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INDEX – GJRMI - Volume 6, Issue 11, November 2017 MEDICINAL PLANT RESEARCH Biotechnology – Research EVALUATION OF ANTIOXIDANT PROPERTY OF HYDRO-ALCOHOL ROOT EXTRACT FROM THYMUS LINEARIS BENTH. AND DIPSACUS INERMIS WALL. IN KASHMIR VALLEY Irfan Wani, Naveed Manzoor, Krishan p Singh*, Anju Pal
121–126
COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – INFLORESCENCE OF LANTANA CAMARA L.* OF THE FAMILY VERBENACEAE PLACE – OFF KANAKAPURA ROAD, BANGALORE, KARNATAKA, INDIA *BOTANICAL NAME VALIDATED FROM www.theplantlist.org AS ON 30/11/2017
Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 11 | November 2017 | 121–126 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
Research Article EVALUATION OF ANTIOXIDANT PROPERTY OF HYDRO-ALCOHOL ROOT EXTRACT FROM THYMUS LINEARIS BENTH. AND DIPSACUS INERMIS WALL. IN KASHMIR VALLEY Irfan Wani1, Naveed Manzoor2, Krishan p Singh3*, Anju Pal4 1,2
Department of science and technology(Microbiology), Shri Venkateshwara UniversityNH-24, Venkateshwara Nagar, Amroha, Uttar Pradesh- 244236. 3 Department of Biotechnology, Shri Vankateshwara University NH-24, Amroha, Uttar Pradesh-244236 4 Dept of Horticulture, G B Pant University of Science & Technology, Pant Nagar, Uttrakhand *Corresponding Author: E-mail ID: krishanpalbtech@gmail.com; Mobile no. 8755675553, 9411904938
Received: 10/9/2017; Revised: 29/10/2017; Accepted: 30/10/2017
ABSTRACT The aim of the study was to determine DPPH activity, reducing power assay and hydrogen peroxide radical scavenging activity of hydro-alcoholic root extract of two medicinal plants viz. Thymus linearis and Dipsacus inermis. In this study, hydro-alcoholic extract were prepared from powdered roots. The hydro-alcoholic root extracts of both plants showed DPPH activity,reducing power assay and hydrogen perioxide scavanging activity when compared to standard ascorbic acid. The results of the study also showed that the hydro-alcoholic root extract of Thymus linearis exhibited greater antioxidant activity than Dipsacus inermis root extract. The present investigation suggests that medicinal plants which possess good antioxidant potential are the best supplements for the diseases associated with oxidative stress.
KEYWORDS: DPPH, Thymus linearis, Dipsacus inermis, Ascorbic acid, Antioxidant activity.
Cite this article: Irfan Wani, Naveed Manzoor, Krishan p Singh*, Anju Pal (2017), EVALUATION OF ANTIOXIDANT PROPERTY OF HYDRO-ALCOHOL ROOT EXTRACT FROM THYMUS LINEARIS BENTH. AND DIPSACUS INERMIS WALL. IN KASHMIR VALLEY, Global J Res. Med. Plants & Indigen. Med., Volume 6 (11): 114–120
Global Journal of Research on Medicinal plants & Indigenous medicine || GJRMI ||
Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 11 | November 2017 | 121–126
INTRODUCTION Antioxidants play an important role as health protecting factor. Scientific evidencesuggests that antioxidants reduce the risk for chronic diseases including cancer and heart disease. Primary sources of naturally occurring antioxidants are whole grains, fruits and vegetables (The Wealth of India, 1988). Plant sourced antioxidants like vitamin C, vitamin E, carotenes, phenolic acids etc. have been recognized as having the potential to reduce disease risk (Indian herbal pharmacopoeia, 2002). Most of the antioxidant compounds in a typical diet are derived from plant sources and belong to various classes of compounds with a wide variety of physical and chemical properties (Sravani & Paarakh, 2012). Free radicals are generated in our body during the normal metabolic processes and during exposure to adverse patho-physiological conditions (Van Langendonckt, Casanas-Roux & Donnez J. 2002). They are unstable species that are able to induce cellular damage in several ways. The most deleterious effects of free radicals is to damage DNA, which is associated with the process of carcinogenesis (Piconi, Quagliaro & Ceriello, 2003). Phytochemicals such as phenolics, carotenoids and dietary fibers are gaining increased attention because of their antioxidant, anticarcinogenic, antimutagenic, and other health promoting properties. Although synthetic antioxidants such as butylatedhydroxy toluene, butylatedhydroxy anisole, gallic acid esters and tertiary butylated hydroquinone have potential to neutralize free radicals, they have been criticized due to possible toxic effects, low solubility along with moderate antioxidant activity. Hence there arises a need to discover new potential natural sources of antioxidants (Block & Langseth, 1994). Several studies have successfully correlated the phenolic content with antioxidant activity. For example, natural phytochemicals present in berry crops, tea, oilseeds, beans, fruits and vegetables, herbs, spices and several medicinal plants are good source of antioxidant activity whichhas been correlated to the plant secondary metabolites such as flavonoids, carotenoids,
alkaloids, tannins and phenolic compounds (Emami, Asili, Mohagheghi et al., 2007). Thymus linearis is a species in the genus of aromatic perennial herbaceous plants and subshrubs to 40 cm tall in the family Lamiaceae, native to temperate regions in Europe, North Africa and Asia. Stems tend to be narrow or even wiry; leaves are evergreen in most species, arranged in opposite pairs, oval, entire, and small, 4–20 mm long, and usually aromatic. Thyme Flowers are in dense terminal heads, with an uneven calyx, with the upper lip threelobed, yellow, white or purple. Dipsacus inermis is a species in the genus Dipsacus which contains approximately 28 to 33 species and belongs to the family of the Caprifoliaceae. Dipsicus inermis is deciduous. The simple leaves are opposite. They are lanceolate with entire margins. Dipsacus inermis produces heads of white tubular flowers from August to September; Dipsicus inermisis native to the Himalaya and Myanmar. The present research work was therefore designed to evaluate the antioxidant activity of the hydro-alcohol root extract of two medicinal plants namely Thymus linearis and Dipsacus inermis. MATERIAL AND METHODS Collection of medicinal plants Fresh roots of Thymus linearis and Dipsacus inermis medicinal plants has been used in the present study and were collected in the month of July 2014 from low altitude Gogaldor Tangmarg Kashmir, India. Each specimen/plant material was appropriately labelled, sample ID, date of collection, locality, and their medicinal uses were recorded. Processing of medicinal plants The roots of plants were used to prepare extracts. The plants collected were washed with water to remove the soil and dust particles. They were dried in thoroughly shaded place, and blended to form a fine powder and stored in airtight containers.
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Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 11 | November 2017 | 121–126
Antioxidant evaluation a) DPPH radical scavenging assay: The antioxidant activity of the extracts were measured on the basis of the scavenging activity of the stable 1, 1- diphenyl 2-picrylhyorazyl (DPPH) free radical according to the method described by Brand Williams with slight modifications (Brand-williams 1995). 1mL of 0.1mM DPPH solution in methanol was mixed with 1mL of plant extract solution of varying concentrations (100, 200, 300, 400, 500 and 600 µg/mL). Corresponding blank sample were prepared and L-Ascorbic acid (1–100 µg/mL) was used as reference standard. Mixer of 1mL methanol and 1mL DPPH solution was used as control. The reaction was carried out in triplicate and the decrease in absorbance was measured at 517nm after 30 minutes in dark using UV-Vis spectrophotometer. The inhibition % was calculated using the following formula. Inhibition % = Ac-As/Ac×100 Where Ac is the absorbance of the control As is the absorbance of the sample b) Reducing power assay The reducing power was determined according to the method of (Oyaizu, 1986). Various concentrations (50, 100, 150, 200, 250 and 300 µg/ml) of hydro-alcohol root extracts (2.5 mL) were mixed with 2.5 ml of 200 mmol/L sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min. After 2.5 mL of 10% trichloroacetic acid (w/v) were added, the mixture was centrifuged at 650 rpm for 10 min. The upper layer (5 mL) was mixed with 5 mL deionised water and 1 mL of 0.1% of ferric chloride, and the absorbance was measured at 700 nm. Higher absorbance indicates higher reducing power. Ascorbic acidwas used as standard. c) Hydrogen Peroxide Scavenging Activity The capability of scavenging hydrogen peroxide by the extract was determined based on the method of (Ruch, Cheng & Klaunig, 1989). Hydrogen peroxide solution (2 mM) was
prepared in 50 mM phosphate buffer (pH 7.4). Aliquots (0.1 mL) of the extracted sample (different concentration of 50, 100, 150, 200, 250, and 300 μg/mL) were transferred into the test tubes and their volumes were made up to 0.4 mL with 50 mM phosphate buffer (pH 7.4). After adding 0.6 mL hydrogen peroxide solution, tubes were vortexed and the absorbance of the hydrogen peroxide at 230 nm was determined after 10 min, against a blank. The abilities to scavenge the hydrogen peroxide were calculated based on the following equation: Scavenging activity (%) = 1 – Absorbance of sample/Absorbance of control × 100 RESULTS AND DISCUSSION Free radicals results in extensive damage to tissues and biomolecules leading to various disease conditions. So the medicinal plants are employed as an alternative source of medicine to mitigate the diseases associated with oxidative stress (Roja & Rao, 2000). In the present study, free radical scavenging activities of the hydro-alcohol root extract of Thymus linearis and Dipsacus inermis were evaluated. Free radicals are involved in many disorders like neurodegenerative diseases, cancer and AIDS. Antioxidants due to their scavenging activity are useful for the management of those diseases. DPPH radical scavenging activity DPPH radical scavenging activity is one of the most sensitive and widely used method for screening the antioxidant activity of plant extract.Table-1 depicts the antioxidant activities of the hydro-alcohol root extracts of Thymus linearis and Dipsacus inermis. It was observed that the extract of Thymus linearis had higher activity than Dipsacus inermis. 100–600 µg/mL of hydro-alcohol root extracts of Thymus linearis and Dipsacus inermis extracts showed reducing activity, although less than that of ascorbic acid. The reducing activity increased with the increased concentration of the extracts. At 100 μg/mL, the absorbance of hydro-alcohol root extracts of Thymus linearis, Dipsacus inermis and ascorbic acid were 57.30, 43.72 and
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Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 11 | November 2017 | 121–126
70.86 respectively, while at 600 μg/mL, the absorbance of hydro-alcohol root extracts of Thymus linearis, Dipsacus inermis and ascorbic acid were 88.14, 79.49 and 98.40 respectively. A higher absorbance indicates a higher reducing power. These results demonstrated that the hydro-alcoholic root extracts of Thymus linearis and Dipsacus inermis had considerable DPPH activity. This method has been used extensively to predict antioxidant activities because of the relatively short time required for analysis. Our results revealed that the hydroalcoholic root
extract of both plants had the free radical scavenging activity when compared with standard Ascorbic acid. The results indicated the protondonating ability of the extracts, which could serve as free radical inhibitors or scavengers and can also be served as primary antioxidants. We observed correlation between polyphenolic contents of the extracts and its anti-oxidant properties. Consequently, this could be exploited as health care supplement (Huang, Ou & Prior, 2005).
Table-1: Antioxidant activities of the hydro-alcohol root extracts of Thymus linearis and Dipsacus inermis. Conc of extracts (µg/mL) 100 200 300 400 500 600
Thymus linearis 57.30 ± 0.78 66.40 ± 0.33 73.33 ± 0.41 79.20 ± 0.36 83.33 ± 0.78 88.14 ± 0.68
Dipsacus inermis 43.72 ± 0.28 49.38 ± 0.39 57.50 ± 0.60 65.64 ± 0.80 71.25 ± 0.84 79.49 ± 0.33
Ascorbic acid 70.86 ± 0.35 78.60 ± 0.55 82.36 ± 0.76 89.47 ± 0.66 93.30 ± 0.43 98.40 ± 0.50
Reducing power assay
Hydrogen peroxide scavenging activity
Hydro-alcoholic root extract of Thymus linearis and Dipsacus inermis showed considerable antioxidant activity compared to Ascorbic acid. At the concentration of 50 μg/mL, the absorbance of hydro-alcoholic root extract of Thymus linearis, Dipsacus inermis and Ascorbic acid were 0.23, 0.14 and 0.52, respectively; while at 300 μg/mL, the absorbance of hydro-alcoholic root extracts of were 1.425, 1.136 and 2.325 (Table-2). The iron reducing capacity of the hydro-alcoholic root extractives of Thymus linearis and Dipsacus inermis were estimated from their ability to reduce the Fe3+-ferricyanide complex to the ferrous form by donating an electron. The reducing ability of the extracts was in the range of 0.145 ± 0.01– 1.425 ± 0.07μm Fe (II)/g (Table-2). In this study, ferrous reducing antioxidant capacity was increased with the increase of phenolic contents. Our results are consistent with the result published previously (Huang, Ou & Prior, 2005).
The extracts were capable of scavenging hydrogen peroxide in an amount dependent manner. The hydro-alcohol root extract of Thymus linearis and Dipsacus inermis exhibited 13.74–48.47% and 10.91–38.89%of scavenging activity on hydrogen peroxide, respectively. On the other hand, using the same amounts, ascorbic acid exhibited 17.65– 59.56% hydrogen peroxide scavenging activity. Results show that the scavenging activity values on hydrogen peroxide of 50 µg of the extracts of Thymus linearis (13.74%) and Dipsacus inermis (10.91%) were less than Ascorbic acid (17.65%). Hydrogen peroxide itself is not very reactive, but it can sometimes be toxic to cells by generating hydroxyl radical in the cells (Halliwell, 1991). Thus, removing of H2O2 is very important for antioxidant defence in cell or food systems.
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Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 11 | November 2017 | 121–126
Table-2: Reducing power of hydro-alcoholic root extracts of Thymus linearis and Dipsacus inermis. Conc of extracts (µg/mL) 50 100 150 200 250 300
Thymus linearis 0.235 ± 0.06 0.390 ± 0.02 0.546 ± 0.06 0.696 ± 0.02 1.039 ± 0.08 1.425 ± 0.07
Dipsacus inermis 0.145 ± 0.01 0.230 ± 0.03 0.398 ± 0.05 0.530 ± 0.03 0.856 ± 0.02 1.136 ± 0.01
Ascorbic acid 0.520 ± 0.01 0.710 ± 0.02 0.990 ± 0.06 1.396 JJ± 0.02 1.949 ± 0.08 2.325 ± 0.07
Table-3: The scavenging ability of hydro-alcoholic root extracts of Thymus linearis and Dipsacus inermis on hydrogen peroxide Conc of extracts (µg/mL) 50 100 150 200 250 300
Thymus linearis 13.74 16.47 19.86 28.71 36.23 48.47
CONCLUSION In the current investigation, the hydroalcohol root extracts of Thymus linearis and Dipsacus inermis in the ratio of 50 : 50 has been selected. The hydro-alcoholic root extracts of both plants showed DPPH activity, reducing power and hydrogen perioxide scavenging activity when compared to standard ascorbic
Dipsacus inermis 10.91 12.75 16.90 21.23 29.12 38.89
Ascorbic acid 17.65 19.37 27.87 34.66 45.92 59.56
acid. The results ofthis study also showed that the hydro-alcoholic root extract of Thymus linearis exhibited greater antioxidant activity than Dipsacus inermis root extract. The present investigation suggests that medicinal plants which possess good antioxidant potential are the best supplements for the diseases associated with oxidative stress.
REFERENCES Block G, Langseth L. (1994). Antioxidant vitamins and disease prevention. Food Technol 48:80–84. Brand-williams W, Cuvelier ME and Berset C. (1995). Use of free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft and Technologie 28:25–30. Emami
SA, Asili J, Mohagheghi Z, Hassanzadeh MK. (2007). Antioxidant activity of leaves and fruits of Iranian
conifers. Evid Based Complement Alternat Med 4:313–319s. Halliwell, B. (1991). Reactive oxygen species in living systems: Source, biochemistry, and role in human disease. American Journal of Medicine 91;14–22. Huang D, Ou B, Prior RL. (2005). The chemistry behind antioxidant capacity assays. J Agric Food Chem 53:1841– 1856.
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Indian herbal pharmacopoeia. (2002). Revised new edn, Indian drug manufacturers association, Mumbai 79–87. Oyaizu M. (1986). Studies on products of browning reactions: antioxidative activities of products of browning reaction prepared from glucosamine. Japanese Journal of Nutrition 44:307– 315. Piconi L, Quagliaro L, Ceriello A. (2003). Oxidative stress in diabetes. Clin Chem Lab Med 41:1144–1149. Roja
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Ruch RJ, Cheng SJ, Klaunig JE. (1989). Prevention of cytotoxicity and inhibition of intracellular
Source of Support: NIL
Conflict of Interest: None Declared
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