INTRODUCTION:

The therapeutic value of Syzygium cuminii (L) skeel Commonly known as ‘’Jamun’’ in the local language has been recognized in different systems of traditional medication for the treatment of different diseases and ailments of human beings1, Such as leaves and seed are used in treatment of Bronchitis, asthma, thirst, biliousness, dysentery, ulcers. The fruit pulp is used in diabetics and also as a blood purifier. Several studies using modern techniques have authenticated its use in diabetes and its complications (nephropathy, cataract, insulin resistance)^1,2

Previously isolated classes of constituent:

Fifteen polyphenols and two Acylated flavonal glycoside are isolated from the leaves3 of Syzygium cuminii (L) Skeel. Essential oil and triterpenoids are isolated from the stem and fruits4. From flower olinolic acid, triterpenoid, ellagic acid and flavanols are isolated in small amount5.The literature survive reveals that there is no substantial work has been carried out on roots of Syzygium cuminii (L) skeel. Hence effort is made to investigate for the first time the roots of Syzygium cuminil

MATERIAL AND METHOD:

All the melting points were recorded by Toshniwal melting point apparatus and were uncorrected. IR spectra of the compounds were recorded using the KBr

pellet method on a Nicolet Avator 330 FTIR, Perkin-Elmer model 700, IR spectrophotometer. 1HNMR spectra of the compounds were taken on AMX 400 (270 MHZ), EM-360 (270 MHZ) NMR spectrometer using CDCl3 as solvent.

Mass spectra were recorded on FABMASS. TLC was carried out using Silica-gel G (Merck). Column chromatography was carried out on Silica-gel (Merck, 70-230 mesh) and cellulose powder (Merck, Bombay) as a stationary phase. All the chemicals and reagents used were obtained in high purity either from S.D. fine chemicals Pvt. Ltd., Bombay, India or E – Merck Pvt. Ltd., Bombay, India.

General experimental procedure⁶:

The shade dried roots were powdered (3 kg), extracted in succession by maceration, The solvents were used according to their increasing polarity i.e Butanol, pet. ether chloroformand ethyl acetate. The residues obtained from different solvents were kept in desiccators and the phytochemical investigation was carried out with Butenolic axtract (the residue obtained from Butanolic extract is 35 gm) It gave positive test for flavonoides . The residue (25 g) was triturated in mortar with CHCl3 (10 ml) and adsorbed onto silica gel (20 g). After evaporation of the solvent it was loaded onto a silica gel column (150 g) prepared in petroleum ether (60-80˚C). The column was eluted first with petroleum ether (60-80˚C), petroleum ether (60-80˚C): benzene graded mixtures (95:5, 90:10, 80:20, 70:30, 60:40 and 50:50), then with benzene followed by graded mixtures of benzene: chloroform (95:5, 90:10, 80:20 70:30, 60:40 and 50:50), chloroform and finally chloroform: methanol (95:5, 90:10, 80:20, 70:30, 60:40 and (50:50). The elutions were monitored by TLC (Silica gel-G; visualization by Vanillin - Sulphuric acid reagent heated at 110ºC). 5 ml of identical elutes were collected each time. TLC monitored elutes were combined and concentrated to 5 ml and kept in a refrigerator. Elution carried out with Benzene graded mixture, benzene: chloroform (95:5) resulted in getting “one” component ScRex-2, the solvent system for monitoring TLC viz., Benzene: chloroform (95:5v/v) Elution carried out with chloroform: methanol (60:40) resulted in getting “one” component ScRex-5, the solvent system for monitoring TLC viz., chloroform: methanol (60:40v/v) Elution carried out with chloroform: methanol (50:50) resulted in getting “one” component ScRex-6a, the solvent system for monitoring TLC viz., chloroform: methanol (50:50v/v) The remaining solvent residue was not analyzed. However the work is going on in this laboratory to analyze the residue

MY R IC ET IN E G L YC OS IDE S cR ex -5

3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-1- benzopyrone-4-one.5-glycosid:

Melting point: 300ºC; Rf value 0.5 (solvent system; C₆H₆: EtOAc 80:20); IR (KBr,_max,cm^-1): 3412 (O-H s,b) and 1690 (C=Os in carboxyl group); ¹HNMR (CDCL3) : δ 0.8- δ 0.48 (number of CH, CH2 protons); MS m/z (rel.int.):661 [M]+(C25H28O10), 599 (30%), 397 (90%)

MYRECETIN GENTOBIOSE ScRex-6b

3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-1-benzopyrone-4-one.gentobios:

Melting point: 350ºC; Rf value 0.42 (solvent 1692cm^-1system; C₆H₆: EtOAc 80:20); IR (KBr,,_max,cm^-1): 3430(O-Hs,in H-bonded alcohol), 2928(m C-H str in alkane), and1692 (s C=O str of carboxylic acid). ¹HNMR (CDCL₃) : δ 0.5- δ 3.9 (number of CH, CH2 protons); MS m/z (rel.int.) 663: [M]+ (C₂₇H₃₅O₁₉),663, 453(90%), 407 (60%), 284 (10%), 248 (70%).

QUER CETIN G LYCOSIDE ScR ex -2

3,5,7-trihydroxy-2-(3,4,-diihydroxyphenyl)-4H-1-benzopyrone-4-one,glycoside:

Meltingpoint: 360ºC; Rf value 0.8 (solvent system; C₆H₆: methanol. 80:20); IR(KBr,,_max,cm^-1): 3420 (s,bO-Hstr of H-bonded alcohol), 2928(m C-H str of alkane),1692 (s C=Ostr of carboxylic acid) (¹HNMR (CDCL₃): δ 2.5- δ 5.4 (number of CH, CH₂ protons); MS m/z (rel.int.) 601: [M]+ (C₂₅H₂₈O₁₇ ) 585 (50%),601(30%),599 (48%), 575 (70%), 453 (35%), 397 (80%)

Evaluation of Antioxidant activity by in vitro Techniques:

Reducing power assay⁷:

Principle:

The reducing power of Butanolic extract of root’s of syzygium cuminii (L) skeel was determined by the method of Oyaizu 1986.Substances, which have reduction potential,react with potassium ferricyanide (Fe³⁺) to form potassium ferrocyanide (Fe²⁺), whichthen reacts with ferric chloride to form ferric ferrous complex that has an absorptionmaximum at 700 nm⁵.

Antioxidant

Potassium ferricyanide + Ferric chloride

Potassium ferrocyanide + Ferrous chloride

Materials required:

1. Potassium ferricyanide (1% w/v)

2. Phosphate buffer (0.2 M, pH 6.6)

3. Trichloro acetic acid (10%)

4. Ferric chloride (0.1%) 5. Ascorbic acid 1%.

Figure 1: Effect of butenolic extract of the root’s of Syzygium cuminii (L) Skeel on reducing power assay:

Tested

material

Concentration

(µg/ml)

% increase in Reducing

power (±SEM)

EC 50

(µg/ml

Ascorbic acid

15.98±0.002

27.41±0.01

41.54±0.009

64.77±0.001

91.91±0.001

5.6 (µg/ml)

Butenolic extract

of the root’s of

Syzygium

cuminii(L)

Skeel

6.13±0.001

13.56±0.006

26.76±0.007

44.33±0.005

75.09±0.004

9.4 (µg/ml)

All values are expressed as mean ± SEM for three tests

Figure 2: Effect of butenolic extract of the root’s of Syzygium cuminii (L) Skeel on DPPH assay

Tested

material

Concentration

(µg/ml)

% D.P.P.H Radical

Scavenging (±SEM

IC 50

(µg/ml)

Ascorbic acid

23.04±0.0006

40.05±0.0003

57.58±0.0002

76.39±0.0009

83.40±0.0001

3 µg/ml

Butenolic extract

of the root’s of

Syzygium cuminii(L)

Skeel

16.62±0.0007

25.82±0.0002

33.78±0.0011

49.17±0.0005

73.44±0.0001

8.2 µg/ml

All values are expressed as mean ± SEM for three tests

Procedure:

Various concentrations of the extracts (1to 16 µg/ml) in 1.0 ml of deionized water were mixed with phosphate buffer (2.5 ml) and potassium ferricyanide (2.5 ml). The mixture was incubated at 50 0C for 20 min. Aliquots of trichloroacetic acid (2.5 ml) were added to the mixture, which was then centrifuged at 3000 rpm for 10 min. the upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and a freshly prepared ferric chloride solution (0.5 ml). The absorbance was measured at 700 nm. A blank was prepared without adding extract. Ascorbic acid at various concentrations (1 to 16 µg/ml) was used as standard. Increased absorbance of the reaction mixture indicates increase in reducing power.

A _test

% increase in Reducing Power ₌ - 1 X 100

A _blank

A test is absorbance of test solution, A blank is absorbance of blank.The antioxidant activity of the root extract was expressed as EC50 and compared with standard.

4.3.2 DPPH assay: (2, 2-diphenyl-1-picrylhydrazyl)⁸:

Principle:

The scavenging reaction between (DPPH.) and an antioxidant (H-A) can be written as:

(DPPH) + (H-A) DPPH-H + (A)

(Purple) (Yellow)

Antioxidants react with DPPH., which is a stable free radical and is reduced to the DPPHH and as consequence the absorbance’s decreased from the DPPH. radical to the DPPH-H form. The degree of discoloration indicates the scavenging potential of the antioxidant compounds or extracts in terms of hydrogen donating ability⁸.

Materials required:

1. Methanolic solution of DPPH (0.1 mM): 39.4 mg of DPPH was dissolved in one liter of analytical grade methanol.

2. Ascorbic acid 1%.

Procedure:

0.1 mM solution of DPPH in methanol was prepared and 1.0 ml of this solution was added to 3.0 ml of extract solution in methanol at different concentration (1-16 µg/ml). Thirty minutes later, the absorbance was measured at 517 nm. A blank was prepared without adding extract. Ascorbic acid at various concentrations (1 to16 µg/ml) was used as standard. Lower the absorbance of the reaction mixture indicates higher free radical scavenging activity. The capability to scavenge the DPPH radical was calculated using the following equation:

A _control– A _test

DPPH Scavenged (%) = X 100

A _control

Where A control is the absorbance of the control reaction and A test is the absorbance in the presence of the sample of the extracts. The antioxidant activity of the root’s extract was expressed as IC50 and compared with standard. The IC50 value was defined as the concentration (in µg/ml) of extracts that inhibits the formation of DPPH radicals by 50

RESULTS and DISCUSSION:

Reducing power assay:

The reductive capabilities of the butanolic extract of root’s of Syzygium cuminii (L) skeel were compared with ascorbic acid for the reduction of the Fe3+ - Fe2+ transformation in the presence of the butanolic extracts of the root. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. However, the antioxidant activity of antioxidants have been attributed to various mechanisms, among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging antioxidant activity. The reducing power of butenolic extract of the root’s of Syzygium cuminii (L) Skeel increased with increasing amount of sample⁹.

For the measurements of the reductive ability, we investigated the Fe3+ / Fe2+ transformation in the presence of the butenolic extract of the root’s of Syzygium cuminii (L) Skeel.The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant.The antioxidant activity has been reported to be concomitant with the development of reducing power. The reducing power of butanolic extract of the root’s of Syzygium cuminii (L) Skeel increased with the increasing amount of sample. All the concentration of butenolic extract of the root’s of syzygium cuminii (L) skeel showed significant activities when compared to standard ascorbic acid. The EC50 values were found to be 5.6 µg/ml and 9.4 µg/ml of butanolic extract of the root’s of Syzygium cuminii (L) Skeel and ascorbic acid respectively See Figure1.

DPPH assay:

The 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical widely used as the model system to investigate the scavenging activities of several natural compounds such as phenolic or crude mixtures such as the butanolic extract of plants. DPPH radical is scavenged by antioxidants through the donation of proton forming the reduced DPPH.The color changes from purple to yellow after reduction, which can be quantified by its decrease of absorbance at wavelength 517 nm. Radical scavenging activity increased with increasing percentage of the free radical inhibition. DPPH is a relatively stable radical. The assay is based on the measurement of the scavenging ability of antioxidants towards the stable radical DPPH which reacts with suitable reducing agent. The electrons become paired off and solution loses color stochiometrically depending on the number of electrons taken up. DPPH was used to determine the proton radical scavenging action of butenolic extracts of the root’s of Syzygium cuminii (L) Skeel, because it possesses a proton free radical and shows a characteristic absorbance at 517 nm. From the present results, it may be postulated that butenoli extract of the root’s of Syzygium cuminii (L) Skeel reduces the radical to corresponding hydrazine when it reacts with hydrogen donors in antioxidant principles¹⁰. DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become stable diamagnetic molecule. The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm, which is induced by antioxidants. Figure 2 illustrates a decrease in the concentration of DPPH radical due to the scavenging ability of the soluble constituents in the butanolic extract of the root’s of Syzygium cuminii (L) Skeel and the standard ascorbic acid, as a reference compound, presented the highest activity at all concentrations. The IC 50 values were found to be 3 µg/ml and 8.2 µg/ml for butanolic extract of the root’s of Syzygium cuminii (L) Skeel ascorbic acid respectively See Figure 2.

ACKNOWLEDGEMENT:

The authors sincerely thanks to Dr.Y.N Sitaram for authentification and Dr M. G. Purohit, for spectral interpretation. NGSMIPS, Mangalore. For constant support and facilities provided to carry out this work. My acknowledgement with deepest appreciation goes to my brothers Moid and thouheed for their moral support and encouragement through my work.

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Received on 18.02.2009 Modified on 30.04.2009

Asian J. Research Chem. 2(2): April.-June, 2009 page 218-221