Asian Journal of Pharmaceutical Science & Technology e-issn: 2248 9185 www.ajpst.com Print ISSN: 2248 9177 TLC AND HPLC FINGERPRINT DEVELOPMENT OF TRACHYSPERMUM AMMI LINN. AND ITS POLYHERBAL MARKETED FORMULATIONS, AJMODADI CHURNA Ramanjaneyulu K 1*, Shrivastava A.K 2, Singh Nitu 3 1 Department of Quality assurance, NIMS Institute of Pharmacy, NIMS University, Jaipur, Rajasthan, India. 2 Department of Pharmaceutical Chemistry, NIMS Institute of Pharmacy, NIMS University, Jaipur, Rajasthan, India. 3 Department of Pharmacognosy, NIMS Institute of Pharmacy, NIMS University, Jaipur, Rajasthan, India. ABSTRACT The most important challenges faced by herbal formulations arise because of their lack of complete evaluation. Evaluation is necessary to ensure quality and purity of the herbal product. The present study deals with the standardization of Trachyspermum ammi and its marketed polyherbal formulations, Ajmodadi churna of four different companies. It contains eleven herbal ingredients in which Trachyspermum ammi is one of the main ingredient and it is used in the treatment of indigestion, constipation and to improve appetite. In this research paper, an attempt has been made to develop standardization method based on the TLC and HPLC fingerprinting profile of Trachyspermum ammi and its marketed polyherbal formulations, Ajmodadi churna of four different companies. The fingerprinting profile of Trachyspermum ammi is taken as a reference standard in comparing with four marketed formulations of Ajmodadi churna. The main objective of the present study was focused on identification of Trachyspermum ammi present in polyherbal formulations of Ajmodadi churna based on TLC and HPLC fingerprinting profiles. Key words: Trachyspermum ammi, Ajmodadi churna, Polyherbal formulation, TLC, HPLC, standardization. INTRODUCTION Herbal drugs have been in use by different civilizations in different parts of the world for Centuries to fight a large number of diseases. India can emerge as the major country and play the lead role in production of standardized, therapeutically effective ayurvedic formulations [1]. Herbal formulations show the number of problems when quality aspect is considered. This is because of nature of the herbal ingredients and different secondary metabolites present therein. Mainly, variation in the chemical profile of the herbal due to intrinsic and extrinsic factors (growing, harvesting, storage and drying processes) [2-4]. Chromatographic fingerprint have been suggested to check for authenticity or provide quality control of herbal medicine [5]. Chromatography has the advantage of separating a complicated system into relatively simple subsystems and then presenting the chemical patterns of herbal medicine in the form of a chromatogram. The World Health Organization (WHO) accepts fingerprint chromatography as an identification and quality evaluation technique for medicinal herbs since 1991 [6]. Fingerprints can be a unique identification utility for herbs and their different species. The Govt. of India has also adopted the fingerprint approach for botanicals because it supports the traditional concept and is easy to practice at different levels of sophistication [7, 8]. Trachyspermum ammi Linn. (Umbelliferae), known in India as Ajowan, is widely distributed in northern part of the India. In India, the fruit are used as remedy for indigestion and colic and also used in poultices to relieve asthma and arthritis. It is also having aphrodisiac properties. It is used in a steeped liquid form against diarrhea and flatulence. It is mostly used for indigestion and dyspepsia [9]. It is the main ingredient of Ajmodadi churna. Ajmodadi churna is a polyherbal ayurvedic medicine used as a carminative and an antispasmodic, is a strong wormifuge, and helps in all painful conditions like sciatica and stiffness in back and also restores normal digestive functions [10]. Trachyspermum ammi is recognized as the marker compound in Ajmodadi churna formulations. There are some reports on the application of TLC (Thin layer chromatography), HPLC (High performance liquid Corresponding Author: Ramanjaneyulu K E-mail: kadari.ramanjaneyulu@gmail.com 40 P a g e
chromatography) methods for the analysis of Trachyspermum ammi, but attempts to apply these techniques for the fingerprinting profiling of Trachyspermum ammi in polyherbal formulations are not available. The main objective of the present study was focused on identification of Trachyspermum ammi present in polyherbal formulations of Ajmodadi churna based on TLC and HPLC fingerprinting profiles. In the present research article TLC and HPLC Chromatogram of Trachyspermum ammi is taken as standard for comparing its fingerprinting profile with four marketed formulations of Ajmodadi churna. These formulations contain Trachyspermum ammi as main ingredient. In this research work qualitative analysis was done based on the fingerprinting profile of standard plant in comparison with its formulations. MATERIALS AND METHODS Chemicals and Reagents The entire chemicals used in the experiment were of analytical grade. All the solvents used in the experiment were procured from RFCL Pvt.LTD, New Delhi, India. Plant material Trachyspermum ammi was (See Figure No.6) procured from the local market of Jaipur, Rajasthan, India from the ayurvedic store Jagram Gangasahay, Tripolian bazaar, Jaipur, shop number.362 and their identity was confirmed by correlating their morphological microscopical characters with their literature review [11]. Preparation of Powder Crude drug has taken and roasted in a stainless steel pan at low temperature till it becomes free from Moisture. The sample of Trachyspermum ammi (seeds) was powdered in a pulverizer and pass through sieve number 80. It is packed in tightly closed containers to protect from light and moisture. Marketed formulations The marketed samples of various brands of Ajmodadi churna i.e., Jamuna pharmaceuticals, Krishna pharmaceuticals, Navjeewan pharmaceuticals & Sadhana chemicals (See Figure No.7) were used in the present research work which were purchased from a registered ayurvedic Pharmacy in Jaipur, Rajasthan. Extraction procedure The powdered churna of Trachyspermum ammi and its marketed formulations of Ajmodadi churna was soxhleted with methanol for 72 hrs. After completion of soxhlation, residue was removed and filtration followed by the evaporation of solvent and extract was concentrated and these extracts were used for TLC and HPLC analysis [12]. Preparation of sample solution 1ml of the methanolic extract was taken and transferred to the 10ml clean and dried volumetric flask and diluted to 10ml with methanol. Then the volume was made up to the mark with methanol. From the above freshly prepared sample, 2ml was pipetted out and transferred to the clean and dried 25ml volumetric flask. Finally the volume was made up to the mark with methanol. INSTRUMENTATION AND CHROMATOGRAPHIC CONDITIONS Thin Layer Chromatography Slurry of silica gel G was prepared in distilled water and poured on glass to form a thin film. The prepared plates were allowed for setting (air-drying). After setting, the plates were kept in an oven at 100 to 120C (30 min) for activation [13]. The extracts of samples were applied to the activated plates (1cm above from the bottom). It was then kept in previously saturated developing chamber containing mobile phase, and allowed to run 3/4th of height of the plate. The developed plate was removed, air dried and kept plate in Iodine vapor chamber and observes spots and Rf values were noted [14] and they were showed in Table 1 in results and discussion. Mobile phase: Toulene: Ethyl acetate (7:3v/v) Spraying reagent : Iodine vapors High Performance Liquid Chromatography In this study, a simple, rapid, precise, accurate and economical high performance liquid chromatography (HPLC) method has been used for qualitative analysis. HPLC chromatograms were obtained when methanolic extracts of Trachyspermum ammi and its marketed polyherbal formulations, Ajmodadi churna was introduced into the sample loop. The HPLC analysis was done by using reverse phased column and by using suitable pump. Fingerprinting profiles of sample was done by using suitable mobile phase at definite flow rate. The column effluent was monitored with detector. The detector was connected to a computer and the data were analyzed by software. The detector detected peaks for standard plant extract and its formulation extracts at particular wave length based on these peaks we have confirmed the presence or absence of Trachyspermum ammi (standard plant) in marketed polyherbal formulations of Ajmodadi churna and they were showed in Figure no-1-5 and Table no-2 in results and discussion. Chromatographic conditions Instrument name : Shimadzu (SPD10AT-VP) HPLC system Software: Empower software Column: Ultracarb C 8 Mobile phase: Methanol: Water: Acetic acid (70:30:0.1% v/v) Sample volume: 20 μl 41 P a g e
Flow rate: 1ml min-1 Total run time: 30 min Detector : U.V- Visible detector Wave length: 280nm RESULTS AND DISCUSSION Methanolic extracts of Trachyspermum ammi and its formulations were subjected for TLC and their Rf values are given in Table No: 1. The standard plant Trachyspermum ammi showed the Rf value of 0.77 which is corresponding to Rf value for thymol which is the major constituent of Trachyspermum ammi and other Rf values are 0.68, 0.64 which may be corresponding to carvacrol, limonene which are the minor constituents of Trachyspermum ammi. Ajmodadi churna manufactured by the Jamuna Pharmaceuticals showed the Rf values of 0.70, 0.69, 0.68 these values are not matched with the Rf value of Trachyspermum ammi, so based on this it was found that it is obsent in Jamuna. The Rf values of Ajmodadi churna manufactured by Krishna Pharmaceuticals, Navjeewan Pharmaceuticals and Sadhana chemicals showed Rf values which are matched with Rf value of Trachyspermum ammi. Based on these Rf values of formulations we had concluded that Trachyspermum ammi is obsent in only Jamuna and other three formulations contains Trachyspermum ammi. Table 1. TLC Rf values for methanolic extract of Trachyspermum ammi and its formulations S.No Methanolic extract Rf values Spots 1 Trachyspermum ammi Spot2 0.68 Spot3S 0.64 Spot1 0.70 2 Jamuna Spot2 0.69 Spot 3 0.68 3 Krishna Spot 2 0.69 Spot 3 0.64 4 Navjeewan Spot 2 0.63 Spot 3 0.60 5 Sadhana Spot 2 0.69 Spot 3 0.63 CHROMATOGRAMS Figure 1. HPLC Chromatogram of Trachyspermum ammi Figure 2. HPLC Chromatogram of Ajmodadi churna (Jamuna pharmaceuticals) 42 P a g e
Figure 3. HPLC Chromatogram of Ajmodadi churna (Krishna Pharmaceuticals) Figure 4. HPLC Chromatogram of Ajmodadi churna (Navjeewan Pharmaceuticals) Figure 5. HPLC Chromatogram of Ajmodadi churna (Sadhana chemicals) 43 P a g e
Table 2. Comparative HPLC Rt and % area values of various Phytoconstituents of Trachyspermum ammi and its formulatioms Trachyspermum Jamuna Krishna Navjeewan Sadhana ammi S. NO R t Range Rt % Area Rt % % % Rt % Area Rt Rt Area Area Area 1 1.4-1.5 --- --- 1.455 0.2 --- --- --- --- --- --- 2 1.6-1.7 --- --- 1.630 95.7 --- --- --- --- --- 3 1.7-1.8 --- --- --- --- 1.722 93.6 --- --- --- --- 4 2.4-2.5 --- --- --- --- 2.455 5.7 --- --- --- --- 5 2.8-2.9 --- --- 2.848 0.2 --- --- --- --- --- --- 6 3.1-3.2 --- --- 3.318 0.1 --- --- 7 3.7-3.8 --- --- --- 3.788 0.6 --- --- --- --- 8 4.1-4.2 4.153 20.83 --- --- --- 4.178 97.25 9 4.2-4.3 --- --- --- --- --- --- 4.427 97.54 --- --- 10 4.7-4.8 4.747 26.50 --- --- --- --- --- --- 11 5.1-5.2 --- --- 5.185 0.3 --- --- --- --- --- --- 12 6.3-6.4 6.487 1.14 --- --- --- --- --- --- --- --- 13 6.4-6.5 6.055 0.71 --- --- --- --- --- --- --- --- 14 6.5-6.6 --- --- --- --- --- --- --- --- 6.532 0.71 15 6.7-6.8 --- --- --- --- 6.750 2.268 --- --- --- --- 16 6.8-6.9 --- --- 6.825 2.1 --- --- --- --- --- --- 17 7.7-7.8 7.723 0.6 --- --- --- --- --- --- --- --- 18 9.9-10 --- --- 9.913 0.2 --- --- --- --- --- --- 19 10.8-10.9 --- --- 10.847 0.3 --- --- --- --- --- 20 11.6-11.7 --- --- --- --- --- --- --- --- 11.640 0.25 21 14.5-14.6 --- --- --- --- --- --- --- --- 14.062 0.49 22 15.1-15.2 --- --- --- --- --- --- --- 15.152 1.29 23 15.6-15.7 --- --- --- --- --- 15.695 0.53 --- --- 24 16.4-16.5 --- --- --- --- 16.40 82.268e- 020.1 --- --- --- --- 25 16.5-16.6 --- --- 16.583 0.3 --- --- --- --- --- --- 26 17.0-17.1 --- --- --- --- --- --- 17.070 1.24 --- --- 27 18.3-18.4 18.342 50.47 --- --- --- --- --- --- --- 28 22.8-22.9 --- --- --- --- --- --- 22.838 0.23 --- --- 29 26.8-26.9 --- --- --- --- --- --- 26.867 0.46 --- --- 30 37.1-37.2 37.190 0.23 --- --- --- --- --- --- --- --- 31 42.7-42.8 42.082 0.12 --- --- --- --- --- --- --- --- In the chromatogram obtained from seeds of Trachyspermum ammi after the methanolic extracts of the principle peak at Rt 4.153 min. corresponding to 20.83% area. Since to be peak of thymol which is the most important ingredient of Trachyspermum ammi while the peak at 4.7 min. corresponding to 26.50% area. Since to be carvacrol and thymol are found in the above values other constituents paracymene, since to have it Rt value 18.3 min. whether corresponding area is 15.47%. This peak may possible resulted peak contains paracymene, terpene, and limonene. While the myrcene possibly appears later shown in the table of chromatogram of Trachyspermum ammi. These active principles are found in the Trachyspermum ammi under RP-HPLC conditions. Ajmodadi Churna is prepared with many polyherbal fruits and seeds with closely related drugs and their Phytoconstituents. All the chromatograms showed peak area upto 97.545-97.25% and therefore the peaks starting from Rt of 4.4 min. and upto near about 4.5 min. and ending at Rt at 7 min. may be of importance, this is cumulative peak of Phenols including thymol, carvacrol and possibly other Phenols from other drugs and other constituents showing the showing the higher values then calculated levels. Herbal formulations do not show most similarity in their HPLC chromatograms. The highest % area peak has been observed at 4.427 min. made for within % area of 97.5 Ajmodadi churna manufactured by Navajeevan Pharmaceuticals. The Rt of 4.178 min with an area of 92.2% 44 P a g e
Ajmodadi churna manufactured by Sadhna Chemicals. Ajmodadi churna manufactured by Jamuna Pharmaceuticals shows the best period of 1.630 min. with an area of 97.5%. Ajmodadi churna manufactured by Krishna Pharmaceuticals shows the best period of 1.722 min. with an area of 93.6%. Therefore we can conclude the peak between 4.0 4.3 is a conclusive peak per standardization of Ajmodadi churna. However the further studies are required to make an exhaustive statement. Ajmodadi churna manufactured by Jamuna and Krishna Pharmaceuticals shows the absence of Trachyspermum ammi. Therefore the possibly the constituents are altered and hence Jamuna and Krishna shows different types of peaks. The characteristic peak was observed in the previous statement which is absent in the Jamuna and Krishna which leads to doubt on quality of drug. The other formulations of Ajmodadi churna that means Sadhna and Navajeevan showed the presence of Trachyspermum ammi in it. Figure 6. Seeds of Trachyspermum ammi Figure 7. Marketed formulations of Ajmodadi churna Jamuna pharmaceuticals Krishna pharmaceuticals Navjeewan pharmaceuticals Sadhana chemicals 45 P a g e
CONCLUSION Therefore on the observation of the results and discussions it can be concluded that the standardization of herb Trachyspermum ammi in formulations can be done on the basis of chromatographic patterns of plant (Trachyspermum ammi) in comparing with its marketed formulations of Ajmodadi churna. Ajmodadi churna manufactured by Jamuna and Krishna Pharmaceuticals shows the absence of Trachyspermum ammi. Therefore the possibly the constituents are altered and hence Jamuna and Krishna shows different types of peaks. The characteristic peak was observed in the previous statement Trachyspermum ammi is absent in the Jamuna which leads to doubt on quality of drug which is may be due to the variations in geographical conditions of various plants present in Ajmodadi churna manufactured by Jamuna and Krishna pharmaceuticals during at the time of collection.the other formulations of Ajmodadi churna manufactured by Navajeevan pharmaceuticals and Sadhna chemicals showed the presence of Trachyspermum ammi in it. ACKNOWLEDGEMENT The authors are thankful to the Dr. Balvir Singh Tomar, Chancellor and Dr. K.C. Singhal, Vice Chancellor, NIMS University, Jaipur for providing necessary facilities to carry out the research work. REFERENCES 1. Organisation Mondiale De La Sante. Quality control methods for medicinal plant materials. Original English, World Health Organisation, 1, 1999, 159. 2. Gilani AH, Rahman AJ. Ethno Pharmacology, 100, 2005, 43-49. 3. WHO. Traditional Medicine Strategy, World Health Organization; Geneva. 2002-2005. 4. Zidorn C, Schubert B, Stuppner H. Biochem Sys and Eco, 33, 2005, 855-72. 5. Layloff T, Scientific Fingerprinting: A Pharmaceutical Regulatory Tool. Pharm.Technol, 15, 1991, 146-148. 6. WHO, Guidelines for the Assessment of Herbal Medicine, Munich, World Health Organization, 1991. 7. CHMP, Guideline on Quality of Herbal Medicinal Products/Traditional Herbal Medicinal Products, Committee for Medicinal Products for Human use, European Medicines Agency Inspections, 2005. 8. Welsh WJ, Lin WK, Tersigni SH, Collantes E, Duta R, Carey MS. Anal. Chem, 68, 1996, 3473. 9. Anilakumar KR, et al. Food and Chemical Toxicology, 47, 2009, 279-82. 10. http://www.ayurvedicdietsolution.com. 11. Anonymous.The Ayurvedic Pharmacopoeia of India Govt. of India M.H & F.W Dept. of Health, 1, 1990, 170-71. 12. Gupta AK. Introduction to Pharmaceutics-1, CBS Publications: 148-155. 13. Kasture AV, et al. Pharmaceutical Analysis. Nirali Prakashan Publications, 2, 9th edition, 2010, 18-25. 14. Skoog DA, Hollar JF, Nieman T. A Principle of Instrumental Analysis, Thomson Learning Inc., Singapore, 1998, 110, 300. 46 P a g e