Hypolipidemic, Hypoglycemic and Antioxidant Activities of Flower Extracts of Allamanda violacea A.DC. (Apocynaceae)

Purpose : To investigate the anti-dyslipidemic, anti-oxidant and anti-diabetic activities of the aqueous extract and solvent fractions of A. violacea flowers. Methods: The aqueous extract was fractionated into petroleum ether, ether, chloroform, chloroform-methanol (4:1) and chloroform-methanol (3:2) fractions. Lipid lowering activity was evaluated in two models, viz, triton WR-1339 - induced hyperlipimea in rats as well as fructose-rich high fat diet. To assess anti-oxidant activity, in-vitro model of non-enzymic superoxide hydroxyl radicals and microsomal lipid peroxidation by non-enzymic inducer was adopted. Hypoglycemic activity was evaluated by sucrose-loaded rat model. Results : Amongst the fractions, ether and chloroform fractions caused marked decrease in the levels of total cholesterol (Tc), triglycerides (Tg), plasma lipids (Pl), and protein by 24, 23, 23 and 22 %, and 24, 22, 23 and 19 %, respectively. In rats fed with high fat diet (HFD), ether and chloroform fractions lowered Tc, Tg and, Pl by 26, 25 and 26 %, and 18, 19 and 20 %, respectively. Significant decrease in superoxide anions, hydroxyl radicals and microsomal lipid peroxidation by ether and chloroform fractions was also observed. Chloroform, chloroform-methanol (4:1) and chloroform-methanol (3:2) fractions showed antihyperglycaemic activity to the extent of 25.2, 21.6 and 23.2 %, respectively. Conclusion: The flowers of this plant, especially the ether and chloroform extracts, may be suitable for anti-oxidant use and lipid management.


INTRODUCTION
Atherosclerosis is the major cause of heart diseases, stroke and death in both developed and developing countries.It is well established that elevated levels of low density lipoproteins constitute primary risk factors for atherosclerosis.Epidemiological studies have indicated that dyslipidemia and coagulation disturbances are among the most significant risk factor for the development of atherosclerotic conditions [1] Current pharmacological treatment of atherosclerosis includes the use of the statin class of 3-hydroxy-3-methyl glutaryl.CoA (HMG.CoA) reductase inhibitors and the fibrate class, peroxisome proliferatoractivated receptor alpha (PPARα) antagonists.They are effective in lowering triglyceride (Tg) and low density lipoprotein (LDL) but most patients still experience coronary events despite statin therapy.In addition, there are reports of undesirable side effects (myopathy) of some 'super statins' whereby the scope for improving the potency of this class of drug may be modest [2].Furthermore, the fibrate class of drugs, which are mostly used to treat hypertriglyceridemia and low HDL cholesterol, requires high doses to show significant efficacy [3].In addition, a combination of fibrate and statin has met with serious safety concerns as exemplified by the withdrawal of cerivastatin in 2001.Besides, hyperglycemia and dyslipidemia, which are the two major components of metabolic syndrome, are also one of the crucial risk factors for cardiovascular diseases [4].Therefore, there is a need for a different class of compounds to treat hyperglycemia and dyslipidemia without the attendant serious side effects.
Oxidative stress has recently been implicated in the pathogenesis of various diseases such as diabetes and coronary artery diseases.Hydroxyl free radical ( .OH) has also been found to be responsible for the peroxidative damage to lipoproteins present in the blood, which in turn are responsible for the initiation and progression of atherosclerosis [5].Therefore, treating both oxidative stress and disorders of lipid metabolism together, may be a novel approach to regress atherosclerosis and other cardiovascular diseases.We reported earlier that some precursors in hormone synthesis, such as, guggulsterone, possess lipid-lowering activity, together with mild anti-oxidant effect and also inhibit oxidative modification of LDL [6].Recently, we also found that some pregnane derivatives exhibited antioxidant and anti-dyslipidemic activities simultaneously [7,8].
A. violacea (purple allamanda, violet allamanda, syn A. blanchetti) is an ornamental plant of Allamanda genus in the Apocyanaceae family.Previous phytochemical examination of this plant indicated the presence of plumericin, isoplumericin and 5, 6-dimethoxycoumarin [9].The ethanol extracts of the roots, leaves and stems of this plant have been reported to posses' cytostatic and cytotoxic activities [10].However, no work has been reported on the flowers of this plant.The present study was undertaken to evaluate the antidyslipidemic, anti-oxidant and anti-diabetic activities of the extract and fractions of the flowers of A. violacea.

Chemicals and reagents
All the chemicals/solvents used were of high purity (AR/GR grade) and all the solvents used were dried by standard procedures.Triton WR-1339 was purchased from Sigma Chemical Company, St Louis, MO, USA and high fat diet (HFD) from Research Diet Inc, New Brunswick, USA (Product code no.D 99122211).Triglyceride (Tg) test kits and total cholesterol (Tc) test kits were purchased from Merck Co.

Experimental animals
Rats (Charles Foster strain, male, adult, body weight 200 -225 g) were kept in a room with controlled temperature (25 -26 o C), humidity (60 -80 %) and 12/12 h light/dark cycle in hygienic conditions.Animals, which were acclimatized for one week before starting the experiment, had free access to normal diet and water ad libitum [7,8].

Preparation of extract
The whole plant of A. violaceae was collected in the month of October 2010 from Lucknow, India.The identity of the plant was confirmed by Dr Tariq Hussain, Head, Department of Taxonomy and Herbarium, National Botanical Research Institute, Lucknow, India, where a voucher specimen, no.97108, was deposited.
The flowers of the plant were shade-dried, powdered, soaked in water overnight and exhaustively extracted by percolation with ethanol of increasing concentration from 50 to 95 % at room temperature [11].The combined alcoholic extract was concentrated in a rotator evaporator under reduced pressure to get the hydroalcohol extract.The hydroalcohol extract was shaken successsively with petroleum ether (Pet Et 2 O, 35 o C, 1000 ml x3), ether (Et 2 O, 1000 ml x3), chloroform (CHCl 3 , 1000 ml x3), chloroform: methanol (CHCl 3 :CH 3 OH, 4:1, 1000 ml x3), chloroform: methanol (CHCl 3 :CH 3 OH, 3:2, 1000 ml x3) for preliminary separation of the constituents of different polarities.The fractionation was carried out at room temperature and the organic layer was removed from the separating funnel only when there was a separation of a clear layer.After fractionation was complete, the residue was discarded.

Phytochemical screening of extract fractions
High performance liquid chromatography (HPLC) analyses was carried out on a
Fractions/extracts were triturated with 0.2 %w/v aqueous gum acacia suspension, and fed orally (100 mg/kg) and simultaneously with triton, after further feeding was stopped.Control and triton groups, which did not receive fraction/extract treatment, received the same amount of gum acacia suspension (vehicle).After 18 h of treatment, the animals were anaesthetized with thiopentone sodium (50 mg/kg) prepared in normal saline and then 2 mL blood was withdrawn from the retro-orbital sinus using glass capillary in EDTA-coated Eppendorf tube (3 mg/ml blood).The blood was centrifuged at 2500 g and 4 o C for 10 min to separate plasma.The plasma was diluted with normal saline (1:3) and used for analysis of total cholesterol (Tc), triglycerides (Tg) and phospholipids (Pl) by standard enzymatic methods [14].
In the chronic experiment, hyperlipemia was produced by feeding with high fat diet (HFD) once a day for 30 days.The fraction/extract was administered (50 mg/kg) orally simultaneously with HFD to the extracttreated groups.The control animals received the same amount of normal saline (2.5 ml/kg) or arachis oil (5 ml/kg).At the end of the experiment, the rats were fasted overnight and blood (2 mL) was withdrawn.The animals were killed and their liver promptly excised.The plasma was analyzed for Tc, Tg, Pl, lecithin-cholesterol acyltransferase activity (LCAT) and post-heparin lipase activity (PHLA) activity.

Assessment of antioxidant activity
Superoxide anions (O -2 ) were generated enzymatically [14] by xanthine (160 mM), xanthine oxidase (0.04 U) and nitroblue tetrazolium (320 μM) in the absence or presence of the fraction/extract (400 μg/ml) in 100 mM phosphate buffer (pH 8.2).The fractions were sonicated well in phosphate buffer before use.The reaction mixtures were incubated at 37 o C and after 30 min, the reaction was stopped by adding 0.

Assessment of antihyperglycaemic activity
Male albino rats of Charles Foster strain with mean body weight of 160 ± 20 g were selected for this study.The blood glucose level of each animal was checked with the help of glucometer (Boehringer Mannheim) after 16 h starvation.The animals showing blood glucose level between 3.33 and 4.44 mM (60 -80 mg/dl) were divided into eight groups of five animals each.The test group was administered a suspension of the extract orally (formulated in 1 % gum acacia) at a dose of 100 mg/kg while the control group received an equal amount of 1 % gum acacia (400 μl) only.A sucrose load (10 g/kg) was given to the animals in both groups orally 30 min later, to evaluate the decrease in the postprandiol rise in blood glucose [15].The blood glucose profile of the rats was determined at 30, 60, 90 and 120 min after extract/vehicle administration with a glucometer.Food, but not water, was withheld from the animals during the course of experimentation [15].

Statistical analysis
Data were analyzed using Student's t-test.Hyperlipidemia groups were compared with control and extract-treated groups.In the other experiments, the extract groups were compared with the reference group.Similarly, the generation of oxygen-free radicals with different solvent fractions was compared with that of the unfractionated extract.Quantitative glucose tolerance of each animal was calculated by trapezoid method using Prism software.(version 3, GraphPad software, Inc).P < 0.05 was used to determine significant difference.

RESULTS
The yield of the various fractions was as follows: petroleum ether (0.052 %), ether (0.115 %), chloroform (0.970 g, 0.092 %), chloroform:methanol ( indicating the presence of flavones/sterols/terpenes in free state or in the form of their glycosides.However, chloroform-methanol (4:1) and chloroformmethanol (3:2) fractions gave only positive Feigl, vanillin perchloric acid and NaOH test, indicating the likely presence of phenols in the free state or in the form of their glycosides.
Varying but significant decrease in lipid levels was noticed following treatment of hyperlipidemic rats with the extracts/fractions of A. violaceae flowers at an oral dose of 100 mg/kg.Ether and chloroform fractions decreased the levels of Tc, Tg, Pl, and protein by 24, 23, 23, 22 % and 24, 22, 23, 19 %, respectively, while petroleum ether, chloroform: methanol (4:1) and chloroform: methanol (3:2) fractions showed mild lipidlowering activity as compared to triton.On the other hand, the standard drug (gemfibrozil), at the same dose (100mg/kg) decreased the plasma levels of Tc, Tg, Pl, and protein by 36, 32, 34, and 27 %, respectively.Post-heparin lipolytic activity was partially activated in the plasma of the hyperlipidemic rats, producing a significant inhibition of 26 %.However, gemfibrozil caused a significant reversal of the lecithincholesterol acyltransferase (LCAT) activity and post heparin lipase activity (PHLA) (Table 1).
In the high-fat diet (HFD) model (Table 2), feeding the rats with high-fat diet once a day for 30 days produced hyperlipidemia as evidenced by increase in the plasma levels of Tc (+  3.There was significant inhibition of superoxide anions by (7 -32 %), hydroxyl radicals (15 -36 %) and microsomal lipid peroxidation (13 -35 %) in non enzymatic system compared to that of extracts.

DISCUSSION
Triton WR-1339 acts as a surfactant, suppresses the action of lipases and blocks the uptake of lipoproteins by extrahepatic tissues, thus resulting in increase in the levels of circulatory lipids [7].Furthermore, triton WR-1339 is known to cause structural modifications in circulatory lipoproteins, which hinder their interaction with capillary lipoprotein lipases.[21]It is probable that the extracts/fractions might have interfered with clustering lipoproteins coated with triton [21].In this way, lipoprotein may get freely catabolized by these enzymes.
The stimulation of plasma lecithin-cholesterol acyltransferase LCAT and hepatic lipases is the mechanism responsible for a significant lowering of β-lipoprotein lipid.Though the ether and chloroform extracts caused significant decrease in the plasma levels of lipids in triton as well as HFD models of hyperlipidemia, their effects were comparatively less than that of the standard drug, gemfibrozil.Hyperlipidemia may also induce other abnormalities such as oxidation of fatty acids, leading to the formation of ketonic bodies as well as liver and muscle resistance to insulin, which initiates the progression of diabetes in patients [7].The effect of these extracts/fractions on triglyceride lowering was also observed through reversal of postheparin treated animals.There is a significant correlation between the ability of tissue to incorporate free fatty acid by hydrolysis of lipoprotein triacylglycerol and the enzyme, lipoprotein lipase [22].The extracts/fractions inhibited cholesterol biosynthesis and potentiated the activity of lipolytic enzymes to early clearance of lipids from circulation in triton-induced hyperlipidemia.

CONCLUSION
Findings from this study suggest that the flowers of A. violacea, in particular, the ether and chloroform fractions, have good potentials for lipid management.However, further investigations to isolate and identify antihyperlipidemic and antioxidant principles in the plant as well as elucidate its mode of action are required.

Table 3 :
Effect of extracts/ fractions of A. violacea flowers on generation of free radical and lipid peroxidation in rat liver microsomes in vitro (% change in values of parameter is shown in parenthesis)  0.001, NS = not significant.Reference (standard drug) compared to the systems without drug treatment.

Table 4 :
Antihyperglycaemic activity (mean ± SEM) of the extracts/fractions of A. violacea flowers in sucrose-loaded rat model b 25 mg/kg dose.Note: AUC was based on fasting blood glucose (FB glucose).