DETERMINATION OF (-) Α-BISABOLOL IN MATRICARIA CHAMOMILE OIL AND IN NANOFORMULATION BY HPTLC METHOD: IT S APPLICATION IN EX VIVO STUDIES

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1 Page2190 Indo American Journal of Pharmaceutical Research, 2015 ISSN NO: DETERMINATION OF (-) Α-BISABOLOL IN MATRICARIA CHAMOMILE OIL AND IN NANOFORMULATION BY HPTLC METHOD: IT S APPLICATION IN EX VIVO STUDIES Zrien Naz 1, Mohd. Shah Faisal 1, Abdul Muheem 1, Surender Singh 2, Roop.K.Khar 3, Farhan J.Ahmad 1* 1 Nanoformulation Research Lab, Faculty of Pharmacy, JamiaHamdard, New Delhi, India. 2 Department of Pharmacology, All India Institute of Medical Sciences, New Delhi, India. 3 Principle, B.S Anangpuria Institute of Pharmacy, Faridabad, New Delhi, India. ARTICLE INFO Article history Received 25/05/2015 Available online 30/06/2015 Keywords HPTLC, (-)-α-bisabolol, Nanoformulation, Skin permeation, Skin deposition. ABSTRACT A simple, accurate sensitive and stability indicating high performance thin layer chromatography (HPTLC) method for the estimation of (-)-α-bisabolol in chamomile oil and in nanoformulation was developed and validated according to ICH guidelines. The quantification of (-)-α-bisabolol was done by applying standard drug solution on precoated HPTLC plates (silica gel 60 F254) that act as stationary phase, kept in twin trough glass chamber presaturated with mobile phase consisting of toluene: chloroform: methanol: acetic acid in the ratio 9.2:0.4:0.4:0.2:0.04 (v/v/v/v). Linear ascending mode was used for the development of chromatograms and spectrodensitometric analysis was done at 430nm. The linear regression analysis data,in concentration range of ng per spot for (-)-αbisabolol (Rf = 0.46±.02), showed best relationship (r2=0.99) with respect to peak area. The limit of detection (8.68 ng per spot) and limit of quantification (26.30 ng per spot) were determined by standard deviation method. Stress degradation studies confirmed that the developed method gave well resolved peaks of the drug and degradation products, hence this can be used to separate pure compounds from impurities.the developed HPTLC method was used for the estimation of (-)-α-bisabolol in bulk drug and in nanoemulsion formulation. It can also be employed for ex-vivo skin permeation and skin deposition studies as the developed method is economical, robust and time saving. Corresponding author Zrien Naz Research Scholar, Department of Pharmaceutics Faculty of Pharmacy, JamiaHamdard, New Delhi zrien.naz@gmail.com Please cite this article in press as Zrien Naz et al. Determination of (-) α-bisabolol in Matricaria chamomileoil and in nanoformulation by HPTLC method: its application in ex vivo studies. Indo American Journal of Pharm Research.2015:5(06). Copy right 2015 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Page2191 INTRODUCTION The German chamomile oil, which is commonly known as the blue oil is obtained by the hydrodistillation of the flower of Matricaria chamomile, family Asteraceae. It mainly consists of sesquiterpene derivatives (75 90%) that impart blue color to the essential oil [1]. It is remarkable that chamomile flower oil contains (-) -α-bisabolol, a natural monocyclic sesquiterpene alcohol (figure 1) obtained as colorless viscous oil as the primary constituent of the essential oil [2]. It possesses anti-irritant, antiinflammatory and anti-microbial activity and is also known to have inhibitory activity on prostaglandins and leukotriene [3, 4].It is remarkable that chamomile flower oil contains (-)-α-bisabolol, a natural monocyclic sesquiterpenealcohol (figure 1) obtained as colorless viscous oil as the primary constituent of the essential oil [2]. It possesses anti-irritant, anti-inflammatory and anti-microbial activities and is also known to have inhibitory activity on prostaglandins and leukotriene [3, 4]. (-)-α-bisabolol inhibited 5- lipoxygenase and cyclooxygenase at an inhibitory concentration of 40 μm and 70-80μM respectively [5]. It is also recommended in cosmetic preparations as an anti-inflammatory compound [6]. Fig: 1 Structure of (-)-α-bisabolol. The current therapeutic management of arthritis is done with a range of pharmacotherapeutic agents like NSAIDS, glucocorticoids, immunosuppressant, is associated with side-effects and toxicity. In order to minimize the side effects and to find an effective management for arthritis so that surgery could be mitigated, research is being focused on herbal agents. Based on the pharmacological activity as an inhibitor of proinflammatory enzymes, (-) -α-bisabolol, synonymously levomenol, is expected to reduce wear and tear in arteries [7].This is also evident from the traditional use of chamomile oil as a constituent in preparations meant to be applied topically to alleviate joint pain. Hence chamomile oil can be used externally in the treatment of arthritis by incorporating it in novel nanoformulations for topical application. The analytical methods for the estimation (-) -α-bisabolol in the German chamomile oil include profiling by HPLC and GC/MS for the determination of its active principle in oil and herbal extracts [8, 9]. The British Pharmacopoeia monograph assesses the chamomile oil qualitatively by TLC and quantified by GC for its content of bisabolol oxides A/Band (-) -α-bisabolol [10]. Thus from the literature survey it is obvious that analytical methods for the quantification of (-) -α-bisabolol in bulk drug and in pharmaceutical formulation are limited and nobody has reported HPTLC method for the same. Moreover the reported methods are costly, time consuming and their application in pharmaceutical formulation requires special preparation for extraction of drug. Herein we reporta stress indicating high performance thin layer chromatography (HPTLC) method in order to determine the content of (-) -αbisabolol in bulk drug, in extracts and in novel nanoformulations. The developed HPTLC method is advantageous over other methods as it helps in quantification of the drug from the nanoformulation and can from biological samples without the application of expensive and tedious extraction processes. Thus this method was used for the determination of (-) -α-bisabolol in ex-vivo skin permeation and skin deposition studies. MATERIALS AND METHODS Materials and Reagents Chamomile oil and (-)-α-bisabolol were purchased from Sigma Aldrich (Germany). Tween 80, and PEG 400 were procured from Hi-Media (Mumbai, India). Toluene and ethyl acetate were purchased from Fisher international. HPTLC, precoated silica gel 60 F 254 plates were purchased E. Merck, Darmstadt, Germany. All other chemicals and reagents were of analytical grade and purchased from Thomas Baker, India. Experimental/ Methodology Equipment The chromatographic system used was CAMAG HPTLC system with Linomat V applicator which was programmed through WINCATS software. A TLC scanner III is attached with the system used for scanning of developed plates. Preparation of the spraying reagent The spraying reagent was prepared by mixing 0.5 ml of anisaldehyde and 10 ml of glacial acetic acid with 85 ml of methanol. Then 5mL of sulphuric acid was added in above prepared mixture [11].

3 Page2192 Chromatography Precoated HPTLC plates (silica gel 60 F254) of size 20 x 20 cm were used for chromatographic development. The twin trough glass chamber (CAMAG) was pre-saturated with a mobile phase consisting of toluene: chloroform: methanol: acetic acid in the ratio 9.2:0.4:0.4:0.2:0.04 (v/v/v/v) for 30mins to ensure uniform distribution of solvent vapors. Samples were applied by using CAMAG Linomat V applicator using microliter syringe, having capacity of 100 µl as narrow bands of 6mm width. Chromatogram was developed up to 80% of plate height through linear ascending development technique, at room temperature 25±2 o C and relative humidity 55±5%. The plate was removed, air dried and then derivatized with anisaldehyde sulfuric acid spraying reagent. The prepared chromatogram was dried in oven at 60 o C for 5 minutes. Densitometric analysis was performed at 430 nm with a Camag TLC scanner III. The slit dimensions were 5 mm 0.45 mm and the scanning speed of 20 mm s -1. Preparation of the standard (-) α-bisabolol solution A standard stock solution was prepared by dissolving 10µL of (-) α-bisabolol in 10mL of toluene to obtain a stock solution of 1000 ng/ml. From the stock solution, different working concentrations ranging from ng/ml were prepared. Calibration curve of standard (-) α-bisabolol Different volumes of stock solution 0.1, 0.2, 0.4, 0.6, 0.8 and 1µL were spotted in triplicate on a TLC plate to obtain concentrations of 100, 200, 400, 600, 800 and 1000 ng per spot of (-)-α-bisabolol. The data of peak areas and corresponding concentrations were treated by linear least-square regression analysis. Method validation Specificity, linearity and robustness In the established method for ascertaining specificity, as per ICH guidelines Q2 [R1], the spot of the standard and the sample was scanned and their R f values were compared to investigate the potential interference. For linearity, accurately known volume, 100µL of (-) α-bisabolol was dissolved in minimum quantity of toluene and volume was made up to 100 ml with toluene in a volumetric flask to obtain a stock solution of 1000ng/mL which was used for analysis of six different concentrations spotted in triplicate on TLC plate to obtain concentrations of 100, 200, 400, 600, 800 and 1000 ng per spot of (-)-α-bisabolol. The densitograms were reported and plotted between the mean peak area as well as peak height of (-)-α-bisabolol (Y-axis) and corresponding concentration (X-axis) at 430nm. Robustness was carried out by introducing small change in the mobile phase volume and duration of mobile phase saturation and the effect on the results were examined. Mobile phase having composition of toluene: chloroform: methanol: acetic acid 9.0:0.6:0.4:0.2:0.04 (v/v/v/v), with presaturation time of 2 hours was tried and chromatograms were developed. Robustness of the method was studied in triplicate at a concentration level of 400 ng per spot. Accuracy, precision and recovery studies Accuracy was studied by performing recovery studies by six replicate analyses of spiked quality control samples at concentrations of 50, 100 and 150% followed by their comparison with the standard sample concentration prepared on the same day. Accuracy was expressed as percentage recovery. Precision measures the level of concord between the single test results in comparison to the standard procedure that is applied to multiple sampling of the same sample. It is measured by intra and inter day measurements of relative standard deviation of mean area. The repeatability was studied by assaying six samples of (-)-α-bisabolol having same concentration applied six times during the same day. From the stock solution of (-)-α-bisabolol having concentration 1000ng/mL, 0.4 µl were taken for applying six times to get a concentration of 400 ng per spot, on separate plates. Intermediate precision was evaluated within the experimental laboratory but carried out with different instrument as well as on different days. Intra-day and interday precision were carried out at three different concentration levels of (-)-α-bisabolol that is 200, 400, 1000 ng per spot respectively for each spot. Single spots of three different concentration levels were applied in triplicate on HPTLC plate. The plates were scanned and quantified for the concentration of (-)-αbisabolol. Detection limit and quantification limit The Limit of detection (LOD) and Limit of quantification (LOQ) were determined by standard deviation method. Blank sample (toluene only) was injected in triplicate and peak area was recorded. The LOD and LOQ were calculated using the slope of the calibration curve and standard deviation of the blank sample using formulae: (1) (2) Where SD is the standard deviation of the blank response and S is the slope of the calibration curve. The LOD and LOQ were determined using a signal to noise approach.

4 Page2193 Stress stability studies of (-)-α-bisabolol The Stress degradation studies of (-)-α-bisabolol in various conditions such as acid, base, peroxide, dry heat, wet heat, sunlight and ultraviolet light were studied by the developed HPTLC method to provide an indication of the stability indicating property and specificity of the proposed method. Acid and base induced degradation Added 5 ml of (-)-α-bisabolol to 50 ml of ethanolic solution of each 1M HCl and 1M NaOH taken separately. These mixtures were refluxed for 3 h at 90 0 C in dark in order to exclude the possible effects of light on degradation. From the resultant solution 2 µl (2000 ng per spot) each was applied on TLC plate in triplicate. Also from the initially prepared stock solution of (-)-αbisabolol 0.2 µl (200 ng per spot) of the standard was applied to compare the R f values. Photochemical degradation and hydrogen peroxide induced degradation Photochemical stability of (-)-α-bisabolol was studied by exposing the drug solution having concentration ng/ml, to direct sunlight and UV radiation at 254 nm for 24 h each. From the resultant solution 2 µl was applied on TLC plate in triplicate along with standard (-)-α-bisabolol solution for degradation studies. For hydrogen peroxide induced degradation to 50 ml solution of standard (-)-α-bisabolol in toluene, having concentration of 2000 ng/ml, 50 ml of 30% v/v hydrogen peroxide was added. Then the solution was heated on boiling water bath for 6 h for the removal of excess hydrogen peroxide. The volume of the resulting solution was made 100 ml with toluene. From each of the resultant solution 2 µl was applied on TLC plate in triplicate along with standard (- )-α-bisabolol solution for degradation studies using proposed method. Dry heat and wet heat induced degradation The standard (-)-α-bisabolol 1mL was stored at C for 3 h then it was dissolved in toluene to get a concentration 2000 ng/ml. Similarly for wet heat induced degradation 1 ml of the sample was kept in boiling water bath for 3 h then it was dissolved in toluene to get a concentration 2000 ng/ml. From each of the resultant solution 2 µl was applied on TLC plate in triplicate along with standard (-)-α-bisabolol solution for degradation studies. Quantification of (-)-α-bisabolol in chamomile oil From the essential oil 100 µl was dissolved in 10 ml toluene to get a stock solution of 10000ng/mL. From this 0.2 µl, was applied in triplicate on TLC plate to get a concentration of 2000 ng per spot of chamomile oil. Standard solution of (-)-α-bisabolol (1000ng/mL), was applied was applied in triplicate to get a concentration of 200ng per spot along with it. Formulation of nanoemulsion The nanoemulsion comprised of miglyol 810n as the oil phase with 2.5% chamomile oil and water as an external phase was formulated by water titration method (12). Tween 80 and ethanol in Smix ratio of 1:1 was selected as surfactant and co surfactant mixture from pseudoternary phase diagram. Ex-vivo skin permeation and skin deposition studies. Animals The animal study was conducted after approved by Intuitional animal ethical committee, central animal house, JamiaHamdard, New Delhi, India. Male Wister rats having average body weight 220±10gm were kept in individual polycarbonate cages under the standard laboratory conditions and having free access to commercial pellet diet (Lipton, India) and tap water ad libitum. The animal house temperature and relative humidity was maintained at (25±2 C) and (50±15%) respectively. All animal experimental procedures were in accordance with the guidelines of the central animal ethics committee. Preparation of the Rat s skin Albino rats obtained from the central animal facility at JamiaHamdard were sacrificed and their abdominal skin was excised and hair was removed. The dermis side was wiped with isopropyl alcohol to remove residual adhering fat. The skin trimmed to surface area cm 2 and mounted on a vertical diffusion sampling cell with receptor capacity of 5 ml for permeation studies. The receptor compartment was filled with acetate buffer ph 5.5 with ethanol in ratio (4:1) and was stirred constantly at a speed of 600 rpm at temperature o C for 8-10 h. The donor cell was filled with 1 ml of nanoemulsion and 0.5 ml of aliquot was collected from the receiver cell at designated time intervals (viz. 0, 1, 2, 4, 6, 8, 10, 12 and 24 h) for 24 h period and replaced immediately with the same volume of fresh media. After appropriate dilutions, the samples were filtered using 0.45μm membrane filter and the amount of drug in the receptor media was analyzed using the developed HPTLC method. Similarly, skin permeation studies with chamomile oil alone was performed. Analysis of (-)-α-bisabolol content by skin deposition studies. After 24 hours, the skin surface was washed, cut into small pieces, homogenized with methanol (5mL) by Teflon homogenizer and left for 6 hours at room temperature. After shaking for 5 minutes, it was centrifuged for 5 minutes at 5000 rpm and supernatant filtered using 0.45μm membrane filter and analyzed for (-)-α-bisabolol content by the developed HPTLC method.

5 Page2194 RESULTS AND DISCUSSION Optimization of mobile phase The HPTLC method was optimized with a view to quantify the content of (-)-α-bisabolol in chamomile oil and in nanoformulations. Initially pure solvents of different polarities like toluene, methanol, chloroform, dichloromethane, n - Hexane etc. were used individually. For better resolution the second level solvents were tried and solvent strength was increased by the addition of polar solvents. In toluene (polarity index: 2.3), chloroform (polarity index: 4.3) was added in ratios of 9:0.4, 9.3:0.7, 9.5:0.5 and chromatograms were developed. Methanol (<1.0 ml) was added to enhance separation. Few drops of ammonia was added for better resolution and acetic acid for achieving separation and sharpness between peaks. The final mobile phase consisting of toluene: chloroform: methanol: acetic acid in ratio 9.2:0.4:0.4:0.2:0.04 (v/v/v/v) was used in the investigation gave compact well resolved spots for (-)-α-bisabolol in different concentration levels at R f value 0.46±.02 (13). Densitometric analysis was carried out in the absorption mode at λ max 430 nm. Calibration curves of standard (-) α-bisabolol The Calibration curve was constructed for (-)-α-bisabolol and the linear regression analysis data showed good linear relationship (r 2 = ) with respect to peak area in concentration range of ng per spot. The values of slope and intercept are and respectively (table 1). Table 1: HPTLC data for linearity and calibration plot by peak area.\ Parameter Linearity Calibration plot Peak area (n=3) Peak height(n=3) Peak area(n=3) Linearity range (ng/spot) Regression equation(y) x x x Correlation co-efficient (R 2 ) intercept slope Standard deviation 5.03% 7.80% 5.67% Confidence limit (95%) Standard error Method validation The method was validated with respect to the parameters like linearity, accuracy, precision, recovery, robustness, LOD and LOQ. The linearity of an analytical method is its ability to elicit tests results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analytes in samples in given range. The linearity by mean area and height was determined using linear least square regression analysis at concentration levels from ng per spots, showed r 2 = and respectively. The values of slopes and intercepts by mean area and height are given in table 1.The proposed method is robust as indicated from the low value of % RSD (0.22) as obtained subsequent to bringing small changes in the mobile phase volume. The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous sample.the intra- and inter-day precision and accuracy of the (-)-α-bisabolol assay determined at 200, 400 and 1000ng/spot is summarized in table 2. Table 2: HPTLC Validation data for precision and accuracy. (-)-α-bisabolol applied (-)-α-bisabolol Precision a (CV, %) Accuracy b (%) (ng/spot) found (ng)±sd Intra-day ± ± ± Inter day ± ± ± a Precision as coefficient of variation (CV, %) = 100 (standard deviation/(-)-α-bisabolol found). b Accuracy = 100 [(-)-α-bisabolol found/(-)-α-bisabolol applied]. The CV for intra-day and inter-day precisions were and %, respectively. The accuracy was in the range %. The low CV values demonstrated precision of the method.the proposed method showed a good recovery of 99.13%, 99.05% % when spiked with 50%, 100%, and 150% of the standard (-)-α-bisabolol respectively (table3). The LOD of the proposed method with signal-to-noise ratio of 3:1 was found to be 8.68 ng per spot and LOQ value with signal-to-noise ratio of 10:1 was found to be ng per spot.

6 Page2195 Table 3: Accuracy by recovery. Excess drug used Theoretical content Content Found ± SD %Recovery %RSD SE ± ± ± ± Forced degradation studies: stability indicating property The chromatograms of the sample degraded with different stress conditions showed well separated spots of the drug as well as some additional peaks at different R f values. The spots of the degraded product were well resolved from the drug spot (figure 2). Fig 2: TLC chromatograms of degradation product of (-)-α-bisabolol on exposure to different stress conditions: A acid; B base; C dry heat; D wet heat; E day light; F UV; G peroxide; H A typical chromatogram of (-)-α-bisabolol (R f 0.46±.02) The peak of (-)-α-bisabolol not significantly shifted in the presence of degraded peaks which indicated stability indicating property of the proposed method. The extent of degradation in different stress conditions has been shown in table 4. Drug recovery was maximum at the level of 74.07% in day light showing that drug is comparatively stable at day temperature. More than 80% degradation was observed in dry heat condition. Sample exposure conditions Table 4: Forced degradation studies of (-)-α-bisabolol. R f of degradation products Average concentration (n=3) ± SD SE % Degraded % Average recovery Acid 1M HCl 0.05,0.1,0.23,0.29,0.36,0.46,0.43,0.85, ± Base 1M NaOH 0.07,0.13,0.26,0.29,0.36,0.46,0.55,0.60, ± day light 0.25, ± UV-254 nm 0.02,0.23,0.29,0.37,0.46, ± Dry heat 0.22,0.30,0.34,0.37,0.46,0.55,0.70,0.74, ± Wet heat 0.20,0.32,0.37,0.46,0.52,0.54,0.59,0.74, ± Peroxide 30%v/v 0.19,0.26,0.34,0.46, ± Quantification of (-) α-bisabolol in chamomile oil and in nanoformulation The proposed HPTLC method was applied for quantitive estimations of (-)-α-bisabolol in Chamomile oil Blue, in marker and in nanoformulation. The marker showed the presence of additional peaks at R f of its isomers (-)-α-bisabolol oxide A/B. The chamomile oil showed the presence of other constituents apart from (-)-α-bisabolol. In nanoformulations additional peaks were observed apart from spot of (-)-α-bisabolol. The presence of (-)-α-bisabolol was confirmed by matching the mean R f value (0.46±.02) from the chromatogram. The content of (-)-α-bisabolol in chamomile oil, standard (-)-α-bisabolol and in nanoformulation was found to be ± 2.01 %, ± 1.96 % and ± 0.06 % respectively (table 5).

7 Page2196 Table 5: Drug Content. Sample Avg % bisabolol content ±SD %RSD chamomile oil ± Bisabolol marker(sigma) ± Nanoemulsion 1.60 ± Analysis of skin permeation and skin deposition studies The permeation ability of the nanoemulsion formulation containing chamomile oil was compared with the oil alone. The results revealed that the permeation rate and permeation coefficient of nanoemulsion NEC-1 through rat skin is significantly (p<0.001) lower in comparison to the oil (CL-0). Skin deposition study was done to see how much drug was retained in skin. NEC-1 showed skin retention of 38.85% which is significantly (p<0.05) higher as compared to CL-0 (17.46%) as is evident in the permeation graph (figure 3). Since the formulation is designed for use in the topical treatment of arthritis slower permeation and more skin retention is required. Fig 3: Permeation graph of nanoformulation NEC-1 and chamomile oil CL-0. CONCLUSION The developed HPTLC method can be effectively used for the quantitative estimation of (-) α-bisabolol in chamomile oil and its nanoformulation. So far analytical method for quantitative estimation of (-) α-bisabolol in chamomile oil have not reported a HPTLC method which could be used to estimate the drug in ex vivo studies.. The proposed method is simple, selective and accurate. It seems to have severed the purpose best as even in case of tissue homogenate it gave well resolved peaks of (-) α-bisabolol. It does not involve any extraction techniques therefore it can be concluded that development of such method is not time saving but also it is economical. In future such researches can help in separation of pure herbal components and can also help to quantify traces of drug in biological samples directly. Authors Statements Conflicts of interest All authors have none to declare. ACKNOWLEDGEMENT The authors are highly grateful to Dr.Sayeed Ahmad (Assistant professor, Pharmacognosy) for guidance on method development.

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