Amritamehari churnam (AC) is an antidiabetic polyherbal formulation constituting of four herbal medicinal plants namely Phyllanthus emblica , Salacia reticulata , Tinospora cordifolia and Curcuma longa . The feasibility of using gamma irradiation at doses between 2.5 and 10 kGy to reduce microbial load and enhance shelf life of this formulation was investigated. The irradiated and non-irradiated products were stored at room temperature (25–32 °C and 50–85% R.H., 1.5 years). Acceptability of the irradiated product was assessed based on sensory, microbial, physical and chemical attributes as well as their antioxidant status. A dose 7.5 kGy was sufficient to maintain microbial quality within acceptable limit up to 18 months of storage. No significant differences in sensory properties were observed between the non-irradiated and irradiated sample. The applied dose did not cause any significant qualitative and quantitative changes in the chemical constituents, antioxidant activities as well as physical properties when measured by EPR spectroscopy.

Herbs are prone to contamination by microorganisms and insect pests during processing and storage. This shortens their shelf life as well as in rare cases cause major illness particularly if the herbs are in combination with food poisoning organisms such as Salmonella and Staphylococcus aureus . Adoption of Good Manufacturing Practices (GMP) can reduce the contamination during processing and storage to a great extent. Conventional decontamination methods using heat and chemicals can reduce contaminant substantially. However these methods also result in changes in flavor as a result of loss/degradation of labile volatile aroma components with a consequent reduction in the quality of the herb. Food irradiation is a relatively new environmental-friendly technology that can aid in enhancing food safety, quality and trade ( Diehl, 1995 ). Available reports have clearly demonstrated that exposure of food commodities to ionizing radiation (gamma/electron beam) is an effective method for disinfestation, decontamination, overcoming quarantine barriers in international trade and for improving nutritional attributes and shelf life ( Hong et al., 2008 ).

Due to their low moisture content irradiation of herbs at higher doses, up to 10 kGy, results in considerable improvement in the microbial quality. Effect of radiation processing on the chemical and microbial quality of herbs has been extensively investigated ( Alothman, Bhat, & Karim, 2009 ). The quality parameters of herbs and spices do not change appreciably after radiation processing. An enhancement in aroma and flavoring properties as well as extractability of active principles has been noted in several cases. This results in positive impact on the quality of the final herbal preparation. Very few reports, however, exists on the effect of radiation processing on the quality of herbal formulations.

Diabetes mellitus is a very common disease that has been implicated to be the main cause of death in human population throughout the world including India. Several antidiabetic drugs and herbal remedies are used for treatment of diabetes. One such promising antidiabetic herbal drug widely used in clinical practice in Ayurveda is herbal formulation-Amritamehari churnam (AC). The product is a mixture of Phyllanthus emblica , Salacia reticulata , Tinospora cordifolia and Curcuma longa . The formulation is prone to microbial contamination on storage and thus has a low shelf life. Radiation processing as a method for extension of shelf life of this product was thus attempted.

AC (4 Kg) samples (mesh size 40) procured from Kottakal Ayurvaidyashala, India, was divided into two sets (1 Kg each). One set was kept as non-irradiated sample. The other set of samples, packed in high density polyethylene bags of 0.12 mm thickness were irradiated in a gamma chamber with cobalt-60 source (GC 5000, BRIT, DAE, Mumbai) to doses of 2.5, 5, 7.5 and 10 kGy at the dose rate of 6.01 kGy/h. Gamma Chamber was calibrated using Fricke dosimeter. Irradiated and corresponding non-irradiated samples were stored under ambient conditions (25–32 °C, 50–85% R.H.) for 2 years. Samples were taken at predetermined storage intervals (every three months) were analyzed in triplicate for the analysis of chemical constituents and antioxidant activities. For EPR studies, powdered samples were grouped into three lots. One served as control, second lot was irradiated at 2.50–10.0 kGy. The third lot was subjected to thermal treatment at 100 °C for 1 h using a laboratory oven to study the induced radicals by thermolysis ( Sanyal, Chawla, & Sharma, 2009 ). During the time kinetics study of the paramagnetic species for a period of 90 d, irradiated, thermally treated and non-irradiated samples were stored inside EPR quartz tube under normal laboratory conditions at ambient temperature (25–32 °C). Three replicates of each sample were evaluated. All chemicals were purchased from Sigma–Aldrich Chemicals, USA. Solvents (s.d Fine Chemicals Ltd., India) were redistilled before use.

Testing was carried out by an experienced sensory panel (15 members) using a 9 point scale with 1, dislike extremely or not characteristic of the product and 9, like extremely or very characteristic of the product. Values less than 4 were considered to be unacceptable to the consumers. Parameters evaluated were color, aroma, texture and overall acceptability. The sensory evaluation was done at every three months of storage intervals.

Standard methods were used to enumerate microorganisms present at each sampling time and treatment for 2 years of storage. The mesophilic bacteria, yeast and mold counts were carried out using Plate Count Agar and the pour plate method. The sample (5 g) was homogenized in 45 ml of sterile physiological saline. Appropriate dilutions were pour plated using plate count agar. The colonies were counted after 24 h of incubation at 37 °C. Total yeast and mold count was performed by pour plate method using potato dextrose agar supplemented with 10% tartaric acid to maintain pH of media to 3.5. Plates were incubated at 27 °C for 48 h. Microbial counts were expressed as cfu g⁻¹ of herbal products. Each analysis was performed in triplicate ( Khurana, Sharma, & Bhaduria, 2011 ).

All samples (50 g each) were defatted with hexane and subsequently extracted with methanol (4 × 250 ml), 80% aq. methanol (4 × 250 ml) and finally with distilled water (4 × 200 ml) in an omnimixer. Three replicates were prepared. The respective extracts were dried in vacuum and made to 10% (w/v) solution of the respective solvents. These extracts were used for chemical analyses as well as antioxidant activity measurements.

TLC densitometry was carried out using toluene:ethyl formate:formic acid (5:4:1) as solvent system on silicagel G plates. Separated bands were visualized by spraying 50% sulfuric acid and heating at 110 °C and identified using standards. Quantitative estimation was performed on a dual wavelength flying spot scanning densitometer, CS-9301PC, Shimadzu, (Kyoto, Japan). Density of the spots of interest was determined in the reflectance mode (529 nm) and concentrations of the samples were obtained from the standard curve (correlation coefficient 0.99) prepared using gallic acid.

Identification and quantification of the major active constituents by HPLC analysis was carried out on a HPLC system (Jasco Corporation, Tokyo, Japan) equipped with a C-18 reverse phase stainless steel column (30 cm × 0.46 cm) and a UV detector. Extracts were eluted with three different solvent systems. [1] Gallic acid and ellagic acid were separated using Solvent A (1.5% H₃PO4) and solvent B (CH₃COOH/CH₃CN/H₃PO₄/H₂0 (20:24:1.5:54.5)) starting with 80% A, linearly decreasing to 33% A after 30 min, 10% after 33 min, and 0% after 39.3 min (269 nm) and estimated using standard curve prepared by gallic acid. [2] Solvent A (H₂0) and solvent B (CH₃CN), with a gradient starting with 100% A, linearly decreasing to 0% A after 25 min, for separation of curcuminoids at 420 nm and using curcumin for preparing standard curve (correlation coefficient 0.99). [3] An isocratic elution using CH₃CN:H₂0 (16:84) at 254 NM to estimate mangiferin using the same compound for the preparation of the standard curve (correlation coefficient 0.99).

Sample (40 μl, 1 mg/ml) was mixed with 200 μl Folin Ciocalteus reagent and 1160 μl of distilled water. After 3 min incubation 600 μl 20% sodium carbonate solution was added to the mixture and kept in dark for 2 h at room temperature and absorbance was measured at 756 nm. Gallic acid was used as a standard and the total phenolic content was expressed as μg of gallic acid equivalents (GAE) per mg of extract ( Gao, Ohlander, Jeppsson, Bjork, & Trajkovski, 2000 ).

Test compound (25 μl, 1 mg/ml) was added to 1 ml of freshly prepared DPPH solution (0.025 gm/L in methanol). The absorbance was measured at 517 nm after 20 min. The calibration curve was plotted with %DPPH^• scavenged versus concentration of the standard antioxidant (Ascorbic acid). The results were expressed as Ascorbic acid Equivalent Antioxidant capacity (AEAC) ( Aquino et al., 2001 ).