INTRODUCTION In India medicinal plants, herbs, species and herbal remedies are known to Ayurveda. Ayurveda is the system of traditional medicine prevalent in India since 2000 BC. Ayurveda the Indian tradititional health care system (ayus-life, veda=knowledge, meaning science of life) is the oldest medical system in the world and being revived in its complete form under the name of Maharishi Ayurveda Glaser 1988. Ayurveda, the traditional form of medicine has been using various herbs for thousand of years such as :- Withania somnifera, Aloe, Ocimum basilicum, Elettaria cardamomum, Cinchona officinalis, Cassia fistula, Casia tora,Castor Datura straamonium, Emblica officinalis, Ephedra vulgaris, Flax seed, Zingiber officinalis, Hibiscus rosa-sinensis, Lemongrass, Tagetes errecta, Morus alba, Nelumbo nucifera, Nymphea lotus, Papaver somniferum, Podium Punica grantaum, Pomegranatum Seed oil, Cydonia oblonga, Rose petals, Sandal wood, Solanum nigrum, Curcuma longa, Vinca rosea, Rauwolfia serpentin. These ayurvedic herbs are very effective in increasing the body resistance and are used in the treatment of various disease. The traditional medicine all over the world in now days revalued by an extensive activity of research on different plant species and their therapeutic principles. Experimental evidences suggest that free radicals (FR) and reactive oxygen species (ROS) can be involved in a high number of disease (Richards and Sharma 1991; Niwa, 1991). Health organizations have approved its efficacy.This system provides an approach to prevention and treatment of different diseased by a large number of medical procedures and pharmaceuticals (Zaman, 1974). In Ayurveda, Withania somnifera a member of family Solanaceae is a valued herb and as such was used and cultivated for centuries in India. Withania somnifera is also known as Ashwagandha ('smells like a horse'), Indian ginseng and winter cherry in the west. Ashwagandha (Withania somnifera, Dunal) is an important medicinal plant cultivated and primarily occupies north-western region Page 17

of Madhya Pradesh about an area of 4000 ha in India (Nigam and Kandalkar, 1995). Withania somnifera has been used for thousands of years and is still regarded as one of the most valuables ayurvedic medicinal plants. Studies indicate that Withania somnifera possesses anti-inflammatory, anti-tumor, anti-stress, antioxidant, immunomodulatory, and rejuvenation properties (Archana and Namasivayam, 1999). This solanaceae plant grows in dry area in sub tropical regions, like India, on the Himalayas above the sea level. Baluchistan, Sri Lanka, and in the Mediterranean area: Sicily and Sardinia (Kapoor, 1990; Kirikar and Basu, 1993). Plant parts used are root, leaf and seed (Kapoor, 1990d; Kirikar and Basu, 1993f). Withania is a small or medium under shrub which attains approximately 150 cm height, erect, grayish, perennial, odour is strong and disagreeably like horse urine. Roots are fleshy, tapering ,whitish, branched at the apex covered with minute stellate hairs. Leaves are simple, 2-6 cm wide, 3-8 cm long, alternate, petiole 1-2 cm long, ovate or elliptic ovate, actute. Flowers are sessile, axilliary clusters, 1 cm long, it blossom nearly throughout the year. Corolla greenish or yellow or white yellowish, 5mm long, lobes lanceolate, acute and thin, calyx is visibly expanded around the fruit. Roots are one or more, tuberous, some long. Fruits are little, globular, red, smooth, covered by a membranaceous closely fitting calyx edible, open at the apex. Seeds are numerous, yellow-white, reniform, laterally compressed, poisonous. (Kapoor, 1990; Kirtikar and Basu, 1993). Chemical constituents present in different parts of plant. In whole plant a Steroidal lactone, the Withanolides G, H, I, J, K, U, structure with 14 (alfa) hydroxy group rather than a 8-14 double bond (Kirson and Gottlieb, 1981), three new anolides II, III, IV (Vande Velde and Lavie, 1981), from Withania somnifera chemotypes III an intermediate in the biosynthesis of withanolide E has been elucidated as (20 R, 22 R) 14 (alfa), 20-dihydroxy-1- oxowitha-2, 5, 16, 24tetraenolid (Vande Velde and Lavie, 1982) are present. Pharmacological action:- W. somnifera produces effects in the Central Nervous System (CNS) by acting via GABA receptor system. In fact it contains Page 18

an ingredient which may posses a GABA mimetic activity (Mehta etal., 1991). Glycowithanolides-sitoindosides VII-X and withaferin A show effects in experimentally validated Alzheimers disease model (include by Ibotenic acid) and inhibit the tolerance to morphine, prolonging morphine including analgesia (Ghosal et al., 1995; Ramarao et al., 1995). Withania prevents myelosopression induced by CPA, AZT, PD and increases immunity (Ziauddin et al., 1996). Withania is anti-inflammatory (carrogenine- induced) and it has heptoprotective effects against alcohol and CC14 (Sudhir et al., 1986). W. somnifera has antigranuloma activity (Al-Hindawi et al., 1992). Withaferin A has anti-tumor effects against Ehrlich ascites carcinoma, Sarcoma-180 (Devi et al., 1993, 1995; Sharad et al., 1996). The plants extract inhibits the aging process (Rastogi and Mehrotra, 1993). Withanolide D exhibits anti-tumor activity against Ehrlich ascites carcinoma and human epidermoid carcinoma of nasopharynx (Rastogi and Mehrotra 1993). W. somnifera prevents LPO induced by pyrogenic substances like lipopolysaccharide (LPS form Klebsiella pneumoniae) and peptidoglycan (PGN da Staphylococcus aureus). Pyrogenic substances like LPS and PGN are known to increase LPO. LPS induced LPO in mice and rabbits (0.2 µg/kg iv), has a peak with in the span of 24th reached in 4-6 after administration. Simultaneous administration of W. somnifera (100mg/kg,po) along with LPS significantly prevents the rise in LPO levels. The same result is obtained with PGN, with the difference that the peak is reached 1-2h after the administration. The antioxidant effects of W. somnifera depends on the presence of steroidal lactones, withanolides, which are the main active components (2.8%) (Dhuley, 1998). Out of the 112 elements in nature, about 80 are metals, most of which are found only in trace amount in the biosphere and in biological materials. There are at least some twenty metals or metal like elements which do give rise to wellreorganized toxic effects in man and his ecological associates (Friberg, et al., 1979; Murti, 1980). These elements include Arsenic, Antimony, Beryllium, Cobalt, Chromium, Cadmium, Lead, Manganese, Mercury, Molybdenum, Nickel and Tin. These metals have been known to be toxic to man for centuries and their carcinogenic activities have also been reviewed by Frust, (1977). Heavy metal Page 19

includes the element with atomic number greater than 20, excluding alkali metals, alkaline earths, lanthanides and actinides. Metallic elements are intrinsic components of the environment. The most common heavy metal contaminants are Cadmium, Chromium, Copper, Mercury, Lead, Zinc, Arsenic, Nickel and Vanadium. Heavy metals form the major group of toxic pollutants among the other pollutants, as these metals tamper the harmony of the ecosystem (Rao and Patnaik, 1999). The accumulation of potentially toxic elements may vary from plant to plant and soil (Kishu, et al., 2000). Agricultural soils are usually rich in heavy metals due to fungicides, herbicides, phosphate fertilizers, organic manures and the decaying plants and animals residue. The use of sewage sludge and waste water for irrigation further increases the concentration of heavy metals in agricultural soils. Agricultural runoff together with soil erosion are the potential sources of heavy metals in aquatic bodies (Agrawal, 2002). World Health Organization (WHO) has recognized health hazards of these metals in food chain even at low concentration (WHO, 1984). The deposition of metals in soil and water find their way to human being via plant uptake process. Uptake and translocation of metals cannot be considered as an isolated action but seems to be affected by several synthetic and antagonistic reactions with other naturally occurring species (Delhaize, et al., 1993). Heavy metal ions such as Cu2+, Zn2+, Mn2+, Fe2+, Ni2+, and Co2+ are essential micronutrients for plant metabolism but when present in excess, these and other non essential metals such as Cd2+, Hg2+, Ag2+, and Pb2+ can become extremely toxic (Williams, et al., 2000). Accumulation of excessive amounts of essential metals due to breakdown or inadequate functioning of the homeostatic excretory mechanism or to excessive absorption from the diet may also lead to toxicity (Agrawal, 2002). Heavy metals like Zinc, Lead, Cadmium, Nickel and Mercury are phytotoxic even in very small amount, which eventually lead to reduced growth, impaired metabolism and lower dry matter production. In recent years, research has been focused on accumulation of heavy metals in crop plants and naturally Page 20

growing weeds. The process of metal uptake and accumulation by different plants depend on the concentration of available metals in soils, solubility sequences and the plant species growing on these soil (Chaney, 1973; Anderson, 1977 c; Pahlsson, 1989; Kufka and Kuras, 1997). At high concentration, these metals become toxic, as do the non-essential metals, causing symptoms such as chlorosis and necrosis, stunting, leaf discoloration and inhibition of root growth (Marschner, 1995; Van Assche, 1999). Heavy metals enter plants mainly through the root system and may cause a range of morphological and physiological disturbances (Pahlsson, 1989). Enormous studies have been carried out by different workers regarding the heavy metal accumulation in crop plants and vegetables (Root, et al., 1975; Beekett and Devis, 1977; Carlson and Bazzar, 1977; Miles and Parker, 1979). Uptake and accumulation of heavy metals eg. Pb2+ and Cd2+ by plants reduce qualitative and quantitative productivity of the species and cause a serious health hazard though the food chain to other life forms. Not only the mature plants, but these metals have also been reported to effect seed germination, seedlings growth and several metabolic processes of the plants (Haung and Bazzar, 1974; Singh, et al. 1988). Among various toxic metals Cd and Ni are recognized as two most hazardous elements, which are not essential for the plant growth but easily taken up, by the plants (Kabata and pendias, 1992). One of the most serious environmental stress is the harmful effect of heavy metals. Cadmium is considered to be one of the most dangerous heavy metal, whose toxic effect on plants and animals is well known. plants often accumulate a large quality of Cd without any poisoning symptoms, which after entering the food chain may endanger the human health as well (Adriano, 1986). The U.S. Environmental protection agency has listed Cd as one of the priority pollutants along with Pb, Ti and Hg (Keith and Telliard, 1979). Cadmium has been placed under the category of 'very toxic and relatively accessible metal' by Wood (1974). the major portion of cadmium ingested into our body is trapped in the kidneys. In addition phosphate fertilizers have been found to contain high levels of Cd (Brummer, 1986; Campbell, 1988; He et al., 1994). Cd released to the environment enters Page 21

biochemical cycles, gets bioconcentrated and may effect human health (eg-ItaiItai, a disease caused by Cd-contaminated rice in Japan) (Rivai et al., 1990; Wagner, 1993; Grotan and Vanbladeren 1994). Cadmium accumulation in soils and crop plants is of increasing concern due to adverse effect on human health and crop production (Wanger, 1993; Hall, 2002). Although Cd is not an essential mineral nutrient for crop plants, it can be easily taken by plant roots when the growth medium contains high level of it. Cadmium concentration in normal plants range from 0.1-2.4 ppm (Alloway, 1990) but at higher concentrations it has been shown to affect plant growth and dry matter yield adversely (Bingham, et al., 1976). Studies have also been carried out to elucidate the biotoxic effects of cadmium in vegetable crop. It has been observed that root crops, such as carrot and potatoes, leafy vegetables and tobacco take up cadmium more readily than most other plants. It has also been observed that oilseeds take up more cadmium than cereals(Rahlenbeck, et al., 1999, Ndiokwere, 1984, Tumbo-Oeri, 1988, Mc Kenna and Chaney, 1995). Investigations reveal that root growth is more affected than the shoot growth in Cadmium supplementation. Other symptoms include chlorosis, excessive yellowing, broadening of leaves and brewing of roots with stunted growth plants (Singh et al., 1988). It was found in recent reports, that Cadmium levels of 11.2 to 56 ppm may be injurious for the rice plants. Cadmium inhibits the activities of enzymes of nitrogen assimilation, nitrate reductase (NR), nitrite redutase (NiR) and glutamine synthetase and thus impairs the process of N assimilation in growing seedlings. Crop plants greatly differ in their uptake and transport of Cd. Differences in Cd uptake and accumulation has been shown both among plant species (Grant et al., 1998; Cakmak et al., 2002b; Ozturk et al., 2003b). The Cd toxicity is the most conspicuously manifested in retardation or complete inhibition of root growth at higher metal levels (Lyon and Beeson, 1948; Rauser, 1973). Plant mechanism effecting the root uptake and shoot transport of Cd can also affect the expression of Cd toxicity in plants, and can decrease the Page 22

yield (Cakmak et al., 2000 b; Kochian et al., 2002; Dunbar et al., 2003). Despite similar Cd concentrations in the leaves or shoots, plant cells can be detoxified by Cd-binding proteins such as phytochelatins or metallothionins (Grant et al., 1988; Cobbett, 2000; Hall, 2002). Agreat deal of interest has been generated to the toxic effect of heavy metals on plants in recent years. Among the heavy metal pollutants, Nickel (Ni) needs special reference for its potential harmful effect on plants. Ni is strongly phytotoxic at higher concentrations. Although Ni was identified as an element as early as 1751, its occurrence in plants was not recognized for a futher hundred years, when Farchhammer (1855) reported Ni in Oak wood. Nickel a natural ubiquitous element of the earth and water (0.001 to 0.003 mg/lt, Snodgrass, 1980 ). Being a trace metal of such vital importance, various studies have been conducted to elucidate the release of Nickle into the environment and further, its uptake by plants and finally their consumption by human being, thus terminating the food chain. The two main sources of Nickle release in to the environment are the industrial processes (Ramteke 2000) and anthropogenic source (Nussey, et al., 2000). Among the heavy metal pollutants, Nickle (Ni) needs special reference for its potential harmful effect on plants. Ni is strongly phytotoxic at higher concentrations. The adverse effect of Nickle on microorganisms, plants and animals are well documented (Komczynske, et al., 1963; Babich and Stotzky, 1983; Smialowicz et al., 1984). The phyto-toxic effect of Nickle are also well known (Narwal, et al., 1991). Nickle enters the plants mainly through the roots when it is taken up by the plants (Barman et al., 2000). High or elevated levels of Nickle may lead to chlorosis, leaf shrinkage and ultimately death of the plants. Nickle toxicity depends on concentration of Nickle, the plant species and the nature of the soil. Foliar accumulation of Nickle has also been reported (Cash and Leone, 1987). The toxic effect of excessive Ni in soils for plants life was reportedly pointed by Haselhoff (1893), who demonstrated its toxicity, to corn and bean plants using solution culture techniques. Since then, large number of reports on Ni Page 23

phytotoxicity have been made. Most of these describe poor growth accompanied by chlorosis due to Ni. Cotton (1930) found that 0.5 ppm Ni produced chlorosis in buckwheat. Brenchley (1938) found 2 ppm Ni to be toxic to bean and barley. Flax (Linum usitatissimun) was found to be especially sensitive, showing toxicity at 0.5-5.0 ppm (Millikan, 1949). Ni also reduced the total chlorophyll content of the leaves of the plants that grew in presence of its inorganic forms in Phaseolus vulgaris (Krupa et al., 1993) and in organic form in cabbage (Molas, 2002). The symptoms of Ni toxicity apperar to be a combination of induced iron-deficiency chlorosis and foliar necrosis. Other toxic symptoms include stunted growth of roots and shoots, deformation of various plant parts, and unusual spotting on leaves and stems. Rauser (1978) studied the early effects of Ni toxicity in bush bean Phaseolus vulgaris, which showed decrease in dry matter production, abnormal vertical orientation of the leaves, abnormal starch and apolar soluble phenolics accumulation. Anderson et al., (1973, 1979) have studied the anatomical developments of Ni toxicity symptoms in the leaves of oat (Avena byzantina) plant. In its free form, however Ni can accumulate in animals and plants is thus then potentially toxic. Vanselow (1966) suggested a minimum of 5ppm Ni in uncontaminated vegetation, however, (Bowen, 1966) suggested that Ni concentrations may vary form 1-2.7 µg/g dry weight. Connar and Shackleete (1975) reported 0.2-4.5 ppm in field crops and vegetable crops. Ni is used as fungicide, especially for the control of cereal rusts (Mishra and Kar, 1974). It is also known to be highly toxic to a wide range of algae (Stokes et al., 1973; Patrick et al., 1975). Ozone (O3) a tri-atomic form of molecular oxygen, is a naturally occurring substance that is vitally important to the welfare of life on our planet. Ozone occurs throughout the atmosphere at all altitudes from ground level to about 100 km. (Approximately 90 percent of atmospheric O3 is concentrated in a broad layer, between about 25 and 50 km above the ground surface. This part of the upper atmosphere is called stratosphere (WHO, 1998). The thin veil, creates by this invisible gas created a shield of great importance for the planet as it absorbs ultraviolet-B (UV-B) radiation emitted by the sun. Ultraviolet-B radiation (UV-B Page 24

: 280-320 nm) comprises a small fraction of the total solar radiation striking the Earth' surface, yet exposure to UV-B at ambient or enhanced level is known to elicit a variety of responses in higher plants (Bornman and Teramura, 1993; Rozema et al., 1997; Xiong and Day, 2001; Caldwell et., al., 1998, 2003). The solar radiation at the top of the Earth's atmosphere contains a significant amount of radiation of wavelengths shorter and more energetic than visible light (400-700 nm). Wavelengths in the range of 100-400 nm constitute the ultraviolet special region. The shortest of these wavelengths are UV-C (100-280 nm), which are completely blocked by atmospheric oxygen (O2) and Ozone (O3). Wavelengths in the UV-B range (280-320 nm) are absorbed efficiently, though not completely by O3, while UV-A wavelengths (320-400 nm) are also absorbed weakly by O3, and are therefore more easily transmitted to the earth's surface (UNEP, 1998). UV-B radiation comprises only a small portion of electromagnetic spectrum, it has a disproportionately high photo biological effect since the energy content of UV-B radiation is greater than the visible radiation, and it is largely absorbed by the two most important molecules-protein and DNA (Ravanat et al., 2001; Tanaka et al., 2002; Van de Poll et al., 2002). Ultraviolet-B has a wide range of deleterious effects on a variety of organisms (Dey et al., 1988; Blaustein et al., 1994; Bothwell et al., 1994). In humans and animals, exposure is principally via the eyes and skin, with effects occurring as a result of the absorption of solar energy by molecules (termed chromophores) present in the tissues/cells. The absorption of light energy leads to changes in these molecules that eventually can result in a biological effect. Enhanced UV-B (eUV-B) radiation causes structural, physiological, biochemical and molecular changes in plants (Teramura and Sullivan, 1994; Rozema et al., 1997; Bjorn, 1996; Tevini, 1999). Data on the potential sensitivity of the plants to UV-B over the last few decades indicate that approximately one third of plant species studied are negatively affected by enhanced UV-B radiation (Sullivan and Rozema, 1999). Page 25

UV-B radiation affects plants in several ways e.g. by impairment of chloroplast function (Bornman, 1989), decrease in protein synthesis (Jordan et al., 1992) and lowering of mRNA levels of photosynthetic genes (Mackerness et al., 1998). UV-B exposure also resulted in upregulation of genes involved in the synthesis of phenolic compounds (Strid et al., 1996). An increase in UV-B radiation has been shown to cause a negative effect on the growth and productivity of wheat, rice and soybean under field conditions (Tevini and Teramura, 1989). Increased UV-B radiation may include detrimental changes to plant anatomical features, photosynthesis, biomass and flowering, although some of the changes may be taken as positive responses for some plants (Cen and Borman, 1993). It has been reported that photosynthesis and photosynthetic productivity of some higher plants are vulnerable to increased UVB radiation (Caldwell et al., 1989, 1995; Tevini and Teramura, 1989; Jordan, 1996). The damage to photosynthetic apparatus may include many aspects such as PS II reaction centre, electron transfer, stomatal conductance, loss of soluble Calvin cycle enzymes etc. (Baker et al., 1997). Larkum et al., (2001) have shown that water oxidizing complex (WOC) is the most sensitive target of UV-B damage in PS II. Growth characteristics such as plant height and leaf area were reduced in UV-B sensitive plants to various extents, depending upon the plant species and cultivars (Lydon et al., 1986). Ultraviolet-B radiation also affected the plant growth and functions mostly through its absorption by proteins, nucleic acids and lipids and the subsequent

deleterious effects on their integrity and function

(Caldwell et al., 1989; Hollosy, 2002). During cultivation of plant, multiple stresses in the form of biotic and abiotic factors play important role in growth, development and production of plants. Therefore, an attempt has been made to study the effects of two major stress factors UV-B and heavy metals (Cd and Ni) in Ashwagandha. Ashwangandha (Withania somnifera) is important medicinal plant, any adverse effect of heavy metals and UV-B on plant may lead to serve health and Page 26

economical consequences. The present work aimed to asses the morphological and physiological effect of two potential stress factors ie. UV-B and heavy metals (Cd and Ni) on Withania somnifera was carried out with the following objectives To evaluate the response of Withania to individual and interactive effects of supplemental UV-B radiations and heavy metals on photosynthetic

(Chlorophyll

and

Caroteniods)

and

Non-

photosynthetic (Anthocyanin and Flavonoids ) pigments.  To evaluate the individual & interactive effects of supplemental UV-B and heavy metals on morphology of the test plant.

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