Shelf Life Evaluation and Comparative HPTLC Profile of Hridya Yoga Churna

Background: Quality, safety and efficacy of medicines used form the back‐ bone of every health care system. Shelf‐life is the length of time, after which all substances start to degrade in their qualities and become unsuit‐ able for consumption or sale. The saviryatavadhi of churna is considered to be 2 months according to ayurvedic classics. The use of modern pack‐ aging technology and preservatives have increased the shelf life period of ayurvedic medicines and the shelf life period of churna is 2 years according to Rule 161‐B, Drugs and Cosmetics Act. To ascertain the quality, hridya yoga churna was taken up to assess its shelf life period. Methods: Accelerated stability study (includes evaluation of organoleptic and physic‐ chemical parameters, assay of active constituents and microbial load) and Comparative HPTLC (High Performance Thin Layer Chromatography) were done to evaluate the shelf life of the Hridya yoga churna. Atomic absorp‐ tion spectrophotometry was done to analyze the heavy metal content in the sample. Results and Conclusion: In the present study the shelf life period of Hridya yoga churna is found to be 3 years and 7 months. Similar Rf values obtained in HPTLC analysis of hridya yoga churna initially and after six months showed the minimum deterioration of the product. The presence of heavy metals namely arsenic, cadmium and lead were not detected and mercury was present within permissible limits.


INTRODUCTION
Shelf life or "Saviryata avadhi" means the time period during which the potency (Virya) of a drug remains unaffected due to environmental factors or from microbial contamination.Different dosage forms have different shelf life period.Pancha vidha kashaya kalpanas (five different dosage forms) are described in therapeutic system of Ayurveda, which is further classified into upakalpanas.These include different dosage forms like Swarasa (juice), Kwatha (decoction), Kalka (paste), Churna (powder), Vati (tablet), Taila (oils), Lepa-malahara (ointment), Asava and Arista (self generated alcoholic preparations), Avaleha (linctus), Bhasma (inci nerated powder), Pisti (paste) etc.Among them some have very short shelf life period, Eg.Swarasa-which is to be used immediately after preparation, and some of them have long shelf life, Eg.Asava, Arishta, Bhasma etc.The information regarding the concept of Shelf life (Saviryatavadhi) for different dosage forms are found scattered in Brhatrayi.According to Acharya Charaka a drug can be utilized for therapeutic purposes until it retains its fragrance, color, taste etc. 1 During the medieval period authors attempted to compile the information regarding the concept of shelflife and Saviryata avadhi of different dosage forms are available in texts like Vangasena Samhita, 2 Sharangadhara Samhita, 3 Yogaratnakara 4 etc.The uses of modern packaging technology and preservatives have increased the shelf life period of ayurvedic medicines.The saviryata avadhi of churna given in Sarngadharasamhita is 2 months. 3Shelf-life defined for Churna according to Rule 161-B, Drugs and Cosmetics Act is 2 years.Stability study provides evidence on how quality of a drug substance or product varies with time under influence of variety of environmental factors such as, temperature, humidity and light and also to establish a retest period for the drug substance or product and recommended storage conditions. 5Considering the developments in pharmaceutical industry an attempt has been made to evaluate the shelf life of Hridya yoga churna of Bhavamisra.

Preparation of the study drug
The individual drugs of the classical hridya yoga were collected and identified.Individual powder microscopy was done at Pharmacognosy unit, IPGT and RA, Jamnagar to prove the authenticity of the drug.All the drugs were powdered separately and the powder was sieved through mesh size #85.All the seven drugs were taken in equal quantity and mixed together to make the formulation.The formulation was packed in air tight food grade containers.Four containers of 50 g each were packed and stored in accelerated stability study chamber.Composition of the hridya yoga churna is given in Table 1.

Accelerated stability study
Accelerated stability study was conducted as per ICH guideline Q1.A(R2). 6Samples were stored at 40°C ± 2°C, and 75 ± 5% relative humidity.Samples were withdrawn at the intervals of 1, 3, and 6 months.Basic analytical parameters including loss on drying at 110°C, 7 ash value, 8 pH value, 9 water soluble extractives, 10 methanol soluble extractives, 11 percentage of tannins, 12 alkaloids 13 and saponins 14 were evaluated at regular intervals.10% degradation was set as the acceptable point to extrapolate the accelerated stability data.Real time aging factor 5 and 3.

Heavy metal Analysis
Heavy metal analysis was performed using Atomic absorption sprectrophotometer as per the methods described in WHO guidelines. 15Atomic absorption spectroscopy (AAS) is a spectroanalytical procedure for the quantitative determination of chemical elements using the absorption of optical radiation (light) by free atoms in the gaseous state.

HPTLC Analysis
The HPTLC (High Performance Thin Layer Chromatography) finger print profile of methanolic extract of Hridya yoga was taken on aluminium plate coated with silica gel 60 F254 (E.Merck) as adsorbent and employing CAMAG Linomat 5 applicator.The mobile phase used was Toluene: Ethyl acetate: Formic acid (10:3:1).For derivatization Anisal-dehyde Sulphuric acid reagent was used and heated at 100 ± 5°C till the spots appeared.The plate was dried and visualized under UV 254 nm, 366 nm and 540 nm.HPTLC finger prints were taken initially and after 6 months.

RESULTS AND DISCUSSION
The main purpose of conducting stability testing of pharmaceutical products is to ensure the efficacy and quality of active compounds in product and to establish shelf life or expiry period.No considerable changes were observed in organoleptic characters (Table 2) and microbial load (Table 5) of the Hridya yoga churna even after 6 months.Results of different physico-chemical parameters (Table 3) and active constituent assays (Table 4) were taken in consideration to evaluate intercept and slope (Table 7).Extrapolated shelf life of Hridya yoga Churna (Table 9) was calculated with 10% degradation rate (Table 8) from physicochemical parameters at accelerated condition 40°C ± 2 and 75% ± 5 RH.On the basis of available data from accelerated stability study, it can be extrapolated that shelf life of Hridya yoga Churna is 42.81 months (3.07 years) for countries which come under climatic zone III & IV.Ayurvedic medicines were alleged to contain heavy metals and for the purpose of establishing the safety and efficacy, heavy metal analysis of the Hridya yoga churna was carried out (Table 6).The presence of arsenic, lead and cadmium were not detected in the Hridya yoga churna.Even though Mercury was detected (.217 ppm), it was within the permissible limits.HPTLC finger print studies of methanolic extract of Hridya yoga were carried out and chromatogram was developed and detected using the UV visible chamber, initially and after 6 months.Seven spots were identi fied at UV 254 nm in the chromatogram as depicted in Figure 1 and 2. The corresponding R f values of the seven components are 0.14, 0.32, 0.46, 0.61, 0.75, 0.82, 0.96 (Table 10).Eight spots were identified at 366 nm      (Figure 3 and 4).The corresponding R f values are 0.14, 0.32, 0.38, 0.42, 0.46, 0.52, 0.69, 0.75 (Table 11).Ten spots were identified at 540 nm (Figure 5 and 6).The corresponding R f values are 0.07, 0.14, 0.23, 0.32, 0.46, 0.52, 0.61, 0.75, 0.90, 0.96 (Table 12).All the spots identified in the hridya yoga extract initially were identified at six months also which shows the minimum deterioration of the product.

CONCLUSION
Shelf-life defined for Churna at Rule 161-B, Drugs and Cosmetics Act is 2 years.In the present study the quality of Hridya yoga churna is assessed by evaluation of shelf life.On the basis of the accelerated stability study conducted the shelf life period of Hridya yoga churna is found to be 3 years and 7 months.Similar R f values obtained in HPTLC analysis of hridya yoga churna initially and after six months showed the minimum deterioration of the product.The presence of heavy metals namely arsenic, cadmium and lead were not detected and mercury was present within permissible limits.
3 was used for extrapolation of shelf life for climatic Zone I & II countries and climatic Zone III & IV countries respectively.India comes under climatic zone III & IV.Number of months when 10% degradation was occurred was calculated using following formula: Months when 10% degradation occurs = [0 month assay value-{(0 month assay value×10/100}]-Intercept Slope

Table 6 : Heavy Metal Analysis of Hridya Yoga Heavy Metal by AAS Results Permissible limits as per WHO
*parts per million.Pharmacognosy Journal, Vol 8, Issue 3, May-Jun, 2016