INTRODUCTION:

Azima tetracantha (Salvadoraceae) is a well known medicinal herb, termed ‘Mulsangu’ in Tamil and 'Kundali' in Sanskrit. Root, root bark and leaves of Azima tetracantha (lam) are used with food as a remedy for rheumatism, diuretic and as stimulant¹. Traditionally Indian medical practitioners use Azima tetracantha (lam) in inflammatory conditions, cough, asthma, small pox and diarrhoea^2,3. The major phyto-constituents reported in Azima tetracantha (lam) are azimine, azecarpin, carpine, isorhamnitine-3-O-rutinoside, friedelin, lupeol, glutinol and β-sitosterol^4,5. Azima tetracantha (lam) is reported to have antifungal⁶antitumour⁷, antidiabetic⁸, antidiarrhoeal⁹ and hepatoprotectiveactivities.

Azima tetracantha (lam) is a low, spinouts, highly branched bush, woody below but with pale green, herbaceous, almost quadrangular young branches. The leaves are in opposite to sub-opposite, decussate pairs. They are shortly petiolate, about 2x4cm long, entire, elliptic, acute, sharply mucronate, rigid, pale green with an acute base. Usually, there are two laterally placed spines in the axil of a leaf. The spines which morphologically represent the first pair of leaves of the auxiliary shoot are about three cm long, more or less, triangular in cross section, very sharp and with an indurate apex. The plant is dioeciously. The flowers are borne in the axils of leaves. Generally, there is cymes of three flowers in the axil of a leaf which is the upper branches, especially of the male plants become greatly reduced or even completely suppressed.

The detection and analyses of phytochemical constituents within medicinal plants extracts have relied on a number of chromatographic and spectrometric techniques such as TLC, UV, IR and NMR¹⁰. Of recent, phytochemical analysis of medicinal plant extracts involve sophisticated techniques that include Gas chromatography instrument coupled with mass spectrometer (GC-MS). GCMS affords direct analysis of the phytochemicals present in medicinal plant extracts¹¹.

MATERIALS AND METHODS:

Collection of plants:

The aerial part (leaves) of Azima tetracantha (lam) was collected from the Panayur area of Madurai, Tamilnadu as raw material, during the second week of February 2015 and a voucher specimen is stored in C.L. Baid Mehta College of Pharmacy (001/ATL/CLBP) and the plant material was authenticated by a renowned botanist. About 500 g of coarse powdered leaf in 2.5 L water is boiled, cooled and filtered. The filtrate is evaporated to dryness in desiccator and stored in refrigerator (Yield- 26.5% w/w). The aqueous extract of Azima tetracantha (lam) (AEAT) was subjected to preliminary phytochemical analysis¹²

Various extraction methods for isolation of constituents:

The whole plant will be subjected to shade drying and extraction with petroleum ether (60-80^oC) chloroform, Ethyl acetate and 80% ethanol in soxhlet apparatus by simultaneous extraction each for 72 hours. Concentrate the solvents in vacuum. The crude solid obtained on evaporation are to be studied for preliminary qualitative phytochemical evaluation.

Phytochemical Screening:

The extract was subjected to phytochemical analysis to test the presence of carbohydrates, glycosides, alkaloids, flavonoids, tannins, sterols, and saponins in leaf extracts.

GC-MS Analysis^13,14

The Gas Chromatography Mass Spectrometry analysis of the extract was performed using GC-MS (Model: GC-MS-QP 2010,Shimadzu, Tokyo, Japan) equipped with a VF 5ms fused silica capillary column of 30m length, carrier gas was Helium (99.99%), used at a constant flow rate of 1.51ml/min. Two micro litre of the sample was injected in a split mode with a scan range of 40 – 1000 m/z. The total running time of GC-MS was 35 min. The relativepercentage of the extract was expressed as percentage with peak area normalization.

Identification of components:

The components in the extract were assigned by the comparison of their retention indices and mass spectra fragmentation patterns with those stored on the computer and also with published literatures. NIST08.LIB, WILEY8.LIB, FA_ME.LIB library sources were used for matching the identified components in the plant material. The name, molecular weight and molecular formula of the components of the test materials were ascertained.

Anti oxidant activity¹⁵

Determination of in vitro anti oxidant activity:

DPPH free radical scavenging activity:

The DPPH assay is based on the reduction of DPPH a stable free radical. Radical scavenging activity of compounds against stable DPPH was determined spectrophotometrically. When antioxidant reacts with DPPH which is a stable free radical becomes paired off in the presence of a hydrogen donor and it is reduced to DPPHH as a result the absorption decreases from DPPH which is clearly identified by colour change from deep violet to yellow colour measured by reading the absorbance at 517 nmusing a UV spectrophotometer. The solution of DPPH is mixed with that of a substance that can donate a hydrogen atom will give rise to the reduced form with the loss of violet colour. A series of test tubes labeled from 1- 5, was taken. To each tube a concentration of 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0 microgram of test solution (extract) was added to 2.0 ml of 0.004% solution of DPPH in methanol. 1ml of methanol and 2 ml of 0.004% DPPH solution was used as experimental negative control. After 30 min of incubation at room temperature, the reduction in the number of free radicals was measured by reading the absorbance at 517 nmusing a UV spectrophotometer. Radical scavenging activity was expressed as the inhibition percentage ¹⁶. The scavenging activity of the samples corresponded to the intensity of quenching DPPH. The standard (positive control) used here was Quercetin.

Absorbance of control – Absorbance of test sample

Percentage= ---------------------------------------------- x100

of inhibitionAbsorbance control.

Xanthine oxidase inhibition assay^17,18

The XO oxidase activity was assayed spectrophotometrically under aerobic conditions. The assay mixture, consisted of 50 microlitres of test solution, 35 micro liter of 0.1mM phosphate buffer (pH=7.5), and 30 microlitres of enzyme solution (0.01 units/ml in 0.1mM phosphate buffer, pH=7.5) and was prepared immediately before use. After pre incubation at 25 ⁰C centigrade for 15 minutes, the reaction was initiated by the addition of 60 micro litre of substrate solution (150mM xanthine in the same buffer). The assay mixture was incubated at 25degree centigrade for 30minutes. Absorbance at 290nm was measured with a spectrophotometer blank was prepared in the same manner. One unit of XO was defined as the amount of enzyme required to produce 1nmol of uric acid/minutes at 25 ⁰C.Xanthine oxidase inhibitory activity is expressed as the percentage inhibition of XO in the above system, calculated as (1-B/A) X 100, where A and B are the activities of the enzyme without and with test material.

Hydrogen peroxide scavenging assay^19,20

A solution of H2O2 (40mM) was prepared in phosphate buffer. The bark extracts at the concentration of 10mg/10μl were added to H2O2solution (0.6ml) and the total volume was made up to 3ml. The absorbance of the reaction mixture was recorded at 230nm in a spectrophotometer (Genesys 10-S, USA). A blank solution containing phosphate buffer, without H2O2 was prepared. Ascorbic acid is used as positive control. The extent of H2O2 scavenging of the plant extracts was calculated as

(A0–A1)

% scavenging of = ----------------------------------- ×100

hydrogen A0

peroxide

A0=Absorbance of control

A1- Absorbance in the presence of plant extract

RESULTS:

GC-MS ANALYSIS:

The GC-MS chromatogram (Figure 1,2) shows the peak area separation. The chromatogram reveals that the chloroform extract and ethyl acetate extract of Azima tetracantha Lamis rich in flavanoids. The analysis revealed the presence of twelve compounds from the chloroform leaf extract of Azima tetracantha (Table 1) and fourteen compounds from ethyl acetate extract (Table 2).

Table 1 : Identification of Compound In Azima Tetracantha Lam Chloroform Extract

S.NO.	R.T	NAME OF THE COMPOUND	MOLECULAR FORMULA	MOLECULARWEIGHT
1	16.12	2,6 dimethyl 4(4 methoxy methyl) phenol	C₁₆H₂₆O₂	250.37644 g/mol
2	17.12	Pentadecanoic acid, 14 methyl, methyl ester	C₁₇H₃₄O₂	270.457 g/mol
3	18.53	8- cyclohexadecen 1-one	C₁₆H₂₈O₂	236.393 g/mol
4	18.83	9-octadecenoic acid 2-methyl ester	C₁₉H₃₆O₂	269.49 g/mol
5.	19.05	Cyclopentane tri decanoic acid, methyl ester	C₇H₁₂O	128.168980 g/mol
6.	19.77	Ethyl 8-methyl 9- tetra decen-1-ol acetate	C₁₇H₃₂O₂	268.4348 g/mol
7.	20.53	Z,Z,4,16- octadecadien-1-ol acetate	C₂₀H₃₆O₂	308.506 g/mol
8.	21.08	1-tetradecene 2-decyl	C₁₅H₂₈	208.38300 g/mol
9.	21.85	Eicosane 2,6,10,14,18 penta methyl	C₂₅H₅₂	352.6804 g/mol
10.	22.72	1-Tricosene	C₂₃H₄₆	322.621 g/mol
11.	23.88	Dodecanoic acid, undecyl ester	C₂₃H₄₆O₂	354.619 g/mol
12.	25.28	Cyclohexane 1,4,didecyl	C₂₆H₅₂	364.702 g/mol

Figure 1

Table 2: Ethylaceteate Extract

S.NO	R.T	NAME OF THE COMPOUND	MOLECULAR FORMULA	MOLECULAR WEIGHT
1	9.22	Ionone	C₁₃H₂₀O	192.3 g/mol
2	12.53	2,5-di-tert-butylaniline	C₁₄H₂₃N	205.34 g/mol
3	16.1	Dodeca 1,6- dien 12-ol,6,10- dimethyl	C₁₄H₂₆O	210.36 g/mol
4	17.05	Cyclo dodecanecarboxylic acid,methyl ester	C₈H₁₄O₂	142.198 g/mol
5	17.73	Hexadecanoic acid, ethyl ester	C₁₈H₃₆O₂	284.484 g/mol
6	18.5	Z,E-2-methyl 3,13-octa decadien – 1-ol	C₁₉H₃₆O	280.488 g/mol
7	18.8	Oleic acid	C₁₈H₃₄O₂	282.46 g/mol
8	19.43	E-8 methyl-9-tetra decen-1-ol acetate	C₁₇H₃₂O	268.4348 g/mol
9	20.47	5-methyl docosane	C ₂₂H₄₆	310.61 g/mol
10	21.08	(Z)-9- octadecenoic acid butyl ester	C₂₂H₄₂O₂	338.5677 g/mol
11	21.8	13- docosenic acid, methyl ester	C₂₃H₄₄O₂	352.5943 g/mol
12	22.67	2- pentacosanone	C₂₅H₅₀O	366.674 g/mol
13	23.75	4- hexadecynoic acid, 2-butoxy, butyl ester	C₂₄H₄₄O₃	380.613 g/mol
14	25.1	14- heptacosanone	C₂₇H₅₄O	394.728 g/mol

Figure 2

In-vitro antioxidant activity:

DPPH free radical scavenging activity²¹

Antioxidants cease the free radical reaction by donating hydrogen from the phenolic hydroxyl groups. The ethanolic extract showed up to 66% inhibition of DPPH free radical at 517 nm at high concentration of 1mg/ml. At the concentrations of 0.3mg/ml, 0.5mg/ml and 1mg/ml, quercetin shows similar activity of about 76% inhibition of DPPH free radicals. This might be due to the complete reaction of quercetin with the DPPH free radicals present in the solution and there are no free radicals remain in the solution to react with quercetin. That is, all DPPH free radicals are consumed at 1mg/ml solution of quercetin. The percentage inhibition of DPPH free radical by ethanol extract in the presence of standard quercetin at 517 nm was recorded in Table 3.

Xanthine oxidase inhibition assay:

The in-vitro xanthine oxidase inhibition of root ethanolic extract revealed significant (69%) inhibitory activity at 30µg/ml and it compared with the standard drug quercetin which showed 70% inhibition at 30µg/ml. (Table 4)

Hydrogen peroxide scavenging assay²²

The crude extract of Azima tetracantha (lam) was capable of scavenging H2 O2 in an amount dependent manner. The scavenging ability of the extract and standard, Ascorbic acid are shown in Table 5. H2 O2 scavenging activity of leaf extract of Azima tetracantha (lam) was closer to that of Ascorbic acid at doses of 100, 200, 300 and 400 μg/ml.

Table: 3 % Percentage inhibition of DPPH free radicals by extract /standard at 517 nm

Particulars	Concentration (microgram/ml)	% of activity(inhibition)
Particulars	Concentration (microgram/ml)	Ethyl acetate Extract	Ethanol extract	Quercetin
Extract	0.1	7.6	7.8	15
	0.2	15	17	24
	0.3	25	26	52
	0.5	54	55	60
	1.0	66	67	76

Table: 4 % Percentage inhibition of xanthine oxidase by extract/standard at 290 nm

Sample	Concentration(30µg/ml)	Antioxidant activity %
Ethyl acetate extract	500ppm	68
Ethanol extract	500 ppm	69
Quercetin(control)	500ppm	70

Table: 5 % Percentage inhibition of Hydrogen peroxide by extract /standard at 230 nm

S.No	Concentration(µg/ml)	% of inhibition
Ethyl acetate extract
1	100	26.55%
2	200	41.90%
3	300	65.43%
4	400	77.50%
Ethanol extract
1	100	27.45%
2	200	40.96%
3	300	66.87%
4	400	78.09%
Ascorbic acid
1	100	76.50%

DISCUSSION:

Free radicals are harmful by-products generated during normal cellular metabolism, which could initiate oxidative damage in the body^23,24. The chemical composition leaf extract of Azima tetracantha (lam) indicates the presence of phenolic compounds including tannins and flavonoids, which are known to possess antioxidant activities. The high phenolic and flavonoid content in the ethanolic extract of leaf extract of Azima tetracantha (lam) may be responsible for its free radical scavenging activity.

DPPH is the agent of choice for many similar studies in evaluating the free radical scavenging activity of natural compounds²⁵. DPPH is a stable free radical that accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The reduction capability of the DPPH radical is determined by the decrease in its absorbance at 516 nm induced by antioxidants.

Xanthine oxidase is a flavoprotein that catalyses the oxidation of hypoxanthine to xanthine and generates superoxide and uric acid²⁶. It has been shown that xanthine oxidase inhibitors may be useful for the treatment of hepatic disease and gout, which is caused by the generation of uric acid and superoxide anion radical²⁷. Flavonoids and Polyphenolic crude extracts have been reported to possess xanthine oxidase inhibitory activity^28-30. Hypouricemic agents are commonly employed for the treatment of chronic gout arthritis, which includes xanthine oxidase inhibitors and uricosuric agents^32-35.

The crude extract of leaf extract of Azima tetracantha (lam) scavenged H2O2 which may be attributed to the presence of phenolic compounds present in the root bark extract are good electron donors, they may accelerate the conversion of H2O2 to H2O³⁶ hence, the crude extract might also help accelerate the conversion of H2O2 to H2O.

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Received on 11.10.2018 Modified on 03.12.2018

Asian J. Research Chem. 2018; 11(6): 857-862.

DOI:10.5958/0974-4150.2018.00150.5