a mechanism-based pharmacological evaluation of efficacy

J Basic Clin Physiol Pharmacol 2016; 27(2): 121–129

*Corresponding author: Dr. Anirban Pal, Central Institute of Medicinal and Aromatic Plants, Council of Scientific and Industrial Research (CSIR), Lucknow 226015, India, Phone: +91 52 227 186 44, Fax: +91 52 223 42666, E-mail: [email protected], [email protected] Vardan Singh: Central Institute of Medicinal and Aromatic Plants, Council of Scientific and Industrial Research (CSIR), Lucknow, India; and Central Drug Research Institute, Council of Scientific and Industrial Research, Lucknow, IndiaAtul Shrivastava, Upma Chaturvedi, Jitendra K. Saxena and Gitika Bhatia: Central Drug Research Institute, Council of Scientific and Industrial Research (CSIR), Lucknow, IndiaJyotshna, Subhash Chandra Singh and Karuna Shanker: Central Institute of Medicinal and Aromatic Plants, Council of Scientific and Industrial Research (CSIR), Lucknow, India

Shiv Vardan Singh, Atul Shrivastava, Jyotshna, Upma Chaturvedi, Subhash Chandra Singh, Karuna Shanker, Jitendra K. Saxena, Gitika Bhatia and Anirban Pal*

A mechanism-based pharmacological evaluation of efficacy of Flacourtia indica in management of dyslipidemia and oxidative stress in hyperlipidemic rats

DOI 10.1515/jbcpp-2015-0017Received February 19, 2015; accepted July 20, 2015; previously published online October 21, 2015

Abstract

Background: Flacourtia indica (Burm. f.) Merr. is a medici-nal plant indigenous to India and is broadly used world-wide for the treatment of a variety of health ailments. The present study was experimented on hyperlipidemic Charles Foster rats with the aim to explore the possible mechanism responsible for the antidyslipidemic activity of the hydromethanolic extract from F. indica leaves (FIL).Methods: Hyperlipidemia was induced by a single intra-peritoneal dose of Triton WR-1339 in Charles Foster rats. The plasma lipid levels were estimated in control and treated groups. The antioxidant potential of F. indica was assessed in both enzymatic and non-enzymatic systems. An acute toxicity study of high-performance liquid chro-matography (HPLC)-fingerprinted extract was carried out in Swiss albino mice.Results: The F. indica extract at a dose of 150 mg/kg signif-icantly lowers the plasma level of total cholesterol (17%), triglycerides (13%), and phospholipids (16%) by increas-ing post-heparin lipolytic activity (19%) and lecithin-cho-lesterol-acyltransferase activity (20%) in Triton-induced

hyperlipidemic rats. In addition, the F. indica extract showed significant in vitro antioxidant and anti-adipo-genic activity. HPLC analysis indicates the presence of fla-vanones and flavones in the extract, and the extract was found to be non-toxic up to a dose of 2000 mg/kg body weight in the acute oral toxicity study.Conclusions: These finding suggest that F. indica holds significant potential in preventing clinical deterioration induced by dyslipidemia along with oxidative stress.

Keywords: acute toxicity study; dyslipidemia; Flacourtia indica; lipid lowering; oxidative stress.

IntroductionCardiovascular diseases, including atherosclerosis, are the leading cause of death worldwide, with hyperlipidemia being one of the central risk factors implicated in the devel-opment of lesions and atherosclerosis progression [1]. Fur-thermore, disorders of lipid metabolism are also associated with overproduction of reactive oxygen species (ROS), thus enhancing oxidative stress [2]. Hydroxyl free radicals (OH●) are potentially involved in the initiation and progression of atherosclerosis in hyperlipidemic individuals via the per-oxidative damage of lipoproteins present in the blood [3]. In addition, enhanced oxidative stress in such individuals further increases the risk of cardiovascular diseases, and thus, the management of dyslipidemia along with oxidative stress might aid in reducing these cardiovascular events efficiently [4]. Modern pharmacological therapies and available lipid-lowering drugs, viz. fibrates, statins, and bile acid sequestrants, are effective but are associated with various side effects [5]. Moreover, some recent studies have also shown that long-term use of cholesterol biosynthesis inhibitors has an adverse effect on brain neurotransmis-sion [6]. Therefore, the search for effective bioactive sub-stances that could efficiently metabolize the lipids along with normalizing the oxidative stress is imperative.

mailto:[email protected], [email protected]

122 Singh et al.: Antidyslipidemic and antioxidant activity of F. indica

Natural products are the most promising source of effective bioactive substances in the treatment of various health ailments. Flacourtia indica (Burm. f.) Merr. (family Flacourtiaceae) is a small bushy tree native to India and possesses worldwide traditional medicinal values [7] in the treatment of various health disorders viz., jaundice, enlarged spleen, cholera, diabetes, and malaria [8]. In recent years, the phytochemical studies of F. indica led to the isolation of phenolic glycosides [9], butyrolactone lignan, sterols, poliothrysoside, coumarins, flavonoids, and condensed tannins [10].

Dyslipidemia is a common incidence generally seen in type II diabetes and contributes a major risk for car-diovascular diseases. Recently, ethanolic extract from the leaves of Flacourtia indica has been reported to possess significant antidiabetic potential [11]. Thus, the present study was designed to evaluate (a) the mechanism-based antidyslipidemic and antioxidant activities of the hydromethanolic extract of F. indica leaves (FIL), (b) acute oral toxicity of the bioactive extract, and (c) chemical fin-gerprinting analysis of the extract by reverse-phase HPLC.

Materials and methodsPreparation of plant extract

Fresh leaves of F. indica were collected in March 2013 from the Kukrail forest near Lucknow, India. The specimen was identified by Dr. S.C. Singh (taxonomist) and deposited at the institutional (CSIR-Central Institute of Medicinal and Aromatic Plants) herbarium with voucher no. 13689. Dry powdered leaves of F. indica (100 g) were extracted with 500 mL of hydromethanolic solvent (1:1), and the solvent was dried under reduced pressure at 1034.21 kPa and 40 °C. The process was repeated thrice for optimum recovery of the crude extract (19.5 g).

Phytochemical analysis of plant extract

Standard phytochemical tests were performed to determine the pres-ence of alkaloids, flavonoids, tannins, saponins, glycosides, terpe-noids, and steroids in the extract [12]. The free radical scavenging activity of the extract studied by using the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) method and obtaining the total phenolic content using the Folin-Ciocalteau reagent. The flavonoid content was estimated through the aluminum chloride method as reported earlier [12].

Drugs and chemicals

All the chemicals were procured from Sigma Chemical Company (St Louis, MO, USA), and the standard pellet diet was purchased from Lipton India Limited (Bangalore, India).

Animals

Adult male rats of Charles Foster strain (age 2–4 weeks old, weight 100–150 g) were bred and maintained in the animal house of the institute and used for the experiment after approval from the Institu-tional Animal Ethics Committee (IAEC/2010/149). The animals were kept in controlled conditions of temperature (25 °C–26 °C), relative humidity (60%–80%), and 12/12-h light/dark cycle (light from 8:00 a.m. to 8:00 p.m.) and provided with standard pellet diet and water ad libitum. After the end of experiments, the animals were sacrificed with an overdose of anesthetic ether.

Induction of hyperlipidemia

The animals were divided into six groups with six animals each: group 1, control animals; group 2, Triton-treated animals; group 3, Triton+FIL (50 mg/kg body weight); group 4, Triton+FIL (100 mg/kg body weight); group 5, Triton+FIL (150 mg/kg body weight); group 6, Triton+standard drug gemfibrozil (50 mg/kg body weight). Hyperlipidemia in rats was induced by an intraperitoneal injection of Triton WR-1339 at 400 mg/kg body weight, prepared in normal saline, which was administered to all the groups except the con-trol group [13]. Simultaneously, the F. indica extract and gemfibro-zil were prepared (macerated) with 0.2% w/w aqueous gum acacia and administered orally at their respective doses. The control and Triton group animals received equal volume of vehicle (gum acacia suspension). Pellet diet was withdrawn after dosing, and the rats were fasted for next 18 h, followed by anesthesia with sodium pen-tothal solution (50 mg/kg i.p.), prepared in normal saline. Blood was collected from the retro-orbital plexus using glass capillary in EDTA-coated tubes (3 mg/mL blood). The blood was centrifuged at 2500 g for 10 min at 4 °C to harvest the plasma for further biochemi-cal analysis.

Plasma lipids and lipoproteins

The levels of total cholesterol (TC), triglycerides (TG), phospho-lipids (PL), and high-density lipoproteins (HDLs) were estimated according to the methods reported earlier [14, 15]. Very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were also evaluated according to the following formulas: VLDL = TG/5 and LDL = TC−HDL−TG/5.

Plasma lipolytic enzymes

Lecithin-cholesterol acyltransferase (LCAT) activity and post-heparin lipolytic activity (PHLA) in plasma were measured according to meth-ods reported earlier [16].

Risk of atherogenicity

The risk for the development of atherosclerosis was expressed in terms of atherogenic index [(TC−HDL)/HDL] and the HDL/LDL ratio.

Singh et al.: Antidyslipidemic and antioxidant activity of F. indica 123

In vitro antioxidant activity

The free radical scavenging efficacy of the F. indica extract (50–150 μg/mL) was determined against the generation of superoxide anions (O2

−) and hydroxyl free radicals (OH●) in both enzymatic and non-enzymatic systems by a method reported earlier [17].

Cell viability assay

The MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide) assay was used to determine cell viability [18]. Briefly, the 3T3-L1 cells (1 × 104/well) suspended in Dulbecco’s modified Eagle medium (DMEM) containing 10% FBS were seeded into a 96-well culture plate and incubated for 24 h under 5% CO2 with or without FIL at 5-, 10-, 25-, and 50-μg/mL concentrations. The following day, 10 μL (5 mg/mL) of MTT was added and incubated for 4 h, and the media was replaced with 150 μL dimethyl sulfoxide (DMSO). The absorbance at 550 nm was measured using spectroscopic plate (ELISA) Reader (Synergy HT, SN. 253580, Biotech Instrument). All the samples were assayed in triplicate to minimize the error.

Anti-adipogenic assay

Two days post-confluency, 3T3-L1 cells were treated with the induc-tion media (10% calf serum/DMEM containing 1 μg/mL insulin, 1 μM dexamethasone, and 500 μM IBMX). After the induction of medium treatment (day 2), the cells were treated with insulin alone (10% calf serum/DMEM containing 1 μg/mL insulin). Complete differentiation was normally achieved after 8 days. To test the effect of FIL on the differentiation of 3T3-L1 preadipocytes to adipocytes, 10- to 50-μg/mL concentrations were used. For the assessment of adipogenesis, the differentiated cells were fixed in 4% w/v paraformaldehyde for 20 min, washed with 1 × phosphate-buffered saline (PBS), and stained with 0.34% Oil Red O in 60% isopropanol for 15 min. The cells were washed thrice with 1 × PBS, and the stain was extracted with 80% isopropanol by keeping it at room temperature for 30 min on an orbital shaker. The optical density (OD) of the extracted dye was read at 520 nm [19].

Acute oral toxicity

The acute oral toxicity of F. indica was carried out in Swiss albino mice to explore its safety profile in accordance with the OECD test guide-line no. 423 (1987). Mice were divided into three groups of six mice in each of both sexes (group 1, vehicle control; group 2, FIL at 1000 mg/kg body weight; group 3, FIL at 2000 mg/kg body weight). The extract was suspended in 0.7% carboxymethylcellulose (CMC in water) and orally administered in a single dose. In parallel, the control animals received only the vehicle (CMC). The animals were monitored every hour for any abnormal symptoms on the day of administration and checked for mortality thereafter until the end of the experiment (day 7). The animals were sacrificed on the seventh day after treatment, and blood and serum samples were collected from all the animals for hematological and biochemical investigations.

HPLC analysis and chromatographic conditions

The chemical fingerprint of the hydromethanolic extract of F. indica was developed by reverse-phase HPLC using a monolith column (Merck 150 × 4.6 mm i.d.) and (A) acidified water, 0.1% AcOH, and (B) acetonitrile-methanol, 50:50 v/v, as gradient elution. The flow rate of mobile composition was 1.0 mL/min, and the column temperature was maintained at 30 °C. The elution conditions were as follows: 0.01 min, 5% B; 10 min, 10% B; 15 min, 22% B; 30 min, 23% B; 40 min, 35% B; 45 min, 40% B; 50 min, 45% B 55 min, 50% B; 60 min, 60% B. The injection volume was 10 μL, and data acquisition was performed in the range of 200–400 nm to monitor the column eluent. The PDA detector was set at 280 nm for the quantitative analysis of the tar-geted compounds in the extract. A representative chromatogram (3D-HPLC fingerprint) of the extract is depicted in Figure 4.

Statistical analysis

All groups were compared by one-way analysis of variance (ANOVA), and the significance of the mean difference between different groups was done by Tukey’s post hoc test. A two-tailed (α = 2) probability, p < 0.05, was considered statistically significant. The number of inde-pendent determinations for in vivo experiments was n = 6 and for in vitro experiments was n = 3.

Results

Phytochemical analysis of plant extract

The results of the phytochemical analysis revealed the presence of the alkaloids, flavonoids, tannins, saponins, glycosides, terpenoids, and steroids in the active extract. RS50 for the DPPH radical scavenging was found to be 43.34±2.65 μg/mL, whereas the total phenolic content was found to be 12.20±1.2 mg gallic acid equivalent/g of dry plant extract. The total flavonoid content was quantified as 2.35±0.2 mg quercetin equivalent/g of dry plant extract.

Triton-induced hyperlipidemia

Plasma lipids and lipoproteins

Acute administration of Triton WR-1339 caused a marked increase in plasma levels of TC (3.1-fold), TG (3.4-fold), PL (2.9-fold), LDL (6.1-fold), and VLDL (3.4-fold) and a sig-nificant decrease in the plasma level of HDL (−42.4%). Treatment with F. indica hydromethanolic extract (FIL) of hyperlipidemic rats at 150 mg/kg body weight dose signifi-cantly lowered the plasma levels of TC (−17%), TG (−13%), PL (−16%), LDL (−22%), and VLDL (−13%) and increased the plasma HDL level (15%) (Figure 1).


Plasma lipolytic enzymes

The acute administration of Triton also caused the inhi-bition of PHLA (−28.1%) and LCAT (−47.3%) activities, whereas the treatment with FIL at 150 mg/kg body weight restored PHLA (19%) and LCAT (20%) activities, which was found comparable to standard drug gemfibrozil (20%) (Figure 2A).

Risk of atherogenicity

Triton-treated animals exhibited higher atherogenic index (5.7-fold) and lower HDL/LDL ratio (87%), which indicates a higher risk for development of atherosclerosis. Treat-ment with FIL at 150 mg/kg body weight significantly reduced the atherogenic index by 26% and increased the HDL/LDL ratio by 19% (Figure 2B).

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Figure 1: Effect of the F. indica extract on plasma lipids and lipoproteins.Data are presented as mean±SE of six animals. The Triton-treated group was compared with control; FIL 50 mg/kg, FIL 100 mg/kg, FIL 150 mg/kg, and gemfibrozil 50 mg/kg. ***p < 0.001, **p < 0.01, * p < 0.05, and p > 0.05 (ns).


Cell viability and adipogenesis

The results of the MTT assay showed that FIL treatment at concentrations between 5 and 50 μg/mL had no sig-nificant cytotoxic effect on 3T3-L1 preadipocytes. As FIL did not show any cytotoxic effect on the proliferation of preadipocytes, we then assessed the effect of FIL on adipo-cytes differentiation. FIL treatment significantly inhibited the differentiation of preadipocytes in a dose-dependent

manner, exhibiting 23.2% inhibition at 50-μg/mL concen-tration (Figure 2C).

In vitro antioxidant activity

The generation of superoxide anions (16% and 22%) and hydroxyl free radicals (14% and 17%) in enzymatic systems were significantly inhibited by FIL at 100- and

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Figure 2: Antidyslipidemic and antiatherogenic effects of F. indica extract; effect on lipolytic enzymes of plasma (A) and risk of atherogenicity (B).Data are presented as mean±SE of six animals. Triton-treated group was compared with control; FIL 50 mg/kg, FIL 100 mg/kg, FIL 150 mg/kg, and gemfibrozil were compared with Triton-treated animals. Effect of the F. indica extract on cell viability and adipogenesis (C). Data are presented as mean±SE of triplicate experiments (n = 3). All groups were compared with control. ***p < 0.001, **p < 0.01, *p < 0.05, and p > 0.05 (ns).


150-μg/mL concentrations, respectively. FIL also inhibited the non-enzymatic generation of superoxide anions (19%) and hydroxyl free radicals (21%) at 150 μg/mL concentra-tion (Figure 3).

Acute oral toxicity

No observational changes, morbidity, or mortality was observed throughout the experimental period up to the dose of 2000 mg/kg body weight. Blood (serum) samples upon analysis showed non-significant changes in all the parameters like total hemoglobin, red blood cell (RBC) count, white blood cell (WBC) count, serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALKP), creatinine, TGs, cholesterol, albumin, and serum protein (Table 1). No significant changes were found in the relative organ weight of the experimental animals.

HPLC analysis and chromatographic conditions

Fourteen major peaks representing phenolics, viz., 7.584, 15.601, 20.07, 23.511, 24.227, 25.073, 26.757, 30.767, 34.661, 49.713, 50.499, 51.526, 52.546, and 54.499 min, were observed in the hydromethanolic extract of F. indica, using monolithic-HPLC methodology. Each of the flavo-noid peaks was well resolved from the neighboring peaks, displaying excellent peak symmetry and separation effi-ciency (Figure 4).

DiscussionHyperlipidemia, along with oxidative stress, has been con-sidered as a more prominent causative factor for the devel-opment of cardiovascular diseases such as atherosclerosis,

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Figure 3: Effect of F. indica extract on in vitro generation of superoxide anions and hydroxyl free radicals in enzymatic and non-enzymatic systems.Data are presented as mean±SE of triplicate experiments (n = 3). All groups were compared with control. ***p < 0.001, **p < 0.01, *p < 0.05, and p > 0.05 (ns).


Table 1: Effect of FIL hydromethanolic extract on different hematological and biochemical parameters in Swiss albino mice.

Parameters Control 1000 mg/kg 2000 mg/kg

Hematological Body weight, g 36.03±2.89 31.09±2.98 30.19±2.12 Total WBC count, thousand cells/mm3 4.28±0.45 5.56±1.41 5.59±0.85 Total RBC count, million cells/mm3 8.17±1.13 7.69±0.74 7.32±.72 Hemoglobin, g/dL 15.18±0.34 13.58±0.37 15.68±0.87

Biochemical SGOT, U/L 32.40±3.84 32.66±2.50 32.50±1.53 SGPT, U/L 11.07±1.84 11.23±1.310 12.60±1.26 ALKP, U/L 310.23±19.87 311.92±46.86 314.04±11.7 Creatinine, mg/dL 0.84±0.04 0.91±0.08 1.03±0.12 Serum protein, mg/dL 1.02±0.08 0.98±0.05 1.03±0.07 Serum bilirubin, mg/dL 0.6±0.05 0.71±0.023 0.69±0.021 Serum TGs, mg/dL 101.01±21.40 118.17±17.29 129.56±20.65 Total serum cholesterol, mg/dL 110.90±5.50 106.44±7.13 123.36±14.20

Data are the mean percentage±SD of six mice (both sexes) in each group. SGOT, Serum glutamic oxaloacetic transaminase.

acute myocardial infarction, hypertension, and coronary heart diseases [20]. In the present study, hydromethanolic extract from the leaves of F. indica was investigated for its antihyperlipidemic activity in Triton-induced hyper-lipidemic rats. Triton WR-1339 (tyloxapol) is non-ionic sur-factant being widely used to investigate the possible mode of action of lipid-lowering drugs/molecules [13]. Triton also inhibits the lipases activity and obstructs the uptake of lipoproteins from circulation by extra-hepatic tissues, which results into higher level of circulatory lipids [21]. The F. indica extract treatment significantly reduced the lipid level in hyperlipidemic animals at a dose of 150 mg/kg body weight. In fasting condition, the only source of serum lipids is endogenous production; thus, the reduction in plasma lipids level by the F. indica extract clearly indicates that it has an effect on endogenous lipid metabolism.

Furthermore, to explore the possible mode of action of the F. indica extract, LCAT activity and PHLA were analyzed in experimental animals. The LCAT and PHLA activities were found to be reduced in Triton-induced hyperlipidemic animals, whereas they increased in the F. indica-treated groups. As LCAT converts cholesterol into a cholesteryl ester (a more hydrophobic form of cholesterol), which is then sequestered into the core of a lipoprotein particle (HDL) [17], a reduced cholesterol and enhanced HDL level was found in F. indica-treated animals. Administration of heparin induces the release of various lipolytic lipases (viz. TG lipase and lipoprotein lipase) located on the surface of endothelial cells, which causes the breakdown of lipopro-teins and lipids [22]. In our study, the enhanced activity of PHLA in F. indica-treated animals may be responsible for

the reduction in the plasma levels of lipids (TG and PL) and lipoproteins (LDL and VLDL). The HDL/LDL ratio and the atherogenic index are the two common indicators that reflect the risk of cardiovascular diseases in an individual. In Triton-treated hyperlipidemic rats, the HDL/LDL ratio was found to be much lower than normal, whereas the F. indica extract enhanced the HDL/LDL ratio by reducing the LDL level and increasing the HDL level in treated rats. The atherogenic index is considered as a better indicator of cardiovascular disease risk than individual lipoprotein concentrations [23]. In our experiments, Triton increased the atherogenic index in hyperlipidemic rats. F. indica, meanwhile, significantly lowered the atherogenic index and increased the HDL/LDL ratio, indicating that F. indica significantly reduces the risk of cardiovascular diseases including atherosclerosis.

Besides in vivo lipid-lowering activity, F. indica also possesses potential antioxidant activities. The DPPH free (stable) radical scavenging activity results revealed that the F. indica extract has significant radical scavenging effi-cacy. As DPPH radical scavenging activity is widely used as a marker to evaluate the antioxidant potential of the plant extracts/molecules, we further explored its antioxidant potential in enzymatic and non-enzymatic systems. The F. indica extract commendably inhibits the generation of O2

− and OH● in a concentration-dependent manner in both enzymatic and non-enzymatic systems (in vitro). Inhibi-tion of the enzymatic system suggests that the F. indica extract has inhibitory effect on the enzymes/agents responsible for the endogenous production of O2

− and OH● radicals, whereas the results of the non-enzymatic system


proved that it also has the efficacy to remove the radicals from the circulation. These results collectively indicate that F. indica may reduce oxidative stress by inhibiting endogenous generation and neutralization of preformed free radicals. Moreover, F. indica treatment significantly increases plasma levels of HDL, which is itself a powerful antioxidant on its own [24]. Furthermore, the effect of the F. indica extract on adipogenesis of 3T3-L1 preadipocytes was also studied because many known lipid-lowering drugs like niacin targets the adipogenesis and inhibits lipid accumulation [25]. The F. indica extract significantly inhibited lipid accumulation in preadipocytes, without causing any effect on the viability of cells. This indicates that F. indica reduces adipogenesis without causing apop-tosis, so there might be a possibility of another mechanism behind its anti-adipogenic activity. In acute oral toxicity, the F. indica extract was found to be well tolerable and did not create any sign of toxicity and is thus considered safe up to a dose of 2000 mg/kg body weight.

Reverse-phase chromatography has been extensively employed for the separation of flavonoids on C8 or C18 columns but rarely on monolithic columns with polar

mobile phases, such as methanol, acetonitrile, tetrahydro-furan, or acetic acid solutions. The chromophoric nature of flavonoids makes them unique to identify based on their UV spectra [26]. The classes of flavonoids that characterize FIL hydromethanolic extract (flavanones, flavones, and, to a lesser extent, flavonols/flavanols) have their maximum absorption at specific wavelength ranges: flavanones (280–290 nm), flavones (304–350 nm), and flavonols (352–385 nm). A representative 3D-PDA HPLC chromatogram (200–400 nm) of F. indica extract (Figure 4) highlights the presence of flavanones and flavones with their characteris-tic UV spectrum plot index. Presence of substantial amount of flavonoids in the active extract may be responsible for the observed antidyslipidemic and antioxidant activities.

ConclusionsThe results of the present study clearly indicate that FIL have significant potential to lower plasma lipids level in hyperlipidemic conditions and also possess potential antioxidant activity. Thus, F. indica may be used as a good

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Figure 4: 3D-HPLC chromatogram of FIL hydromethanolic extract (100 mg/mL).The upper plot comprises the characteristic UV-VIS spectrum of major peaks corresponds to the flavonoidal-glycosides group of compounds present in the extract.


herbal candidate for the treatment of cardiovascular dis-eases and related complications. This preliminary work will be also helpful in the further characterization of active extract to obtain effective phytomolecules (responsible for the observed activity) with their possible mode of action.

Acknowledgments: The authors are grateful to the direc-tor of CSIR-CDRI and CSIR-CIMAP for providing the neces-sary research facilities to carry out this work. S.V.S. is also thankful to the Indian Council of Medical Research (ICMR), India, for the award of Senior Research Fellowship.Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.Research funding: None declared.Employment or leadership: None declared.Honorarium: None declared.Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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a mechanism-based pharmacological evaluation of efficacy

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