1. INTRODUCTION. Herbal medicines have been used in India for thousands of years and is

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1 1. INTRODUCTION Herbal medicines have been used in India for thousands of years and is increasingly been used worldwide during the last few decades as evidenced by rapidly growing global and national markets of herbal drugs 1. The global pharmaceutical market was US $9 billion in the year According to WHO estimates, the present demand for medicinal plants is ~US $14 billion a year and by the year 2050 it would be ~US $5 trillion. Due to high prices and harmful side effects of synthetic drugs, people rely more on herbal drugs and this trend is growing, not only in developing countries but in developed countries too. India has 2.4% of world s area with 8% of global biodiversity. The forests of India are estimated to harbor 90% of India s medicinal plants. In India, around 25,000 effective plant based formulations are used in traditional and folk medicine. More than 1.5 million practitioners are using the traditional medicinal system for health care in India. It is estimated that more than 7,800 manufacturing units are involved in the production of natural health care products and traditional plant based formulations in India, which requires more than 2,000 tons of medicinal plant raw materials annually 1. Ashwagandha, botanical name - Withania somnifera Dunal belonging to family Solanaceae, is one of the most valuable, widely used medicinal herb. Roots and leaves of this plant are proven to have a wide range of pharmacological activity 2. Ashwagandha is commonly known as Indian Ginseng. It is considered as health care food supplement. There are a number of marketed formulations containing Ashwagandha. The plant is indigenous to India and it grows throughout the country. Several types of alkaloids, withanolides, glycosides, glucose and free amino acids are Department of Pharmacognosy, KLE University s College of Pharmacy, Bangalore 1

2 the major chemical constituents of this plant 3. Withaferin A is commonly used as a marker compound to evaluate and standardize Ashwagandha 4. Kalmegh, botanical name - Andrographis paniculata Nees belonging to family Acanthaceae, is another valuable, widely used medicinal herb. Whole plant is of medicinal value 5. The plant is commonly known as King of bitters. A number of formulations containing Kalmegh are available in the market. Plant is cultivated widely in India. Kalmegh contains bitter principles Andrographolide, 14-deoxy Andrographolide, neoandrographolide, andropanoside, andrographiside and flavonoids. Andrographolide is commonly used as a marker compound to evaluate Kalmegh 6,7. There is a huge demand for both these medicinal plants 8,9. Both these plants are used as anti-cancer drugs and is specially used to treat immunocompromised patients. With the advent of science in herbal field, there is a resurgence of interest in the herbal medicines in recent years. During the last few decades research on indigenous drugs has made swift stride. Superiority of the herb is based on the microbial quality, amount of active constituents present and its pharmacological activity. A considerable amount of work has been done to study the potentialities of herbal medicines. Research on herbal drugs involves a multidimensional study with a combination of fields of biology, chemistry, pharmacology, microbiology and other collateral disciplines to prove quality, safety and efficacy of the drug. Unfortunately, the number of reports of people experiencing negative effects, caused by the use of herbal drugs, has also been increasing. There may be various reasons for such problems. Poor quality of herbal medicines is mainly due to insufficient attention being paid to the quality assurance and control of these products. Although WHO 10 has developed guidelines for the quality control of herbal drugs Department of Pharmacognosy, KLE University s College of Pharmacy, Bangalore 2

3 which provide a detailed description of the techniques and measures required for the appropriate cultivation and collection of medicinal plants, there is still a lacuna between this available knowledge and implementation, because farmers and other relevant persons like producers, handlers and processors of herbal drugs are not much aware of WHO s guidelines and they continue their work as before without any quality control measures which results in inferior quality of herbal drugs with lots of contaminants like heavy metals, pesticides and microbes. Herbal medicines may be associated with a broad variety of microbial loading and exert an important impact on overall quality of herbal products and preparations Generally, herbs are valued for their distinctive aroma, colour, flavor and their pharmacological activity. But they are often contaminated with high levels of bacteria, molds and yeasts. If untreated, the herbs will result in rapid spoilage of the foods and can also result in serious food-borne illness. Post harvest and postprocessing herbs are always contaminated with microorganisms from the plants themselves, soil, water, air and dust. A wide spectrum of microorganisms and microbial loads has been previously reported in medicinal plants Risk assessment of the microbial load of medicinal plants has therefore become an important subject in the establishment of modern Hazard Analysis Critical Control Point (HACCP) schemes or International Standards Organization (ISO) standards. The herbs could be contaminated by human handlers during harvesting or processing. According to WHO and European Pharmacopoeia 21,22, herbal drugs must meet the modern hygienic standards, which aim at low microbial load or the absence of pathogenic microorganisms. WHO, USFDA, European Scientific Cooperative on Phytomedicine (ESCOP) have published standard sets of guidelines to address the concerns 10,23. As plant materials are highly susceptible to microbial contamination 11, many herbal drugs Department of Pharmacognosy, KLE University s College of Pharmacy, Bangalore 3

4 are failing to compete in International market due to high microbial load 1,24,25. According to international requirement, Microbial quality is extremely important to be achieved - to make plant materials suitable for human use and commercialization. Presence of microorganisms such as Staphylococcus, E.coli, Pseudomonas, Salmonella and other bacteria and fungus craft serious health hazard if present beyond acceptable limit If such products are consumed, it will pose deleterious affects on patients specially, immunocompromised patients 31. Moreover, Ashwagandha and Kalmegh are anti-cancer drugs, hence the microbial quality of these plants becomes a major concern. Hence, there is an imperative need for microbial decontamination of these two medicinal plants. Sterilization is a process by which all forms of microbial life are inactivated so that they are not able to reproduce or survive. Various methods available for sterilization are dry heat, moist heat, gas (ethylene oxide - ETO) and ionizing radiation. Although, there are many methods of microbial decontamination, the most appropriate one is the use of ionizing radiations. A) Dry Heat Sterilization It is carried out by maintaining the product at c for a period of not less than two hours. This technique is suitable for heat resistant products, such as glassware, metal ware and some pharmaceuticals. B) Moist Heat Sterilization - It is carried out by air free, saturated steam under pressure, at not less than c for a minimum holding time of 15 minutes. The products suitable for this technique are surgical instruments and thermostable healthcare products. C) Ethylene oxide (ETO) ETO sterilization is generally employed for heat and radiation sensitive materials. Ethylene oxide is used in either pure form or admixed with inert gases. However, the chemicals used as sterilants destroy a wide range of Department of Pharmacognosy, KLE University s College of Pharmacy, Bangalore 4

5 pathogens and typically the same properties make them harmful to humans. ETO sterilization is widely used by medical device manufacturers for large scale sterilization, but is becoming less popular in hospitals, because of the well-known effects caused by it. D) Radiation Sterilization Gamma rays emanating from artificially produced radioisotopes (such as Cobalt 60 or Caesium 137), high energy electrons from accelerators or High energy X rays from X ray machines are used for sterilization. The major advantage of radiation is that only one parameter, i.e. the time of irradiation, needs to be controlled. The process is considered far safer compared to ETO. Some of the products which can be sterilized by gamma radiation are Antibiotic powders, Ayurvedic products, herbal products, ointments, surgical blades, sutures, pharmaceutical raw materials etc. Dry heat and moist heat sterilization have their own disadvantages as some microorganisms survive at the exposed temperatures. And moreover, they are suitable only for thermo stable products. Effectively ETO and Radiation are the main industrial methods of sterilization. There is a growing concern over the safety of industrial use of ETO. When an ETO sterilizer is opened, the release of the gas from the packages can give rise to aerial concentration of as much as 400ppm which is far in excess of the limit of 50ppm fixed by the occupational safety and health administration, USA, and is carcinogenic. They also produce acute toxicity including irritation of the skin, conjunctiva and nasal mucosa. The problem of residuals, necessitates their determination in the ETO sterilized products before release. It is in the light of aforementioned considerations that both Food and Drug administration (USA) and the Environmental Protection Agency (EPA) are questioning the efficiency and safety of ETO as sterilizing agent. Department of Pharmacognosy, KLE University s College of Pharmacy, Bangalore 5

6 Some European countries have restrictive regulations that do not admit ETO process. For all practical purposes the ETO method of sterilization is being replaced by ionizing radiation. Radiation sterilization using ionizing radiation is becoming popular. The sterilization is achieved either by using gamma radiations or high electron beams from machine sources that accelerate electrons to high energy levels (5-10 MeV) or X-ray Machines of energies less than 5 MeV. These radiations are highly penetrating and readily pass through majority of products. Ionising radiation, preferably treating with gamma irradiation is considered to be most suitable technique for microbial decontamination of medicinal plants 32. As with other decontamination processes such as pasteurization and sterilization technologies involving the input of thermal, mechanical or photonic energy, the objective of processing with ionizing radiation is to destroy pathogenic and spoilage microorganisms without compromising the safety and quality of the medicinal plant. All these processes produce physical and chemical changes, but the extent of these changes differs significantly. Depending on the type of energy, its penetration into medicinal plant and the amount of energy ultimately deposited differs. Several different chemical bonds in the constituents are broken or formed, leading to either desired or undesired effects. Hence, each plant has to be checked for the radiation dose, which is required for microbial decontamination. Gamma irradiation is better than ethylene dioxide treatment (Food and Environmental Protection Section Joint FAO/IAEA Division Nuclear Techniques in Food and Agriculture International Atomic Energy Agency, 1999) 33. In comparison to thermally sterilized products, the extent of chemical change in radiation-sterilized products is relatively small and uniform. Gamma irradiation as a microbial decontamination process for medicinal Department of Pharmacognosy, KLE University s College of Pharmacy, Bangalore 6

7 plants is technically feasible, friendly enough to environment and very effective to resolve technical problems in trade and commercialization. Benefits include precise dosing, rapid processing, uniform dose distribution, system flexibility and dosimetric release. It can also control a variety of microorganisms and thus improve the quality of plant materials. Comparison of different methods of sterilization with ionizing radiation is shown in Table 1. Table 1: Comparison of different methods of sterilization with Radiation Sterilization Process. Factor Dry heat Moist heat Ethylene oxide Radiation Conditions for sterilization ETO Concentration: 900mg/l Radiation Dose: 25kGy (2.5Mrad) Temperature: Temperature: Temperature: NA C C 55 0 C Relative Relative Relative NA humidity: NA humidity: Yes humidity: 50 60% Pressure : Pressure : Pressure : NA NA psi 10psi Time: 2 hrs. Time: 30 mins. Time: 6 hrs. Depends Time Yes Yes Yes Yes Temperature Yes Yes Yes No Pressure/ Vacuum Yes No Yes No Humidity No NA Yes No Post sterilization degassing period Choice of packaging Sterilizing the product packed in the shipping pack NA Yes Yes No (7 14 days) Narrow Narrow Narrow Wide Not possible Not possible Not possible Possible Type of process Batch Batch Batch Continuous Mode of killing of microorganisms Oxidation Hydrolysis Alkylation Biological damage of DNA Penetration power Low Low Low Very high Inactivation factor Residual toxicity Nil Nil Yes, ETG (toxic) ETO & ETCH (carcinogenic) Nil Department of Pharmacognosy, KLE University s College of Pharmacy, Bangalore 7

8 Radiation sensitive products, having low or susceptible bio burden, can be sterilized effectively at lower dose. If lower irradiation doses are used additional microbiological monitoring of the product before irradiation would be necessary in order to assess the adequacy of the procedure. Hence, research must be done on each medicinal plant to find out the radiation sensitivity to effectively kill the microorganisms present. The choice of a sterilizing radiation dose is based on the initial microbial load (bio burden) coupled with desired sterility assurance level. The high initial investment in gamma radiation facilities is justifiable since the method promotes greater safety to the personnel and environment. The calculation of absorbed dose is based on dosimetric methods with Chemical dosimeters such as ceric-cerous sulphate, dichromate dosimeters and others. Cericcerous sulphate dosimeters are used for routine monitoring and quality control of the irradiated products. The dosimeters are placed in maximum and minimum dose positions of the product boxes and it is ascertained that a minimum dose of 25kGy is imparted in the products. The use of biological indicators based on Bacillus pumilus can also be used as a measure of effectiveness of radiation sterilization. Gamma sterilization represents nearly 50% of total sterilization needs of several developed countries. In India, only an estimated 15% of the healthcare products are currently sterilized using gamma radiation, indicating a good scope for expansion of gamma radiation facilities in the country. If one has to consider some of the disadvantages, there are only a few in case of gamma radiation based sterilization. One is that the process of radioactive decay is spontaneous and therefore can not be switched off like EB or X-ray machines, the facility must be in continuous operation in order to make use of the naturally decaying Department of Pharmacognosy, KLE University s College of Pharmacy, Bangalore 8

9 radio element. Second, there are still some materials which do not withstand the dose of 25kGy and are susceptible to damage. Unlike chemical fumigants, gamma irradiation does not leave any harmful toxic residues and is more effective. It is efficient and can be used to treat pre-packed commodities. Since gamma rays have high penetrating power, medicinal plants can be irradiated after packaging, irrespective of the size of the carton. Poor post harvest practices including inadequate storage and preservation facilities, as well as adverse climatic conditions, cause heavy losses in India's agricultural products. Irradiation promises to offer an effective means for minimizing these losses, thereby increasing their availability, and helps the product to compete in global market. Export development authorities, commodity boards, food industry, farmers, traders, exporters of agricultural commodities, ayurvedic practitioners, health care providers and consumers can be benefited from the use of radiation processing technology. The prevalence of a variety of climatic conditions puts India in a supreme position with respect to richness of medicinal flora. As such, India should occupy a significant position in the world trade of botanical drugs. India should focus on developing different methods of microbial decontamination on valuable plants like Ashwagandha and Kalmegh in order to improve the quality of herbal drugs. Further the treated medicinal plants should be checked for their integrity in terms of their microbial and medicinal quality, safety and efficacy over a period of storage, scientifically through detailed pharmacognostical, physicochemical, phytochemical, toxicological, pharmacological and microbiological investigations. In order to push India as a significant player in the global market, suitable processes should be adopted to improve the microbial quality and further the herbal products should be standardized as per WHO guidelines 10. Department of Pharmacognosy, KLE University s College of Pharmacy, Bangalore 9

10 Literature survey reveals that there are no research on efficacy of gamma irradiation on microbial safety and medicinal quality of Ashwagandha and Kalmegh. Hence an attempt is made to study the efficacy of gamma irradiation on microbial safety and medicinal quality of these two plants. The decontamination method proposed to be applied to reduce the microbial load in samples of Ashwagandha and Kalmegh will be checked for stability upto 12 months of storage at room temperature based on pharmacognostical, physicochemical, phytochemical, pharmacological, toxicological and microbiological analysis. Department of Pharmacognosy, KLE University s College of Pharmacy, Bangalore 10