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ISSN: 0974-6943 Kusum S. Akki et al. / Journal of Pharmacy Research 2009, 2(4),752-755 Available online through www.jpronline.info

Research Article

Phytochemical investigations and in vitro evaluation of Nyctanthes arbor-tristis leaf extracts for antioxidant property Kusum S. Akki*, G Krishnamurthy1 and H. S. Bhoja naik2 *Department of Pharmacognosy& Phytochemistry,K.L.E.S’s College of Pharmacy,Vidyanagar, Hubli Karnataka,India. 1Department of Chemistry, Sahyadri Science College, Shimoga, Karnataka, India. 2Department of Industrial chemistry, Kuvempu University, Jnana Sahyadri Shimoga ,Karnataka, India. * For Correspondence:Kusum S. Akki E-mail:[email protected] Received on:17-12-2008; Accepted on :22-02-2009 ABSTRACT The decoction of the leaves of Nyctanthes arbor-tristis Linn. widely used in Ayurvedic system of medicine for the treatment of sciatica, arthritis, fevers and various painful conditions.The leaf was studied for Pharmacognostic evaluations, including examination of morphological and microscopic characters, determination of leaf constants, ash values and extractive values. The dried leaves of Nyctanthes arbortristis were subjected to prilimiary phytochemical screening by extracting exhaustively the crude drug with alcohol in a Soxhlet extractor. The extract was concentrated using a rotary flash evaporator, residue was dried in a dessicator over sodium sulfite to give a semisolid mass. This alcoholic extract was further fractionated in to Pet ether, ethyl acetate, butanol and aqueous fractions. Preliminary Phytochemical investigations showed the presence of Flavonoids, Carbohydrates, Alkaloids, Phytosterols, Phenolic Compounds and Glycosides. Free radical scavenging potential of the different extracts of leaves of Nyctanthes arbor-tristis, was evaluated in vitro by using diphenyl-picrylhydrazyl(DPPH) assay. In this method the antioxidants present in the plant extracts reacted with DPPH, which is a stable free radical and converted it to 1,1-diphenyl-1,2-picryl, hydrazine which is measured at 517 nm. The scavenging effect of plant extracts and standard (ascorbic acid and BHT) on the DPPH radical decreases in the following order: ascorbic acid > Butanol > Ethyl acetate >BHT > Pet ether and ascorbic acid was found to be 93.88% at concentration of 10 µg, BHT, Butanol, Ethyl acetate and Pet ether was found to be 97.42%, 95.22% 84.63% and 82.04% at concentration of 100 µg respectively. In the present study, different extracts of Nyctanthes arbor-tristis leaves showed concentration dependent free radical scavenging activity. Key words: Antioxidant, Nyctanthes arbor-tristis, flavonoids INTRODUCTION Nyctanthes arbor-tristis Linn. ( Oleaceae ) is a large shrub which is widely cultivated throughout India as a garden plant. The bitter leaves are used in Ayurvedic system of medicine for the treatment of rheumatism, sciatica, diuretic and intestinal worms. The powdered seeds are recommended for the treatment of scurvy1,2. The reported phytoconstituents of the leaves are arborsides A, B, C , flavonol glycosides, astragalin and nicotiglorin. Flavonoids are large group of compounds occurring abundantly in plants. They occur as glycosides and contain several phenolic hydroxyl groups on their ring structure. Many flavonoids are found to be strong free radical scavengers and antioxidants.3 The present work has been designed to set some diagnostic indices for identification and preparation of monograph of the leaves and since leaves contain flavonoids an attempt has been made to evaluate antioxidant potential of the extracts from the leaves of Nyctanthes arbor-tristis .

Pharmacognostic Screening: Organoleptic, macroscopic 4 and microscopic 5 characters were studied as described in quality control methods. Proximate values such as Extractives values 6, moisture content7 and ash values 8. were carried on powdered crude drug of the leaves of Nyctanthes arbor-tristis. Preparation of extracts: Shade dried & powdered leaves were subjected to exhaustive soxhlet extraction with alcohol and further fractionated with Pet ether, Ethylacetate and Butanol. Phytochemical screening:

MATERIALS AND METHODS

Qualitative chemical tests of all these extracts were carried out 9,10.

Plant Material: The leaves of Nyctanthes arbor-tristis were collected in the month of June/July, from from local areas of Hubli Dharwad and was authenticated by Dr. B.D. Huddar, Head, Department of Botany, Kadasiddheshwar Arts College and H.S. Kotambari Science institute, Hubli.

Journal of Pharmacy Research

Antioxidant activity- free radical scavenging on DPPH Radical: For the present study the samples were prepared in different concentrations i.e. 10-100µgm in AR grade methanol. The samples of above concentrations were mixed with 3ml of 100µM of DPPH .The absorbance of the resulting solutions and the blank (with same chemi-

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ISSN: 0974-6943 Kusum S. Akki et al. / Journal of Pharmacy Research 2009, 2(4),752-755 Kusum S. Akki etal., Phytochemical investigations and in vitro evaluation of Nyctanthes arbor-tristis leaf extracts for antioxidant property

TableNo: 1 Leaf surface datas of Nyctanthes arbor-tristis leaves Sl.No. 1 2 3 4 1 2

Parameter Stomatal index Upper epidermis Lower epidermis Vein islet no Vein termination no Palisade ratio Upper epidermis Lower epidermis

Determined Value Stomata absent 5.5-6.5 14-16 11-14 3-5 3-4

Table No: 3. Qualitative chemical analysis of various extracts of Nyctanthes arbor-tristis leaf extracts . Nature Total Pet. Ethyl n-Butanol Alcohol Ether acetate Alkaloids + — + — Sterols + + — — Carbohydrates + — + + Tannins + — + + Proteins + — — + Glycoside + — + + Flavonoids + — + +

Table No: 2. a. Extractive values, b. Moisture content and c. Ash Table 4-Antioxidant Activity 0f Nyctanthes arbor-tristis Leaf extracts values of Nyctanthes arbor-tristis leaves Sl.No. a 1 2 b c 1 2 3 4

Parameter Determined Extractive values Value % w/w Alcohol soluble extractive value 21.22 Water soluble extractive value 13.78 Moisture content 12.98 Ash Values Total ash 7.98 Acid insoluble ash 5.23 Water soluble ash 2.997 Sulphated ash 8.54

samples

Concentration

Pet ether Ethyl acetate Butanol Butylated Hydroxy Toulene (BHT) Ascorbic acid (Asc.acid)

- 10 – 100 µg - 10– 100 µg - 10 – 100µg - 10 – 100µg - 1– 10µg

Table 5. Free Radical Scavenging Activity on DPPH Radical Percentage free radicals scavenging activity Conc. µg

Pet ether

Ethylacetate

Butanol

Butylated Hydroxy Toulene

Conc µgm

Ascorbic acid

10 20 30 40 50 60 70 80 90 100 IC50

44.88±0.108 49.53±0.574 58.69±0.496 65.45±0.636 67.85±0.488 69.42±0.266 74.86±0.113 79.67±0.253 80.37±0.571 82.04±0.273 20.39 µg

49.18±0.210 59.08±0.180 64.54±0.365 68.82±0.289 83.64±0.333 84.02±0.648 88.65±0.257 90.79±0.267 94.61±0.323 95.22±0.176 11.29µg

49.24±0.231 61.54±0.290 64.86±0.134 75.69±0.328 83.52±0.298 88.53±0.195 93.06±0.338 94.83±0.929 96.18±0.457 97.42±0.696 10.71µg

49.03±0.033 54.53±0.206 57.39±0.347 60.99±0.305 65.17±0.220 69.22±0.108 70.37±0.256 76.39±0.253 79.40±0.049 84.63±0.253 12.52µg

1 2 3 4 5 6 7 8 9 10 IC50

48.54±0.216 53.48±0.173 58.74±0.318 63.27±0.226 69.21±0.046 70.57±0.225 75.29±0.090 79.38±0.040 87.51±0.361 93.88±0.084 1.4µg

Values are mean ± SEM of triplicate determinations,IC 50 - Inhibition Concentration (the concentration producing 50% of maximal inhibition)

cals except sample) were recorded after 20mins at room temperature. BHT and ascorbic acid was used as the standard. The disappearance of DPPH was read spectrophotometrically at 517nm. Radical Scavenging Capacity (RSC) in percent was calculated. From the obtained RSC values the IC50 were calculated, which represents the concentration of the scavenging compound that caused 50% neutralization.11 RESULTS AND DISCUSSION Organoleptic and macroscopic characters:

Microscopic characters: The cells of upper epidermis are thick walled somewhat straight and devoid of stomata and of lower epidermis are smaller in size with numerous anomocytic stomata, Presence of long unicellular trichomes with pointed apex, multicellular trichomes with blunt apex and glandular trichomes. Under epidermis are two rows of palisade cells followed by 7-9 rows of spongy parenchyma traversed by vascular strands and encircled by parenchymatous sheath. In the midrib region it consists of vascular bundles which are collateral closed and also present are starch grains and occasionally few lignified fibres.

Leaves of Nyctanthes arbor-tristis. are simple, 5-15 cm long, 2.5-7.5

cm width, ovate, acute to acuminate; both surfaces Leaf Surface Data: rough, margin entire or distinctly toothed base round to someResults of Leaf Surface Data’s are as Tabulated in table 1. what cuneate, venation reticulate, lateral veins 3-6 pairs, petiole 0.5-1.5 cm long, odour indistinct , bitter and astringent taste.


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Free Radical Scavenging Activity on DPPH Radical

1 0 0 9 5 9 0 8 5

% INHIBITION

8 0 7 5 7 0 6 5 6 0 5 5

P E B B

5 0 4 5

E T U H

T E T H E R H Y L A C E T A T T A N O L T

4 0 0

2 0

4 0

60

8 0

100

C O N C E N T R A T I O N I N M I C R O G R A M S

Fig.1. Free radical Scavenging activity of extracts on DPPH radical

Free Radical Scavenging Activity on DPPH Radical

100

90

% INHIBITION

80

70

60

ASCORBICACI 50

40 0

2

4

6

8

10

CONCENTRATION IN MICROGRAMS Fig.2. Free radical Scavenging activity of ascorbic acid on DPPH radical Antioxidant activity: Proximate values: Results of Extractive values, Moisture content and Ash values of shade dried Nyctanthes arbor-trists leaves are as tabulated in Table : 2 Preliminary Phytochemical Investigations: Preliminary phytochemical analysis are as tabulated in Table : 3


Antioxidant activity of various extracts was performed by using free radical scavenging on DPPH Radical and it was observed that free radicals were scavenged by extracts in a concentration dependent manner. The maximum % inhibition on DPPH was 82.04 (Pet ether), 94.61 (ethylacetate), 97.42 (Butanol) and 84.63 (BHT) at a concentration of 100 µg. and ascorbic acid showed 93.88 at a concentration of 10 µg [Table 4& 5]. IC50 Values of pet ether, ethylacetate, Butanol, BHT, Ascorbic acid was 20.39µg, 11.29µg, 10.71µg, 12.52µg and 1.4µg respectively. The

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Butanolic extract shown the maximum free radical scavenging capacity as compared to other extracts [Fig.1.& 2]. REFRENCES:

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Source of support: Nil, Conflict of interest: None Declared


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