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INDEX – GJRMI - Volume 6, Issue 10, October 2017 MEDICINAL PLANT RESEARCH Biotechnology – Research EVALUATION OF ANTIOXIDANT PROPERTY OF HYDRO-ALCOHOL ROOT EXTRACT FROM INULA RACEMOSA AND PHYTOLACCA ACINOSA IN KASHMIR VALLEY Naveed Manzoor, Irfan Wani, Krishan p Singh*, Anju Pal
114–120
COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – FLOWER OF PUNICA GRANATUM L.* OF THE FAMILY LYTHRACEAE PLACE – OFF KANAKAPURA ROAD, BANGALORE, KARNATAKA, INDIA *BOTANICAL NAME VALIDATED FROM www.theplantlist.org AS ON 31/10/2017
Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 10 | October 2017 | 114–120 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 INULA RACEMOSA AND PHYTOLACCA ACINOSA IN KASHMIR VALLEY Naveed Manzoor1, Irfan Wani2, Krishan p Singh3*, Anju Pal4 1,2
Department of science and technology (Microbiology), Shri Venkateshwara University NH-24, Venkateshwara Nagar, Amroha, Uttar Pradesh- 244236. 3 Department of Biotechnology, Shri Venkateshwara 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 hydro-alcholic root extract of Inula racemosa and Phytolacca acinosa were investigated for their in-vitro antioxidant activities.Hydro-alcohol extract were prepared from powdered roots. The hydro-alcoholic root extracts of both plants showed DPPH activity, reducing power and hydrogen peroxidescavenging activity when compared to standard ascorbic acid. Further, Phytolacca acinosa exhibited greater antioxidant activity than Inula racemosa 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. KEY WORDS: DPPH, Inula racemosa, Phytolacca acinosa, Antioxidant activity.
Cite this article: Naveed Manzoor, Irfan Wani, Krishan p Singh*, Anju Pal (2017), EVALUATION OF ANTIOXIDANT PROPERTY OF HYDRO-ALCOHOL ROOT EXTRACT FROM INULA RACEMOSA AND PHYTOLACCA ACINOSA IN KASHMIR VALLEY, Global J Res. Med. Plants & Indigen. Med., Volume 6 (10): 114–120
Global Journal of Research on Medicinal plants & Indigenous medicine || GJRMI ||
Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 10 | October 2017 | 114–120
INTRODUCTION Medicinal plants play a pivotal role in the health care of ancient and modern cultures. Ayurveda, the Indian system of medicine mainly uses plant based drugs or formulations to treat various human ailments as they contain the components of therapeutic value (WHO, 1993).In addition, plant based drugs remain an important source of therapeutic agents because of the availability, relatively cheaper cost and non-toxic nature when compared to modern medicine (Agbor and Ngogang, 2005). Numerous medicinal plants are used in the cellular and metabolic disease treatment such as diabetes, obesity, cancer, etc. There are some speculations that the generation of free radicals inside the body in some physiological conditions results in the cellular changes and development of disease, which could be neutralized by the antioxidants from different medicinal plants. Several studies have shown that plant derived antioxidant neutraceuticals scavenge free radicals and modulate oxidative stress-related degenerative effects (Ames 1993 and Joseph 1999). Previous research reports suggest that higher intake of antioxidant rich food is associated with decreased risk of degenerative diseases particularly cardiovascular diseases and cancer (Thatte, Bagadey and Dahanukar, 2000). About 5% or more of the inhaled oxygen (O2) is converted to reactive oxygen species such as O2-, H2O2, and OH. Antioxidants can act by scavenging reactive oxygen species, inhibiting their formation, binding transition metal ions and preventing formation of OH and/or decomposition of lipid hydro-peroxides, which could lead to the repairing of damages (Gupta and Sharma, 2006). Inula racemosa is a stout herbaceous alpine perennial, 1.5m tall, with very large basal leaves and usually terminally borne, yellow flower heads. It is an Asian plant in the Daisy family native to the temperate and alpine Western Himalayas of Xinjiang, Afghanistan, Kashmir, Nepal and Pakistan. The plant is a stout shrub, bearing large leaves arranged in a racemose manner. The stem is grooved and all
vegetative parts are scabrid tomentose. Lower leaves are narrowed to a winged leaf stack. Upper leaves are lanceolate and stem clasping. The flowers are large, shady yellow daisies produced in mid to late summer. They are borne on apical spike like cluster. The fruits, slender achenes, 0.4 cm long, bearded with 0.75 cm long pappus hairs. Root stock branched; fresh roots are irregularly fusiform (20–25 × 5 cm). Sometimes a number of roots are found in the collar zone, though usually few occur in each clump. These roots have a dull brownish skin with yellowish colour inside. They possess a sweet and somewhat camphoraceous odour and have a bitter taste (Chopra, Nayar and Chopra, 1956). Phytolacca is a genus of perennial plants na tive to North and South America, East Asia and New Zealand. Some members of the genus are known as pokeweeds or similar names such as pokebush, pokeberry, pokeroot or poke sallet (Ed Dinger, 2011). The genus comprises about 25 to 35 species of perennial herbs, shrubs, and trees growing from 1 to 25 m (3.3 to 82.0 ft) tall. Habitat of the herb is Valleys, hillsides, forest under stories, forest margins and roadsides at elevations of 500–3400 metres. It is also found in cultivated land houses, moist fertile lands and as a weed.They have alternate simple leaves, pointed at the end, with entire or crinkled margins; the leaves can be either deciduous or evergreen. The flowers are greenish-white to pink, produced in long racemes at the ends of the stems (Quattrocchi and Umberto, 2000; Flora of china, 1832; Flora of North America, 1753). The present study was designed to evaluate the antioxidant activity of the hydro-alcohol root extract of Inula racemosa and Phytolacca acinosa. MATERIAL AND METHODS Collection of medicinal plants Fresh roots of Inula racemosa and Phytolacca acinosa medicinal plants were collected in the month of July 2014 from low altitude Gogaldor Tangmarg Kashmir, India
Global Journal of Research on Medicinal plants & Indigenous medicine || GJRMI ||
Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 10 | October 2017 | 114–120
Each specimen/plant material was labelled, numbered with the date of collection, locality, and their medicinal uses were recorded. Processing of medicinal plants The root of plants was used to prepare extracts for the study. The plants collected were washed with water to remove the soil and dust particles. Then they were dried in thoroughly shaded place, and blended to form a fine powder and stored in airtight containers. Antioxidant evaluation a) DPPH radical Scavenging Assay: The antioxidant activity of the extracts was 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(MacBride, 1937) with slight modifications. 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 was prepared and L-Ascorbic acid (1–100 µg/ml) was used as reference standard. Mixture 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 (Oyaizu1986). Various concentrations (50, 100, 150, 200, 250 and 300 ug/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 and 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 and Klaunig, 1989). A 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 There is an increasing evidence that indigenous antioxidants may be useful in preventing the deleterious consequences of oxidative stress and there is increasing interest in the protective biochemical functions of natural antioxidants contained in spices, herbs and medicinal plants (Osawa, 1994; Noda Y et al., 1997).In the present study, free radical scavenging activities of the hydro-alcohol root extract of Inula racemosa and Phytolacca acinosa 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 widely used method for screening the antioxidant activity of plant extract. DPPH stable free radical method is a sensitive way to
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Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 10 | October 2017 | 114–120
determine the antioxidant activity of plant extracts. Table-1 shows the antioxidant activities of the hydro-alcohol root extracts of Inula racemosa and Phytolacca acinosa assessed using the DPPH radical scavenging activity. We observed that the extract of Phytolacca acinosa had higher activity than that Inula racemosa. 100–600 µg/mL of hydroalcohol root extracts of Inula racemosa and Phytolacca acinosa extracts showed reducing activity, although less than that of Ascorbic acid, a reference antioxidant, the extractives increased the reducing activity with the increased concentration of the extracts. At 100 μg/mL, the absorbance of hydro-alcohol root extracts of Inula racemosa, Phytolacca acinosa and ascorbic acid were 49.30, 62.72 and 70.86 respectively, while at 600 μg/mL, the absorbance were 91.14, 83.49 and 98.40 respectively (Table-1). A higher absorbance indicates a higher reducing power. These results demonstrated that the hydro-alcoholic root extracts of Inula racemosa and Phytolacca acinosa 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 hydro-alcoholic root extract of both plants had the free radical scavenging activity when compared with standard Ascorbic acid (Figure 1). The results indicated the protondonating ability of the extractives which could serve as free radical inhibitors or scavengers and can also be served as primary antioxidants. The work performed adequately demonstrates that there exists correlation between polyphenolic contents of the extractives and its anti-oxidant properties. Consequently, this could be exploited as health care supplement (Noda et al., 1997). Reducing power assay The reducing powers of hydro-alcoholic root extractives of two different plants were shown in Table-2. Hydro-alcoholic root extract of two different plants showed considerable antioxidant activity compared to Ascorbic acid (standard).
At the concentration of 50 μg/mL, the absorbance of hydro-alcoholic root extract of Inula racemosa, Phytolacca acinosa and ascorbic acid were 0.25, 0.19 and 0.52, respectively. At 300 μg/mL, the absorbance of hydro-alcoholic root extracts of Inula racemosa, Phytolacca acinosa and ascorbic acid were 1.225, 1.936 and 2.325 (Table-2). The extractives were found to increase in antioxidant activity with the increasing concentration of the extracts. The iron reducing capacity of the hydro-alcoholic root extractives of Inula racemosa and Phytolacca acinosa were estimated from their ability to reduce the Fe3+ferricyanide complex to the ferrous form by donating an electron. The reducing ability of the extractives was in the range of 0.190 ± 0.06– 1.936 ± 0.01μm 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 and Prior, 2005). Hydrogen peroxide scavenging activity The scavenging ability of hydro-alcoholic root extracts of Inula racemosa and Phytolacca acinosa on hydrogen peroxide is shown Table 3 and compared with ascorbic acid standards. The extracts were capable of scavenging hydrogen peroxide in an amount dependent manner. The hydro-alcohol root extract of Inula racemosa and Phytolacca acinosa exhibited 10.34-36.55 µg/mL and 13.89-45.32 µg/mL respectively. On the other hand, using the same amounts, ascorbic acid exhibited 17.65 - 59.56 µg/mL hydrogen peroxide scavenging activity. Results show that the scavenging activity values on hydrogen peroxide of 50 µg of the extracts of Inula racemosa and Phytolacca acinosa decreases than that of Ascorbic acid in the descending order. Hydrogen peroxide itself is not very reactive, but it can sometimes be toxic to cell because of it may give rise to hydroxyl radical in the cells (Halliwell 1991). Thus, the removing of H2O2 is very important for antioxidant defence in cell or food systems.
Global Journal of Research on Medicinal plants & Indigenous medicine || GJRMI ||
Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 10 | October 2017 | 114–120
Table-1: Absorbance of hydro-alcohol root extracts of Inula racemosa, Phytolacca acinosa and ascorbic acid for antioxidant activity Conc extracts (µg/mL) 100
of Inula racemosa
Phytolacca acinosa Ascorbic acid
49.30 ± 0.78
54.72 ± 0.28
70.86 ± 0.35
200
55.40 ± 0.33
64.38 ± 0.39
78.60 ± 0.55
300
62.33 ± 0.41
69.50 ± 0.60
82.36 ± 0.76
400
70.20 ± 0.36
79.64 ± 0.80
89.47 ± 0.66
500
77.33 ± 0.78
83.25 ± 0.84
93.30 ± 0.43
600
84.1g4 ± 0.68
89.49 ± 0.33
98.40 ± 0.50
Figure-1: Free radical scavenging activity when compared with standard Ascorbic acid 120
Inula racemosa
Phytolacca acinosa
%age of activity
100 80 60
82.36
78.6 70.86 54.72 49.3
64.38
69.5 62.33
Ascorbic Acid
93.3 89.47 83.25 79.64 77.33 70.2
98.4 89.49 84.14
55.4
40 20 0 100
200
300
400
500
600
Conc of extracts (mg/mL)
Table-2: Reducing powers of hydro-alcoholic root extractives of Inula racemosa and Phytolacca accinosa Conc of (µg/mL) 50 100 150 200 250 300
extracts Inula racemosa 0.190 ± 0.06 0.340 ± 0.02 0.646 ± 0.06 0.986 ± 0.02 1.039 ± 0.08 1.225 ± 0.07
Phytolacca acinosa
Ascorbic acid
0.250 ± 0.01 0.532 ± 0.03 0.918 ± 0.05 1.030 ± 0.03 1.656 ± 0.02 1.936 ± 0.01
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
Global Journal of Research on Medicinal plants & Indigenous medicine || GJRMI ||
Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 10 | October 2017 | 114–120
Table-3: Scavenging ability of hydro-alcoholic root extracts of Inula racemosa and Phytolacca acinosa on hydrogen peroxide Conc of (µg/mL) 50 100 150 200 250 300
extracts Inula racemosa 10.34 14.11 17.76 21.63 27.23 36.55
CONCLUSION In conclusion, the hydro-alcoholic root extracts of both plants showed DPPH acticity, reducing power assay and hydrogen perioxide scavanging activity when compared to standard ascorbic acid. The results ofthis study also
Phytolacca acinosa
Ascorbic acid
13.89 16.45 20.90 26.89 33.76 45.32
17.65 19.37 27.87 34.66 45.92 59.56
showed that the hydro-alcoholic root extract of Phytolacca acinosa exhibited greater antioxidant activity than Inula racemosa 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.
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Global J Res. Med. Plants & Indigen. Med. | Volume 6, Issue 10 | October 2017 | 114–120
MacBride JF. Flora of Peru.(1937). Publications of Field Museum of Natural History, Botanical Series. 8:553–556. Noda Y, Anzai-Kmori A, Kohono M, Shimnei M, Packer L. (1997) Hydroxyl and superoxide anion radical scavenging activities of natural source antioxidants using the computerized JES-FR30 ESR spectromoter system. Biochem Mol Biol Inter. 42:35–44. Osawa T. (1994). Post harvest biochemistry. In:Uritani I, Garcia VV, Mendoza, EM, editors. Novel neutral antioxidant for utilization in food and biological systems. Japan: Japan Scientific Societies Press. 241–251. 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.
Source of Support: NIL
Quattrocchi, Umberto (2000). CRC World Dictionary of Plant Names. 3 M-Q. CRC Press. 2000;2065. ISBN 978-0-84932677–6. Ruch RJ, Cheng SJ, Klaunig JE. (1989). Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis. 10:1003–1008. Thatte U, Bagadey S, Dahanukar S. (2000). Modulation of programmed cell death by medicinal plants. Cell Mol Biol. 46:199–214. World health organization regional office for the western pacific. (1993). Research guidelines for evaluating the safety and efficacy of herbal medicines. Manila, World Health organization regional office for the western pacific.
Conflict of Interest: None Declared
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