Amla (Emblica officinalis) is one of the oldest oriental medicines mentioned in Ayurveda as potential remedy for assorted ailments. It is an ingredient of many Ayurvedic formulations which are widely used in the Ayurvedic system of medicine. Various formulations of amla like amla churna, amalaki rasayan and triphala churna are believed to increase defense against diseases. Use of these formulations is increasing day by day. Increase in demand of these formulations due to their various therapeutic uses led to the availability of a number of brands in the market. Being a plant based formulations these are highly vulnerable to adulteration and contamination that can finally alter the efficacy of the product and may pose serious health risks for consumers.
Studies of chemical and biochemical properties of these formulations are important to ensure quality control of these formulations. Hence, in the present investigation studies on chemical and biochemical properties of various marketed formulations of Emblica officinals were performed to assess the quality of various market formulation of Emblica officinals. Three formulations of Emblica officinals i.e. amla churna, amalaki rasayan and triphala churna from five reputed brands were analyzed for physico-chemical parameters like loss on drying, ash, acid-insoluble ash, water and alcohol soluble extractive to verify the compliance as per Ayurvedic Pharmacopoeia of India (API) guidelines.Quantitative phytochemical estimations were performed to estimate the total phenolics and total flavonoids content in these formulations. Biochemical studies were carried out to confirm antioxidant , anti-inflammatory and anti diabetic activities of these formulations. This study will enhance the faith on Ayurvedic and herbal formulations to fight against diseases. In this study various physicochemical and phytochemical parameters were studied to assess the quality of the marketed product. Morphological and microscopic characteristics of the Triphala powder samples showed the adulteration of powdered endocarp of the ingredients.
A variation was also observed in pH and moisture values. Excluding one or two samples, ash values were found within permissible limits. Samples of all categories of manufacturers were found contaminated with various fungal species and the majority of them exceeding the permissible limit of 103spores/g for the medicinal formulation of internal uses as set by the World Health Organization. A remarkable variation in therapeutically important phytoconstituents was also observed among the samples of popular brands. Findings of this study suggest for formulation of stringent quality control guidelines for herbal formulations so that maximum benefits can be obtained from these traditional formulations.
The world is witnessing an unprecedented growth in the usage of herbal products. India is a mother hub for natural herbs based science. Herbal drug technology is used for converting botanical materials into medicines, where standardization and quality control with proper integration of modern scientific techniques and traditional knowledge is important. Ayurveda is a well known Indian traditional health care system. Several medicinal plants are used in the preparation of Ayurvedic formulations named Rasayana, recommended for their interesting antioxidant activities (Reference). Phyllanthus embelica Linn. (syn. Emblica officinalis), commonly known as Amla and Indian gooseberry is widely used in Ayurvedic system of medicine.
Amla belongs to the family of Euphorbiaceae and is known for its medicinal and therapeutic properties from ancient time in India and also considered as a wonder fruit for health conscious population (Reference). Amla tree grows in the mixed-deciduous dry forests of India, from northwest Himalayas (Jammu and Kashmir, Himachal Pradesh and Uttaranchal) to eastern Himalayas in Assam, Meghalaya, Manipur and Tripura. It is a seasonal fruit and is highly perishable. Its storage life in atmospheric conditions after harvesting is only 5 to 6 days (Pathak et al., 2009). It is used as ingredient in many herbal formulations. In Ayurvedic system it is often used in the form of Triphala, Amla churna and Amalaki rasayan. Triphala is an herbal formulation containing fruits of Emblica officinalis, Terminalia chebula and Terminalia belerica in equal proportions.
Amalaki Rasayana is a classical Ayurvedic medicine prepared from Amalaki (Embelica officinalis). It is basically Amla powder processed in Amla juice for enhancing its therapeutic properties. Amalaki Rasayana strengthens each and every part of body due to its rejuvenating property. This medicine is not only rejuvenating but curative also. It boosts immunity and balances Vata-Pitta and Kapha and hence gives relief in diseases caused due to vitiation of any of the humor. Amalaki Rasayan imparts energy. As the name itself suggests it is a Rasayana drug of Ayurveda. Amla churna is fine powder of dries fruits of Emblica officinalis and is used as food supplement as well as in the form of medicine. The efficacy of any formulations depends on the quality of the formulation. Reproducible efficacy and safety of herbal products is based on reproducible quality. Standardization and quality control of these formulation is important in justifying their acceptability in any system of medicine. Also If herbal formulations are to be regarded as rational drugs, their pharmaceutical quality must be properly studied (Bauer et al., 1994).
Material and Reagents
Folin ciocalteau reagent, sodium carbonate, sodium nitrite, methanol, aluminum chloride, potassium acetate, bovine serum albumin (BSA), acetyl salicylic acid, gallic acid, quercetin, 1,1-diphenyl picrylhyrazyl (DPPH), potassium ferricyanide, ferric chloride, sodium phosphate, ammonium molybdate, sulfanillic acid (SA), ascorbic acid, diamine tetraacetic acid (EDTA), ferrozine, sodium nitroprusside (SNP), napthyldiamine dichloride (NED), sodium nitro-pruside (SNP), Ascorbic acid, tris-HCl, sodium acetate and ferrous chloride were purchased from Merck. Ethanol, petroleum ether, methanol, hydrochloric acid (HCl), sulfuric acid (H2SO4), chloroform, ammonia, glacial acetic acid, sodium hydroxide, di-sodium phosphate, phosphoric acid and acetone were of AR grade purchased from Loba Chemie.
Instruments and Conditions
UV-Visible analysis was performed by using the Shimadzu-1800s (range from 190 – 1100 nm) at Regional Ayurveda Research Institute for Drug Development, Gwalior. Quartz cuvettes were used in whole experiment having capacity of 3 mL Analytical balance, muffle furnace, water bath and pH meter were used in experiments.
Total 15 formulations (Five different brands of Amla Churna, Amalaki Rasayan and Triphala Churna from) were collected from local market of Gwalior District, Madhya Pradesh. Coding and the details of formulations are given in Table -1. The samples were stored at room temperature (25 ± 2) °C in order to be used conveniently in the study.
Preparation of Aqueous Extract
For the analysis of biochemical properties (Total Phenolic content , Total Flavonoid content ,Antioxidant activity, Anti-inflammatory activity and Antidiabetic activity), an aqueous extract of each formulation was prepared by 4 g of sample mixed with 100 mL of double distilled water. Extract was filtered by using Whatman filter paper and stored in fridge (4 °C) for further analysis.
Determination of Physicochemical Parameters
Determination of the pH
One gram of powdered sample was added to 100 mL of distilled water, stirred and filtered. pH of this filtrate was measured by using the calibrated pH meter.
Determination of Loss on Drying at 105 °C
4 gram of the sample was weighed accurately in a previously weighed petridish. Then it was heated in an oven at 105 ºC for 5 hours. Weight was taken after cooling the petridish in desiccators. The procedure was repeated till constant weight was obtained. Loss on drying was calculated using the following formula: Percentage of Loss on drying at 105°C = R/S×100 (where R; weight of sample after heating at 105 oC and S; weight taken initially).
Determination of Total Ash
4 gram of the air dried sample was weighed accurately in a previously ignited and tarred Silica dish. The material was spread evenly and ignited in a muffle furnace at 600 ºC until it is white, indicating the absence of carbon. Then the dish was cooled in a desiccator and weighed. If carbon free ash cannot be obtained by this manner, the residue on the cooled dish was moisten with about 2 mL of water or a saturated solution of Ammonium nitrate, dried on a water-bath, and then ignited in the muffle furnace up to constant weight. The total ash percentages were calculated using the following formula: Total ash % = R/S×100 (where R; weight of ash and S; weight of sample).
Determination of Acid insoluble-ash
Total ash of the sample as described above was determined out first. To the dish containing the total ash, 25 mL of 1:5 hydrochloric acid in five portions of 5 mL was added each time, boiled gently for 5 minutes and filtered. The insoluble matter was collected on an ash less filter paper (Whatman No. 41), and washed with distilled water until the residue was free from acid. The filter paper containing the insoluble matter was transferred to the original dish, dried and ignited to constant weight. After cooling the dish in desiccators, the weight was taken. The percentage of Acid insoluble – ash was calculated using the following formula: Acid insoluble ash % = R/S×100 (where R; weight of acid insoluble ash and S; weight of sample).
Determination of Water Extractive Value
Accurately 4 g of the sample was weighted in a glass stopper flask. 100 ml of distilled water was added and shaked occasionally for 6 hours and then allowed to stand for 18 hours. Filtered rapidly taking care not to lose any solvent . 25 ml of the filtrate was pipette out in a pre-weighed 100 ml beaker and evaporated to dryness on a water bath. Kept in a hot air oven at 105 oC for 6 hours, cooled in a desiccator and weighed . Experiment was repeated twice, and average value was taken. Percentage of water-soluble extractive = (Weight of the extract × 10000/ 25 × Weight of the sample taken).
Determination of Alcohol-soluble extractive
Accurately 4 g of the sample was weighted in a glass stopper flask. 100 ml of Alcohol (approximately 95%) was added ad shaked occasionally for 6 hours and then allowed to stand for 18 hours. Filtered rapidly taking care not to lose any solvent . 25 ml of the filtrate was pipette out in a pre-weighed 100 ml beaker and evaporated to dryness on a water bath. Kept in a hot air oven at 105 oC for 6 hours, cooled in a desiccator and weighed . Experiment was repeated twice, and average value was taken. Percentage of alcohol-soluble extractive = (Weight of the extract × 10000/ 25 × Weight of the sample taken).
Total Phenol Content
Total phenol content of aqueous extract was analyzed using Folin-ciocalteu assay [Singleton et al;1965]. Briefly, 1 mL of sample of varying concentrations was incubated in 5 mL of Folin-ciocalteu reagent and 4 mL of 1 mol/L Na2CO3. After 15 min of incubation, absorbance was measured at 765 nm by spectrophotometer (Shimadzu UV-8400). Gallic acid dissolved in 50% ethanol was used as standard. The total phenolic content was reported in terms of µg of gallic acid equivalents/g of formulations (GAEs).
Total Flavonoids Content
The total flavonoid content was estimated by using the aluminum chloride colorimetric assay (Zhishen et al; 1999)Briefly, 0.3 mL of 5% (w/v) NaNO2 was added to every 4 mL of extract solution of varying concentrations. After 5 min of incubation, 0.3 mL of 10% (w/ v) AlCl3 was added. After 6 min, 2 mL of 1 mol/L NaOH was added. The total volume was made up to 10 mL with distilled water. After shaking for 1 min, the absorbance was noted at 510 nm. The total flavonoids content was reported in terms of µg of quercetin equivalents/g of formulations.