in vitro)

S. No. Formulation codes Zone of inhibition (mm)±SD (n=3)
P.acne S.epidermidis
1 F1 19.2±0.05 19.3±0.05
2 F2 19.7±0.05 19.8±0.05
3 F3 19.5±0.05 19.7±0.05
4 F4 19.6±0.05 19.5±0.05
5 Marketed formulation 20±0.05 20±0.05

Table 7: Percentage inhibition of optimized formulations

S. No. Formulation codes P.acne S.epidermidis
A.I. P.I. (%) A.I. P.I. (%)
1 F1 0.96 96 0.965 96.5
2 F2 0.985 98.5 0.99 99
3 F3 0.97 97 0.985 98.5
4 F4 0.98 98 0.97 97
5 Marketed formulation 1 100 1 100

Fig. 5: Anti-acne activity profile of optimized formulations (in vitro).

DISCUSSION

The F2 gel containing Aloe vera mucilage powder (0.4%) and carbomer (0.4%) shows higher release of herbal actives. It showed maximum anti-oxidant (IC50 value 0.407 mg) amongst the four gels. Significant anti-acne activity against P. acne and S. epidermidis was showed by F2 when compared with the marketed synthetic gel (Clindac gel).

CONCLUSION

The studies conclude that all four herbal gels showed anti-oxidant and anti-acne activity. In addition to the promising biological activity it was also observed that the varying concentrations of gelling agent played an important role in the release of the activephyto-constituents.

ACKNOWLEDGEMENT

We are thankful to Micro Bio Laboratory for providing facilities to conduct anti-acne activity. We would like to sincerely thank the Management, Principal and Director of Mumbai Education Trust’s Institute of Pharmacy for providing us the infrastructure for carrying out the research work. Our sincere thanks to Gayatri Herbals Pvt Ltd, Kamal Arochem for providing gift samples.

CONFLICT OF INTERESTS

Declared None

REFERENCES

  1. Ray C, Trivedi P, Sharma V. Acne and its treatment lines. Int J Res Pharm Biosci 2013;3(1):1-16.
  2. Vats A, Sharma P. Formulation and evaluation of topical anti acne formulation of coriander oil. Int J Pharm Pharm Sci Res 2012;2(3):61-6.
  3. Daud SF, Pandey G, Joshi M, Pathak R, Wankhede SA. Study of antibacterial effect of some selected essential oils and medicinal herbs against acne causing bacteria. Int J Pharm Sci Invent 2013;2(1):27-34.
  4. Kumar SM, Chandrasekar JN, Nanjan MJ. Herbal remedies for acne. Nat Prod Resour 2005;4(4):328-34.
  5. NS, AT, NL. Anti-oxidant activity of Trigonella forneum graecum using various in vitro and ex vivo models. Int J Pharm Pharm Sci 2011;3(2):97.
  6. Mistry K, Mehta M, Mendpara N, Gamit S, Shah G. Determination of antibacterial activity and MIC of crude extract of Abrusprecatorius L. Adv Biotech 2010;10(02):25-7.