|Year : 2013 | Volume
| Issue : 1 | Page : 10-14
Quality parameters, fatty acid profiling and estimation of umbelliferone in grahaṇimihira tailam: An ayurvedic oil preparation
M Deepak, Prakash U Kumar, B Nitha, Indira Balachandran, AB Rema Shree
Drug Standardisation Division, Centre for Medicinal Plants Research, Arya Vaidya Sala, Kottakkal, Malappuram, Kerala, India
|Date of Web Publication||18-Jun-2014|
A B Rema Shree
Centre for Medicinal Plants Research, Arya Vaidya Sala, Kottakkal Malappuram, Kerala
Source of Support: Department of AYUSH, Ministry of Health
and Family Welfare, Govt. of India., Conflict of Interest: None
Background: Grahaṇimihira tailam is an unexplored ayurvedic oil preparation which consists of 34 ingredients. The efficacy of this traditional ayurvedic medicine is undisputable. Proper clinical standardization of this formulation will go a long way in securing greater recognition for it. The main objective of this study was to develop standardization parameters for the formulation in a multidisciplinary way.
Materials and Methods: A simple and efficient method for the quantification of umbelliferone by high performance thin layer chromatography was developed and validated. Presence of the major fatty acids and their percentage were assessed by using gas chromatography-mass spectrometry (GC-MS). Various physio-chemical parameters, microbiological load, aflatoxins and mineral oil were also evaluated. Spread plate method was used for checking microbial contamination.
Results: The results were validated as per standard protocols. Quantitative estimation revealed the percentage of umbelliferone to be in the range of 0.88-0.98 (w/w). GC-MS analysis of sample led to the identification of 14 fatty acids, in which linoleic acid was obtained as the major fatty acid. Microbes, aflatoxins and mineral oils were found to be absent in the tailam.
Conclusion: The results which give the quantitative estimates of various physico-chemical parameters can be adopted to establish new standards for analysis of batch-to-batch variation and this data will facilitate shelf life studies in the future.
Keywords: Fatty acids, gas chromatography-mass spectrometry, grahanimihira tailam, high performance thin layer chromatography, physico-chemical parameters, umbelliferone
|How to cite this article:|
Deepak M, Kumar PU, Nitha B, Balachandran I, Rema Shree A B. Quality parameters, fatty acid profiling and estimation of umbelliferone in grahaṇimihira tailam: An ayurvedic oil preparation. Ancient Sci Life 2013;33:10-4
|How to cite this URL:|
Deepak M, Kumar PU, Nitha B, Balachandran I, Rema Shree A B. Quality parameters, fatty acid profiling and estimation of umbelliferone in grahaṇimihira tailam: An ayurvedic oil preparation. Ancient Sci Life [serial online] 2013 [cited 2020 Dec 4];33:10-4. Available from: https://www.ancientscienceoflife.org/text.asp?2013/33/1/10/134557
| Introduction|| |
Medicines in ayurveda are characterized by their state, appearance and action. Oil preparations have occupy a prime position among these medicines.  Grahan.imihira tailam (GRM) is a liquid preparation which uses 34 ingredients with Sesame oil as the basic ingredient. It is a medicated oil, light yellowish color with no specific odour. The major therapeutic uses reported are diarrhea, malabsorption syndrome, hiccup, fever, cough, dyspnea/asthma, jaundice, increased frequency and turbidity of urine, wrinkles on the skin, graying of hair, thrist, emesis, vertigo, pain in abdomen, piles, excessive flow of urine, edema, pain/colic, emaciation, oligospermia, threatened abortion and abortion.  The involvement of a large number of ingredients in their preparation makes all ayurvedic medicines difficult to study. It is due to this and due to inherent variability of chemical constituents of ingredients it is difficult to establish quality parameters. However, standardization of GRM is desirable for its greater recognition and acceptance. The present study has been undertaken to develop standardization protocols for the tailam with respect to the presence of major group of compounds and physico-chemical characters. The main objectives of the study include: Design of a simple and efficient high performance thin layer chromatography (HPTLC) quantification method for umbelliferone, an active coumarin and a reported antioxidant,  which has type-2 diabetes  and cancer  prevention activities. Umbelliferone is found as one of the major compounds in the tailam by thin layer chromatographic analysis, it was found stable through the preparation processes of tailam. Hence the compound was used as an analytical marker i.e., an effective tool in routine lab analysis and a measure of batch-to-batch variation. Identification of main fatty acids present in GRM was found as an important aspect to analyze the chemical composition. The percentage of major fatty acids present in GRM was analyzed and characterized by advanced gas chromatography-mass spectrometry (GC-MS) technique. The standardization study of GRM can never be achieved by one or two parameters and hence is preferable to achieve it in a multidisciplinary way. With this in mind, the study covers the chemical, biochemical and microbiological aspects of GRM. Tests for major phyto-constituents, various physico-chemical values, mineral oil, aflatoxin and microbiological screening were done. These studies are found to be essential in order to assess the quality and purity of the drugs. The HPTLC methods were standardized and validated according standard protocols.
| Materials and methods|| |
Three lab batches of tailam (GRM I, GRM II, GRM III) 500 ml each, were obtained from R and D division, Arya Vaidya Sala Kottakkal, Kerala. Three batches were made uniformly for examining the batch-to-batch variations. All solvents and chemicals used were of AR-grade and were obtained from Merck, Mumbai (India). Standard umbelliferone was purchased from Sigma Aldrich, Bangalore, India.
Quantification of umbelliferone
On a boiling water bath, 5 g of GRM was refluxed with 50 ml of 90% methanol by connecting a condenser and cooling it on a refrigerator for 1 h and filtered. This procedure was repeated thrice for complete extraction. These extracts were combined and the solvent was evaporated under reduced pressure and made up to 10 ml. sample solution which was used for HPTLC quantification, total phenolics assay and for preliminary phytochemical tests.
Preparation of standard
A stock solution of umbelliferone (0.1 mg/ml) was prepared by dissolving 1 mg of accurately weighed sample in methanol and making up the volume up to 10 ml. The stock solution was further diluted with methanol for working standard solution of 0.01 mg/ml.
HPTLC quantification was performed using CAMAG Switzerland. Stationary phase was aluminum backed pre-coated silica gel plates Merck 60 F 254 ( 0.2 mm thickness). Samples and standard were applied to the plate as bands at 10 mm from the bottom of the plate and 15 mm from the sides using CAMAG ATS 4. Development of the plate was carried out by CAMAG ADC 2. The plates were run up to 80 mm in ascending mode with solvent system Hexane: Acetone (6:4 v/v) at room temperature (28 ± 2°C) in a Twin Trough Chamber which was previously saturated with mobile phase. After development, the air-dried plate was scanned at 325 nm in CAMAG TLC SCANNER 3 using Deuterium lamp with Camag winCATS software. Nearly 60% of relative humidity.
Method validation and recovery
Limit of detection (LOD) and limit of quantification were analyzed for precision and sensitivity method. The accuracy of the method was established by performing recovery experiments at three different levels using the standard addition method.
GC-MS analysis of fatty acids
1 g of the sample was refluxed with 100 ml of 2% ethanolic potassium hydroxide for 1 h. The solvent was removed and diluted with water and washed with n-hexane two to three times to remove unsaponifiable matter. The aqueous portion containing saponified matter was collectedand acidified with dilute hydrochloric acid. The free fatty acids were extracted with diethyl ether two to three times. The combined ethereal layer was dried over anhydrous sodium sulfate. The solvent was removed. The mixed fatty acids were refluxed with methanol (100 ml) and concentrated sulfuric acid (5 ml) for 4 h. Methanol was removed and a saturated solution of potassium carbonate was added until the effervescence ceased. This was shaken with diethyl ether in a separating funnel. The collected ether layer containing methylated fatty acid was collected over anhydrous sodium sulfate. The solvent was filtered and. The methylated fatty acids were dissolved in a 10 ml volumetric flask and made up to the volume.
Source used was electron impact ionization. HP-5 MS column of 30 m length and 0.25 mm inner diameter was used. Carrier gas was helium with flow rate 1 ml/min. Column was programmed 150-180° C at 2° C rise per min, 200-250° C at 5° C rise per min. Injection temperature and interface temperature were 250° C and 270° C. Injection volume was 1 μl with split ratio 1:10. Electron volt and Detector volt were respectively 70 eV and 1.6 kV.
Various physico-chemical parameters which are specific for tailam preparations such as Saponification value, iodine value, acid value and peroxide value were evaluated. Saponification value is determined by titrating against hydrochloric acid. Iodine value was calculated by iodimetric titration. Acid value was evaluated by potassium hydroxide titration method. Peroxide value was determined by iodimetric method by titrating against sodium thio sulfate. All these parameters were determined as per pharmacopoeia of India guidelines.  Methanolic extract was subjected to qualitative phyto-chemical tests for screening major phyto-constituents such as carbohydrates, glycosides, flavonoids, alkaloids, sterols, phenolic compounds, tannins, saponins and amino acids. 
Tests for total phenolics, afflatoxin and mineral oil
The total phenolics assay was done as per Folin-Ciocalteu method.  Total phenolics content is expressed as mg Gallic acid Equivalents. Aflatoxin assay was carried out using HPTLC with Standards B1, B2, G1 and G2 and developed in the solvent system Chloroform: Acetone: Isopropyl alcohol (85:10:5).  Mineral oil presence in the sample was tested by Holde's method. 
Micro flora analysis
For detecting the presence of bacteria and fungi, 1 ml of GRM was aseptically transferred in to 9 ml of sterile distilled water and shaken vigorously. Appropriate dilutions were made and 0.1 ml from dilution was transferred aseptically to the surface of appropriate growth media, nutrient agar for bacteria and sabourauds dextrose agar for fungi. Triplicates of each sample were incubated at 37°C for bacteria and at room temperature for fungi. After incubation, the plates were observed visually and under the microscope for the detection of microbial growth. The plates were examined mainly for the presence of common contaminating bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella typhi, Pseudomonas sp., Klebsiella sp., Proteus sp. and also for common contaminants in the air such as aerobic spore forming bacilli. The presences of fungal contaminants such as Aspergillus niger, Aspergillus flavus, Penicillium sp., Rhizopus sp., were examined. 
| Results and Discussion|| |
The solvent system selected for the quantification after trials, Hexane: Acetone (6:4 v/v) was found to be a suitable system for getting desirable resolution. To identify the purity of umbelliferone in the sample, Rf and spectral data of umbelliferone in standard and sample compound were compared. TLC profile of sample with standard is shown in [Figure 1]. The peak purity of the umbelliferone was assessed by comparing the spectra, viz. peak start and peak apex and peak end positions of the spot. The Rf value and spectra of umbelliferone in the sample was found matching with the standard. Densitometric scanning profile at 325 nm of the sample and standard are given in [Figure 2]. To confirm the identity of the standard in the sample, the band of umbelliferone was scraped out and purified by recrystallization and a mass spectra analysis was performed. The mass obtained was 162.03, which is the same of that of umbelliferone.
|Figure 1: Thin layer chromatography profile of samples and standard. 1-3 test solution of grahaṇimihira tailam, 4-umbelliferone standard|
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|Figure 2: High performance thin layer chromatography densitometric scan of samples and standard at 325 nm|
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Calibration curve for umbelliferone was found to be linear in the range of 10-100 ng/spot in the analysis. Equation for the calibration curve is Y = 269.633 + 44.131X. A good correlation factor, r = 0.99986, was obtained between the standard and sample. Standard deviation obtained was 0.95%. The CV% obtained was 1.10. The amounts of umbelliferone found in samples were respectively 0.98, 0.88 and 0.93 (w/w). LOD and limit of quantitation were respectively 3 and 5 ng/ml. The value of mean percentage recovery was found to be 98.97, which indicates the accuracy of the chromatographic method [Table 1].
The GC-MS analysis of the saponified and methylated sample leads to the identification of 14 fatty acids. In this, linoleic acid (43%) was present in a higher amount when compared to others. Palmitic acid was the next major fatty acid (20.6%) in sample. The other major fatty acids are respectively myristic acid (8.9%), lauric acid (5.9%), eicosanoic acid (5.7%) and capric acid (3.4%). 99.18% of the total fatty acids were identified and 0.820% was left unidentified [Table 2].
Results of physico-chemical tests are given in [Table 3]. pH value of GRM shows its slight acidic nature. Saponification value is in the range of sesame oil. Iodine value of GRM was seen to be below the range of sesame oil. Peroxide value of GRM was less than one and it is an indication of low oxidation rate in GRM. Acid value is near four and it shows that fatty acid content in the formulation to be in range. Acid value and peroxide values are acceptable only if those values are below 6. Here the low acid and peroxide values showed that the GRM was fresh and of good quality. These values are specific for each tailams and can be used for the routine quality analysis and batch variations. The results of screening tests for major phytochemical groups show that the formulation contains major compounds such as alkaloids, carbohydrates, glycosides, sterols, phenolic compounds, tannins and flavanoids especially flavones in lower concentrations. However, major chemical groups such as amino acids, proteins and saponins were absent in GRM.
The total phenolic content by Folin-Ciocalteu method shows that sample (I) contains 0.445, sample (II) contains 0.441 and sample (III) contains 0.446 mg GAE/g. These results show that the tailam contains low level of poly phenolic compounds.
Aflatoxin assay with standards B1, B2, G1 and G2 by HPTLC method revealed their absence in the samples. The results reveal that samples are not contaminated with fungal species, especially those producing aflatoxin. Test for mineral oil in samples shows no turbidity during the experiment. The results indicate that GRM samples are free from adulteration with mineral oil, some of which have been proven to be carcinogens.
The results of the analysis for microbial contamination are presented in [Table 4]. No contaminating bacterial or fungal growths were observed in plates loaded with any of the dilutions after proper incubation. The study also reveals that the tailam is not contaminated with any of bacterial or fungal strains.
| Conclusion|| |
A simple, rapid HPTLC method for quantification for umbelliferone present in GRM tailam has been developed and validated as per standard protocols. The results were reproducible and consistent. The method can be utilized for the routine analysis and also for stability checking. Fatty acid profiling of the tailam was carried out for identification of major compounds and to analyze their respective percentage. Results of various physico-chemical parameters are enough to establish the quality standards for future reference and these will serve as an effective tool for stability studies. Absence of mineral oils, aflatoxins and microbes indicates the quality and purity of the formulation and it was essential to maintain safety.
| Acknowledgments|| |
The authors are greatly thankful to Department of AYUSH, ministry of health and family welfare, New Delhi and Arya Vaidya Sala, Kottakkal for providing financial support and facilities.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]