研究用高效薄层色谱法从刀豆中定量分离鸭嘴花碱和胡椒碱

1、International Journal of Pharmaceutical Research2010, Volume 2, Issue 3, 14-17ISSN 0975-2366Research ArticlePatel RK, Kanani RJ, Patel VR*2, Patel MG3 1S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Kherva (Mehsana), Gujarat, India. 2Baroda College of Pharmacy, Paru

2、l Group of Institutes, Limda (Vadodara), Gujarat, India. 3Parul Institute of Pharmacy, Parul Group of Institutes, Limda (Vadodara), Gujarat, India.*Corresponding Author: E-mail: vish_rx, Phone: +91-9428386569.Received: 13/02010, Revised: 13/05/2010, Accepted: 01/06/2010ABSTRACTVasavaleha is a tradit

3、ional Ayurvedic oral Herbal formulation consisting of five herbs, Vasaka (Adhatoda vasica Nees.), Pippali (Piper longum Linn.), Sugar, Ghee and Honey. It is available as a popular proprietary, from most manufacturers of ayurvedic drugs. A selective, precise and accurate High Performance Thin Layer C

4、hromatography (HPTLC) method has been developed for the simultaneous quantification of Vasicine and Piperine in Vasavaleha as well as its bulk drug. The method employed TLC aluminum plate precoated with silica gel 60 F254 as a stationary phase. The solvent system consists of Dioxane: Toluene: Ethyl

5、acetate: Methanol: Ammonia (1.5:2:1:1:0.3 % v/v). This system was found to give compact spot for Vasicine and Piperine. Densiometric analysis was carried out in the absorbance mode at 285 nm. The linear regression anal-ysis data for the calibration plot showed good linear relation with r2 = 0.992 an

6、d 0.993 with respect to peak area for Vasicine and Piperine respectively, in concentration range 2-10 µg/spot. The method was validated for precision, recovery, Limit of Detection and Limit of Quantification. The proposed HPTLC method was found to be simple, precised and accurate and can be use

7、d for the quality control of the raw materials as well as formulations.KEY WORDS: Vasavaleha, Vasicine, Piperine, HPTLC.mens 7-8. Vasicine reference standard was procured from INTRODUCTONStandardization and analysis of the chemical marker Pioneer Enterprise, Mumbai and Piperine reference stan-of the

8、 Ayurvedic and other poly herbal formulation is al-dard was procured from Sigma Aldrich, Mumbai. Analyti-ways very big problem. Quantitative estimation of chemi-cal grade dioxane, toluene, ethyl acetate, methanol and cal markers of each ingredient in the poly herbal prepara-ammonia were obtained fro

9、m Merck chemical, India. tion required ideal separation technique by which thesemarkers are separated with highest purity and with least Instrumentationinferences from each other 1. For botanicals and herbal Analysis was performed on 10cm x 10cm plates cut preparations, there is a requirement for sc

10、ientific proof and from 20cm x 20cm aluminum-backed silica gel 60 F254 plates (E. Merck). Samples were applied to the plates by clinical validation with chemical standardization, biologi-means of a Linomat-V automatic spotter with the aid of cal assays, animal models and clinical trials 2.Hamilton 1

11、00 µl syringe. TLC plates were developed in Vasavaleha is a traditional Ayurvedic oral Herbalflat bottom twin trough chamber. Densitometry was per-formulation consisting of five ingredients, Vasaka (Adha-formed with a TLC scanner-3 with Win-CATS 4 software toda vasica Nees.), Pippali (Piper lon

12、gum Linn.), Sugar,resident in a Pentium IV computer. Ghee and Honey. It is available as a popular proprietary,from most manufacturers of ayurvedic drugs. It is mainlyused in cough, dysppnoea, asthma, tuberculosis, bleeding Sample preparationThe method of extraction of markers from Vasavaleha disorde

13、r, intercostals neuralgia, pleurodynia and anginadeveloped in laboratory was followed for sample prepara-pectoris.tion. 5 gm of Vasavaleha was taken in 100 ml of volume- In the present study, we report the development of atric flask and 50 ml of water was added. Content was simple, optimized and val

14、idated HPTLC method for themixed well and made alkaline with 0.1N KOH and allowed simultaneous estimation of Vasicine and Piperine in Vasa-to stand for 1 hour. 25 ml of chloroform was added to this valeha. Two chemical markers were selected, one fromand frequently shaken for 1 hour and allowed to st

15、and for each medicinal herbs used as raw materials, Vasicine from24 hrs. Transferred the content in separating funnel and Vasaka and Piperine from Pippali, for the quantificationmixed thoroughly. The chloroform layer was separated and purpose because these markers are responsible for the phy-rest of

16、 the solution was again extracted with more quanti-siological action of the plants 5-6. The method was vali-ties of chloroform at least three times. Combined chloro-dated on the basis of its selectivity, linearity, precision,form extract was evaporated and residue was redissolved in accuracy, limit

17、of detection (LOD) and limit of quantifica-25 ml methanol. The resulting solution was filtered by tion (LOQ) according to ICH requirements.Whatman filter paper and volume was reduced to 10 ml involumetric flask. This solution was used for the analysis of MATERIAL AND METHODboth the alkaloids in Vasa

18、valeha. 1 gm of powder of eachsample of Vasaka leaves powder and Pippali fruit powder Vasavaleha and its individual components were pro-was refluxed separately with 15 ml of methanol for two cured from a local market in Ahmedabad city, Gujarat andhours. Filtered, evaporated and dissolved the residue

19、 in 10 were authenticated by comparison with herbarium speci-1114 | IJPR | July-September Patel et al / International Journal of Pharmaceutical Research 2010 2(3) 14-17ml methanol. Prior to use, all samples were filtered through a 0.45 µm nylon membrane filter.Calibration Analysis was perf

20、ormed on 10 cm × 10 cm precoated silica gel 60 F254 TLC plate of uniform thickness Plates were prewashed by development with methanol then dried in an air. For preparation of the standard, curve 2 to 10 µl Linomat-V spotter overspotted volumes of each of the TLC standard solutions of Vasic

21、ine and Piperine. The plate was dried in air and densitometric scan was performed at 285 nm with Linomat-III Scanner. A calibration equation relat-ing to the standard concentration to scan areas was deter-mined by the use of a linear regression program on a per-sonal computer.Estimation of markers i

22、n Vasavaleha From the sample solutions, 5.0 µl was applied on the precoated silica gel plate and process was repeated to de-velop and scan the plate as mentioned above. The amount of Vasicine and Piperine in the samples was calculated from the calibration equation generated in previous chapter

23、by using the average area of triplicate sample aliquots.Validation parameter The method was validated according to ICH guideline for linearity, precision, accuracy, selectivity, limit of detec-tion and limit of quantification 9. Specificity of an analyt-ical method is its ability to measure the anal

24、yte accurately and specifically in the presence of component that may be expected to be present in the sample matrix. Test and stan-dard solution of Vasicine and Piperine were spotted on the TLC plate, developed and scanned as described above. The test chromatogram was compared with the standard. Li

25、-nearity of the method was performed by analyzing standard solution of Vasicine and Piperine by the proposed method in concentration range 2 to 10 µg/spot. Accuracy of the proposed method was determined by recovery study. Re-covery study was carried out by adding three different quantities of V

26、asicine and Piperine (1, 2 and 3 mg/gm) to preanalyzed sample of Vasavaleha. All the procedure was repeated five times as discussed above in sample prepara-tion. From the linear regression percentage recovery of Vasicine and Piperine were determined. Precision was determined by repeatability, intra

27、day and inter day repro-ducibility experiment of the proposed method. Intra day repeatability was evaluated by preparing and analyzing the standard solution of the drug six times in a day. The inter day reproducibility was determined by analyzing freshly prepared solution once a day over six consecu

28、tive days under same operative condition. Limit of detection and Quantification of Vasicine and Piperine were calculated visually by error and trial.RESULT AND DISCUSSIONQuantification of markers in Vasavaleha The two markers those were used for quantification were found in Vasavaleha. Vasicine and

29、Piperine formed a dark zone in a short UV light at 254 nm and showing brown red zone after sprayed with Dragendorffs reagent with an Rf of 0.45 ± 0.0051 and Rf of 0.83 ± 0.0065 respec-tively. The mobile phase comprised of Dioxane: Toluene: Ethyl acetate: Methanol: Ammonia (1.5:2:1:1:0.3) w

30、as used for the development of TLC plate which gave betterresolution of all the compounds among the mobile phase tried. The chromatogram of Vasavaleha was quantified with respect to Vasicine (10.78 ± 0.25 % w/w) and Piprine (6.12 ± 0.21 % w/w). The chromatogram of mixture of Vasicine and P

31、iperine standard markers (Figure 1) and Vasavaleha (Figure 2) were shown complete separation of two markers from other constituents. The results obtained are shown in Table 1.Table 1: Quantification of Vasicine and Piperine in Vasavaleha and its Ingredients by HPTLC.Sample Amount of Vasi-Amount of P

32、ipe-cinea (% w/w) rinea (% w/w)Vasaka powder 0.42 ± 0.023- Pippali powder - 1.52 ± 0.014 Vasavaleha10.78 ± 0.256.12 ± 0.21aMean ± SD (n=6)Figure 1: HPTLC Chromatogram of standard Vasicine and Piperine.Figure 2: HPTLC Chromatogram of Vasavaleha.Method validation for HPTLC fin

33、gerprinting method Linear correlation was obtained between peak areas and concentrations of Vasicine and Piperine in the range of 2-10 µg/spot. Characteristic parameters for regression equ-ation and correlation are given in Table 2. The linearity of the calibration graphs was validated by the h

34、igh value of correlation coefficients of the regression. The percent re-IJPR | July-September | 15Patel et al / International Journal of Pharmaceutical Research 2010 2(3) 14-17covery obtained was 91.11 ± 0.12 to 93.59 ± 0.11 % for that the proposed method is precise. The limit of detection

35、 Vasicine and 89.63 ± 0.14 to 92.89 ± 0.12 % for Piperine. (LOD) and Limit of Quantification (LOQ) of the drug was The results of recovery study are given in Table 3. For both calculated by signal to noise ratio. LOD and LOQ for Va-Vasicine and Piperine, Relative standard deviation of all

36、sicine and Piperine were found 0.4 µg/spot & 1 µg/spot the parameters is less than 2 % for the degree of repeatabil-and 0.5 µg/spot & 1.2 µg/spot respectively. Chromatogramity indicating the high repeatability of the proposed me-of Vasaka leaves (Figure 3) and Pippali fru

37、it (Figure 4)thod. The results of method precision data are given in was also taken and quantified as a standardized tool of raw Table 4 for Vasicine and Piperine. Value of % Relative material. Standard Deviation (RSD) of intra-day and inter-day revealTable 2: Regression Parameters for the analysis

38、of Vasicine and Piperine by HPTLC MethodParameterRange Slope InterceptRegression coefficient Regression EquationValue for Vasicine 2-10 (µg/spot) 1725 4578 0.992Y = 1725x + 4578Value for Piperine 2-10(µg/spot) 1237 1634 0.993Y=1237x + 1634Table 3: Recovery study of Vasicine and Piperine in

39、 Vasavaleha by HPTLC.Theoretical amount of drug taken (mg/gm)Amount of drug added(mg/gm)Practical amount of drug found (mg/gm)% Recovery ± S.D.(n=3)Vasicine Piperine Vasicine Piperine Vasicine Piperine Vasicine Piperine 2.15 1.22 1 1 2.87 1.99 91.11 ± 0.12 89.63 ± 0.14 2.15 1.22 2 2 3

40、.79 2.84 91.32 ± 0.15 88.19 ± 0.15 2.15 1.22 3 3 4.82 3.92 93.59 ± 0.11 92.89 ± 0.12Table 4: Precision of the Intra-daily and Inter-daily HPTLC measurement for Vasicine and Piperine.Intra-daily Inter-dailyCompound Content RSD Content RSD(% w/w) (%) (% w/w) (%) 10.78 ± 10.63

41、±Vasicine 1.54 3.120.25 0.31 6.12 ± 5.93 ±Piperine 2.17 2.210.21 0.12Mean ± SD (n=6) bSample were analyzed six times a day cSample were analyzed once a day over six consecutive daysFigure 4: HPTLC Chromatogram of Pippali.CONCLUSION The results indicate that Vasavaleha contains a

42、num-ber of markers that may be responsible for the therapeutic activity. The HPTLC method developed will assist in the standardization of Vasavaleha using biologically active chemical markers. The proposed HPTLC methods for si-multaneous estimation of Vasicine and Piperine from Va-savaleha, seems to be accurate, precise, reproducible and repeatable. It is the first attempts, when both the markers in Vasavaleha were simultaneously estimated and compared for the respective raw materials. Also profiles of the indi-vidual components in Vasavaleha have been re