INTRODUCTION:
Melia azedarach Linn. (M azedarach) a deciduous tree, belongs to Family Meliaceae, Genus Melia1,2 is commonly known as China berry, Indian lilac, margosa tree, Persian lilac, pride of India, sichuan, pagoda-tree, bead-tree, syringe berry tree, umbrella tree, white cedar 3,4. This species, native to Asia, particularly India, is cultivated and naturalised as an ornamental avenue tree in different parts of the world 5,6 . The plant and its various parts have been widely used and have been scientifically validated for various pharmacological effects viz., anthelmintic, antibacterial, antifungal, anti-inflammatory, antimalarial, antiproliferative, antiprotozoal, antiviral agent 7,8,9. The plant is reported to exhibit immunomodulatory, cytotoxic, nematicidal, antiulcer, cathartic, emetic activities 10,11. A large number of phytoconstituents, melianin, meliatoxin meliantriol, nimbinene, azaridine, meliacin, quercetin, rutin, vanillic acid kaempferol, rutin, margosine, lupeol, β-sitositosterol, kaempferol, isoquercetin, quercetin, rutin, glutamic acid, threonine, methionine, leucine, lycine and proline etc. have been reported in M azedarach 12.
In spite of its several medicinal potentials, no systematic report on lipid lowering activity of M azedarach is available in the literature. The aim of the present work is to identify and quantify few phytochemicals of M azedarach and to evaluate the antioxidant capacity using different in vitro assays including DPPH, ABTS, O phenanthroline and total antioxidant assay as well as to evaluate lipid lowering activity in vivo methods.
MATERIAL AND METHODS:
Plant material
Leaf (twig) of M azedarach was collected from Tumkur, Karnataka, India, during September and October, 2009 and was authenticated by Dr. Gopalakrishna Bhat, retired Professor, Department of Botany, Poorna Prajna College, Udupi, Karnataka. A voucher specimens of the plant material vide No PP 595 have been deposited in the Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal, India.
Preparation of extracts
The shade dried leaves of M azedarach were ground to a powder, extracted with different solvents of increasing polarity viz., petroleum ether, chloroform, ethyl acetate and methanol by hot percolation method in a Soxhlet extraction apparatus. Aqueous extract of the powder was prepared by cold maceration process and all the extracts were concentrated in vacuo in a rotatory flash evaporator and were preserved for testing. The extracts were subjected to qualitative tests and for quantification of phytoconstituents.
Determination of total phenolic content
Total phenolic content was estimated by Folin-Ciocalteu colorimetric method using gallic acid as a standard phenolic compound13, 14. In this method, 100 µL of the extracts of M azedarach samples (1 mg/mL) were mixed with 900 µL of distilled water and 5 mL of 0.2 N Folin-Ciocalteu reagents. After 5 min, 4 mL of saturated sodium carbonate (75 g/L) was added. The absorbance of the resulting blue-coloured solution was measured at 765 nm after incubation at 30 °C for 1.5 h with intermittent shaking. Quantitative measurements were performed based on a standard calibration curve (10, 20, 40, 80, 160 and 320 µg/mL of gallic acid in 95% methanol). The results were expressed as gallic acid equivalents (GAE) in milligrams per gram of dry material using the following formula.
Where, T = Total content of phenolic compounds, mg/g plant extract, expressed as GAE, C = Concentration of gallic acid established from the calibration curve mg/mL, V = Volume of extract in mL, M = Weight of plant extract in g
Determination of total flavonoid content
Total flavonoid content was determined using quercetin as standard15,16. Solution of aluminium trichloride (5 mL of 2%) was mixed with the same volume of M azedarach sample (1 mg/mL). Absorbance at 415 nm was measured after 10 min against a blank sample consisting of 5 mL of sample solution and 5 mL of methanol without aluminium trichloride. The total flavonoid content was determined using a standard curve of quercetin (1–64 µg/mL). The average of three readings was expressed as quercetin equivalents (QE) (mg quercetin/g of dry weight basis).
Determination of Tannin content
Tannin content of the samples was determined by using standard tannic acid solution (1 mL, concentration 0-1000 µg/mL in distilled water) was pipetted into 10 mL standard volumetric flasks containing 7.5 mL of water. Folin-Denis reagent 0.5 mL and 1.0 mL of sodium carbonate solution was added and diluted to the mark with water. This solution was mixed well and the absorbance was measured at 760 nm after 30 min. Absorbance was plotted against concentration of tannic acid. Determination of tannin in the sample was carried in the same manner as standard and total tannin content was expressed in mg tannic acid equivalent (TAE)/g dry weight of powder. The blank consisted of all the reagents without the sample17.
In vitro antioxidant study
DPPH radical scavenging assay
The free radical-scavenging activity was measured by radical-scavenging ability using the stable radical DPPH•. 18,19
Different extracts of M azedarach (1mL) was mixed with 1mL of solution of DPPH (0.1 mM). A blank control was also prepared with an equal amount ethanol and DPPH. After 20 min incubation in the dark, absorbance was recorded at 517 nm. Ascorbic acid was used as the reference compound for comparison. The percentage of scavenging by the sample was calculated using the following formula. Results were expressed as the sample concentration able to decrease 50% the quantity of DPPH free radical (IC50).
ABTS free radical scavenging assay
Freshly prepared ABTS solution was used for the assay. Different extracts of M azedarach (1mL each) was allowed to react with 1 mL ABTS solution and absorbance was measured at 734 nm using a spectrophotometer and methanol was used as control. The ABTS scavenging capacity of the extract was compared with that of standard. The scavenging was calculated in percent by the same formula given in DPPH assay20.
Reduction of ferric ions by ortho-phenanthroline colour method
To measure the capacity of reduction of ferric ion by extracts of M azedarach, 0.2 mL standard / extract (various concentration 2-1000 µg/mL), 0.2 mL 1, l0-Phenanthroline-iron (III) colour reagent, 0.6 mL methanol and 4 mL water were mixed and incubated at 50 °C for 30 min. The absorbance was read at 510 nm against reagent blank21,22.
The scavenging was calculated in percent by the formula
Test - Control
% Scavenging
= X 100
Control
Determination of total antioxidant capacity
The assay is based on the reduction of Mo (VI) to Mo (V) by the antioxidant compounds and subsequent formation of a green phosphate/Mo (V) complex23.
The sample solutions, 0.1 mL aliquot of each of the different extracts of M azedarach (100 µg/mL) were mixed with 0.3 mL of reagent solution containing 0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate. The tubes were capped and the reaction mixture was incubated in a water bath at 95 °C for 90 min. Absorbance of each mixture solution was measured at 695 nm against a blank after cooling. The blank solution contained all the reagents excepting the test sample. The antioxidant activity was expressed as equivalents of trolox (mg/g of dry extract) 24.
Assessment of M azedarach for lipid lowering activity
Animals: Male Wistar Albino rats, obtained from Central Animal Research Facility (CARF) Manipal University, Manipal, after obtaining ethical committee clearance from the Institutional Animal Ethics Committee of KMC, Manipal. No. IAEC/KMC/38/2010-2011 dated May 29, 2010. Animals were acclimatized to the experimental room having temperature 25 ± 2 °C, controlled humidity conditions and 12 h light-dark cycle. The rats were fed with commercially available rat standard pelleted diet and water ad libitum. Study was conducted in accordance with the CPCSEA guidelines.
Acute toxicity
The study was conducted in Wistar Albino rats as per OECD guidelines 423.
In vivo lipid lowering activity of different extracts were carried out by two animal models namely
1. Triton WR -1339 induced hyperlipidemia (Acute model)25, 26, 27.
2. High fat diet induced hyperlipidemia (Chronic Model) in rats28.
1. Triton WR -1339 induced hyperlipidemia (acute model)
The experimental animals were randomised based on their body weight. The experimental groups consisted of 13 groups, each containing six rats. The groups are normal control, hyperlipidaemic (positive) control, test group from 3 to 12 receiving orally doses of 100 mg/kg and 200 mg/kg of different extracts of M azedarach and group 13 was treated with standard drug (atorvastatin, 0.4 mg/kg). The normal control group and hyperlipidaemic control groups received only vehicle. The treated groups, both test and standard were, given prophylactic dose of test drugs at the dose of 100 mg/kg and 200 mg/kg and atorvastatin 0.4 mg/kg p.o respectively for seven consecutive days. On 8th day, Triton WR-1339 reagent (200 mg/kg i.p) was injected to the overnight fasted animals belonging to all the groups except normal control group, immediately after above oral treatment to induce hyperlipidemia. Blood samples were collected by retro orbital puncture at 24 h post administration of Triton WR-1339 under light anaesthesia and the serum was separated at 1359 g for 10 min at 4 °C and used for determination lipid profiles.
2. High fat diet (HFD) induced hyperlipidemia (chronic model)
Preparation of High Fat Diet (HFD)
610 g of NPD, 5 g of deoxycholic acid, 5 g cholesterol and 90 g of fructose was mixed in a tray and dough mass made by adding 280 g of coconut oil. It was then converted in to ball of uniform size, stored in refrigerator before usage.29,30
Experimental
Animals were randomised and grouped based on their body weight before feeding the HFD. One group of animals were fed with normal pellets and served as normal control (Group 1). The remaining animals were fed with HFD, for four weeks for the induction of hyperlipidaemia. At the end of fourth week blood was withdrawn from retro orbital plexus under light anaesthesia and serum was analysed for lipid profile and based on the cholesterol level animals were re-randomized into 5 groups. The normal control group and hyperlipidaemic control groups received only vehicle. The animals received respective treatments from 4th to 8th weeks. On 30th day, (8th week), blood was withdrawn under light anaesthesia from overnight fasted animals by retro-orbital plexus and serum was separated by centrifugation at 1359 g for 10 min at 4 °C temperature and subjected for fasting lipid profiles analysis.
From the collected blood serum, the biochemical markers like high density lipoprotein (HDL), triglycerides (TG), total cholesterol (TC), Very low density lipoproteins (VLDL) were estimated by using commercial kits (Roche Diagnostics GmbH, Mannheim, Germany) and protocol from manufacturer using auto analyser (Cobas c111, Roche Labs) in FIST-DST Lab, MCOPS, Manipal.
Statistical analysis
All the data were expressed as mean ± SEM and analyzed by one way analysis of variance (ANOVA) followed by Dunnett’s comparison Test using Graph Pad Prism version 5.00 for Windows, Graph Pad Software, San Diego California USA, www. graphpad. com p<0.001 was considered as statistically significant.
RESULTS AND DISCUSSION:
Total flavonoid, Total phenolic and Tannin content of the samples was determined and presented in the Table 1 and Table 2.
Phenolic compounds in plants are reported to possess antioxidant activity 31,32. Total phenolic contents were determined and found to be more in aqueous extract followed by ethyl acetate, acetone, methanol, chloroform and petroleum ether extracts (Table 1).
Flavonoids are another group of bioactive compounds found in nature and possess antioxidant activity. Highest activity was observed with ethyl acetate extract followed by acetone, chloroform, methanol, aqueous, and petroleum ether extracts.
Tannin contents were determined and found to be belonging to both hydrolysable tannins, non-hydrolysable types. The total tannins (TAE)/g dry weight of powder is (Table 2.)
Table 2. Content of tannin in M azedarach (TAE/g dry weight of powder)
|
Hydrolysable Tannins |
91.48±0.27 |
|
Non Hydrolysable tannins |
90.47±0.86 |
|
Total tannins |
181.95±1.03 |
Table 1. Content of Phenolic and flavonoid in M azedarach
|
Extracts |
Phenolics (mg GA)/g |
Flavonoids (mg QE/g) |
|
Petroleum ether |
0.86±0.27 |
0.12±0.95 |
|
CHCl3 |
2.59±1.31 |
139.17±1.06 |
|
Acetone |
2.85±0.93 |
147.83±0.94 |
|
Ethyl acetate |
3.32±0.63 |
156.91± 0.86 |
|
Methanolic |
2.68±0.28 |
53.35±1.82 |
|
Aqueous |
14.84±0.98 |
2.864±0.92 |
The different extracts of the leaves of M azedarach were evaluated for antioxidant activity. The free radicle scavenging activity is associated with phenolic compounds and mainly flavonoids. Highest phenolic content was found in aqueous extract and highest flavonoid content found in ethyl acetate extract (Table 1).
The assessment of antioxidant activity showed that all extracts were capable to scavenge the radicals. In DPPH radical scavenging assay the ethyl acetate extract exhibited significant activity (27.67±1.6 µg/mL) compared to other extracts. In the ABTS assay ethyl acetate extract exhibited significant activity (48.89±.69 µg/mL), whereas, methanolic, chloroform, aqueous and acetone extract have shown less activity. In reduction of ferric ions by ortho-phenanthroline colour method, methanolic extract exhibited significant activity with (IC50 98.18±0.81 µg/mL), whereas, other extracts were less active. The results are tabulated in table 3.
Figure 1. Total antioxidant capacity of M azedarach
Table 3. IC50 values of different extracts of M azedarach (µg/mL)
|
Extracts |
DPPH |
ABTS |
Ortho-phenanthroline |
|
Petroleum ether |
>1000 |
>1000 |
393.86±1.02 |
|
CHCl3 |
436.35±1.34 |
152.72±0.62 |
162.10±0.65 |
|
Ethyl acetate |
27.67±1.6 |
48.89±.69 |
214.49±0.82 |
|
Acetone |
770.8±0.97 |
336.27± 0.89 |
110.36±1.06 |
|
MeOH |
81.75±0.95 |
89.63± 0.43 |
98.18±0.81 |
|
Aqueous |
331.17±0.86 |
309.63±1.02 |
247.87±0.91 |
In the assessment of total antioxidant capacity, chloroform extract showed highest total antioxidant capacity (Trolox Equivalent) 76.92mg/ml, whereas other extracts have showed less activity. The results are tabulated in the Figure 1.
The acute toxicity test was performed as per OECD guidelines adoption 423 in overnight fasted Wistar albino rats at 2000 mg/kg body weight. Oral administration of different extracts showed neither any sign of clinical abnormality nor any mortality. Hence the sealing doses were considered safe for each extract. One tenth and 1/20th of the safe dose was selected for lipid lowering activity.
Lipid profile in serum indicates the increased triglyceride and cholesterol levels were significantly reduced by treatment. Triglyceride and cholesterol levels were significantly increased in triton treated animals to control rats. Results were presented in Table 4.
Table 4. Effect of different extracts of M azedarach on serum lipid parameters in Triton WR-139 induced hyperlipidemia in rats
|
Group |
Total Cholesterol |
TG |
HDL |
|
|
Normal |
56.9 ± 2.79 |
59.27±5.9 a |
39.23±1.99 a |
|
|
Triton |
233.8±32.65 |
710±64.69 |
15.28±1.94 |
|
|
Atorvastatin |
67.27±4.053a |
56.4±6.67 a |
34.83±14.71a |
|
PE 100 |
174.8±16.32 |
481.9±41.38 |
26.3±6.129 |
|
PE 200 |
182.3±8.66 |
471.3±26.11 |
24.51±9.29 |
|
CHL 100 |
197.6±13.26 |
608.6±87.59 |
28.32±2.61 |
|
CHL 200 |
160.7±11.37 |
636.9±85.4 |
27.05±3.74 |
|
EA 100 |
177.3±8.43b |
574.9±85.94 |
28.15±13.51 |
|
EA 200 |
166±5.62 |
474.4±88.81 |
31.22±2.41b |
|
MeOH 100 |
141.6±15.79 a |
309.8±24.6 a |
29.13±1.55 |
|
MeOH 200 |
136.9±11.56 a |
241.1±36.85 a |
32.43±3.06 b |
|
AQ 100 |
145.2±13.44 a |
298.2±47.55 a |
34.68±5.90 a |
|
|
AQ 200 |
115.2±8.10 a |
173.6±35.24 a |
28.8±2.468 |
|
Data are means ± SEM. a p<0.001, b p<0.01, c p<0.05 When compared with disease control group
Administration of TritonWR-1339 to normal rats in the dose of 200 mg/kg caused significant increase in lipid profile such as TG, TC (p<0.001) as compared to normal control rats. Treatment with standard drug (Atorvastatin 0.4mg/Kg) significantly decreased the level of TG, TC (p<0.001). Pre-treatment of aqueous (AQ) 100 and 200 mg/kg and methanolic extract (MeOH) 100 and 200 mg/kg decreased the level of TG, TC, (p<0.001) which was increased due to triton administration, whereas pre-treatment with petroleum ether (PE), chloroform (CHL), ethyl acetate (EA) extracts did not modify the triton induced hyperlipidemia. However, pre-treatment with M azedarach extracts have shown significant increase in HDL compared to triton induced hyperlipidaemic group.
Table 5. Effect of different extracts of M azedarach on serum lipid parameters in on HFD fed hyperlipidaemic rats
|
Groups |
TC |
TG |
HDL |
|
Normal |
52.74 ± 1.94 a |
59.73 ± 3.50a |
47.94± 2.55a |
|
HFD |
91.16 ± 4.50 |
135.9 ± 10.23 |
31.73± 0.12 |
|
Atorvastain |
67.06 ± 2.09a |
80.65 ± 3.94a |
41.26± 4.14a |
|
AQ 100 |
72.78 ± 2.95b |
76.9 ± 4.14a |
38.77± 2.03b |
|
AQ 200 |
68.32 ± 3.79a |
84.05 ± 5.59a |
36.33± 4.01 |
|
MeOH100 |
74.59 ± 1.05c |
91.62 ± 4.34b |
33.21± 0.14 |
|
MeOH200 |
76.22 ± 2.77c |
93.1 ± 9.63b |
37.28±4.05 b |
Data are means ± SEM. a p<0.001, b p<0.01, c p<0.05 When compared with disease control group
Chronic administration of HFD to normal animals causes significant rise in the level of TG, TC as compared to normal control rats. Simultaneous administration of aqueous (AQ), methanolic (MeOH) extracts in the dose of 100mg/kg and 200 mg/kg p.o significantly decreased the TG, TC (p<0.01, p<0.001) as compared to HFD treated rats. Treatment with reference drug (Atorvastatin 0.4 mg/Kg) also decreased the level of TG, TC (p<0.001) caused due to HFD treatment (Table no 5). However pre-treatment with M azedarach extracts have not shown significant increase in HDL compared to HFD induced hyperlipidaemic group.
CONCLUSION:
The present study focuses on the estimation of phytochemicals, in vitro antioxidant study and lipid lowering activities of different extracts of M azedarach. Ethyl acetate and methanolic extracts showed significant antioxidant activity. The present study offers data for supporting the use of M azedarach extracts as natural antioxidant agents and this plant represent an important source of flavonoid, phenolic compounds. Experimental results suggest that M azedarach has the potential to be a candidate as a lipid lowering agent. beneficial effects may be due polyphenols and related compounds present in them. Mechanism of action needs to be envisaged.
ACKNOWLEDGMENTS:
The authors thank AICTE, New Delhi for the financial support. Authors also thank DST, FIST, authorities of Manipal University, Manipal College of Pharmaceutical Sciences, Manipal for providing all the facilities to carry out research work.
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Received on 08.08.2013 Modified on 12.09.2013
Accepted on 22.10.2013 © RJPT All right reserved
Research J. Pharm. and Tech. 6(11): November 2013; Page 1195-1199