Chapter 8 TLC Densitometric Quantification of Vasicine, Vasicinone and Embelin from Adhatoda zeylanica leaves and Embelia ribes fruits 8.1 INTRODUCTION With the global increase in the demand for plant derived medicine, there is a need to ensure the quality of the herbal drugs. Plants are a complex mixture of a variety of chemical constituents that can vary considerably depending on genetic and environmental factors, method of cultivation, time of collection, post-harvest processing, etc. This inherent variability in the chemistry may adversely affect the efficacy of medicinal plants. Hence it is a pre-requisite to ensure that the plants used for therapy or for research purposes are of a quality that gives the desired efficacy and that the quality is maintained at each re-collection of the plant materials. To meet this requirement it is essential to establish qualitative and quantitative chemoprofiles of the samples. The present chapter describes the TLC fingerprint profile of the methanolic extract of Adhatoda vasica leaf and Embelia ribes fruit, co-chromatography with the bioactive compounds vasicine, vasicinone and embelin respectively and its quantification using TLC densitometry. 8.2. EXPERIMENTAL 8.2.1 Chemicals All solvents and chemicals used were of analytical grade. 8.2.2 Apparatus (a) Spotting device CAMAG Linomat V Automatic Sample Spotter (b) Syringe 100 µl (Hamilton) (c) TLC Chamber CAMAG glass twin trough chamber (20 10 4 cm) (d) Densitometer CAMAG TLC Scanner 3 linked to wincats software 132
(e) TLC plates precoated silica gel 60 F 254 TLC plate (E. Merck, Cat. no. 1.05554.0007) (0.2 mm thickness) 8.2.3 Plant material collection and authentication The leaves of Adhatoda zeylanica and fruits of Embelia ribes were collected from Gujarat, India. Their authenticity was confirmed by the taxonomist Dr. Sheetal Anandgiwala of our department and a voucher specimen was deposited at the Department of Pharmacognosy and Phytochemistry, B. V. Patel Pharmaceutical Education & Research Development (PERD) Centre, Ahmedabad, India. The material was stored in air tight containers at room temperature until use. 8.2.4 TLC analysis of Adhatoda zeylanica leaves 8.2.4.1 TLC fingerprint profiling Preparation of sample solution 1 g of the powdered plant material was extracted with methanol (25 ml 2) under reflux on a water bath for 30 min. The extracts were filtered, pooled, concentrated and the volume was made up to 25 ml with methanol. Preparation of standard solutions 2 mg of vasicine (purity 98.5 %) standard was dissolved in methanol and the volume was made up to 25 ml in a volumetric flask. 2 mg of vasicinone (purity 97.5 %) standard was dissolved in methanol and the volume was made up to 25 ml in a volumetric flask. Mobile phase Ethyl acetate: Methanol: Ammonia (8: 2: 0.2) Procedure 10 µl each of the sample solution and the standard solutions were applied on a precoated silica gel 60 F 254 TLC plate (E. Merck) of uniform thickness (0.2 mm) and the plate was developed in a twin trough chamber containing mobile phase Ethyl acetate: methanol: ammonia (8: 2: 0.2) up to a distance of 8 cm at 25 ± 2 C temperature and 40 % relative humidity. The plate was dried at room temperature and scanned at 292 nm for vasicine and 233 nm for vasicinone. 133
The plate was observed under UV light at 254 nm and the plate was derivatized with modified Dragendorff s reagent (Wagner and Bladt, 1996). The R f and colour of the resolved bands were noted. 8.2.4.2 Estimation of vasicine and vasicinone using TLC densitometric method Preparation of sample solution 1 g of the powdered plant material was extracted with methanol (25 ml 2) under reflux on a water bath for 30 min. The extracts were filtered, pooled, concentrated and the volume was made up to 25 ml with methanol. Preparation of standard solutions of vasicine and vasicinone A) Standard solution of vasicine- 5 mg of vasicine standard was dissolved in 25 ml of methanol in a volumetric flask. From this stock solution standard solutions of 320-960 µg/ml were prepared by transferring aliquots (1.6 to 4.8 ml) of stock solution to 10 ml volumetric flasks and adjusting the volume to 10 ml with methanol. B) Standard solution of vasicinone - A stock solution of vasicinone was prepared by dissolving 2 mg of accurately weighed vasicinone in methanol and making up the volume to 25 ml with methanol. From this stock solution standard solutions of 80-480 µg/ml were prepared by transferring aliquots (0.5 to 3 ml) of stock solution to 10 ml volumetric flasks and adjusting the volume with methanol. Calibration curve for vasicine and vasicinone 10 µl of each of the standard solutions of, vasicine and vasicinone were applied in triplicate on TLC plate. The plates were developed in a solvent system of Ethyl acetate : Methanol : Ammonia (8 :2 :0.2 v/v) at 25±2 C temperature and 40 % relative humidity, upto a distance of 8 cm. After development, the plate was dried in air and scanned at 292 nm for vasicine and 233 nm for vasicinone. The peak areas were recorded. Calibration curves of vasicine and vasicinone were prepared by plotting peak areas vs concentration. Quantification of vasicine and vasicinone in the sample 10 µl of sample solution was applied in triplicate on a TLC plate. The plate was developed and scanned as described above. The peak areas were recorded. The amount 134
of vasicine and vasicinone in sample was calculated using the linear regression equation derived from the calibration curve of vasicine and vasicinone respectively. 8.2.4.3 Method validation International Conference on Harmonization (ICH) guidelines were followed for the validation of the analytical procedures (CPMP/ICH/281/95 and CPMP/ICH/381/95). The method was validated for precision, repeatability and accuracy. Instrumental precision was checked by repeated scanning of the same spot of vasicine (400 ng) and vasicinone (160 ng) seven times and was expressed as coefficient of variance (% CV). The repeatability of the method was affirmed by analyzing 400 ng/spot of standard solution of vasicine and 160 ng/spot of standard solution of vasicinone after application on the TLC plate (n = 6) and was expressed as % CV. Variability of the method was studied by analyzing aliquots of standard solution of vasicine (320, 400, 480 ng/spot) and vasicinone (160, 240, 320 ng/spot) on the same day (intra-day precision) and on different days (inter-day precision) and the results were expressed as % CV. Accuracy of the method was tested by performing recovery studies at three levels (50 %, 100 % and 125 % addition). The percent recovery and the average percent recovery were calculated. For the determination of limit of detection and limit of quantification different dilutions of the standard solutions of vasicine and vasicinone were applied along with methanol as the blank and determined on the basis of signal to noise ratio. 8.2.5 TLC analysis of Embelia ribes fruit 8.2.5.1 TLC fingerprint profiling Preparation of sample solution 1 g of the powdered plant material was extracted with methanol (25 ml 2) under reflux on a water bath. The extracts were filtered, pooled, concentrated and the volume was made up to 25 ml with methanol. Preparation of standard solutions 2 mg of embelin (purity 95 %) standard was dissolved in methanol and the volume was made up to 25 ml in a volumetric flask. Mobile phase n Propanol : n Butanol : Ammonia (7 : 1 : 2) 135
Procedure 10 µl each of the sample solution and the standard solutions were applied on a precoated silica gel 60 F 254 TLC plate (E. Merck) of uniform thickness (0.2 mm) and the plate was developed in a twin trough chamber containing mobile phase n Propanol : n Butanol : Ammonia (7 : 1 : 2) up to a distance of 8 cm at 25 ± 2 C temperature and 40 % relative humidity. The plate was dried at room temperature and scanned at 333 nm for embelin. The plate was observed under UV light at 254 nm and the R f of the resolved bands were noted. 8.2.5.2 Estimation of embelin using TLC densitometric method Preparation of sample solution 1 g of the powdered plant material was extracted with methanol (25 ml 2) under reflux on a water bath for 30 min. The extracts were filtered, pooled, concentrated and the volume was made up to 25 ml with methanol. Preparation of standard solution of embelin A stock solution of embelin was prepared by dissolving 2 mg of accurately weighed embelin in methanol and making up the volume to 25 ml with methanol. From this stock solution standard solutions of 80-480 µg/ml were prepared by transferring aliquots (0.5 to 3 ml) of stock solution to 10 ml volumetric flasks and adjusting the volume with methanol. Calibration curve embelin 10 µl of the standard solution embelin was applied in triplicate on TLC plate. The plates were developed in a solvent system of n Propanol : n Butanol : Ammonia (7 : 1 : 2) at 25±2 C temperature and 40 % relative humidity, up to a distance of 8 cm. After development, the plate was dried in air and scanned at 333 nm for embelin. The peak areas were recorded. Calibration curve of embelin was prepared by plotting peak areas vs concentration. Quantification of embelin in the sample 10 µl of sample solution was applied in triplicate on a TLC plate. The plate was developed and scanned as described above. The peak areas were recorded. The amount 136
of embelin in sample was calculated using the linear regression equation derived from the calibration curve of embelin. 8.2.5.3 Method validation International Conference on Harmonization (ICH) guidelines were followed for the validation of the analytical procedures (CPMP/ICH/281/95 and CPMP/ICH/381/95). The method was validated for precision, repeatability and accuracy. Instrumental precision was checked by repeated scanning of the same spot of emeblin (320 ng) was expressed as coefficient of variance (% CV). The repeatability of the method was affirmed by analyzing 320 ng/spot of standard solution of embelin after application on the TLC plate (n = 6) and was expressed as % CV. Variability of the method was studied by analyzing aliquots of standard solution of embelin (160, 240, 320 ng/spot) on the same day (intra-day precision) and on different days (inter-day precision) and the results were expressed as % CV. Accuracy of the method was tested by performing recovery studies at three levels (50 %, 100 % and 125 % addition). The percent recovery and the average percent recovery were calculated. For the determination of limit of detection and limit of quantification different dilutions of the standard solutions of vasicine and vasicinone were applied along with methanol as the blank and determined on the basis of signal to noise ratio. 8.3. RESULTS AND DISCUSSION 8.3.1 TLC fingerprint profiling of Adhatoda zeylanica leaves The constituents other than the biomarkers may be contributing to the therapeutic effect of the plant either by synergizing or adding to the effect of the active ingredients, or by antagonizing their adverse effects, or by potentiating their bioavailability (Hamburger and Hostettman, 1991) Therefore, as far as the quality of a medicinal plant drug is concerned, a systematic consideration of all its phytoconstituents is as important as the quantification of the active constituents present in it. TLC fingerprint profile is a systematic documentation of all the constituents of a sample extract resolved in a given chromatographic system. It provides a semi-quantitative sketch of the chemoprofile of the plant extract. The methanolic extract of Adhatoda zeylanica leaf was used for TLC fingerprint profiling and co-tlc with vasicine and vasicinone. The methanolic extract showed the 137
best possible resolution of its constituents on precoated silica gel TLC plates, when developed in mobile phase containing Ethyl acetate : Methanol : Ammonia (8 :2 :0.2 v/v). The developed TLC plate, when observed under UV light showed bands at 254 nm and R f value of which are mentioned in (Table 8.1; Figure 8.1). Table 8.1: TLC details of sample solution of Adhatoda zeylanica leaf. Under UV light at 254 nm (R f value) 0.13 0.27 0.62 (Vasicine) 0.72 0.81 (Vasicinone) 0.92 8.3.2 Quantification of biomarkers vasicine and vasicinone 1 2 3 4 5 1, 2 3 4 5 Figure 8.1 TLC profile of Adhatoda zeylanica leaf. 1: Vasicine standard (0.8µg) ; 2: Vasicinone standard (0.8µg); 3,4,5: Sample solution (0.4µg, 0.8µg, 1.6µg) As far as the analysis of herbal raw materials and herbal preparations is concerned, HPTLC has an edge over other instrumental analytical techniques because it is simple, economical and requires minimum sample clean-up. That is why HPTLC has emerged as an efficient tool for the phytochemical evaluation of herbal drugs (Rajani et al., 2001; Quality Standards of Indian Medicinal Plants, 2003). A densitometric technique scanned at 298 nm and 233 nm was therefore adopted for the quantification of vasicine and vasicinone respectively in the leaf of Adhatoda zeylanica leaf using HPTLC. Of the various solvent systems tried, the one containing Ethyl acetate: Methanol: Ammonia (8 : 2 : 0.5 v/v) gave the best resolution of vasicine (R f = 0.62) and vasicinone (R f = 0.81) in the presence of other compounds in the sample extract. The methods were validated in terms of precision, repeatability and accuracy (Table 8.2). The linear relationship between peak area and amount of vasicine applied was found only within the range of 320-960 ng/spot with correlation coefficient 0.999 and standard deviation 3.62. The linear relationship between peak area and amount of vasicinone applied was found only within the range of 80-480 ng/spot with correlation coefficient 0.999 and standard deviation 3.62.The intra-day and inter-day precision 138
expressed as % CV (Table 8.3) indicate that the proposed method was precise and reproducible. The limit of detection for vasicine was found to be 40 ng and the limit of quantification was found to be 80 ng. The average of percentage recovery at three different levels was found to be 99.14 % (Table 8.3). The limit of detection for vasicinone was found to be 20 ng and the limit of quantification was found to be 40 ng. The average of percentage recovery at three different levels was found to be 98.45 % (Table 8.4). The amount of vasicine and vasicinone in Adhatoda zeylanica leaf was found to be 1.25 % (w/w) and 0.039 % (w/w) respectively as quantified by the proposed method. A B Figure 8.2: A. TLC densitometric scan at 298 nm of Adhatoda zeylanica leaf methanolic extract (blue line) along with vasicine standard (purple line). B. Overlay of UV absorption spectra of the vasicine in the sample track with the vasicine standard A 4444444444444444444444444444444444444 B Figure 8.3: A. TLC densitometric scan at 233 nm of Adhatoda zeylanica leaf methanolic extract (orange line) along with vasicinone standard (pink line) B. Overlay of UV absorption spectra of the vasicinone in the sample track with the vasicinone standard 139
Table 8.2: Method validation parameters for the quantification of vasicine and vasicinone by the proposed method Parameters Vasicine Vasicinone Instrumental precision 0.39 1.55 (% CV, n = 7) Repeatability of standards 0.13 1.62 (% CV, n = 6) Accuracy 99.14 98.45 (average % recovery) Limit of detection 40 ng 20 ng Limit of quantification 80 ng 40 ng Specificity Specific Specific Linearity (Correlation coefficient) 0.999 0.999 Range (ng/spot) 320-960 80 480 Table 8.3: Intra-day and Inter-day precision of vasicine and vasicinone Marker compound Concentration Intra-day Inter-day (ng spot -1 ) Precision Precision Vasicine Vasicinone 320 0.57 0.42 400 0.47 0.30 480 0.68 0.25 160 1.51 1.55 240 0.92 1.55 320 0.65 1.07 140
Table 8.4 Recovery study of vasicine and vasicinone by the proposed TLC densitometric method Marker compound Amount present in the sample (µg) Amount added (µg) Amount found a (µg) Recovery a (%) Average recovery (%) Vasicine Vasicinone 1250 625 1855.58±2.22 98.96±0.46 1250 1250 2480.8±5.99 99.23±1.07 1250 1562.5 1852±14.13 99.24±0.69 39 19.5 47.90±0.01 98.25±0.54 39 39 76.85±0.01 98.52±1.01 39 48.75 86.79±0.02 98.90±0.73 99.14 98.45 a Mean ± Standard deviation, (n = 3). 8.3.3 TLC fingerprint profiling of Embelia ribes fruits The methanolic extract of Embelia ribes fruit leaf was used for TLC fingerprint profiling and co-tlc with embelin. The methanolic extract showed the best possible resolution of its constituents on precoated silica gel TLC plates, when developed in mobile phase containing n-propanol : n-butanol : Ammonia (7 : 1 : 2 v/v). The developed TLC plate, when observed under UV light showed bands at 254 nm and R f value of which are mentioned in (Figure 8.4). Table 8.5: TLC details of sample solution of Embelia ribes fruit Under UV light at 254 nm (R f value) 0.35 (Embelin) 0.40 0.62 0.68 1 2 Figure 8.4: TLC profile of Embelia ribes fruit. 1: Embelin; 2: Sample solution 141
8.3.4 Quantification of biomarker embelin Of the various solvent systems tried, the one containing n-propanol: n-butanol: Ammonia (7 : 1 : 2 v/v) gave the best resolution of embelin (R f = 0.35) in the presence of other compounds in the sample extract. The methods were validated in terms of precision, repeatability and accuracy (Table 8.6). The linear relationship between peak area and amount of embelin applied was found only within the range of 320-960 ng/spot with correlation coefficient 0.993 and standard deviation 4.12. The intra-day and inter-day precision expressed as % CV (Table 8.7) indicate that the proposed method was precise and reproducible. The limit of detection for embelin was found to be 20 ng and the limit of quantification was found to be 40 ng. The average of percentage recovery at three different levels was found to be 99.47 % (Table 8.8). The amount of embelin in Embelia ribes fruit was found to be 3.21 % w/w as quantified by the proposed method. A B Figure 8.5 A. TLC densitometric scan at 337 nm of Embelia ribes fruit methanolic extract (blue line) along with embelin standard (purple line). B. Overlay of UV absorption spectra of the embelin in the sample track with the embelin standard. 142
Table 8.6: Method validation parameters for the quantification of embelin by the proposed method Parameters Embelin Instrumental precision 0.83 (% CV, n = 7) Repeatability of standards 0.46 (% CV, n = 6) Accuracy 99.47 (average % recovery) Limit of detection 20 ng Limit of quantification 40 ng Specificity Specific Linearity (Correlation coefficient) 0.993 Range (ng/spot) 80 480 Table 8.7 Intra-day and Inter-day precision of embelin Marker compound Embelin Concentration (ng spot -1 ) Intra-day precision a Inter-day precision a 160 1.27 1.59 240 0.45 1.11 320 0.55 0.93 Table 8.8 Recovery study of embelin by the proposed TLC densitometric method Marker compound Amount present in the sample (µg) Amount added (µg) Amount found a (µg) Recovery a (%) Average recovery (%) Embelin 3210 1605 4793.5 ± 2.22 99.55 ± 0.46 3210 3210 6381.9 ± 5.99 99.40 ± 1.07 3210 4012.5 7184.2 ± 4.13 99.46 ± 0.69 99.47 a Mean ± Standard deviation, (n = 3) 143
8.4 CONCLUSION The developed HPTLC methods for the quantification of vasicine, vasicinone and embelin were found to be simple, precise, specific and sensitive. The amount of vasicine and vasicinone in Adhatoda zeylanica leaf was found to be 1.25 and 0.039 % (w/w) respectively. The amount of embelin in Embelia ribes fruit was found to be 3.21 % (w/w). 144