Cardiovascular diseases, including atherosclerosis, are the leading cause of death worldwide, with hyperlipidemia being one of the central risk factors implicated in the development of lesions and atherosclerosis progression . Furthermore, disorders of lipid metabolism are also associated with overproduction of reactive oxygen species (ROS), thus enhancing oxidative stress . Hydroxyl free radicals (OH●) are potentially involved in the initiation and progression of atherosclerosis in hyperlipidemic individuals via the peroxidative damage of lipoproteins present in the blood . 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 . Modern pharmacological therapies and available lipid-lowering drugs, viz. fibrates, statins, and bile acid sequestrants, are effective but are associated with various side effects . Moreover, some recent studies have also shown that long-term use of cholesterol biosynthesis inhibitors has an adverse effect on brain neurotransmission . Therefore, the search for effective bioactive substances that could efficiently metabolize the lipids along with normalizing the oxidative stress is imperative.
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  in the treatment of various health disorders viz., jaundice, enlarged spleen, cholera, diabetes, and malaria . In recent years, the phytochemical studies of F. indica led to the isolation of phenolic glycosides , butyrolactone lignan, sterols, poliothrysoside, coumarins, flavonoids, and condensed tannins .
Dyslipidemia is a common incidence generally seen in type II diabetes and contributes a major risk for cardiovascular diseases. Recently, ethanolic extract from the leaves of Flacourtia indica has been reported to possess significant antidiabetic potential . 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 fingerprinting analysis of the extract by reverse-phase HPLC.
Materials and methods
Preparation 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 presence of alkaloids, flavonoids, tannins, saponins, glycosides, terpenoids, and steroids in the extract . 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 .
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).
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 Institutional 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 control group . Simultaneously, the F. indica extract and gemfibrozil 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 pentothal 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 biochemical analysis.
Plasma lipids and lipoproteins
The levels of total cholesterol (TC), triglycerides (TG), phospholipids (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 methods reported earlier .
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.
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 .
Cell viability assay
The MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine cell viability . 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.
Two days post-confluency, 3T3-L1 cells were treated with the induction 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 .
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 guideline 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 targeted compounds in the extract. A representative chromatogram (3D-HPLC fingerprint) of the extract is depicted in Figure 4.
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 independent determinations for in vivo experiments was n=6 and for in vitro experiments was n=3.
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.
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 significant 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 significantly 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 inhibition 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. Treatment 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).
Cell viability and adipogenesis
The results of the MTT assay showed that FIL treatment at concentrations between 5 and 50 μg/mL had no significant 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 adipocytes differentiation. FIL treatment significantly inhibited the differentiation of preadipocytes in a dose-dependent manner, exhibiting 23.2% inhibition at 50-μg/mL concentration (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 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 concentration (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 flavonoid peaks was well resolved from the neighboring peaks, displaying excellent peak symmetry and separation efficiency (Figure 4).
Hyperlipidemia, along with oxidative stress, has been considered as a more prominent causative factor for the development of cardiovascular diseases such as atherosclerosis, acute myocardial infarction, hypertension, and coronary heart diseases . In the present study, hydromethanolic extract from the leaves of F. indica was investigated for its antihyperlipidemic activity in Triton-induced hyperlipidemic rats. Triton WR-1339 (tyloxapol) is non-ionic surfactant being widely used to investigate the possible mode of action of lipid-lowering drugs/molecules . 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 . 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) , 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 lipoproteins and lipids . 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 . 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 efficacy. 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). Inhibition 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 . 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 . 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 apoptosis, 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, tetrahydrofuran, or acetic acid solutions. The chromophoric nature of flavonoids makes them unique to identify based on their UV spectra . 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 characteristic UV spectrum plot index. Presence of substantial amount of flavonoids in the active extract may be responsible for the observed antidyslipidemic and antioxidant activities.
The 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 herbal candidate for the treatment of cardiovascular diseases 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.
The authors are grateful to the director of CSIR-CDRI and CSIR-CIMAP for providing the necessary 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.
Celermajer DS, Chow CK, Marijon E, Anstey NM, Woo KS. Cardiovascular disease in the developing world: prevalences, patterns, and the potential of early disease detection. J Am Coll Cardiol 2012;60:1207–16.CrossrefGoogle Scholar
Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 2004;114:1752–61.Google Scholar
Grundy SM, Cleeman JI, Merz CN, Brewer HB Jr, Clark LT, Hunninghake DB, et al. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines. Circulation 2004;110:227–39.Google Scholar
Saleem S, Haider S, Naqvi F, Tabassum S, Haleem DJ. Long term administration of HMG-CoA-reductase inhibitor (simvastatin) affects brain serotonin neurotransmission in male rats. J Basic Appl Sci 2011;7:79–83.CrossrefGoogle Scholar
The wealth of India, raw materials. New Delhi, India: Council of Scientific and Industrial Research, 1956.Google Scholar
Sashidhara KV, Singh SP, Singh SV, Srivastava RK, Srivastava K, Saxena JK, et al. Isolation and identification of β-hematin inhibitors from Flacourtia indica as promising antiplasmodial agents. Eur J Med Chem 2013;60:497–502.Web of ScienceGoogle Scholar
Madan S, Pannakal ST, Ganapaty S, Singh GN, Kumar Y. Phenolic glucosides from Flacourtia indica. Nat Prod Commun 2009;4:381–4.Google Scholar
Satyanarayana V, Krupadanam GL, Srimannarayana GA. A butyrolactone lignin disaccharide from Flacourtia ramontchi. Phytochemistry 1991;130:1026–9.Google Scholar
Singh V, Singh M, Shukla S, Singh S, Mansoori MH, Kori ML. Antidiabetic effect of Flacourtia indica Merr in streptozotocin induced diabetic rats. Global J Pharmacol 2011;5:147–52.Google Scholar
Soni A, Sosa S. Phytochemical analysis and free radical scavenging potential of herbal and medicinal plant extracts. J Pharmacog Phytochem 2013;2:22–9.Google Scholar
Kuroda M, Tanzawa K, Tsujita Y, Endo A. Mechanism for elevation of hepatic cholesterol synthesis and serum cholesterol levels in Triton WR-1339 induced hyperlipidemia. Biochem Biophys Acta 1977;489:119–25.Google Scholar
Parekh AC, Jung DH. Cholesterol estimation with ferric acetate-uranium acetate and sulfuric acid, ferrous sulfate reagents. Anal Chem 1970;42:1423–7.Google Scholar
Rice LB. Determination of triglycerides (enzymatic method). Clin Chem 1970;31:746–50.Google Scholar
Mays PA, Felts JM. The functional status of lipoprotein lipase in rat liver. Biochem J 1968;108:483–7.Google Scholar
Shrivastava A, Chaturvedi U, Singh SV, Saxena JK, Bhatia G. Lipid lowering and antioxidant effect of miglitol in triton treated hyperlipidemic and high fat diet induced obese rats. Lipids 2013;48:597–607.CrossrefWeb of ScienceGoogle Scholar
Swarnkar G, Sharan K, Siddiqui JA, Chakravarti B, Rawat P, Kumar M, et al. A novel flavonoid isolated from the stem-bark of Ulmus wallichiana Planchon stimulates osteoblast function and inhibits osteoclast and adipocyte differentiation. Eur J Pharmacol 2011;658:65–73.Web of ScienceGoogle Scholar
Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801–9.Google Scholar
Schotz MC, Seanu A, Page IH. Effect of triton on lipoprotein lipase of rat plasma. Am J Physiol 1957;188:399–402.Google Scholar
Applebaum DM, Goldberg AP, Pykalisto OJ, Brunzell JD, Hazzard WR. Effect of estrogen on post-heparin lipolytic activity. Selective decline in hepatic triglyceride lipase. J Clin Invest 1977;59:601–8.Google Scholar
Nwagha UI, Ikekpeazu EJ, Ejezie FE, Neboh EE, Maduka IC. Atherogenic index of plasma as useful predictor of cardiovascular risk among postmenopausal women in Enugu, Nigeria. Afr Health Sci 2010;10:248–52.Google Scholar
Tomas M, Latorre G, Senti M, Marrugat J. The antioxidant function of high density lipoproteins: a new paradigm in atherosclerosis. Res Esp cardiol 2004;57:557–69.Google Scholar
Fujimori K, Amano F. Niacin promotes adipogenesis by reducing production of anti-adipogenic PGF2α through suppression of C/EBPβ-activated COX-2 expression. Prostaglandins Other Lipid Mediat 2011;94:96–103.Web of ScienceGoogle Scholar
Mabry TJ, Markham KR, Thomas MB. The ultraviolet spectra of flavones and flavonols. In: The systematic identification of flavonoids. Berlin Heidelberg: Springer-Verlag, 1970.Google Scholar
About the article
Published Online: 2015-10-21
Published in Print: 2016-03-01