Effect of Water Stress and Vermicompost in ...

Effect of Water Stress and Vermicompost in Physiological…

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12 Effect of Water Stress and Vermicompost in Physiological, Biochemical and Phytochemical Changes in Ashwagandha (Withania Somnifera Dunal.) *Aloka Kumari1 and R. K. Khajuria2 and K.A.Suri2

ABSTRACT A net house experiment was conducted to study the influence water stress effects on physiological, biochemical and phytochemical variations of Withania (Solanaceae), a tropical medicinal herb were studied. Exposure of plants to water stress led to noticeable decrease in leaf area, 1. Plant Systematics Research Centre, University Department of Botany, T.M.Bhagalpur University, Bhagalpur-812007, India. 2. Indian Institute of Integrative Medicine, Natural Product Chemistry Division, Regional Research Laboratory, Canal Road, Jammu Tawi-180001, India Corresponding Author: Aloka Kumari; Address: Plant Systematics Research Centre, University Department of Botany, T.M.Bhagalpur University, Bhagalpur812007, India. Tel.: 09973394775; E-mail: [email protected]; [email protected]

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Recent Advances in Medicinal Plants and Their Cultivation

photosynthetic pigments, root and shoot lengths and photosynthetic activity. Plants maintained high content of total chlorophyll (Chl), root and shoot lengths, leaf area and photosynthesis. The experiment was laid out in a Randomized Block Design (RBD) parameters replicated thrice. As a result, comparative study of control, moderate stress (under shade condition) and water stress with vermidose 1t/hac. (Cow dung + Vegetable wastes + Eisenia foetida) recorded double yield. Significantly higher 12deoxiwithanostramonolide was observed under water stress condition while, there withanolide concentration decreases at different stages of plants. Present paper deals with the physiological, biochemical as well as phytochemical aspects of plant. Keywords: Withania somnifera, Vermicompost, Biochemical, Physiological, Phytochemical, Water Stress. INTRODUCTION The global market of medicinal plant is over 60 billion US $ per year. India, at present export herbal materials and medicines to the tune of Rs. 446.3 crores, as against Rs. 20,000 crores from China. The export potential of the country can be raised to about Rs. 3000 crores by the end of the year 2005. An estimated survey indicated that the use of herbal medicine will reach to the tune of three trillion US $ during 2050, (http://www.kurzweilai.net). Currently, WHO encourages, recommends and promotes the inclusion of herbal drugs in national health care programmes, because such drugs are easily available at a reasonable price within the reach of common man and as such are time tested and thus considered to be much safer than the modern synthetic drugs. Ashwagandha, Withania somnifera Dunal (Solanaceae), known as “Indian ginseng” is a reputed medicinal herb grown for its roots in arid and semi-arid regions of many countries. It belongs to the family Solanaceae. It is a very important herb in Ayurveda, the traditional Indian medicine. Pharmacological activities of this plant include physiologic and metabolic restoration, antiartheritic, antiaging, nerve


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tonic, cognitive function improvement in geriatric states and recovery from neurogenerative disorders like convulsions, t ardive dyskinesia (Bhattacharya et. al, 2002; Dhuley, 2000). It is used for treating tumours, inflammation. It has anti cancer activity, antistress effect. It stimulates immune system and is known to improve memory. Compounds known as withanolides are believed to account for the multiple medicinal applications of Ashwagandha. Withaferin-A is therapeutically active withanolide reported to be present in leaves. A decoction of Ashwagandha root is useful as nutrient and health restorative to the pregnant and old people. Ashwagandha ghrita promotes the nutrition and strength of children and for curing sterility in women. Roots of this plant form ingredients of many herbal formulations prescribed for musculoskeletal conditions, nervous system and as a general health tonic for elderly persons and lactating mothers (Chaterjee et.al, 1995; Bone, 1996). Steroidal alkaloids and lactones in a class of constituents called withanolides are the major active principles of W. somnifera roots. Despite its drought tolerance capacity, prolonged soil moisture deficit inhibits the growth and development of the crop and adversely affects the crop yield potential and plant productivity. Understanding the effect of long duration water stress on the active principle content and root quality will help to augment functional characterization and biochemical integration of molecular and genetic data. The objectives of this study were to understand the response of field grown W. somnifera to water stress and the changes in root secondary metabolites productivity. The effect of water stress is usually perceived as a decrease in photosynthesis and growth, and is associated with alteration in carbon and nitrogen metabolism (Mwanamwenge, et. al., 1999). The objectives of this study were to determine the effect of water stress in respect of vermicompost its physiological, biochemical and phytochemical aspects. MATERIALS AND METHOD Plant Collection: Wild variety seeds of Withania somnifera were collected from Banka district 40 km away

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from the study area and planted in University Department of Botany, T. M. Bhagalpur University dried for a week at room temperature (25-+2° C) and stored in screw capped bottles under ambient condition before experiment during the following April,2010. Best quality of seeds have been selected by germination test (Peter, 2000) and Tetrazolium test (Grabe, 1970). Plants were transplanted after three weeks in 30 x 45 cm spacing in field. Experiment was conducted in Post Graduate Botany Department, T.M, Bhagalpur University, Bhagalpur from 2009 to 2011. At the initial stage of flowering and fruiting when active constituents present in high level plants collected from field for morphological and biogeochemical analysis have been experimented. Morphological and Physical Analysis of Plants: On the basis of best quality physical and chemical profiles of Vermicompost @ 1 t/hac. (Cow dung + Vegetable wastes + Eisenia foetida) have been selected for field treatment. Seeds of Withania somnifera were grown in red loam soil. The plants were grown for 60 days in the nursery bed of 2 X 2 after surface sterilization using 0.1% HgCl2 under green house conditions. VMC was obtained from P. G. Deptt. of Botany, T.M.Bhagalpur University, Bihar, India and added to the soil @ 1 t/hac . Then they were transplanted into polypots and were arranged in a randomized block design with a photoperiod of 10-11 hrs/day. To study the effect of water stress condition on these plants, three treatments were established. T1 = Control plants (watered daily) T2 = Moderate Water Stress (Under shade condition) T3 = Water Stress + Vermicompost The data on the growth parameters viz. plant height, number of leaves, number of branches, plant spread, leaf area and biochemical parameters was collected 180 days after sowing after stress induction. The plants were uprooted and washed carefully with water to clear away the adhered soil particles. The lengths of roots and shoots of T1, T2 and T3 plants were observed with meter scale. Shoots were severed from the roots and fresh weights of shoots and roots


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Tennants (1975) recorded using electronic balance. They were dried in oven at constant temperature of 600 C for 48 hrs. The dry weight of the root and shoot was determined. The leaf area of the plants grown under different treatments was estimated using Grid line intersect method. Number of leaves was also measured manually. Total chlorophyll content of plants was extracted and analyzed according to Arnon (1949). Estimation of total soluble sugar (Dubois et.al.,1956) and estimation of reducing sugar was done. Concentrations of total protein content of the plants grown under different treatments were estimated according to Lowry et al. (1951). Total phosphorus of different parts of plants samples were estimated by Banik and Dey (1981). Sample Extraction and HPLC Analysis: Accurately weighed 30 gm of per sample (root and leaf power) add five times HPLC grade methanol:water (1:1) was refluxed for 10 hours. A repeat this process three times. After pooling out three liquid fractionate weight empty round flask then put this liquid fractionate in that round flask and dry in Rotavapor (R-210). It was then concentrated on in rotary vacuum evaporator at 61-65ÚC. The collected concentrated material was purified with the help of chemicals, after purification 20ml methanol was added and filtered with Millipore. The extracted sample was ready for further analysis (Misra et. al, 2010). The analytical HPLC experiments were performed with a water HPLC system consisting of an Agilent series 1100 instrument equipped with a binary pump, an autosampler, an automatic electronic degasser, an automatic thermostatic column oven, a diode array detector operating at 225 nm, and a computer with Chemstation software for data analysis. High purity nitrogen from nitrogen from a nitrogen generator was used as a carrier gas. Separations were carried out with Column RP-18 with reagent methanol: water (3:2) as an eluent at flow rate of 0.7ml/min. and the column temperature was maintained at 30ÚC. RESULTS AND DISCUSSION After explanting the test plants, soil sample showed improved level of OC, total N, total P and total K in the range

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of 148.76%-625.81%, 151.83%-454.93%, and 268.26%1998.92% respectively over control sample. Following are the landmarks of the present investigation that high drug yielding plants species opening new possibilities of their cultivation. In present status we found that vermicompost (1 t/hac.) enhances the rate of alkaloid percentage (Table 3) doubles data furnishes morphological effects. In our present research work growth and biochemical activity of W.somnifera is maximum in T3 plants compared to all other treatments. The growth parameter decreases under moderate stress condition while enhances under stress condition with Vermicompost treatment such as shoot and root length, number of leaves, fresh and dry weight of W.somnifera (Table. 1). The root length was significantly high in T3 plant compared to other treatments. Also supports chlorophyll content, the rate of total sugar, reducing sugar, phosphate, proteins content (Table 2) and alkaloid contents (Table 3). This might be due the better availability of nutrients from organic and foliar source of nutrients and effective conservation of nutrients such as Fe, Mg and Zn at site of photosynthesis into pigments. The present study has created an interesting data with respect to plant growth, yield characters and biochemical analysis. As evidenced from the work of Xu et. al.(2000) and Hartwingon and Evan (2000), this may be due to effective micro-organism enhances the production of phytohormones like auxins and gibberellins that might have stimulated the growth by increasing the plant height, number of branches. Humic acid influences plant growth through modifying the physiology of plants and by improving the physical, chemical and biological properties of soil (Dursun et.al., 1999). Humic acid provides carbon as an energy source to nitrogen fixing bacteria and thus proves its biological function (Vaughan, 1974). The natural bioregulator in moringa leaf extract also increased the dry matter production registered increased yield compared to control. Leaf growth allows plants to increase their photosynthetic capacity while root growth allows plants to further exploit


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the soil for water and nutrients. But the existence of balance between different organs in stress situations is explained by differential sensitivities of growth of roots and shoots to water stress. At the same cell water potential leaf growth will be reduced more than root growth. It may also be that ABA plays a role in inhibiting shoot growth, while maintaining root growth (Hsiao and Xu, 2000). Water stress decreases leaf area which reduces the intercepted solar radiation (Trilica and Singh, 1979), which affects the biomass and yield of plants. In the present study there was also decrease in root & shoot lengths as well as root and shoot fresh and dry weights in severe water stress conditions than when compared with control. But in T3 plants where there was supplementation of VMC to the plants there was increase in the values than at moderate stress conditions when grown to maturity. In the present study the root/shoot dry weight ratios of control plants were less than the VMC induced treatments and T3 plants. The chlorophyll content in the control increased at the end of the last harvest due to well watering. In the water stress treated plants, chlorophyll decreased gradually. In VMC induced stress treated plants chlorophyll content increases. The same path is followed in the case of chlorophyll ‘a’ and chlorophyll ‘b’ content. Soil water deficits generally reduce leaf area and affect the biochemical processes that result in reduced photosynthesis and concentration of storage carbohydrates (Trilica and Singh, 1979). In the present study, the amount of non-reducing sugars increased gradually in water stress treated plants. This says that utility of sugars by the plant during the stress was reduced hence T2 plants exhibited almost equal amount of sugar to that of T1 plants. But control plants showed decreased non-reducing sugars indicating normal utilization of sugars. In our study there was increase in protein content in control plants and decreased gradually in stress treated plants. The lowering of protein contents in water stress plants were attributed to enhanced proteolytic activity (Ramiz and Mehraj, 2004).

Recent Advances in Medicinal Plants and Their Cultivation Table 2. Biochemical studies of Withania somnifera under different water stress condition with Vermicompost treatment (Value of Mean±SE of 10 samples; T1= Control; T2 = Moderate stress condition (under shade stress) and T3 = Stress Condition + Vermicompost treatment; *=p