Int J App Pharm, Vol 10, Issue 6, 2018, 229-233Original Article
FORMULATION AND DEVELOPMENT OF TOPICAL ANTI ACNE FORMULATION OF SPIRULINA EXTRACT
BADDURI NIHAL, N. VISHAL GUPTA, D.V.GOWDA*, MANOHAR M.
Department of Pharmaceutics, JSS College of Pharmacy, Sri ShivarathreeshwaraNagara, Mysuru, JSS Academy of Higher Education and Research, JSS Medical Institutions Campus, Sri Shivarathreeshwara Nagara, Mysuru 570015, Karnataka, India
Email: dvgowda@jssuni.edu.in
Received: 29 Mar 2018, Revised and Accepted: 04 Oct 2018
ABSTRACT
Objective: The objective of this research was to formulate and evaluate anti-acne ointment of C-phycocyanin(C-PC) extracted from spirulina.
Methods: C-PC was successfully extracted from spirulina by using sonication and cold-maceration processand further purified by dialysis method. By employing disc diffusion and agar dilution method, antimicrobial activity and minimum inhibitory concentration(MIC) ofC-PCasdetermined against Propionibacterium acne(P. acne)and Staphylococcus epidermidis(S. epidermidis).Further, the two different formulationswere prepared by using water soluble and oleaginous bases,and the formulations were characterized for particle size, viscosity, pH, consistency, drug diffusion, antimicrobial activity, and antioxidant effect and stability studies.
Results: C-PC showed MIC value of 1.5±0.1 mg/ml and 1.8±0.2 mg/ml against P. acne and S. epidermidis respectively. The developed formulation hada globule diameter of 5.44 mm, pH of 6.8±0.09, the viscosity of175±0.2cps, spreadabilityofan 8.6±0.12g. cm/sec andhadgood consistency. Both formulations were found stable among which, formulation B(FB) had maximum drug content of 95±0.6% and drug release was up to 92±0.8%.
Conclusion: Theprepared topical C-PC ointment can be successfully employed in the treatment of acneagainstP. acne and S. epidermidis.
Keywords:Acne vulgaris, Antibacterial activity, MIC, Spirulina, Ointment, In vitro activity
© 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijap.2018v10i6.26334
INTRODUCTION
Acne is a long-term skin disease that arises when hair sacs are blocked with departedskincells[1].It is categorized byspots, blackheads, whiteheads, redness,inflammations oroilyskin[2,4].It can be of the inflammatory and non-inflammatory type of acne. Non-inflammatory includes blackheads, whiteheads while inflammatory includes pimples that are red and swollen. Skin areas with comparativelyhigherinoil glands such asthe face, superior part of the upper body, and posterior majorly affected[5].Thesubsequentpresencemay lead to nervousness, reduced confidence and downheartedness[6,7]. Genetics and increased sex hormones during adolescence in both the gendersare the primary causes of acne in 78% of cases [3,10]. However, the role of food and smoking is still unclear [8,9].P. acneand S. epidermidisare the two major gram-+ve bacterial species responsible for acne vulgaris. The former one is ananaerobic, rod-shaped bacteria which lives at the base of the hair folliclebreaksdown sebum to consume as food, as bacteria increases, it causes inflammation which results in an immune response. Whereas the laterinduces acne together with other skin bacteria.
Spirulina is amicroscopic filamentous marinecyanobacterium of genus spirulina, especially Arthrospiraplatensis that is used as a dietarysupplementwhich consists of 70% proteinbyweight[11].Of these proteins, the phycobiliproteins (antenna-like proteins involved in light harvesting) named allophycocyanin, C-PCand phycoerythrin. C-PC is a holoproteinand alsoknownasphycobiliproteins[12,14]. It is responsible for most of the natural benefits being delivered by spirulina. C-PC and β-carotene that have potent antioxidant, antimicrobial and anti-inflammatory activities,exerts strong free radical scavenging activity,inhibits pro-inflammatory cytokine formation, such as TNFα, suppresses cyclooxygenase-2 (COX-2) expression and decreases prostaglandin E2 production [13].Due to increased instances of resistance of acne-inducing bacteria towards the synthetic drugs, the alternate system of medicine for the treatment of acne have been investigated and adopted. Among the alternate systems of medicine, the topical therapeutic agents are more convenient for application. Topical acne treatment is occupying the upper position as they are safe, dilute, patient familiar, economical, easily available and multifunctional. Hence spirulina is natural andpossessing both anti-inflammatory and antioxidant properties, the present study aims to explore its application in the treatment of acneand also evaluate the antimicrobial property of the spirulina containing C-PCagainst acne-causing species.
MATERIALS AND METHODS
Spirulina was procured from Genius natural herbs Pvt. Ltd. Coimbatore, India. Paraffin hard, cetostearyl alcohol and liquid paraffin are obtained from Loba Chemie. Pvt. Ltd, Mumbai. Wool fat and white soft paraffin were procured from Rajesh chemi. Pvt. Ltd,Mumbai. PEG400, PEG4000, ammonium sulfate and stearylalcohol were procured fromMerckspecialties. pvt,Ltd. Mumbai.The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular indicator was procured from geneiPvtLtdBangalore. P. acne and S. epidermidis were procured from NCL, Pune.
Extraction of protein
Cold maceration and sonication are twomethods used for isolation of C-PC. In cold maceration process 1:25(w/v) spirulina dry powder:waterat 40 °C for 24 h. In the sonicationprocess, 1:25 (w/v)of Spirulina dry powder in H2O was sonicated at 40 kHz for 45 min. Thesemi-liquid mixture obtained was subjected for centrifugation at 1000rpm for 20 min at 4 °C. The precipitate (ppt) was discardedand the supernatant layerwas collected. The pH was set to pH 7.0 for subsequent stages [17].
Purification
(NH4)2SO4 precipitation
To attain 50% saturation, (NH4)2SO4 solution was added to 100 ml extract with non-stop stirring. The solutionwas thenkeptside for 120 min. Later was centrifuged at 1200rpm for 30 min. The blue ppt found was liquefied in 0.005 M Sodium-phosphate buffer (pH-7.0)[17]. The concentration was calculated by Boussiba and Richmond method [16] and purity by Bennett and Bogorad method[15].
Dialysis and gel filtration
The solution was dialyzed overnight at 4 °C counter to one liter of 0.005 M sodiumphosphate buffer (pH-7.0). The obtained solutionwas filtered by fleetingovera Sephadex G-25 column (12 × 2 cm).Elements were collected at a flow rate of 0.5 ml/min[17].
Electrophoresis in polyacrylamide gel
In a vertical chamber using 12%SDSPAGE [19].The sample obtained after dialysis was subjected to electrophoresis and gel filtration. Molecular indicatorswerea protein marker widerange, (Myosin 205 kDa, Phosphorylase B 97.4 kDa Bovine serum albumin 66 kDa, Ovalbumin 43 kDa, Carbonic anhydrase 29 kDa, Soybean trypsin inhibitor 20.1 kDa, Lysozyme 14.3 kDa, Aprotinin 6.5 kDa, Insulin 3.5 kDa.) The gel was marked by 0.1% Coomassie Brilliant G250 solution after electrophoresis [18].
Antioxidant activity of C-phycocyanin
Determination ofthe activity of C-PC-from spirulina sample was done by electron spin resonance(ESR)spectroscopy [20]. The ESRspectrometerBruker 300E registers all ESRspectra. Theantioxidant activity of C-PC was found in the concentrationrange of 0.05–0.3 mg ml−1. 2,2-diphenyl-1-picrylhydrazyl (DPPH)artificial constant was taken as standard.The enactment of ESR quantitieswas held in the following conditions: field modulation 100 kHz, modulation amplitude 0.256 G, receiver gain 2×104, the time constant 40.96 m, conversion time 327.68 m, centre field 3440.00 G, sweep width 100.00 G, x-band frequency 9.45 GHz, power 7.96 mW, at 25 °C. The antioxidantactivity of C-PC was defined as: AADPPH = 100 × (ho − hx)/ho [%], In the ESR spectrum of DPPH radicals of the blank and probe, ho and hx are the height of the 2nd peak respectively.
Preparation of the ointment
Two formulations FA andFB consisting ofoileogenous base and water base respectively were prepared (table 1 and 2).
Table 1:Formulation chart ofoleaginous base
| Ingredients | Quantity in % | |
| Paraffin hard | 5 | |
| Wool fat | 10 | |
| Cetostearyl alcohol | 10 | |
| White soft paraffin | 50 | |
| Liquid paraffin | 15 | |
| Extract(C-PC) | 10 |
Ointment base was prepared by adding cetostearylalcohol and wool fat in melted hard paraffin.Finally,white soft paraffin was added and mixed thoroughly onheatingmantleand kept aside.Other ingredients wereweighedaccordingly and added to the above mixture(step1).
A measuredquantity of extract(C-PC) and liquid paraffin were poured into a motorpestleandstirredin one direction by adding the required quantity of the ointment base until a homogenous product is obtained. The prepared formulations were stored in ointment tubes for further studies.
Table2: Formulation chart of the water-soluble base
| Ingredients | Quantity in % |
| PEG400 | 12 |
| PEG4000 | 18 |
| Stearyl alcohol | 28 |
| Extract | 10 |
| Glycerine | 17 |
| Water | q. s |
The required quantity of the ingredients and extract were weighed and stirred thoroughly at a low temperature until the uniform base is formed.The volume was made up to the required quantity with water and stored in an ointment tubeat room temperature until further studies were carried out.
Evaluation test
Physical parameters and identification test
The preparation was mixed with water,and the odour was observed. By placing the formulation against white background colour was observed. By applying the formulation on the hand, greasiness was observed and identification was done visually by placing it in white background.
Uniformity of weight %
Randomly filled 20tubes were weighed. The tubes were emptied, washed with alcohol and dried. The weight of the empty tubes was measured using a digital weighing balance (Shimadzu digital weighing balance model no;BL220H). The difference between the weights was calculated as the net weight of the ointment tube. The average net weight of 20 tubes was noted[21].
Globule diameter
With the help of projection microscope, SIPCON SP, 585. The average diameter was calculated.
pH
Digital pH meter ELICO LI120 (type 003) was employed. About 10g of the formulation was diluted and exposed to pH measurement.
Loss on drying
The ointment was taken in a dry Petri dish on a water bath and dried for 100 °C. Loss on drying was determined[21].
Spreadability
Between 2 glass slides 1g of, the ointment was placed and 100g of weight(m) was placed above the plates.The extra was scrapped off after removing the weight. Lesser the time is taken (t)is taken separation of the two slides, the better is the spreadability(S) [22].
Consistency or hardness of ointment
Three samples were melted and filled into containers without air bubbles and sheared for 5 min then stored at 25±0.5 °C for 24 h. samples were tested using Penetrometer by adjusting the temperature at 25+0.5 °C and position was adjusted such that its tip just touches the surface of the sample and was released for 5 sec and penetration depth was measured and repeated with other samples[23].
Test of the rate of penetration
The rate of penetration can be predictable with the help of flow-through diffusion cell. Animal skin of certain known area should be removed and knotted to the receptacleexisting in a flow-through diffusion cell and placed in a liquid bath. A known amount of the formulation is smeared on the skin,and the amount of drug penetrated into the fluid is measured by collecting aliquots at regular intervals and measured byusing a spectrophotometer[24].
Test of content uniformity
The gross weight of 10 formulation tubes was measured. The outcomesobtainedshould tie each other and with the consideredamount.
Viscosity of ointment
The sample was taken in a dry 250 ml beaker, by using spindle nos. 1 to 4. OF CAP-2000 Brookfield viscometer DV-2Ⅱ+PRO (model no; LR99102) viscosity was determined [25].
Microbiological studies
The antibacterial action of both the FAandFBconsisting C-PCagainstP. acne and S. epidermidis were estimated by disc diffusionmethod and the zone of inhibition was measured with zone reader. By using an agar dilution method,MIC was measured. Nutrient agar media was used at 37 °C+2 °C for 2 d [26].
Diffusion study
3 gm of the formulation was placed in the donorsection of the modifiedkiescarychein diffusion cell, Electro lab diffusion cell (model no;EDC-07). The receptor section containing 22 ml of phosphate buffer pH 7.4 was in contact withthe complete surface of the cellophane membrane. And magnetic stirrer was continuously stirred at 100rpm at temperature 37±0.5°C. 3.14 cm2is the surface areaof-of accessible for diffusion. The experiment was carried out by maintaining the sink condition for 5 h,andthe sampling interval was 30 min. The content of C-PC was determined.
Stability studies
Ointment tubewere stored at altered temperature condition viz. 25 °C±2 °C/60%RH±5℅RH, 30 °C+2 °C/65℅ RH+5℅RH, 40 °C+2 °C/75℅ RH+5℅RH over 180days in a thermal humidity chamber, thermolabs, Mumbai and studied for various parameters of the formulations [27].
RESULTS AND DISCUSSION
Table3:Identification of ingredients in both FAֶand FB
| S. No. | Identification | Specification |
| 1 | Hard Paraffin | White Colour |
| 2 | Wool Fat | Light Yellow |
| 3 | Cetostearyl Alcohol | Color Less |
| 4 | White Soft Paraffin | White Color |
| 5 | Peg 400 | Color Less |
| 6 | Peg 4000 | Color Less |
| 7 | Extract | Intense Blue |
Fig.1: Antioxidant activity of C-PC (n=3, mean±SD)
Table4:Estimation of variousparameters for anti-acne formulation
| Evaluation parameters | FA | FB |
| Explanation | Colour-intense blue Odour-waxy |
Colour-intense blue Odour-odourless |
| Uniformity of weight | Obey with standard | Obey with standard |
| Globule diameter | 5.29 mm | 5.44 mm |
| pH | 6.1±0.06 | 6.8±0.09 |
| Loss on drying | 35℅w/w | 47℅w/w |
| Consistency | Good | Good |
| Viscosity | 198±0.4 cps | 175±0.2 cps |
| Spreadability | 8.1±0.11 g. cm/sec | 8.6±0.12g. cm/sec |
*n=3, mean±SD, The consistency of FB was superiorto FA. The FA was found to be more viscous (198±0.4cps) than FB (175±0.2cps).Thus the FB has betterspreadabilitycharacters (8.6±0.12g. cm/sec) than FA (8.1±0.11g. cm./sec).
Antioxidative activity
C-PCshowed 100% action at the concentration of 0.15 mg ml−1. Spirulina has the action which is analogous to that of Limnothrix. The (EC50) value of C-PC was about 0.08 mg ml−1 which is slightlygreater, butequivalent tothe activity of rutin at an EC50 value of 0.055 mg ml−1fig. 1.
Antioxidative activityof C-PC. The bars designate the standard error of 3dimensions.
From the outcomes, it was found that FBdiffusing 0.7 cm length after 1hr whereas FA diffuses 0.5 cm after 1hr and In vitro drug diffusion for FB was found to be 92% after 5thhrand for FA it is88 %. The results are shown in fig. 2.
Fig.2: Diffusion study of formulation A and B (n=3,mean±SD)
The antimicrobial activity of anti-acne ointment in FA and FB were determined. The zones of inhibition of P. acneandS. epidermidisaredescribed in table 5. Comparatively, FB was found to be superiortoFA.
Table5: Antimicrobial action of anti-acne ointment
| The diameter of zoneof inhibition (mm)* | MIC |
| Formulation | P. acne |
| A | 23.4±1.0 |
| B | 26.1±1.2 |
*n=3, mean±SD
Table 6: Stability parameters
| Parameters (after 3rd week) | FA | FB |
| Temp | 25˚ C | 30˚C |
| pH* | 6.6±.01 | 6.6±.02 |
| Spreadability(g/sec)* | 38.6±.2 | 36.8±.3 |
| Consistency | 165 | 163 |
| Globule diameter | 4.13 | 5.11 |
*n=3, mean±SD, from the above outcomes, it is evidentlymanifest that there were nofluctuations in the evaluationparameters of both the formulations. Comparatively,FB was found to be furtheracceptable thanFA.
DISCUSSION
Antibiotics like tetracycline, salicylic acid etc. show resistance and side-effects, C-PC being natural would be comparatively safe for treating acne. Studies have stated that the aqueous coriander extract had MIC values of 1.7 mg/ml and 2.1 mg/ml,zone of inhibition of 21.5±1.4 mm and 20.6±1.09 mm against P. acne and S. epidermidis respectively. Whereas in our present study, the aqueous extract of spirulina has MIC values of 1.5±0.1 mg/ml and 1.8±0.2 mg/ml, the zone of inhibition of 26.1±1.2 mm and 24.6±1.6 mm respectively. The results indicate that aqueous spirulina extract has better antimicrobial property than aqueous coriander extract [22].
CONCLUSION
Based on the study results,it is concluded that the spirulina extract possesses anti-acne property. And formulation comprising water-soluble base was superior to the oleaginous base, due to the complete solubility of the extract in water.
ACKNOWLEDGMENT
The authors are grateful to the JSS College of Pharmacy, Mysore, India, for giving permission to carry out their research work.
AUTHORS CONTRIBUTIONS
All the author have contributed equally
CONFLICT OF INTERESTS
The authors declare that there is no conflict of interests regarding the publication of this paper
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