Journal of Medicinal Plants Research Vol. 6(5), pp. 657-661, 9 February, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.257
ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper
Evaluation of antioxidant activities of Withania
somnifera leaves growing in natural habitats of
North-west Himalaya, India
R. K. Sharma*, S. S. Samant, P. Sharma and S. Devi
Pant Institute of Himalayan Environment and Development, Himachal Unit, Mohal-Kullu-175 126, Evaluation of antioxidant properties of medicinal plants from Indian Himalayan region has been very
rarely carried out. Withania somnifera
L. is one of the commercially available and most preferred
medicinal plants in the Himalayan region due to its aphrodisiac property and potential to cure various
diseases. The review of literature has indicated that the antioxidant activities of W. somnifera
have not
been carried out for so long. Therefore, in the present study an attempt was made to evaluate the
antioxidant properties of W. somnifera
collected from two different habitats that is, forest and roadside
at Kullu, north-west Himalaya. The total phenolic and flavonoid contents and DPPH (1, 1-Diphenyl-2-
pycrylhydrazyl) scavenging potential of leaves extract of W. somnifera
varied significantly between the
habitat (p<0.05
). The results further showed that the DPPH scavenging potential of leaves extract at
forest site was found significantly higher that is 51% over the roadside. The study indicates that
antioxidant activities of leaves of W. somnifera
L. varies habitat to habitat and antioxidant properties
decrease if plants are exposed to vehicular pollution. Therefore, plantation and cultivation of this
species in vehicular pollution free areas are suggested.
Key words:
Withania somnifera, phenolics, flavonoids, 1, 1-Diphenyl-2-pycrylhydrazyl (DPPH), habitats.


Many plant species are found rich in novel antioxidant
various health problems (for example, carcinogenesis, compounds (Pourmorad et al., 2006) and the activities of coronary heart disease, etc.) related to advancing age antioxidants in a plant depend on phenolic compounds (Cadens and Davies, 2000). Recent studies have clearly (Pietta, 2000). Medicinal plants have been recognized as shown potential of plant products to serve as antioxidant a potential source of natural antioxidants throughout the against various diseases induced by free radicals (Hou et world. Among the various medicinal plants, few endemic al., 2003). Minimizing anitoxidative damage through species are of particular interest as they are commonly scavenging free radicals may be one of the most being used for producing raw materials or preparations important approaches to prevent the human being from containing phytochemicals with significant antioxidant these ageing associated disease and health problems. capacities (Exarchou et al., 2002). Naturally produced Since antioxidants have the capacity to terminate the reactive oxygen species (ROS) can attack cell direct attack of ROS and free radicals mediated oxidative component and creates several types of biological reactions and appear to be or primary importance in the damage (Hutadilok-Towatana et al., 2006). Free radicals prevention of these diseases and health problems. Many (FR) or ROS capable of causing damage to studies have reported the biological activities of phenolics deoxyribonucleic acid (DNA) have been associated with as a potent antioxidant and free radical scavengers (Kahkonen et al., 1999; Sugihara et al., 1999). Motor vehicles have been closely related with increasing air pollution levels in urban areas responsible for more than *Corresponding author. E-mail: [email protected] Tel: 60% of the air pollution of an urban environment (Singh et al., 1995). Depending upon the fuel type, the main exhaust emissions are oxides of nitrogen (NOx), oxides The effect of methanol extracts of leaf samples on scavenging of carbon, oxides of sulphur (SOx), carbon particles, DPPH radical was estimated using the method of Liyana-Pathirama heavy metals, water vapour and hydrocarbons including and Shahidi (2005). A solution of 0.135 mM DPPH in methanol was prepared and 5 ml of the solution was mixed vigorously with 1ml of aldehydes, single and poly aromatic hydrocarbons, leaf extract in methanol. This mixture was left for 30 min in dark at alcohols, olefins, alkylnitriles besides a number of room temperature. The absorbance of mixture was measured using secondary pollutants such as ozone, etc., causing Spectrophotometer (Ultraspec 2100 Pro) at 517 nm. The ascorbic serious environmental and health impacts. W. somnifera acid or butylated hydroxyl toluene (BHT) solutions were used as L. commonly known as Ashwagandha has been a reference solution. The DPPH free radical scavenging activity was calculated using following equation. commercially viable medicinal shrub in Ayurvedic and Indigenous Systems of Medicines for many centuries in DPPH radical scavenging activity (%) = (O.DC– O.DS) × 100 / O.DC India (Jaleel et al., 2008). It is also known as the Indian Ginseng due to its ability of curing a large number of Where O. DC is the absorbance of DPPH radical + methanol; O. DS is the absorbance of DPPH radical + leaf extract/standard. diseases (Samant et al., 2008). The free radical scavenging potential of the W. somnifera from different habitats of the Himalaya has not been reported yet. Statistical analysis
Therefore, an attempt has been made to evaluate the antioxidant potential of the methanol extracts of the Means and standard error for the each site were analysed. The leaves of W. somnifera collected from two different significance differences between the sites were analysed using Student’s T-test at probability levels (0.05, 0.01). All statistical habitats that is, forest and roadside at Kullu valley of the analyses were performed by using SPSS software, version 12. RESULTS AND DISCUSSION
Chemicals were purchased from Sigma and Merck Chemical The contents of the total phenolics and flavonoids, and Company India. All the chemicals and reagents were of the DPPH scavenging potential of the methanol extracts of fresh leaves of W. somnifera L. are presented in Table 1 and Figure 1, respectively. The results showed that methanol extracts of leaves of W. somnifera L. collected Sampling and analysis

from the roadsides had significantly higher contents of Five mature plants, each of W. somnifera having heights of 60 cm total phenolics and flavonoids (100.10±0.90 and 92.88 ± were collected in March 2010 from the roadside habitat of NH-21 at 1.12 mg g-1 fresh leaf, respectively) as compared to forest a distance of 50 m both sides of the road and from forest habitat, (Table 1). On the other hand, DPPH radical scavenging 300 to 500 m away from the NH-21. The numbers of vehicles potential was found significantly higher (p<0.05) in the running on NH-21 were at Kullu averaged from 2,900 per day leaf extract of plants collected from forest as compared to during 6 am to 6 pm (Kuniyal et al., 2007). The samples were washed with running tap water and air dried. The leaves were roadside (Table 1). The present study suggests that separated and used for chemical analysis. For extraction, 1g of leaf plants exposed to vehicular pollution have higher was weighed and crushed in 10 ml of 80% methanol using a mortar contents of total phenolics and flavonoids, but have a and pestle, and were kept at 0°C for 24 h. Finally, the supernatant lower DPPH radical scavenging potential. The higher was taken and final volume was maintained to 10 ml by adding 80% contents of both total phenolics and flavonoids in the leaf methanol and stored in refrigerator for further analyses. The amounts of total phenolics in methanol extracts of leaf samples extracts of plants collected from road side may be ascribe were determined by the modified method of Wolfe et al. (2003). An to increased levels of free radicals due to emission of aliquot (1ml) of the extract was mixed with 1ml of Folin-Ciocalteu pollutants from running vehicles. Under normal conditions Phenol Reagent (previously diluted with double distilled water in 1:1 free radicals usually do not showed any harmful action v/v) and 2 ml of 2% of sodium carbonate. The final volume was because they are unstable, and change to a non radical maintained to 10 ml by double distilled water and whole mixture was then heated at 80°C for 30 min or till the blue color appeared. The absorbance of blue color solution after cooling at room The reduced form of phenolic compound acts as temperature was determined using Spectrophotometer (Ultraspec antioxidant while the oxidized one (phenoxy radical) may 2100 Pro) at 650 nm. The content of the total phenolics was produced cytotoxic effects, which is toxic to a living expressed in mg tannic acid g-1 fresh leaf. A standard curve was system because of their ability to initiate free radical also prepared using different concentrations of the tannic acid. chain reaction in membrane and their propensity to cross- Total flavonoids contents in the methanol extracts of the leaf link with a variety of molecule. An increase of phenolic samples was quantified using the modified method of Ordon-Ez et al. (2006) based on the formation of complex flavonoid-aluminum content was correlated to increase in activity of enzyme and absorbance of yellow color was determined using involved in phenolic compound metabolism under stress Spectrophotometer (Ultraspec 2100 Pro) at 420 nm. An amount of condition and increase in flavonoid concentration is 1 ml of aliquot was mixed to 1 ml of 2% ethanolic aluminium mainly the result of conjugated hydrolysis. The earlier chloride (AlCl3) vigorously. The reaction mixture was left for 1 h at studies have clearly shown that the antioxidants such as room temperature. Total flavonoid contents were calculated as mg quercetin g-1 fresh leaf using standard curve prepared from different peroxidase activity, phenolics, superoxide dismutase and glutathione reductase increased significantly due to Table 1. Total phenolics, flavonoid contents and DPPH inhibition potential of methanol extracts of the fresh leaves
of W. somnifera collected from roadside and forest of north-west Himalaya.
Methanol extracts
Forest site
Values are means ± S.E. of five replicates. The significant differences between polluted and non-polluted sites were analyzed using the student’s t-test. Level of significance: **p≤0.01, *p≤0.05.
Figure 1. DPPH radical scavenging activity of methanol extracts of the leaves of W. somnifera
collected from roadside and forest habitats of north-west Himalaya.
ozone and heavy metals in plants (Tiwari et al., 2010; emission which is present in the form of enzyme in Sharma et al., 2010). Mir et al. (2009) have reported plants. These enzymes provide additional defense increased levels of total flavonoids and phenolic content against oxidative stress and keep the metabolic activities. in selected medicinal plants growing on roadsides. Under stress conditions, enzyme activities increase in the Vehicular emissions have reduced the chlorophyll and plants. The continued exposure of soil to vehicular protein content and reduced leaf areas in roadside plants pollution may get rich in toxic chemicals such Cd, Pb, (Wagh et al., 2006). Photosynthetic pigments content etc., which may be ascribed to increased levels of total phenolics and flavonoids in roadside plants. The free Decreased in chlorophyll level under a stress condition radical scavenging potential of medicinal plants mainly may be due to reduction in pigment biosynthesis or depends on its active ingredients. The phenolic enzymatic chlorophyll degradation and slight reduction in compounds have antioxidant activity due to their redox carotenoides may be due to their protective role against properties, which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet Vehicular emission can lead to oxidative stress and oxygen, or decomposing peroxides. Total phenolics are causing significant decrease to photosynthetic system. also effective free radical scavengers and have Similarly protein content is also reduced under vehicular antioxidative property. The positive correlations between Table 2. DPPH scavenging capacities of methanol extracts of the fresh leaves of W. somnifera L. collected from
roadside and forest of north-west Himalaya.

Ascorbic acid
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