topical anti-acne microemulsion formulation of essential oil

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DEVELOPMENT AND VALIDATION OF NOVEL HPTLC METHOD FOR ANALYSIS OF LEMONGRASS OIL&FORMULATION AND CHARACTERIZATION OF TOPICAL ANTI-ACNE MICROEMULSION OF LEMONGRASS OIL Presented by MD FAIYAZUDDIN M. Pharm (Quality Assurance) Department of Pharmaceutics Faculty of Pharmacy Jamia Hamdard New Delhi- 110062 Supervisor Dr. Sanjula Baboota M. Pharm., Ph.D. Senior Lecturer Department of Pharmaceutics Faculty of Pharmacy Jamia Hamdard New Delhi- 110062

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Acne, caused by P. acnes is the most common cutaneous disorder of multifactorial origin with a prevalence of 70-85% in adolescents Acne occurs due to blockage of oil glands of the skin. Oil glands produce an oily substance, sebum which is generally released onto the skin surface through the pore, when the pores become blocked, the sebum is trapped inside the oil gland and bacteria (Propionibacterium acnes, a normal skin bacteria) is produced causing inflammation. Topical therapy is employed as first-line treatment in mild acne, whereas for moderate and severe acne, systemic therapy is required in addition to topical therapy. NDDS can play a pivotal role in improving the topical delivery of anti-acne agents by enhancing their dermal localization and reducing their side effects INTRODUCTION

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Fig. 2: Stages of acne. (A) Normal follicle; (B) open comedo (blackhead); (C) closed comedo (whitehead); (D) papule; (E) pustule. Fig. 1: A generalized case of Acne vulgaris showing infection caused by P. acnes

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A) Antibiotics 1. Cleocin T© (Clindamycin) 2. Emgel© (Erythromycin) 3. Benzamycin© (Erythromycin/benzoyl peroxide) 4. Azelex© (Azelaic acid) B) Retinoids Avita© (Tretinoin) Tazorac© (Tazarotene) Differin© (Adapalene) Some of topical formulation available for the treatment of acne vulgaris.

Why switchover to herbal formulation : 

Why switchover to herbal formulation All existing formulations in the market (mentioned in previous slide) have enormous side effects including rashes, wrinkling, erythema, skin eruption, resistance and granulation effects. Herbal formualation was therefore an another hit option to bypass these side effects and to provide natural essence to the skin. Therefore it was proposed to develop an essential oil based herbal anti-acne drug delivery system to treat severity of acne vulgaris. Due to having an excellent antibacterial property, Lemongrass oil was chosen for the development of anti-acne microemulsion formulation.

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Therefore the aim of our present study was to identify a natural compound that has potential application in the treatment of acne vulgaris and to develop a novel drug delivery system for the same. Lemongrass oil, which contains citral as the major constituent has an antibacterial activity. Due to its excellent antibacterial/anti-acne nature it can be used to treat acne vulgaris. A topical microemulsion based drug delivery system for lemongrass oil was prepared and evaluated. For analytical purposes, HPTLC method was developed and validated. AIMS OF THE STUDY

OBJECTIVES OF THE STUDY : 

OBJECTIVES OF THE STUDY To develop a novel HPTLC method for analysis of lemongrass oil. To validate the developed HPTLC method as per ICH Guideline. To develop thermodynamically stable microemulsion for lemongrass oil. To optimize the formulation on the basis in vitro release and skin permeation studies. To perform the antibacterial studies.

RATIONALE OF THE STUDY : 

RATIONALE OF THE STUDY WHY LEMONGRASS OIL ? Lemongrass oil has been reported to be very effective in the treatment of acne. Has been found to have in vitro antibacterial activity equivalent to that of Penicillin, particularly against gram-positive organism. Lemongrass oil has antibacterial and antifungal activities due to presence of citral. No validated HPTLC method reported for the estimation of lemongrass oil. No developed microemulsion based topical formulation of Lemon grass oil for the treatment of acne vulgaris.

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High performance thin layer chromatography (HPTLC) Sophisticated analytical technique with improved quality of sorbents and optimized techniques. Gives fast result with high resolution. Widely used for herbal drug analysis and their quality assessment. One of the most widely used techniques for rapid identification of drugs and formulations. Equally applicable to drugs in their pure state and to those extracted from pharmaceutical formulations or from biological samples. WHY HPTLC ?

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WHY MICROEMULSION ? Microemulsions offer several advantages over the conventional topical drug delivery sources Thermodynamically stable Leading to longer shelf life Act as supersolvents, improving the solubility and thermodynamic activity of the drug Enhances the percutaneous uptake of the drugs Small particle size large interfacial area can be quickly released Hydrophilic and lipophilc domains Act as potential reservoir of the drugs, through which pseudo-zero order kinetics can be obtained Ease of preparation

PLAN OF WORK : 

PLAN OF WORK 1. DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD a) Physicochemical properties b) Qualitative analysis c) Development of HPTLC method. d) Validation of developed HPTLC method 2. FORMULATION AND CHARACTERIZATION OF TOPICAL MICROEMULSION OF LEMON GRASS OIL a) Formulation of Microemulsion b) Evaluation of Microemulsion c) In-vitro antimicrobial studies (anti-acne studies)

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PHYSICAL CHARACTERIZATION OF LEMONGRASS OIL Physical properties: Appearance Fluid liquid Color Deep yellow to orange-brown. Aroma Fresh-grassy lemon-type. B.P. 228º C Solubility Soluble in methanol, slightly soluble in ethanol (95%), paraffin oil but practically insoluble (> pH 7) in water and buffer, pH 1% ethanolic solution of lemongrass oil has pH 5.5

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1. LINEARITY Mean area calculation for citral and neral for linearity VALIDATION OF DEVELOPED METHOD

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Linearity curve for citral Equation: y = 5.4003x + 1.5213 Slope =5.4003 Correlation co-efficient = 0.9933 Linearity curves for citral and neral Linearity curve for neral Equation: y = 6.6029x + 2388.8 Slope =6.6029 Correlation co-efficient = 0.9937 The linearity for ten concentration levels from 1000-10000 ng/spot of Citral and Neral were obtained 0.9933 and 0.9937. The method showed good linear relationship.

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Height and Area values: 1800 ng/spot of standard citral (Spot applied six times)a Height and Area values: 1880 ng/spot of standard neral (Spot applied six times)a 2. Precision (System Repeatability)

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Citral=1880 ng/spot (n=6) Neral=1800ng/spot (n=6) Chromatograms for Precision

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Height and area values Std. Citral=1800 ng/spot (Spot sacanned six times)b Height and area values Std. Neral=1880 ng/spot (Spot sacanned six times)b

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Precision (Method Repeatability) Height and Area values for citral

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Mean, % RSD and SEM values for Precision of Citral (Area) Method Rept. Mean, % RSD and SEM values for Precision of Citral (Ht)/ Method Rept.

3. Recovery Studies : 

3. Recovery Studies Peak obtained with 100% percent extra addition of standard drug

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3D Spectral view for bioactive citral in lemongrass oil with different concentrations at 595 nm

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A novel, simultaneous, precise, accurate densitometric method was developed and validated by HPTLC for the analysis of lemongrass oil and its formulation. The mobile phase selected was toluene: ethyl acetate (8.5: 1.5, v/v) and solvent used for preparation was toluene and λmax of lemongrass oil was obtained at 595 nm. Calibration curves were prepared for citral, neral and geranial at 225-3600 ng/spot, 470-3760 ng/spot and 500-25000 ng/ spot respectively with R2 values of 0.9916, 0.9970 and 0.9868 (height basis), 0.9970, 0.9935 and 0.9888 (area basis). The linearity of the method determined at concentration levels from 1000-10000 ng/spot and the R2 values for citral and neral were obtained 0.9933 and 0.9937. Method’s precision studies were carried out by repeatability and intermediary precision both for citral and neral, %RSD for both drugs were obtained as 3.47, 1.94 and 2.57 for citral and 2.97, 5.52 and 4.96 for neral. Accuracy was determined by recovery using HPTLC method. The percent recovery for citral was 95.30-107.81%. Conclusion (HPTLC)

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4. Estimation of citral in lemongrass oil by HPTLC method The present developed HPTLC method was employed for estimation of citral in lemongrass oil by using area data, assessed by calculation given below. Average area 11559.4 mm By using standard plot equation of area for the estimation of citral, concentration obtained is1.269µl/ml. Actual concentration of citral = Conc. x Dilution Factor x Specific gravity = 1.269µl/ml. x 500 x 894 = 576.24 mg/ml Actual content (gm/100ml) =56.72% (Literature reported= 60-80%)

FORMULATION OF MICROEMULSION : 

FORMULATION OF MICROEMULSION 1. Microemulsion component selection a) Lemongrass oil b) Surfactants Tween 80® and Tween 20® c) Co-surfactants Transcutol-P® and Ethanol. d) Distilled Water 2. Construction of Pseudo Ternary Phase Diagram Aqueous titration method Sixteen different combinations of oil and Smix, 1:9, 1:8, 1:7, 1:6, 1:5, 2:8 (1:4), 1:3.5, 1:3, 3:7 (1:2.3), 1:2, 4:6 (1:1.5), 5:5 (1:1), 6:4 (1:0.7), 7:3 (1:0.43), 8:2(1:0.25), 9:1 (1:0.1).

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Drug, surfactants and cosurfactants grouped in different combinations (Lemongrass oil).

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( a)Tween 20: ethanol (1:0) (b)Tween 20: ethanol (1:1) ( c)Tween 20: ethanol (2:1) (d)Tween 20: ethanol (3:1) ( e)Tween 20: Transutol P (1:0) ( f)Tween 20: Transutol P (1:1) PSEUDO-TERNARY PHASE DIAGRAMS

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(g)Tween 20: Transutol P (2:1) (h)Tween 20: Transutol P (3:1) ( i) Tween 80: ethanol (1:0) (j) Tween 80: ethanol (1:1) ( k) Tween 80: ethanol (2:1) (l) Tween 80: ethanol (3:1)

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( m) Tween 80: Transutol P l (1:0) (n) Tween 80: Transutol P (1:1) ( o) Tween 80: Transutol P l (2:1) (p) Tween 80: Transutol P (3:1) Pseudo-ternary phase diagrams of all groups indicating o/w microemulsion region of Lemongrass oil, Tween 80, Tween 20 (surfactant) Ethanol and Transcutol-P (cosurfactant) at different Smix ratios indicated in Figures (a-p). Group I (a-d), Group II (e-h), Group II (i-l) and Group II (m-p).

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Composition of selected microemulsion formulations from different phase diagrams

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3. Physical stability studies for prepared microemulsion of Lemongrass oil Thus, the selected formulations were subjected to :a) Heating cooling cycleSix cycles between refrigerator temperature (4ºC) and 4ºC with storage for 48 hrs.b) Centrifugation studies3500 rpm for 30 minutes. C) Freeze thaw cycleThree freeze thaw cycles between -21ºC and 25ºC for 48 hrs.

4. Characterization Of Lemongrass Oil Microemulsion Formulations : 

4. Characterization Of Lemongrass Oil Microemulsion Formulations A) In vitro skin permeation studies Apparatus: Keshary-Chien diffusion cell Permeation media: Acetate buffer (pH=5.5) + Ethanol (40:60) Skin: Abdominal region of albino rats Assembly temp: 37 ± 0.5 ºC Analysis: Wincat HPTLC instrument at 595 nm.

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Comparative in vitro skin permeation profiles of Lemongrass oil from different microemulsion formulations

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Prepared microemulsion formulation of lemongrass oil

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Histogram for Flux and Permeability In vitro skin permeation studies were performed to compare the release of drug from four different microemulsion formulations (LG1 to LG4) [as other formulations were unstable and failed during stress testing] and Lemongrass per se (LG). In vitro skin permeation was highest in formulation LG3 and LG4 and lowest permeation profile. The formulation LG1 showed intermediate skin permeation profile.

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B) Droplet size analysis Droplet size of the microemulsion was determined by photon correlation spectroscopy (Dynamic Light Scattering Instrument) that analyzes the fluctuations in light scattering due to Brownian motion of the particles. Normally the droplet size increased with the increase in concentration of oil in the formulations. The droplet size of the formulation LG3, containing 15% of oil was 156 ± 0.24 nm. The polydispersity values of the formulations were 1.018 ± 0.021 which indicates uniformity of droplet size within the formulation.

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C) Transmission electron microscopy TEM CM-10 (Philips, Netherlands). Transmission Electron Microscope (TEM) was employed for the microscopic evaluation of optimized formulations at 60-80 KV at different magnifications (1550x, 2150x, 4600x, 21500x and 44000x). TEM Photomicrograph of microemulsion formulation LG3 was interpreted for particle size and distribution. It was finally conclude that the particle were spherical in shape and finley distributed with nano range of particles. Due to spherical shape and nano-size the drug permeation was very good.

D) In Vitro Antibacterial Study : 

D) In Vitro Antibacterial Study The microbiological assay of the optimized microemulsions were tested against Propionibacterium acnes using Cylinder-plate or Cup-Plate method, compared for growth inhibition of bacteria by a measured concentration of Lemongrass oil microemulsion w.r.t. standard preparations of antibiotic with known activity. i) Materials and Methods Test organisms: Propionibacterium acnes. Standard compound: Clindamycin discs. Test samples: LG1- LG4 were diluted to concentrations of 10, 25, 50, 75 and 100 μg/ml in DMSO. Media: Dehydrated brain heart infusion agar (BHIA) was prepared with 5% human blood in deionized distilled water with pH=7.4  0.2 (at 25˚C). Method of preparation of test organisms: The test organisms were maintained on slants of medium and transferred to a fresh slant and incubated at 370 C for 48 hrs.

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*Values are zone of inhibition in mm including the diameter of bore (5mm) ii) Experimental Cup and plate method Using Clidamycin discs as standards, different dilutions of optimized microemulsions (LG1-LG4)/(S1-S4) were prepared in DMSO and poured onto bored holes (5 in each) and then incubated for (48 hr+370+anaerobically). After 48 hrs, the plates were examined for diameter of zones of inhibition. Interpretation of results All the microemulsion formulations were examined for antibacterial activity (anti-acne activity) against P. acnes. Microemulsion formulations showed extremely significant anti-acne activity as compared to standard “Clindamycin”.

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50 μ g/ml against P. acnes 75 μ g/ml against P. acnes 100 μ g/ml against P. acnes Standard Clindamycin discs against P. acnes

Anti-acne activity of microemulsion of lemongrass oil S-1, S-2, S-3, S-4, (μg/ml) w.r.t. Clindamycin against P. acnes : 

Anti-acne activity of microemulsion of lemongrass oil S-1, S-2, S-3, S-4, (μg/ml) w.r.t. Clindamycin against P. acnes

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The developed HPTLC method was found to be accurate, precise, specific and reproducible and excellent method for the quantification of Lemongrass oil. Combination of Tween 80 and Ethanol gave extensive region of microemulsions and the selected formulations were subjected to heating cooling cycle, centrifugation, and freeze thaw cycle and found that formulations from Group II (Tween 80 and Ethanol) passed the test. Droplet size increased with the increase in concentration of oil in the formulations and found to be 156 ± 0.24 nm with low polydispersity. In vitro skin permeation studies were performed and found highest in case of formulation LG3 and lowest for LGl. The skin permeation profiles of LG3 and LG4 were not significantly different (p < 0.05), probably due to the mean size of internal phase droplets, which were significantly smaller in microemulsions. Permeability parameters like steady state flux (Jss) and permeability coefficient (Kp) were significantly increased and found maximum for LG3. In vitro anti-microbial studies showed highest value of zone of inhibition value for LG3 as compared to others. SUMMARY

CONCLUSION : 

CONCLUSION The validated developed method is unique of its own kind and never reported before. It simultaneously determine lemongrass oil content in bulk drug on the basis of citral, neral and geranial. Prepared microemulsion formulation using lemongrass oil was found to have good permeability therefore an excellent dosage form to deliver the drug. Formulation was characterized as well screened for P. acnes and revealed that it contains immense potential to control acne vulgaris.

REFERENCES : 

REFERENCES Alvarez-Figueroa, M.J., Blanco-Mendez, J. (2001). Transdermal delivery of MTX: ionophoretic delivery from hydrogels and passive delivery from microemulsions. Int. J. Pharm. 215, 57-65. Badgujar, Y.B., Jain, P.S., Talele, G.S., Surana, S.J. (2005). HPTLC method for determination of carvedilol in tablet dosage forms. Indian drugs. 42(8), 511-515. Baroli, B., Lopez-Quintela, M.A., Delgado-Charro, M.B., Fadda, A.M., Blanco-Mendez, J. (2000). Microemulsions for topical delivery of 8-methoxsalen. Int. J. Pharm. 69, 209-218. Barry, B.W. (1991). The LPP theory of skin penetration enhancement. In Vitro Percutaneous Absorption: Principles, Fundamentals and Applications. Bronaugh, R.L.and Maibach, H.I. (eds.).CRC press, Orlando, FL, 165-185.