Indian Journal of Natural Products and Resources Vol. 6(1), March 2015 pp. 51-55

Spectroscopic study of antioxidant property and trace elements of spinosa Roxb. ex Link leaves

N K Sharat Singh1, W Radhapiyari2, Md. A H Choudhury3, Th. Brojendro Singh3*, Kh. Bonny3, Th. Sobita Devi4, R K Rajeshwari Devi3 and N Rajmuhon Singh1 1Department of Physics, Manipur University, Canchipur, Imphal-795 003, India 2IBSD, Takyelpat, Imphal-795001 3Oriental College, Takyelpat, Imphal-795001 4D M College of Science, Imphal-795001 1Department of Chemistry, Manipur University, Canchipur, Imphal-795 003

Received 10 February 2014; Accepted 1 November 2014

Antioxidant property of Meyna spinosa Roxb. ex Link leaf was determined by using UV-Visible and Electron Paramagnetic Resonance (EPR) spectroscopy. Further the trace elements present in it were determined by using Atomic Absorption Spectroscopy (AAS). The concentration of the antioxidant activity, IC50 was found to be 563.23 µg/mL and the trace elements detected were Fe, Zn, Cu, Mo, Cr, Mn. Role of antioxidants and trace elements were discussed with reference to the traditional knowledge.

Keywords: Meyna spinosa, Spectroscopy, Antioxidant, Trace elements. IPC code; Int. cl. (2014.01)−A61K 36/00

Introduction radical, stop the free radical from forming in the Man has been dependent upon nature for survival first place and minimize the damage caused by free since time immemorial. and derived radicals3 A free radical is any chemical products are part of health care since human capable of independent existence that possesses civilization. Prior to the development of modern on or more unpaired electrons. Due to their medicine, the traditional systems of medicine that unsaturation with electrons, they are extremely have evolved over the centuries within various reactive. They can be generated by photolysis, communities are still maintained as a great traditional thermolysis, radiolysis, in oxido-reduction processes, knowledge base in herbal medicines1. Many workers enzymatic processes in vivo, by the influence of had been engaged in the extraction of biochemicals, ozone, etc. In addition free radicals are formed structure analysis and antioxidant property of in human cells during normal metabolism and take them. Antioxidants are useful for their anti-ageing and place in many biological reactions4. Uncontrolled anti-disease properties. There is relation among free and abnormal free radical reactions can cause radicals and quality of life and health but some deterioration of physical, biological and health foodstuffs have paramagnetic properties naturally and characteristics of food, pharmaceutical and cosmetic they show a single Electron Paramagnetic Resonance products. Excessive quantity of free radicals in (EPR) spectral signal2. So, the natural antioxidant human body can produce oxidative stress and properties of herbs are recommended strongly now-a- lead to many pathological states such as mutagenesis days. Antioxidants are molecules which can safely and carcinogenesis, aging, rheumatism, arthritis, interact with free radicals and terminate the chain arteriosclerosis, necrosis, diabetes, etc. The deficiency reaction before vital molecules are damaged. or excess of trace elements leads to various Antioxidants may diminish the energy of the free complications and metabolic disorders in human beings5. —————— *Correspondent author: Family plants are trees, shrubs or E-mail: [email protected] infrequently herbs comprising about 450 genera and 52 INDIAN J NAT PROD RESOUR, MARCH 2015

6500 species, including lianous forms. Leaves are Plant extract simple and usually entire and are opposite or 200g of shade-dried and pulverized leaves were sometimes whorled; stipules are present and subjected to extraction in a Soxhlet apparatus with interpetiolar. Meyna spinosa Roxb. ex Link belongs one litre of methanol boiling it at 48°C for 72 h. to family Rubiaceae and its local name (Manipuri) is Extraction was conducted until no more coloured Lam Heibi. This herb is popular among the people of matter was extracted. Solvent extracted mixture was Asia for its curability of hepatic disorder, evaporated to dry state using a rotary evaporator gastrointestinal problems, skin infection; it is under reduced pressure at 40°C. Dried extract was refrigerant and abortifacient6. Fruits of this medicinal then kept in tightly fitted stopper bottles and stored plant are reported to contain sugar, gum and tannic at -4°C. acid whereas seeds contain esters of palmitic acid, stearic and oleic acids. Leaves are used in bone DPPH radical scavenging assay fracture and in the treatment of diphtheria7. The Scavenging activity of extracts on DPPH was Ayurvedic and plant based remedies are a determined using the method followed by Okada and 10 part of cultural heritage of India and so is in the Okada . This method depends on the reduction of North-Eastern region8,9. purple DPPH to a yellow coloured diphenyl In Manipur, North-Eastern state of India, leaf of picrylhydrazine. Determination of the disappearance this plant is used as traditional salad, particularly by of free radicals was done using the Meitei community. Decoction of its leaves is also UV-visible spectrometer (Model: Cary 100 Bio). The used for traditional treatment of inflammation, and as remaining DPPH which showed maximum absorption a hair lotion. Fruit is used in traditional skin lotion at 517 nm was measured. Methanol extract sample’s and as an edible item by the Manipuris. So, the stock solution (1.0 mg/mL) was diluted to final present objectives of this study are to evaluate the concentrations of (0.6 .0.5, 0.4, 0.3, 0.2, 0.1, 0.05 and trace elements by Atomic Absorption Spectroscopy 0.01) mg/mL in ethanol. One millilitre of a 0.3 mM (AAS) and the antioxidant property by using EPR and DPPH ethanol solution was added to 2.5 mL of UV-visible spectrometer of the M. spinosa leaf. sample solution of different concentrations. These are test solutions. One mL of ethanol was added to Materials and Methods 2.5 mL of sample solution of different concentrations. These are blank solutions. One mL DPPH solution Plant materials plus 2.5 mL of ethanol was used as a negative control. M. spinosa (Plate 1) leaves were collected from The blank for this solution is ethanol. As DPPH is Imphal West District, Manipur and washed with sensitive to light, it iwas exposed to the minimum distilled water to avoid from possible environmental possible light. These solutions were allowed to react contaminants like dust and other unwanted at room temperature for 30 minutes. The radical substances. Plant sample was identified by The scavenging activity, expressed as percentage of Department of Botany, D.M. College of Science and inhibition was calculated using the equation: deposited (Acc. No. OC-00103) at bio-hub Centre, Oriental College, Imphal. Inhibition Concentration (%) = 100 – [(absorbance of sample – absorbance of blank) × 100/absorbance of control].

Tests were done in triplicate and IC50 values were calculated by linear regression of plots, where the abscissa represents concentration of the tested plant extract and ordinate as the average percent of scavenging capacity. Concentration of sample required to scavenge 50% of DPPH (IC50) was determined.

Electron Paramagnetic/Spin Resonance spectrometer (EPR) analysis

This extract was used for analysis of antioxidant Plate 1—Meyna spinosa Roxb. ex Link property by using Electron Paramagnetic/Spin SINGH et al: SPECTROSCOPIC STUDY OF MEYNA SPINOSA ANTIOXIDANT PROPERTY 53

Resonance spectrometer (Model: JEOL, JES-FA concentration 50, IC50 value was found to be 200 ESR spectrometer, X-band microwave unit; 563.23 ± 0.22 µg/mL which is responsible for 50 % TEMPOL was used as field marker). All EPR inhibition of free radicals. Scavenging activity is measurements were performed at room temperature 2.45 ± 0.12 µg/mL. IC50 value of methalonic extract on Varian X Band EPR Spectrometer, operating of leaves was reported to be 16.4 ± 0.41 µg/mL by at a microwave frequency of 9443.847 MHz, with earlier workers16. The difference is attributable to the 100 kHz field modulation. collection of samples from different geographical locations. Protection of UV radiations with the help of Atomic Absorption Spectrometer (AAS) analysis antioxidants17-19 corroborates idea of traditional use of Leaves were dried at 65°C in an oven for 24 h and M. spinosa as a skin lotion. subsequently ground by an agate mortar. Powders were converted into liquid form with acid digestion Some of the essential trace elements detected so far process and used for elemental analysis by Graphite in M. spinosa leaf are: Fe, Zn, Cu, Mo, Cr, Mn and Furnace - Atomic Absorption Spectrometer (Model: corresponding concentrations were found to be Analytic Jena vario-6). 1.02±0.005, 0.434±0.014, 2.30±0.02. 0.011±0.001, 4.27±0.097, 0.014±0.002 ppb, respectively. These Results and Discussion elements can have their definite nutritive roles as As free radicals are responsible for diseases in the well as antioxidant activity. Zinc is essential to all human body, presence of it in our day to day edible organisms and it is an important trace element having items has been found very interesting for the a definite role in metabolism, growth and researchers all around the world. On the other hand development. It is an essential component of over search for antioxidants in the food items as well as in 200 enzymes, viz. cytosolic superoxide dismutase, medicinal plants had been pursued for many decades. having both catalytic and structural roles. Zinc Detection of the EPR signal in the EPR spectrum of deficiency is characterised by recurrent infection, lack 20 M. spinosa leaf powder emphasises the presence of of immunity and poor growth . Low intake of zinc 21 free radicals in it. The weak signal was found to be leads to coronary artery disease . Clinical materials with g = 2.03871. Origin of this singlet line may be prove that Zn can have good effect on eliminating 22,23 due to free radicals of semi-quinones, lignin or due to ulcer and promoting healing of wounds . Iron is phenols especially flavonoids11-15. essential for human body in production of The sharp peak in the spectrum is due to the effect haemoglobin, in oxygenation of red blood cells. It is of quartz tube holding the powder sample. From this needed for healthy immune system and for energy experiment it is concluded that some free radicals are production. Severe iron deficiency results in anaemia present in the leaves of M. spinosa, containing and red blood cells that have a low haemoglobin antioxidants. The EPR spectrum of 2, 2-diphenyl-1- concentration. It is responsible for the catalase picrylhydrazyl (C18H12N5O6), abbreviated as DPPH, a enzyme. stable free radical, was taken. For taking the EPR Copper is involved in the oxidation of Fe+2 to Fe+3 spectrum, an amount of 12 mg of DPPH is dissolved haemoglobin formation and it is an important catalyst in 10 mL of methanol which is also used as stock for iron absorption. Copper deficiency may be a risk solution and kept in a dark chamber as DPPH is very factor for cardiovascular disease. When copper sensitive to light. The spectrum was taken within 15 deficiency occurs symptoms include neutropenia, minutes for 1.5 mL stock solution. cardiac disorders, osteoporosis and anaemia24. It is Further, an amount of 3 mL of stock solution is responsible for cytochrome oxidases and cytosolic mixed with 50 mg of plant extract and EPR spectrum superoxide dismutase enzymes. Chromium is was taken again. Obviously no signal was found to be essential functioning as a glucose tolerance factor. It lost. This is due to fact that the free radicals of DPPH is also used for insulin signalling for biological role molecules are absorbed by antioxidants of the M. and thus sugar metabolism and diabetes22.The spinosa extract by chemical reaction leading to the functional values of manganese are as Lewis acid and conclusion that this plant is a good scavenger of free catalyst for oxidation. It is essential to all organisms, radicals due to the presence of antioxidants. activates numerous enzymes, viz. mitochondrial Total antioxidant activity was observed with the superoxide dismutase. Molybdenum is used as help of the UV-visible spectrometer and inhibition nitrogenase and nitrate reductase enzymes by plants in 54 INDIAN J NAT PROD RESOUR, MARCH 2015

nitrogen fixation. Out of the above mentioned six 3 Zeynep Y, Bunyamin K and Abdulhalik K, An EPR study elements, elements like copper, manganese, zinc are of gamma irradiated medicinal plants: Cross seeds and 25 Mistletoe, Gazi Univ J Sci, 2011, 24(2), 203-207. reported to be antioxidants . Further, iron is treated 26 4 Jasna M C, Sonja M D, Gordana S C, Vesna T T and as antioxidant . Now-a-days chromium is also used Zoranka N M, ESR studies of antioxidative activity of in antioxidant formulation22. The antioxidant property different Elecampane (Inula helenium L.) extracts, APTEFF, of the elements may be attributed due to existence of 2002, 33 1-174. them as various enzymes in the leaves of M. spinosa. 5 Sharat N K S, Devi C B, Sony T S and Rajmuhon N S, Trace elements of some selected medicinal plants of Manipur, Lastly, daily intake requirements of some of the trace Indian J Nat Prod Resour, 2010, 1(2), 227-231. 28 elements by our human body is given as follows : 6 Bora P J and Kumar Y, Floristic diversity of Assam: Study of Iron, 10 mg/ day for male and 15 mg for female; Pabitora Wild Life Sanctuary, Delhi, Daya Publishing House, Zn, 15 mg/ day; Mn, 2.5 – 5.0 mg/ day; Cr, 2003, p.18. 7 Yusuf M, Begum J, Yahab M A and Chowdhury J U, 0.05-0.2 mg/ day; Cu, 2-3 mg/day. 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