Food Chemistry 120 (2010) 825–830

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Food Chemistry

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Umbelliferone – An antioxidant isolated from Acacia nilotica (L.) Willd. Ex. Del.

Rajbir Singh a, Bikram Singh c, Sukhpreet Singh b, Neeraj Kumar c, Subodh Kumar b, Saroj Arora a,*

a Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar 143 005, Punjab, Indiab Department of Chemistry, Guru Nanak Dev University, Amritsar 143 005, Punjab, Indiac Division of Natural Plant Products, Institute of Himalayan Bioresource Technology, Palampur, H.P. 176 061, India

a r t i c l e i n f o

Article history:Received 19 March 2009Received in revised form 18 September2009Accepted 10 November 2009

Keywords:Acacia niloticaAntioxidative assaysNatural plant productsFree radicalsUmbelliferone

0308-8146/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodchem.2009.11.022

* Corresponding author. Tel.: +91 94 17 28 54 85;E-mail address: jrosh1@rediffmail.com (S. Arora).

a b s t r a c t

The bark and leaves of Acacia nilotica are consumed for their promising medicinal properties in severalparts of the world. The aerial portion, including flowers, is used as fodder for animals. This study aimedto isolate the functional components of A. nilotica and to check their antioxidant activities in vitro. In thefractionation of methanol extract, a fraction, AN-2, was isolated, which was identified by spectroscopictechniques, namely NMR and mass spectroscopy to be a coumarin derivative, i.e. umbelliferone. The anti-oxidative activities, including the DPPH�, deoxyribose (site and non-site specific), chelating power, reduc-ing power and lipid peroxidation assays, were studied in vitro and performed in the Dept. of Botanical andEnv. Sciences, GNDU, Amritsar. It was found that the antioxidative effect of umbelliferone was dose-dependent up to 100 lg/ml and then levelled off with no further increase in activity. This is the firstreport of the isolation and antioxidant potential of umbelliferone from A. nilotica.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Acacia nilotica (L.) Willd. Ex Del., is a medicinal tree, familyMimosaceae, known to be rich in phenolics, consisting of con-densed tannin and phlobatannin, gallic acid, (+) – catechin, (�) epi-gallocatechin-7-gallate, and has been used for treatment of viral(colds, bronchitis), bacterial (diarrhoea), amoeboid (dysentery),fungal, bleeding piles and leucoderma diseases (Bhargava, Srivast-ava, & Kumbhare, 1998). The leaves and flowers of this evergreentree are also used as fodder for animals. In a previous study, inthe Genetic Toxicology Laboratory, it has been shown that the A.nilotica plant had antimutagenic and cytotoxic activities (Kaur,Micheal, Arora, Harkonen, & Kumar, 2005). As most of the antimu-tagenic compounds act via scavenging of free radicals, we plannedto investigate the antioxidant activity of the functional compo-nents present in the extract from A. nilotica.

Increasing evidence from epidemiological and biological studieshas shown that reactive oxygen species (ROS) are involved in avariety of physiological and pathological processes (Puddu, Puddu,Cravero, Rosati, & Muscari, 2008; Riccioni, Mancini, Di IIio, Bucciar-elli, & D’Orazio, 2008). Plant- and food-derived antioxidants areimplicated in the prevention of cancer and aging by destroying oxi-dative species that initiate carcinogenesis through oxidative dam-age of DNA (Lori, Rowe, & Paul, 2008). The supplementation of

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functional food with antioxidants, which inhibit the formation offree radicals, can lead to prevention of some diseases.

In this paper, we report the isolation of the compound umbellif-erone from A. nilotica and demonstrate its antioxidant properties,using a battery of in vitro assays, namely DPPH�, deoxyribose (siteand non-site specific), chelating power, reducing power and lipidperoxidation assays.

2. Materials and methods

2.1. Chemicals

20-20-Diphenyl-1-picrylhydrazyl (DPPH�), and 2-thiobarbituricacid were obtained from Sigma Chemical Co. (St. Louis, MO, USA)and deoxyribose was obtained from Lancaster Synthesis Inc.(Windham, USA). All other chemicals were of analytical gradeand procured from the Ranbaxy Fine Chemicals Ltd. (New Delhi,India).

2.2. Plant material

The bark material was collected from a tree, in the month ofNovember, growing on the main side of Bebe Nanki Girls Hostel–II, Guru Nanak Dev University Campus, Amritsar, located at 31�37051.91N; 74� 49034.28E with loamy soil texture. A voucher spec-imen (No. 6421 dated 12-01-2007) has been deposited at the her-barium of Department of Botanical and Environmental Sciences,Guru Nanak Dev University, Amritsar, Punjab, India. The bark

826 R. Singh et al. / Food Chemistry 120 (2010) 825–830

material was washed with tap water (thrice) to remove soil parti-cles, dried in an oven at 40 �C for 24 h and ground to a fine powder.

2.3. Isolation of coumarin fraction

The various extracts of A. nilotica were made by soaking the barkpowder in different solvents with increasing order of polarity(Flow chart 1). After filtration of various solvents from the superna-tant, crude extracts (CE) were obtained by evaporating in a vacuumrotary evaporator. Crude methanol extract, mixed with Celite ‘545’was further subjected to column chromatography (75 � 3.5 cm)over acidic alumina (Brockman’s activity) with a gradient of hex-ane/ethyl acetate (95/5), (90/10), (75/25), (50/50), (25/75) andethyl acetate/methanol (90/10), (80/20), (70/90), (60/40), (50/50)as eluent to give a total of 650 fractions. Fractions 56–154, elutedwith 25% ethyl acetate in hexane, were dried and rechromato-graphed with hexane–ethyl acetate in an increasing gradient elu-tion. The fraction eluted with hexane–ethyl acetate (5%) yieldedwhite solid residue (45 mg, 0.18%) which was named AN-2.

2.4. Identification of coumarin fraction by NMR and mass spectroscopy

The NMR (1H and 13C) experiments were performed on a BrukerAvance-300 spectrometer. The chemical shifts were measured inCD3OD and are expressed in d (ppm). Mass spectra were recorded

Bark powder

Residue

Extraction with hexane (thrice) 24 h at room temperature

Residue

Extraction with chloroform (thrice) 24 h at room temperature

Residue

Extraction with ethyl acetate (thrice) 24 h at room temperature

Residue

Extraction with acetone (thrice) 24 h at room temperature

Residue

Extraction with methanol (thrice) 24 h at room temperature

Residue

Extraction with water (thrice) 24 h at room temperature

Flow chart 1. Extraction of various extracts of Acaci

on a QTOF-Micro of Waters Micromass. Melting point was deter-mined on a Barnstead Electrothermal 9100.

2.5. Determination of total phenolics

The total phenolic content (TPC) of the methanol extract wasdetermined by the method of Folin–Ciocalteu (Kujala, Loponen,Klika, & Pihlaja, 2000), as gallic acid equivalents (GAE) in milli-grammes per gramme of sample.

2.6. Antioxidant testing assays

2.6.1. DPPH�- scavenging assayThe hydrogen donating or radical-scavenging ability of AN-2

was measured using the stable radical DPPH� following the methodgiven by Blois (1958) with modification. In the assay, 300 ll of AN-2 (1–50 lg/ml) were added to 2 ml of DPPH�, (0.1 mM in methano-lic solution). The change of colour of the reaction mixture was thenread at 517 nm against the blank, which did not contain the testcompound, AN-2. The percentage DPPH� inhibition ability of thesample was calculated by the formula:

% Inhibition ¼ ðB0� B1=B0Þ � 100

B0 is the absorbance of blank; B1 is the absorbance of reactionmixture.

Hexane extract

Chloroform

Ethyl acetate extract

Acetone extract

Methanol extract

Water extract

Subjected to Column Chromatography

a nilotica in increasing order of solvent polarity.

R. Singh et al. / Food Chemistry 120 (2010) 825–830 827

2.6.2. Reducing power assayThe reducing power of AN-2 was determined by the method of

Oyaizu (1986) with modifications. In this assay, to a mixture ofphosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricya-nide [K3Fe(CN)6] (2.5 ml, 1%), different concentrations of AN-2(1 ml each) were added. The mixture was incubated at 50 �C for20 min. Aliquots (2.5 ml) of trichloroacetic acid (10%) were addedto the mixture, which was then centrifuged for 10 min at 1036g.The supernatant (2.5 ml) was mixed with distilled water (2.5 ml)and FeCl3 (2.5 ml, 0.1%), and the absorbance was measured at700 nm in a spectrophotometer. Increased absorbance of the reac-tion mixture indicated increased reducing power.

2.6.3. Deoxyribose degradation assayThe site-specific and non-site-specific deoxyribose assays were

performed, following the method of Halliwell, Gutteridge, and Aru-oma (1987) and Arouma, Grootveld, and Halliwell (1987) withslight modifications. In the non-site-specific deoxyribose assay,Haber–Weiss reaction buffer [10 mM FeCl3, 1 mM EDTA (pH 7.4),10 mM H2O2, 10 mM deoxyribose, and 1 mM L-ascorbic acid] wasmixed with different concentrations of AN-2 up to a final volumeof 1.0 ml. Then incubation of the mixture was done at 37 �C for1 h and it was heated at 80 �C for 30 min with 1 ml of 2-TBA (2-thiobarbituric acid) (0.5% 2-TBA in 0.025 M NaOH, 0.02% BHA)and 1 ml of 10% trichloroacetic acid (TCA) in a water bath for45 min. The absorbance of the mixture was measured spectropho-tometrically at 532 nm after cooling. A site-specific assay was per-formed, following slight modifications in which the EDTA wasreplaced with the same volume of the phosphate buffer (pH 7.4),i.e. [10 mM FeCl3, phosphate buffer (pH 7.4), 10 mM H2O2,10 mM deoxyribose, and 1 mM L-ascorbic acid]. The percentageof inhibition was calculated employing the same formula as givenfor the DPPH�-scavenging assay.

2.6.4. Chelating effects on ferrous ionsThe chelating effect on ferrous ions was determined according

to the method of Dinis, Madeira, and Almeida (1994) with somemodifications. In this assay, to a mixture of 1.75 ml of methanoland 0.25 ml of 250 mM FeCl2, the different concentrations of AN-2 (0.25 ml each) were added. Then, 0.25 ml of 2 mM ferrozinewas added, which was kept at room temperature for 10 min beforedetermining the absorbance of the mixture at 562 nm in a spectro-photometer. The chelating effect (%) was calculated from the for-mula as given for the DPPH�-scavenging assay.

2.6.5. Lipid peroxidation by thiobarbituric acid (TBA) assayThe reaction of TBA with malondialdehyde (MDA) forms a

diadduct, a pink chromogen, which can be detected spectropho-tometrically at 532 nm (Halliwell & Guttridge, 1989). The per-fused liver was isolated from normal male rats (250 g) and10% (w/v) homogenate was prepared with homogenizer at 0–4 �C with 0.15 M KCl. The homogenate was centrifuged at800g for 15 min and clear cell-free supernatant was used forthe study of in vitro lipid peroxidation. To the mixture of0.15 M KCl (1 ml) and rat liver homogenates (0.5 ml) and differ-ent concentrations of AN-2 (10–250 lg/ml), were added 100 llof 0.2 mM ferric chloride, to initiate the peroxidation reaction.The reaction was stopped by adding 2 ml of ice-cold HCl(0.25 N) containing 15% trichloroacetic acid (TCA), 0.38% TBA,and 0.5% BHT after incubation at 37 �C for 30 min. The reactionmixtures were heated at 80 �C for 60 min. The samples werecooled, centrifuged and the absorbance of the supernatantswas measured at 532 nm and the percentage of inhibition wascalculated by the formula:

% Inhibition of lipid peroxidation ¼ ðB0� B1=B0Þ � 100

B0 is the absorbance of negative control; B1 is the absorbance ofreaction mixture.

2.7. Statistical analysis

The data, presented as means ± SE of three independent exper-iments and IC50 (50% inhibitory concentration) values were calcu-lated from regression lines. The significance was checked at the 5%level (P 6 0.05) by employing the student’s t-test.

3. Results

3.1. Structure elucidation of compound

The bioactive methanol extract of dried powdered bark of A.nilotica was fractionated using repeated acidic alumina columnchromatography which afforded 650 fractions (100 ml each). Frac-tions 56–154 were further purified by silica gel recolumn chroma-tography and the chromatographic purification of these fractionsled to the isolation of compound ‘AN-2’ in 0.18% yielded (45 mg).Negative electrospray ionisation quadrupole time-of-flight massspectrometry (ESI-QTOF-MS) of this compound resulted in amolecular ion peak at m/z 161.18 [M�H]�, indicating the molecularformula to be C15H10O6 (Fig. 1).

In the 1H NMR spectrum of the compound, six distinct peakswere observed. A broad singlet at d 4.53 was assigned to the OHproton. Two doublets at d 6.12 and 7.79 with coupling constantof 9.3 Hz were assigned to the protons attached at the C-3 and C-4 positions. Similarly, two other doublets, at d 6.73 (J = 8.4 Hz), d7.39 (J = 8.7 Hz) and a singlet at d 6.64, were assigned to the pro-tons attached to C-6, C-5 and C-8, respectively. The 13C NMR spec-trum of AN-2 showed nine distinct signals. A peak at d 162.3 wasassigned to the carbonyl function (C-2) of coumarin derivativeswhile a downfield signal at d 161.7 revealed the presence of a hy-droxyl function at the C-7 position. Other signals at, d 102.0, 110.9,111.7, 113.1, 129.2, 144.6 and 155.8 were attributed to C-8, C-3, C-4a, C-6, C-5, C-4 and C-8a, respectively. Using spectroscopic tech-niques, namely 1H NMR, 13C NMR and mass, the sample was iden-tified as umbelliferone (Table 1).

3.2. Total phenolic content (TPC) and anti-free radical activity ofumbelliferone

The total phenolic contents of the extracts were checked as itwas expected that the extracts having good antioxidant activitywould be rich in phenolic compounds. As the methanol extractexhibited a maximum antioxidant activity, so the TPC was also ob-served to be very high, i.e. 945 mg/g as gallic acid equivalents. Thein vitro antioxidant activity of the umbelliferone is categorised intofour categories, namely >25% weak, >25–50% moderate, >50–75good, >75% strong. It is also important to mention here that, inall the in vitro antioxidant testing assays, umbelliferone showedgood antioxidant activity. All the values of antioxidant activitieswere considered to be significant at P 6 0.05.

Umbelliferone showed excellent DPPH�-scavenging activity,even at very low concentrations, as shown in Fig. 2. The additionof umbelliferone led to a change in colour, with a very fast reactionup to a concentration of 50 lg/ml concentration. The umbellifer-one exhibited good potential of 59.6 ± 1.6% (IC50, 36.1 lg/ml) at50 lg/ml concentration, and beyond this concentration scavengingeffect became almost stable as there was no significant increase inthe inhibitory potential. Reducing power assay showed the umbel-liferone reduced the Fe3+ to ferrous ions (Fe2+) by 64.8 ± 1.1% (IC50,77.8 lg/ml) at 150 lg/ml concentration (Fig. 3). There was onlyslight difference in antioxidant activities of umbelliferone between

Fig. 1. Structure and mass spectrum of umbelliferone (AN-2).

Table 11H and 13C NMR spectral data for compounds AN-2 (umbelliferone); dppm (multi-plicity, coupling constant).

Carbon AN-2 (CD3OD) Literature data (DMSO-d6)

1H (m, J value) 13C 13C

2 162.3 161.33 6.12 (d, 9.3 Hz) 110.9 111.34 7.79 (d, 9.3 Hz) 144.6 144.54a – 111.7 111.45 7.39 (d, 8.7 Hz) 129.2 129.76 6.73 (d, 8.4 Hz) 113.1 113.87 – 161.7 160.48 6.64 (s) 102.0 102.18a – 155.8 155.5

0

20

40

60

80

100

1 5 10 15 20 25 30 35 40 45 50Concentration (µg/ml)

% D

PPH

inhi

bitio

n

Fig. 2. Scavenging of the DPPH radical of AN-2.

0

20

40

60

80

100

10 25 50 100 150Concentartion of extract

% re

duci

ng p

ower

Fig. 3. Reducing potential of AN-2.

0

20

40

60

80

100

1 10 20 30 40 50 60 70 80 90 100Concentration (µg/ml)

% d

eoxy

ribos

e in

hibi

tion

Fig. 4. Scavenging of hydroxyl radicals by AN-2 in site-specific deoxyribosedegradation assay.

828 R. Singh et al. / Food Chemistry 120 (2010) 825–830

site- and non-site-specific assays (Figs. 4 and 5). Umbelliferonewas a strong chelating agent (Fig. 6), as it exhibited a strong poten-tial of 58.8 ± 0.9% with an IC50 value of 178 lg/ml at 200 lg/ml

concentration. Fig. 7 shows that the umbelliferone exhibited inhi-bition of LPO in a dose-dependent manner, i.e. 11.1–67.2 ± 1.8% at10–250 lg/ml concentration (weak – good).

0

20

40

60

80

100

1 10 20 30 40 50 60 70 80 90 100Concentration (µg/ml)

% d

eoxy

ribos

e in

hibi

tion

Fig. 5. Scavenging of hydroxyl radicals by AN-2 in non-site-specific deoxyribosedegradation assay.

0

20

40

60

80

100

25 50 100 125 150 175 200Concentration (µg/ml)

% c

hela

ting

pow

er

Fig. 6. Chelating power potential of AN-2.

0

20

40

60

80

100

10 50 100 150 200 250Concentration (µg/ml)

% L

PO in

hibi

tion

Fig. 7. Lipid peroxidation inhibition (LPO inhibition) potential of AN-2.

R. Singh et al. / Food Chemistry 120 (2010) 825–830 829

4. Discussion

As the methanol extract of A. nilotica was found to have thehighest amount of TPC, as well as high percentage yield (69.9),chromatographic purification on a precoated Kieselgel 60 F254plate (0.2 mm thick; Merck, India), showed the presence of a largenumber of components. The repeated silica gel column chromatog-raphy of the methanol extract led to the isolation of a white solidcompound and this was found to be analytically pure by thin-layerchromatography. The sample was identified as umbelliferone usingspectroscopic techniques 1H NMR, 13C NMR and mass. The assign-ment of the structure is in consonance with the previous report ofEl-Sayed, Al-Said, El-Feraly, and Ross (2000). For bioevaluationstudies, this umbelliferone sample was used, as it is known forits potent anti-rheumatic, antipyretic and analgesic activities (Mol-nar & Garai, 2005). Kosalec, Pepeljnjak, and Kustrak (2005) re-ported that the ethanolic extract of anise-fruits (Pimpinellaanisum L., Apiaceae) contains umbelliferone as its major compoundand the extract showed antifungal activity against all the species ofdermatophytes investigated.

A critical analysis of the previous results obtained in differentassays showed that the methanol extract of A. nilotica was compar-atively more effective than were other extracts of the same plant.In an attempt to identify the antioxidant principle in these extractsthe total phenolic content was determined, as these compoundsare nucleophillic in nature and exhibit important antioxidativeactivities (Singh, Singh, Kumar, & Arora, 2007; Singh et al., 2008;Yildirim et al., 2000). The total phenolic contents were 945, 780,610, 400, 165 and 110 mg gallic acid equivalents (GE) in eachgramme of the plant extract for methanol, acetone, ethyl acetate,water, chloroform and hexane, respectively. Numerous in vitrostudies have shown that polyphenols from red & white wine, rape-seed, pine bark extract, coffee, cocoa and extracts of medicinalplants are recognised bioactive components with antioxidantproperties. Thus, they are viewed as promising therapeutic drugsfor free radical pathologies (Kaur, Kaur, Kumar, Singh, & Kumar,2009; Singh et al., 2007; VanderJagt, Ghattas, VanderJagt, Crossey,& Glew, 2002; Vuorela et al., 2005). In this study, the ability ofumbelliferone to scavenge free radicals was further confirmed byemploying a battery of in vitro assays.

The relatively stable DPPH radical has been widely used to testthe ability of compounds to act as free radical-scavengers or hydro-gen donors and thus to evaluate the antioxidant activity (Jao & Ko,2002). Antioxidants donate hydrogen to free radicals, leading tonon-toxic species and therefore to inhibition of the propagationphase of lipid oxidation. Results shown in Fig. 2 revealed thatumbelliferone at 50 lg/ml exhibits strong DPPH radical-scaveng-ing activity (59.6 ± 1.6%). Methanol extract may contain com-pounds other than umbelliferone that act as electron donorswhich could react with free radicals to convert them to more stableproducts and terminate the radical chain reaction.

Different studies have indicated that antioxidant activity andreducing power are related (Duh, 1998; Duh, Tu, & Yen, 1999).The reducing power of umbelliferone, as a function of concentra-tion, is shown in Fig. 3. In this assay, the yellow colour of the testsolution changes to various shades of green and blue, depending onthe reducing power of each concentration. The presence of reduc-ers (i.e. umbelliferone) causes the reduction of the Fe3+/ferricya-nide complex to the ferrous form. Therefore, measuring theformation of Perl’s Prussian blue at 700 nm can monitor the Fe2+

concentration (Ferreira, Baptista, Vilas-Boas, & Barros, 2007). Thereducing power of umbelliferone increased with concentration.

Hydroxyl radical is the most reactive free radical and it can beformed from superoxide anion and hydrogen peroxide in the pres-ence of metal–ions, such as copper or iron. When a hydroxyl radi-cal reacts with aromatic compounds, it can attach across a doublebond, resulting in a hydroxyl cyclohexadienyl radical. The hydroxylradicals, generated in the Fenton reaction, degrade deoxyriboseinto malonaldehyde and, upon heating this mixture under acidicconditions, malonaldehyde will produce pink-coloured chromo-gens by reacting with thiobarbituric acid. The % of deoxyribodedegradation was assessed by detecting these chromogens specto-metrically at 532 nm (Halliwell et al., 1987). The resulting radicalcan undergo further reactions, such as reaction with oxygen, togive peroxyl radical, or decompose to phenoxyl-type radicals bywater elimination. In addition, this radical species is consideredto be one of the quick initiators of the lipid peroxidation (LPO) pro-cess, abstracting hydrogen atoms from unsaturated fatty acids. Theinhibition of hydroxyl radical, exhibited by umbelliferone(63.6 ± 3.47%), was closer to that of peptide isolated from hoki (Joh-nius belengerii) frame protein by gastrointestinal digestion (Kim, Je,& Kim, 2007).

It is self-evident that the strong reductive capacity of antioxi-dants may also affect ions, especially Fe2+ and Cu2+. Iron is anessential mineral for normal physiology, but an excess of it may re-sult in cellular injury. If they undergo the Fenton reaction, these re-

830 R. Singh et al. / Food Chemistry 120 (2010) 825–830

duced metals may form highly reactive hydroxyl radicals andthereby contribute to oxidative stress (Hippeli & Elstner, 1999).The resulting oxy radicals cause damage to cellular lipids, nucleicacids, proteins and carbohydrates and lead to cellular impairment.Since ferrous ions are the most effective pro-oxidants in a food sys-tem, the good chelating effect would be beneficial and removal offree iron from circulation could be a promising approach to preventoxidative stress-induced diseases. When iron is chelated, it maylose pro-oxidant properties. Hence, we herein tested the chelationof Fe2+, by the umbelliferone in a competition assay with potas-sium ferricyanide. Interestingly, as seen in the Fig. 6, the antioxi-dant factors of the umbelliferone were found to be capable ofbinding Fe2+ ions by 58.8 ± 0.9% at 200 lg/ml, as evidenced bythe loss of absorption at 700 nm. In this assay, umbelliferone inter-fered with the formation of ferrous and ferrozine complex, sug-gesting that it has chelating activity and is able to captureferrous ion before the formation of ferrozine.

In order to determine if the extracts were capable of reducingin vitro oxidative stress, the traditional lipid peroxidation assay,that determines the production of malondialdehyde and related li-pid peroxides in liver homogenate, was carried out. Thiobarbituricacid-reactive substances are produced as by-products of lipidperoxidation induced by the ferrous sulphate: ascorbate system.Initiation of a peroxidation sequence in a membrane or polyunsat-urated fatty acid is due to abstraction of a hydrogen atom from thedouble bond in the fatty acid. The free radical tends to stabilize bya molecular rearrangement to produce a conjugated diene, whichthen readily reacts with an oxygen molecule to give a peroxy rad-ical (Jadhav, Nimbalkar, Kulkarni, & Madhavi, 1996). The inhibitoryeffects of various concentrations of umbelliferone against ferroussulphate and ascorbic acid-induced ghost lipid peroxidation areshown in Fig. 7. Umbelliferone showed the highest inhibition ofghost lipid peroxidation, about 67.2 ± 1.8% at a concentration of250 lg/ml.

5. Conclusion

In this study, A. nilotica is found to be rich in umbelliferone(0.18% with amount of 45 mg/g) and also showed a remarkableantioxidant activity. It is therefore, imperative to study the plantmaterial for its potential as a scavenger of free radicals.

Acknowledgements

This research was supported by a Senior Research FellowshipExtended (ACK. No.: 212241/2K7/1) provided to Dr. Rajbir Singhfrom CSIR India. The authors are grateful to Dr. P.S. Ahuja Director,IHBT (CSIR) India, for providing laboratory facilities. We are alsograteful to Mr. Dharuv and Mr. Ramesh, (Technical assistants),NPP Division, IHBT, Palampur, for providing technical expertise.

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