Antihyperlipidemic Potential of Polyphenol and Glycoside Rich Nerium oleander Flower against TritonWR-1339-Induced Hyperlipidemia in Experimental Sprague Dawley Rats

Nerium oleander Linn. (NO), an evergreen cardiac glycoside-rich shrub is used as folkloremedicine in China to treatmany diseases. It exhibits a wide spectrum of bioactivities but there were not much scienti�c reports on the bioactivity of N. oleander �owers. In the present study, we have evaluated the toxicity pro�le of the 50% hydroethanolic extracts of Nerium oleander �owers (ENO) using in vitro brine shrimp lethality assay and MTT cytotoxicity assay and in vivo acute toxicity test as per the OECD guidelines. e antihyperlipidemic activity of the ENOwas also studied using TritonWR-1339-induced hyperlipemic rats and compared with standard Atorvastatin. In vitro brine shrimp, MTT cytotoxic assay, and in vivo acute toxicity assays showed a wide safety margin which has been evidenced through its lethal concentration (LC50: 795.46 μμg/mL) and growth inhibition (GI50: 993.60 μμg/ml) values. Plasma lipids and lipoproteins were signi�cantly elevated by the intraperitoneal in�ection of TritonWR 1339 in hyperlipidemic rats at 6th and 24th hour. ENO pretreatment showed a signi�cant ameliorative action on elevated lipids and lipoproteins in a dosedependent manner when compared to standard. Altogether, the results prove that Nerium oleander �owers are not toxic at the tested doses and exhibit antilipimic activity.


Introduction
World Health Organization (WHO), American Heart Associations, and epidemiologists around the globe have been pronouncing an alarm on the rapid increase in the burden of heart diseases for the past 20 years [1].Experimental and epidemiological studies have shown that the plasma hyperlipidemic state could contribute to the development of atherosclerosis and cardiovascular-system-related diseases.Cardiovascular diseases (CVDs) are the most common cause of morbidity and mortality in many of the developing countries [2][3][4].A logical strategy, to prevent or to treat atherosclerosis and reduce the incidence of cardiovascular disease events, is to target hyperlipidemia either by drugs or dietary intervention [3,5].Based on this criterion, a number of plants have received attention and have been shown to lower plasma lipid levels [6].Earlier studies also reveal that the consumption of polyphenols and glycosiderich compounds/plants will reduce the risk of hyperlipidemia [7][8][9][10].
Nerium oleander Linn is one such polyphenol-and glycoside-rich evergreen shrub belonging to the Apocynaceae, an important Chinese folk medicine [11].In early times, it is assumed that all parts of the oleander plant are highly toxic to humans, animals, and certain insects due to the presence of cardiac glycosides mainly nerine and oleandrin at higher concentration [12,13].Nowadays, apart from its toxicity, an assortment of pharmacological activities such as antinociceptive, anti-in�ammatory, antibacterial, anticancer, CNS depressant activity, antidiabetic, rodenticides, piscicidal, pesticides, insecticides, abortifacients, among others, have been screened and reported in leaves, stems, and roots of Nerium oleander except �owers [14][15][16][17][18]. Apart cardiac glycosides, �owers of Nerium oleander contains triterpinoids, ursolic acid, steroids, polysaccharides, among others, reported to have various biological activities [14,19,20].Moreover, our earlier �nding con�rms the presence of phytochemical constituents in N. oleander �ower extract (ENO) [21] as well as the cardioprotective and antioxidant potential of ENO against isoproterenol-induced myocardial oxidative damage in experimental rats [22].ese �ndings prompted us to screen this investigation.Hence, in the present investigation, we have studied the toxicological pro�le and antihyperlipidemic potential of hydroethanolic extracts of Nerium oleander (ENO) �owers in experimental animals.4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) and DMEM were obtained from Himedia, Mumbai.Atorvastatin standard was obtained from a registered pharmacy, Sri Ramachandra Pharmacy, Chennai, India.Solvents and all other chemicals used were of analytical grade.

Plant
Material.About 20 kg of fresh �owers of N. oleander (Apocynaceae) was obtained commercially in Chennai, Tamilnadu, and India.It was identi�ed and authenticated by a quali�ed plant taxonomist Professor V. Jayaraman, Director, Plant Anatomy Research Centre, Tambaram, Tamilnadu, India.e �ower was shade air dried and coarsely powdered.About 100 gm of the powder was packed in a Soxhlet extractor with 500 mL of 50% ethanol and extraction was carried out for 18-20 h.e hydroethanolic extract (ENO) of Nerium oleander �ower was concentrated to dryness under reduced pressure and controlled temperatures (40-50 ∘ C) in a rotary �ash evaporator.e powdered extract was stored in an air tight container and used for further studies.In Vitro Brine Shrimp Lethality Assay.Brine shrimp lethality bioassay was carried out according to the principle and protocol previously described by Nkengfack et al., [23].In the experiment, 0.5 mL of the various concentration of ENO (1.5-1000 g/0.5 mL) in triplicates was added to 4.5 mL of brine solution and maintained at room temperature for 24 h under the light and the surviving larvae were counted.Experiments were conducted along with control (vehicle treated).e percentage lethality was determined by comparing the mean surviving larvae of the test and control tubes.LC 50 (concentration at 50% lethal effect) values were determined from the 24 hours count using the best-�t line probit analysis method described by Finney [24].
In Vitro Cytotoxicity Study: MTT Assay.e MTT assay was performed to study the antiproliferative effect of ENO using the earlier protocol [25,26].Lung adenocarcinoma cells (A549 Cells) were treated with different concentrations of ENO (100-900 g/mL) for 24 h.At the end of the treatment, media from control and drug-treated cells were discarded.20 L of MTT containing DMEM (0.5 mg/mL) was added to each well.Cells were then incubated at 37 ∘ C for 4 h in a CO 2 incubator.Spectrophotometrical absorbance of the purple blue formazan dye was measured using an ELISA reader (BIORAD) at 570 nm.e average values were determined from the triplicate readings, and the average value was subtracted from the blank.e MTT reagent yields low background absorbance values in the absence of cells.Optical density of each sample was compared with control optical density and graphs were plotted.e plot of the data obtained in absorbance against number of cells provides a curve with a linear portion.us, both stimulation and inhibition of cell proliferation was measured.GI 50 (50% growth inhibition) values were determined from the maximum percentage of inhibition: In Vivo Acute Oral Toxicity of ENO.e acute toxicity studies were performed in female Sprague Dawley rats as per the OECD guidelines-423.Acute toxicity was performed for three dose of ENO (50, 300, and 2000 mg/kg b.wt).e drug was suspended in 0.5% w/v sodium carboxyl methyl cellulose (CMC) and was prepared freshly each time.Aer the initial weights (150-200 g) were taken, the animals were distributed randomly into four groups ( = /group) for conducting the study.Group I animals receiving the vehicle (0.5% CMC) served as controls and Group II, III, and IV receiving ENO at 50, 300, and 2000 mg/kg b.wt, respectively.Aer a 12 h fast, the drugs were administered by intragastric route to all the animals.
Animals were observed individually aer drug dosing for signs and symptoms such as ataxia, tremors, sleep, coma, among others, at 0, 0.5, 1, 2, 4, 6 hr (with special attention) and then once a day for the next 14 days.Observations also included changes in respiratory, circulatory, autonomic and central nervous systems, as well as somatomotor activity (clinical �ndings such as ataxia, tremors, convulsion, hair loss, salivation, diarrhoea, lethargy, lacrimation, sleep, coma, etc.) and behaviour pattern.Aer drug administration, followed by measurement of body weight once in a week, any mortality and behavioral screenings were recorded for 24 h for the next 14 days.e results are expressed as mean ± SEM of three animals.

Antihyperlipidemia
Triton WR-1399-Induced Hyperlipidemia.Hyperlipidemia was induced in experimental rats using Triton WR 1339 by following the earlier method [27][28][29].Triton (Superinone, Triton WR-1339), is a polymer of p-isooctylpolyoxyethylenephenol and formaldehyde, a nonionic surfactant.Aqueous solution of Triton WR 1399 was made in normal saline and injected at a dose of 250 mg/kg., i.p. e test drug ENO doses 10, 30, and 100 mg/kg were selected as per the results obtained in our earlier studies [21,22].Atorvastatin (ATV) tablet was used as a standard at a dose of 30 mg/kg.All the drugs were suspended in 0.5% w/v sodium carboxyl methyl cellulose (CMC) and were prepared freshly each time.e drugs were administered by intragastric route.
Experimental Design.Aer the acclimatization period, animals were divided into nine groups of six rats each: Group I: Normal, Group II: Triton induction, Group III: Triton induction plus ATV (30 mg/kg/day, for 2 weeks) pretreated group, Group IV, V, VI: Triton induction plus ENO (10 mg/kg/day, for 2 weeks) pretreated group, ENO (30 mg/kg/day, for 2 weeks) pretreated group, ENO (100 mg/kg/day, for 2 weeks) pretreated group respectively, Group VII: ENO (100 mg/kg/day, for 2 weeks) alone treated group.Aer pretreatment with ENO and ATV for a period of 2 weeks, the animals were kept in wire-�oored cages throughout the day to limit coprophagia.ey were starved for 24 hours before the experiment but allowed free access to water [30].Blood samples were collected from overnight fasted rats aer 1 hr aer the last test drug treatment to respective groups for 0th day.
Aer 1 hr of initial blood collection (0 hr) from respective groups, they were intraperitoneally injected with Triton WR 1339 (250 mg/kg) except Group I and VII animals.e body weight of the rats was individually recorded weekly once.Feed and water were changed every day.At the end of 6, 24, and 48th hr aer injection of Triton WR-1339, all the rats were anesthetized for the blood collection.Blood was collected through sino-orbital puncture using EDTA as anticoagulant and the plasma separated was stored at −80 ∘ C and used for the lipid and lipoprotein estimation.(2)

Statistical Analysis.
All data are reported as mean ± standard error mean (SEM).Statistical analysis was done using Graph pad prism 4.0 for windows package.One-way analysis of variance (ANOVA) was performed followed by post-hoc Tukey's test.e  0.0 and   0.01 values were set to access signi�cant protection in treatment groups.

Toxicological �ro�le. Figure 1 illustrated the in vitro
toxicity pro�le of the ethanolic extract of N. oleander �ower which was performed using Artemia nauplii (brine shrimp).Based on the ability of ENO to kill the laboratory-cultured brine shrimp, the toxic nature of ENO was identi�ed.Maximum mortalities were obtained at a concentration of 1000 g/mL whereas; low mortalities were seen at 30 g/mL concentration.LD 0 values were obtained from the best-�t line by plotting concentration verses percentage lethality in probit analysis.
Figure 2 gives an antiproliferative effect of ENO at various doses (10-900 g/mL) against A549 cells (lung adenocarcinoma cell line) for 24 h using MTT assay.ENO exhibits a dose-dependant inhibitory effect on the growth of lung carcinoma cells and the growth inhibition of the cells ( 2 = 0.98) is directly proportional to the dose.ENO exhibits greater cytotoxic effect against A549 cell at 900 g/mL concentration with a GI 0 value of 993.60 g/mL.
e in vivo toxicity pro�le of ENO was monitored aer single oral administration of ENO to female rats at three doses 50, 300, and 2000 mg/kg b.wt, ( = /dose) for a period of 14 days.ENO at all doses did not show any remarkable change in the body weights of the rats (data not shown).ere were no treatment-related mortality and clinical �ndings such as ataxia, tremors, convulsion, hair loss, salivation, diarrhoea, lethargy, lacrimation, sleep, coma, among others (data not shown).All experimental rats were found to be normal throughout the study.e LD 0 of the ENO was found herein to be greater than 2000 mg/kg b.wt when administered once orally via gastric intubation in 12 h fasted female Sprague Dawley rats.F 1: Brine shrimp lethality bioassay-probit analysis of ENO.Different concentrations of ENO (1-1000 g/mL) was tested against brine shrimp for 24 h.

Antihyperlipidemic Potential of ENO.
In the current study, the antihyperlipidemic potential of ENO was also tested using male SD rats.Body weight of the rats was found to be insigni�cant in all the experimental groups (data not shown).e level of total cholesterol, triglycerides, HDL, LDL and VLDL at 0th, 6th, 24th, and 48th h of the triton induced and ENO treated animals were examined in plasma and all the results are shown in Tables 1 and 2, respectively.e levels of total cholesterol, triglycerides, LDL, and VLDL index were found to be normal at 0th hour of all experimental animals before triton induction (Data not shown).However, they were found to be signi�cantly (  0.01) high at 6th h aer triton induction in the triton-induced untreated animals with concomitant decrease in HDL when compared to normal control animals.Similarly, at 24th h, the levels of total cholesterol, triglycerides, LDL, and VLDL were found to be at peak with decrease in HDL whereas at 48th h they were found to be nearer to normal in triton-induced untreated animals.Rats pretreated with ENO (10, 30, and 100 mg/kg) for 14 days signi�cantly prevented the changes in lipid and lipoprotein levels in the plasma aer triton induction compared to triton-induced untreated animals.e reduction in the total cholesterol, TGs, LDL, and VLDL levels by ENO was found to be in a dose-dependent manner and they were found to be comparable with Atorvastatin.Alternatively, the levels of lipid and lipoproteins in ENO (100 mg/kg) alone treated rats were found to be insigni�cant when compared to normal rats.

Discussion
Herbal drugs have been the integral part, in one form or another, of several indigenous therapeutic systems including traditional medicine and Indian System of Alternative Medicine [32].Many numbers of medicinal plants and their active constituents play a role in the prevention and treatment of metabolic disorders like coronary heart disease and diabetes [33][34][35].In traditional medicine, various parts of N. oleander have been used as a cardiotonic agent [36], rodenticides, piscicidal, pesticides, insecticides, and also used as remedies for indigestion, fever, ringworm, malaria, leprosy, venereal diseases, and as abortifacients [14].In early times, all parts of the oleander are believed as poisonous to living things but off-late a number of pharmacological activities have been studied and reported by researchers [15].Any compound to be considered as a drug toxicity studies   is imperative.Hence, assessment of the toxicity pro�le of N. oleander �ower was considered a necessity.
In the present study, both in-vitro and in-vivo toxicity studies were done to identify the LD 50 value of ENO and at which dose it is found to be nontoxic.Brine shrimp lethality bioassay which is considered as a useful tool for the assessment of toxicity of herbal extracts [37], in the present investigation the degree of lethality was found to be directly proportional to the concentration of the extract.e LD 50 was found to be 795.46g/mL which reveals it is reduced toxicity against brine shrimps.Similarly, ENO exhibits a dose-dependant inhibitory effect against the growth of lung carcinoma cells A549 cells.e MTT cell proliferation assay measures the cell proliferation rate and conversely, the reduction in cell viability.e in vitro results concluded that the drug at lower concentrations is found to be safer but at higher concentrations it exhibits cytotoxic effect.is cytotoxic and antiproliferative effect of ENO at higher concentrations might be due to the presence of cardiac glycosides which further con�rms its anticancer activity [38].In a similar manner, ENO-treated animals were found to be normal throughout the study in in vivo acute toxicity study and its LD 50 was found to be >2000 mg/kg b.wt.ese results summarizes that ENO possess a wide safety margin which has been evidenced through its LD 50 and GI 50 values.
ough there was a large class of hypolipidemic drugs available worldwide for the treatment, none of them is fully effective, absolutely safe, and free from side effects [39].Hence, efforts are being made to �nd out safe and effective agents that may be bene�cial in correcting the lipid metabolism and preventing cardiovascular diseases.Earlier, we have studied the cardioprotective and antioxidant potential ENO against isoproterenol-induced myocardial oxidative stress, ENO exhibits better free radical scavenging potential and cholesterol reducing activity [22].Further, to validate the antihyperlipidemic property of ENO, triton WR-1339, a nonionic surfactant, was used in this study to induce hyperlipidemia in experimental animals and compared with standard atorvastatin, a potent lipid-lowering agent which inhibits an early step in the biosynthesis of cholesterol, thereby reducing the plasma cholesterol concentration.eir higher level may decrease the levels of triglycerides along with the cholesterol [40].
Stanley [41] suggested that intravenous or intraperitoneal injection of Triton WR 1339 increases hepatic cholesterol synthesis by increasing HMG CoA reductase activity, the �rst committed enzyme of the HMG-CoA reductase pathway in rodents within 24 hours which resulted in the increased plasma cholesterol and TG concentrations for upto 36 h.Our results are in corroboration with that of previous reports [28,42].Triton causes structural modi�cations in the circulatory lipoproteins and suppress the action of lipases especially lipoprotein lipase activity and as a consequence block the uptake of circulating lipids by extra hepatic tissues, and, in turn, resulting in increased blood lipid concentration [29].Signi�cant increase in the level of cholesterol in the tritoninduced animals might also be due to the increased activity of HMG CoA reductase.
Triton induction was also reported to interact preferentially with HDL, changing the size and density of lipoproteins which used as substrates for the enzyme LCAT, enzyme activity decreased in parallel to the displacement of apo A-1 [43].In the present study, reduction in HDL-C in tritoninduced animals is agreeable with the earlier statement.In our study, the changes in plasma lipid and lipoprotein levels induced by triton WR-1339 can be resisted by ENO (10 and 30 mg/kg) and atorvastatin (30 mg/kg) pretreatment during triton induction.Substantially, ENO treatment boosted the HDL levels at doses of 10, 30, and 100 mg/kg which might be through the inhibition of the activity of HMG CoA reductase and the stimulation of LCAT activity.Similarly, ENO results were found to be comparable with atorvastatin.Atorvastatin is a competitive inhibitor of HMG-CoA reductase, our results corroborate with this statement.
Moreover, ENO was found to be rich in glycosides and polyphenols as evidenced by its concentration and characterization in our earlier investigations [21,22].Glycosides and polyphenolic compounds are reported to inhibit HMG CoA reductase activity [44][45][46].Likewise, �avonoids may decrease the risk of cardiovascular disease by increasing the two ratios HDL/TG, HDL/LDL which may hasten removal of cholesterol from peripheral tissues to liver for catabolism and excretion [7,9].

Conclusion
In conclusion, the results of the present study demonstrated that the ethanolic extract of N. oleander �ower (ENO) was found to be safer at lower concentrations.e antihyperlipidemic activity of ENO is strengthened by its phytoconstituents and can be considered as a potent lipid-lowering and antioxidant agent.ese bene�cial activities may contribute to its cardio protective and antiatherosclerotic property.

T 2 :
Effect of ENO and atorvastatin on lipoproteins in plasma of triton administered rats.Groups HDL-C (mg/dL) LDL-C (mg/dL) (0.01, triton-treated rats versus triton + Atorvastatin and triton + ENO-treated rats- Drugs and Chemicals.A549 cells were procured from Central Institute for Brackish water aquaculture with passage number 10 as a gi sample.Triton WR 1399 was obtained from Sigma Chemical Co., St. Louis, MO, USA.MTT(3-( Toxicological Pro�le.e toxicological pro�le of the hydroethanolic extracts of Nerium oleander (ENO) �owers using in vitro and in vivo methods was studied are as follows.
T 1: Effect of ENO and atorvastatin on total cholesterol and triglycerides in plasma of triton administered rats.