Indian Journal of Traditional Knowledge Vol. 14(4), October 2015, pp. 620-625 Simultaneous determination of ursolic acid and eugenol from Ocimum sanctum L. cultivated by organic and non-organic farming Prakash R Itankar a, Mohammad Tauqeer a, *, Jayshree S Dalal b & Pranali G Chatole a a Department of Pharmaceutical Sciences, Pharmacognosy and Phytochemistry, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur 440 033, Maharashtra, India; b Agnihotri College of Pharmacy, Ramnagar, Wardha 442001 Maharashtra, India E-mails:mohdtauqeer06@gmail.com; prakashitankar@hotmail.com; jayshree_2405@rediffmail.com; pranalichatole@gmail.com Received 20 May 2015, revised 7 September 2015 Worldwide revolution for the improvement of people safety is gaining momentum; hence drug safety for the subject becomes even more prominent in the present day scenario. Sothat, organic farming becomes a topic of general interest all over the globe. In the present study an attempt has been made to develop a simple, precise, rapid and cost-effective high performance thin layer chromatographic (HPTLC) method for simultaneous quantitative estimation of ursolic acid and eugenol in organically and non-organically cultivated Ocimum sanctum L.. The aqueous extracts of organically (OOS) & non-organically (NOS) cultivated Ocimum sanctum were investigated for its phyto-constituents using HPTLC studies. Linear regression data for the calibration curves of standards, viz. ursolic acid and eugenol showed a good linear relationship over a concentration range of 0.2-1.4 µg/spot, 1.0-7.0 µg/spot respectively with the correlation coefficient of 0.99909 and 0.99914 respectively and thus exhibits good linearity between concentration and area. The Ocimum sanctum (OOS) confirmed its better quality with reference to its higher contents of ursolic acid (0.17) and eugenol (1.77). The proposed HPTLC method was found to be rapid, simple and linear for quantitative estimation of ursolic acid and eugenol in the extracts. Keywords: Eugenol, HPTLC, Ocimum sanctum L., Organic, Non-organic, Ursolic acid IPC Int. Cl. 8 : A61K 36/00, C01, C07, A01, H01L 51/00 Biological Diversity Act (2002) and Rule (2004) enforced the noble thought of protecting our biodiversity especially crude drugs from wild origin 1. Therefore, it has become essential criterion for all the herbal industries and those working on medicinal plants, to produce these crude drugs by cultivation in the fields. A series of food scares and the controversy surrounding genetically modified crops have prompted heated debate about the safety and integrity of our food and herbal medicines. Against this background, demand for organically grown food has been growing rapidly 2. Increasing consciousness about conservation of environment as well as health hazards associated with agrochemicals and consumer s preference to safe and hazard-free food are the major factors that leads to the growing interest in alternate forms of Agriculture in the world. Organic agriculture is one among the broad spectrum of productive methods that are supportive to the environment. The demand for organic food is steadily *Corresponding author increasing both in the developed as well as developing countries with an annual average growth rate of 20 25 % 3. Until now this perception that, organically grown food and medicinal plants are better for you appears to have been largely based on intuition rather than conclusive evidence. Therefore, in the present study, an attempt has been made to develop a simple, rapid and accurate HPTLC method for estimation of ursolic acid and eugenol in aqueous extracts of organically (OOS) & non-organically (NOS) cultivated Ocimum sanctum. These constituents are considered to be the active components and can be considered as marker compounds. The analytical method is also validated for specificity, sensitivity, accuracy, precision, LOD and LOQ in accordance with ICH guidelines. Methodology Cultivation and harvesting The land for the organic treatment was selected and converted (period 2.5 yrs before 1 st harvest) 4 and the parallel area with marked buffer zone was selected for
ITANKAR et al.: SIMULTANEOUS METHOD VALIDATION OF URSOLIC ACID & EUGENOL IN OCIMUM SANCTUM 621 conventional treatment. The seeds were procured from Organic India Pvt. Ltd. Lucknow and cultivated by adopting the complete randomized statistical design in twelve replicates of each treatment. Two different treatments were utilized for organic and non-organic (in terms of fertilizer and insecticide/fungicide) 5,6,7. Aerial parts of Ocimum sanctum [OOS & NOS] were harvested at the time of maturity (flowering) and Botanically authenticated from the Department of Botany, Rashtrasant Tukadoji Maharaj, Nagpur University, Nagpur. Voucher specimens (specimen no. 9784) have been deposited for future reference. The aerial parts were cut into small pieces and lyophilised (Lyodel-00-12, Chennai, India). The material was pulverized, again lyophilized and stored in air tight containers for further use. Extraction and qualitative phytochemical screening The decoctions were prepared by extracting the well dried crude powder of different plant material (100 gm each) using an optimized method, with 1000 ml of Milli Q (MQ) water (BioAGE Direct Ultra, Punjab, India) at 80 o C for about 1hr with constant stirring. The aqueous portion was collected and the residual was extracted again with another 1000 ml of MQ water. The resulting cooled aqueous extracts were collected, combined, filtered by gauze, concentrated in vacuo (25 o C) and then lyophilized for drying. The final yield of OOS and NOS, were 21.44, 24.09 % w/w, respectively. The extracts were screened for the presence of polyphenols, saponins, unsaturated sterols, triterpenes, alkaloids, flavonoids, protein/amino acids and carbohydrates and/or glycosides with thin layer chromatography (TLC). Thin layer plates precoated with silica gel G (Merck, 0.25 mm thickness) were used. Development was carried out with different solvent systems such as ethyl acetate: methanol : water (100:13.5:10, v/v/v), ethyl acetate: formic acid : acetic acid : water (100:11:11:26), chloroform : methanol : water (70:30:4,), toluene : ethyl acetate : diethylamine (70:20:10), toluene: ethyl acetate: formic acid (6:3.5:0.5), pet ether: acetone (08:02), hexane: ethyl acetate (7.5: 2.5), toluene: ethyl acetate: glacial acetic acid (9:1:0.4), benzene: methanol (7.5:2.5) and ethyl acetate : methanol : water : acetic acid (65:15:15:10). After development of chromatogram in the solvents the plates were dried and sprayed with FeCl 3, AlCl 3, ninhydrin, vanillin-sulphuric and antimony trichloride for the detection of polyphenols, flavonoids, protein/amino acids, unsaturated sterols and triterpenes respectively. While detection of saponins, carbohydrate and/or glycosides was carried out using anisaldehyde-sulphuric acid, p-anisidine-hydrochloride and naphthoresorcinol reagent, respectively and visualization was carried out under visible and UV light (λ: 366 nm) (Fig. 1) 8,9. HPTLC quantification of ursolic acid and eugenol (Method Validation) The analytical method was validated for specificity, sensitivity, accuracy, precision, LOD and LOQ in accordance with ICH guidelines (ICH: Q2 (R1)) 10. Equipment and chromatographic condition A CAMAG HPTLC system equipped with a sample applicator Linomat V using 100 µl syringe and connected to a nitrogen tank; twin trough plate development chamber; CAMAG TLC scanner-3 with wincats software. The HPTLC plate, precoated with silica gel G 60 F 254 size 20 X 10 cm (Merck Ltd. Germany) was used. Six tracks of samples and fourteen tracks of standards were applied according to following settings: bandwidth 5 mm; distance between bands 4 mm. The plate was developed to 8 cm in a twin trough glass chamber, saturation time 30 min, separation technique ascending, scanning mode Absorbance/Reflectance and temperature 20±5 o C 11,12,13. Chemicals Standard Ursolic acid (Yucca Enterprises, Mumbai), Eugenol (Natural Remedies) and AR grade chemicals were used. Fig.1 Qualitative phytochemical screening of the plant extracts Represent (a) Presence of polyphenols: sprayed with Ferric chloride solution at visible light; (b) Saponins: sprayed with Anisaldehyde-sulphuric acid solution at visible light; (b) Sterols: sprayed with Vanillin-sulphuric acid solution at visible light; (C) Carbohydrates: sprayed with p-anisidine-hydrochloride solution at visible light; (D) Flavonoids: sprayed with Aluminium trichloride solution at 366 nm.
622 INDIAN J TRADIT KNOWLE, VOL. 14, No. 4, OCTOBER 2015 Preparation of standards and sample The stock solution of ursolic acid 0.2 mg/ml was prepared by dissolving the 2 mg of urolic acid in 10 ml of methanol. Similarly stock solution of eugenol 1 mg/ ml was prepared by dissolving the 10 mg of eugnol in 10 ml of methanol. The different amounts of standards, viz. 1 to 7 µl were applied. The OOS and NOS extracts 10 mg/ ml were prepared and different concentrations (1, 2 and 3 µl) of each was applied on HPTLC plate using a Camag Linomat V sample applicator. Calibration curve Calibration curve was constructed according to requirement of ICH guidelines. Each concentration was applied to a plate (20 X 10 cm) in triplicates of 5 mm band length with a distance of 4 mm between each two bands. The distance from the plate side edge was 13 mm and from the bottom of the plate was also 10 mm. The application rate was 5 µl/s, standard zones were quantified by linear densitometric scanning using Camag TLC scanner. Deuterium lamp was utilized as a source of radiation. Evaluation was done using linear regression analysis via peak areas and calibration curve was prepared by plotting peak area vs. concentration applied. Linearity The linearity of the HPTLC method was evaluated by analysing a series of different concentrations of the standards (Ursolic acid and Eugenol), where each concentration was applied in triplicate. Linear regression data for the calibration curves of standards showed a good linear relationship. Specificity The specificity of the method was determined by analysing the drug standard and test samples. The peak for test sample was confirmed by comparing its R f and spectrum with those of the standard. System precision The system precision was assessed by determination of six different concentrations of standards each applied in triplicate. Method precision (repeatability) Repeatability (precision) was determined by repeated analysis of standard samples using the same equipment, same analytical procedures and same laboratory and on the same plate. Repeatability of measurement was determined by spotting 2 µl of standard drug solution on HPTLC plate, after development spot was scanned six times without changing position. The % RSD was determined. Limits of detection and quantification Limit of detection and limit of quantification were validated based on signal to noise ratio where the minimum concentration at which the standard solutions can be reliably detected was recorded. Limits of detection (LODs) and limits of quantification (LOQs) were calculated using the expressions 3.3σ/s and 10σ/s, respectively, in which σ is intercept standard deviation and s is the slope of calibration curve. Recovery study The accuracy of proposed method was evaluated by addition of standard drug solution to extract at three different concentration levels at 50, 100, and 150 % of linearity. This parameter shows the proximity between the experimental values and the real ones. Results and discussion Qualitative phytochemical screening Both extracts showed the presence of carbohydrates, proteins, phytosterols, polyphenols, flavonoids and saponins (Fig. 1). Calibration curve and linearity Standard ursolic acid (R f : 0.49, Figs. 2 & 3), eugenol (R f : 0.76, Figs. 2 & 4) showed single peak in HPTLC plate and chromatogram. A calibration curve was constructed by plotting peak area against concentration applied Figs. 5 & 6. The results of regression analysis are shown in Table 1. The linear regression data for the calibration curves of standards ursolic acid and eugenol showed a good linear relationship over a concentration range of 0.2-1.4 µg/spot, 1.0-7.0 µg/spot respectively with the correlation coefficient of 0.99909 and 0.99914, respectively and linear regression equation was found to be: y = 5.734x-592.931, y = 567.949x-225.681, respectively, where y is the spot area and x is the concentration of the analyte. The 3-D chromatographs of all calibration concentrations and samples (OSS and NOS) are shown in Fig. 7. Specificity and sensitivity It was observed that other constituents present in the formulations did not interfere either with the peak of standards therefore the method was specific. The spectrum of respective standards and respective spots
ITANKAR et al.: SIMULTANEOUS METHOD VALIDATION OF URSOLIC ACID & EUGENOL IN OCIMUM SANCTUM 623 present in the samples was found to be similar or overlapped (Figs. 8 & 9). Limit of detection where the minimum concentration at which the standards solution can be reliably detected was recorded as 0.077 mg/spot, Fig. 6 Calibration curve of Eugenol. Fig. 2 HPTLC plate showing isolation of constituents Where HPTLC plate at visible light, sprayed with anisaldehyde sulphuric acid reagent representing OOS, NOS, UA and EU as organic & non-organic Ocimum sanctum extracts, ursolic acid and eugenol respectively. Fig. 7 3-D chromatographs of all calibration concentrations (UA & EU) and samples (OOS and NOS) Fig. 3 Scan densitogram of Ursolic acid Fig. 8 Overlapping spectra of Ursolic acid Fig. 4 Scan densitogram of Eugenol Fig. 5 Calibration curve of Ursolic acid. Fig. 9 Overlapping spectra of Eugenol
624 INDIAN J TRADIT KNOWLE, VOL. 14, No. 4, OCTOBER 2015 0.47 mg/spot and minimum concentration at which the analyte can be reliably quantified was found to be 0.234 mg/spot, 1.44 mg/spot for ursolic acid and eugenol respectively and was found to be highly sensitive for its determination. Accuracy The developed method showed high and consistent recoveries at all studied levels. The results obtained from recovery studies are presented in Table 2. The mean % recovery ranged from 99.16 % to 101.69 % for ursolic acid and 99.92 % to 102.11 % for eugenol. Additionally, the obtained recoveries were Table 1 Results of regression analysis of linearity data and sensitivity of Ursolic acid and Eugenol Parameter Ursolic acid Eugenol Linearity range (mg/spot) Detection wavelength Mobile phase 0.2-1.4 µg/spot 1.0-7.0 µg/spot 560 nm 560 nm Toluene: Ethyl acetate: Glacial acetic acid (09:01:0.4) Rf value 0.49 0.76 Regression equation y = 5.734x- 592.931 Slope 5.734 567.949 Intercept 592.931 225.681 Correlation coefficient Limit of detection (mg/spot) 0.99909 0.99914 0.077 0.477 Limit of 0.234 1.44 quantitation (mg/spot) Intraday 1.12 1.15 precision (RSD, %)** Interday precision (RSD, %)** 1.25 1.37 **Average of six determinations. Toluene: Ethyl acetate: Glacial acetic acid (09:01:0.4) y = 567.949x-225.681 found to be normally distributed with low % RSD ( 1.261) at 50, 100 and 150 % concentration levels. Precision % RSD values for inter-day variation were not more than 0.43 and 0.10 for intra-day variation were not more than 0.37 and 0.80 for ursolic acid and eugenol, respectively. Low % RSD values indicated the good repeatability and intermediate precision of the method as shown in Table 3. Analysis of ursolic acid in organic and non-organic sample of Ocimum sanctum The above validated method was used for the analysis of the ursolic acid and eugenol in the organic and non-organic sample of Ocimum sanctum, results presented in Table 4. The good performance of the method indicated the suitability of this method for routine analysis of mentioned constituent s determination in pharmaceutical dosage forms. Advantages of developed method The literature survey revealed that there is no official HPTLC method reported in major pharmacopeias like USP, EP, BP and IP for determination of ursolic acid and eugenol in pharmaceutical formulation. It can be seen that this simple and reproducible HPTLC method has enough sensitivity to evaluate preformulation samples, stability samples and pharmaceutical dosage form of ursolic acid and eugenol at low cost. Thus this presented work will meet the industrial and research laboratories requirements. A new, simple, and sensitive HPTLC method has been successfully developed and validated for determination of ursolic acid and eugenol in bulk and pharmaceutical dosage form. The method was found to be accurate, precise, and reproducible. Thus this developed and validated method will help the industries as well as researchers for their sensitive Marker Ursolic acid Eugenol Initial amount (µg/spot) Amount added (µg /spot) Table 2 Results of recovery study Amount of marker added in extracts (µg /spot) Amount Recovered*(%) Recovery +SD*(%) % RSD 0.368 50 % 0.184 101.6963 1.398205 0.249073 0.368 100 % 0.368 99.16072 1.080877 0.148101 0.368 150 % 0.552 100.1045 1.245903 0.135283 1.7 50 % 0.85 99.92044 0.021845 0.857348 1.7 100 % 1.7 102.1188 0.057963 1.669417 1.7 150 % 2.55 101.2991 0.044897 1.042862
ITANKAR et al.: SIMULTANEOUS METHOD VALIDATION OF URSOLIC ACID & EUGENOL IN OCIMUM SANCTUM 625 Table 3 Results of precision study Marker Concentration Interday Intraday % Recovery (±SD) %RSD % Recovery (±SD) %RSD Ursolic 800ng 98.63+1.69 0.43 98.89+1.48 0.37 acid Eugenol 4µg 100.39+2.13 0.10 98.59+1.77 0.80 Table 4 Results of analysis of Ursolic acid and Eugenol in organic and non-organic formulation Extracts Marker Quantity (gm/100gm) OOS Ursolic acid 0.17 Eugenol 1.77 NOS Ursolic acid 0.16 Eugenol 0.66 determination of ursolic acid and eugenol rapidly at low cost. Therefore, the present study scientifically validated the perception, organically grown food and medicinal plants are better for you in terms of level of phytoconstituents using Ocimum sanctum. Acknowledgment Authors are thankful to CEO, Organic India Pvt. Ltd. Lucknow for providing seeds. The authors are grateful to Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur for Memorial Research Fellowship. References 1. Hasan MR & Solaiman AHM, Efficacy of organic and organic fertilizer on the growth of Brassica oleracea L. (Cabbage), Int J Agric Crop Sci, 4 (3) (2012) 128-138. 2. Holdan P, Organic Farming, Food Quality and Human Health: A Review of the Evidence, (Association Soil, United Kingdom, Bristol), 1945, 07-09. Available from: http://www.soilassociation.org/linkclick.aspx?fileticket=cy 8kfP3Q%2BgA%3D&tabid=388. 3. Ramesh P, Singh M & Subba Rao A, Organic farming: Its relevance to the Indian context, Curr Sci, 88 (4) (2005) 561-564. 4. Yadav AK, Training Manual Certification and Inspection Systems in Organic Farming in India, (Government of India, Ministry of Agriculture, Department of Agriculture and Cooperation, National Centre of Organic Farming, Ghaziabad, India), 2011, 17-18. 5. Farooqi AA & Sreeram BS, Cultivation of Medicinal and Aromatic Crops, (Universities Press Pvt. Ltd, Hyderabad, India), 2004, 205-209. 6. Itankar PR, Tauqeer M & Chatole P, Morpho-physiological traits, proximate, nutritional, anti-nutritional and phytochemical analysis of organic & non organic Krishna Tulsi, In: Health Challenges and Ayurveda, Proc 6 th World Ayurveda Congress & Arogya Expo, 2014, 300. 7. Itankar PR & Tauqeer M, Proximate, toxicological and HPTLC analysis of organically and non-organically grown Momordica charantia, In; 1 st International Congress of the Society for Ethnopharmacology on Globalizing Traditional Medicine: Present and Future Prospects, 08-09 th March 2014 (Faculty of pharmacy, Sri Ramachandra University, Porur, Chennai, India), 66-67. 8. Itankar PR, Lokandhe SJ, Verma PR, Arora SK, Sahu RA, et al., Antidiabetic potential of unripe Carrisa carandas Linn. fruit extract, J Ethnopharmacol, 135 (2011) 430-435. 9. Harbone JB, Phytochemical Methods to Modern Techniques of Plant Analysis, (Chapman and Hall, London), 1976, 38-80. 10. ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology Q2 (R1), 2005. 11. Vyas J, Itankar P, Tauqeer M, Kelkar A & Agrawal M, Development of HPTLC method for estimation of piperine, guggulsterone E and Z in polyherbal formulation, Pharmacog J, 5 (2013) 259-264. 12. Stahl LE, Thin Layer Chromatography: A Laboratory Hand Book, 2 nd edn, (Springer, New York), 1969, 52-105, 857. 13. Sonawane SD, Nirmal SA, Patil AN & Pattan SR, Development and validation of HPTLC method to detect curcumin and gallic acid in polyherbal formulation, J Liq Chromatogr Relat Technol, 34 (2011) 2664-2673.