Identification of bioactive compounds with anti-atherosclerotic properties from Emblica officinalis fruit

Sinjitha, S. Nambiar (2015) Identification of bioactive compounds with anti-atherosclerotic properties from Emblica officinalis fruit. PhD thesis, University of Mysore.

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Abstract

Atherosclerosis is a disease of the arteries produced as a result of inflammation. This leads to plaque formation in arteries causing a progressive narrowing of the arterial lumen. This eventually leads to blockage of arteries and complete cessation of blood supply to the heart, culminating in heart attacks or myocardial infarction. Atherosclerosis starts with the oxidation of low density lipoproteins which are then taken up by macrophages. These macrophages then get converted to foam cells which lose their ability to migrate away resulting in the accumulation and proliferation of these cells. These cells form the building blocks of atherosclerotic plaque and create an inflammatory situation in the arteries that lead to the secretion of cytokines that cause the vascular smooth muscle cells residing in the tunica media to migrate in to the tunica intima, proliferate, secrete cytokines and augment the process of plaque formation and plaque enhancement. Various plants and their phytochemicals have been found to reduce the formation of plaque by different mechanisms. The present study investigated the anti-atherosclerotic effect of Emblica officinalis by employing macrophage and smooth muscle cell lines to examine the effect of the extracts on various stages of plaque formation. Initially, five sequential extracts-methanol, acetone, ethyl acetate, hexane and water extracts of 4 plants- Emblica officinalis fruit, Trigonella foenum graecum leaves, Ocimum sanctum leaves and Scoparia dulcis leaves were screened for their antioxidant and anti-atherosclerotic potential using assays analyzing the conditions during atherosclerosis such as nitric oxide scavenging assay, superoxide anion scavenging assay, erythrocyte membrane stabilization ability, total antioxidant capacity, low density lipoprotein (LDL) oxidation inhibition capacity in addition to cholesterol efflux assay and ox-LDL induced foam cell inhibition assay in RAW 264.7 macrophage cell line. It was found that Emblica officinalis fruit methanol and water extracts showed the highest activity in all these assays with an IC50 of 15 μg/ml and 4 μg/ml in superoxide scavenging assay and nitric oxide scavenging assay respectively. Emblica officinalis methanol and water extracts showed the highest percentage inhibition of LDL oxidation and oxidized LDL (ox-LDL) induced foam cell inhibition compared to other plants’ extracts. These two extracts of Emblica officinalis also caused about 2 fold (the highest degree compared to other extracts) increase in cholesterol efflux in RAW 264.7 macrophages showing that out of the five solvent extracts of the four plants tested, E. officinalis had the highest anti-atherosclerotic potential. The extracts tested here were prepared from oven-dried plants. Next, the different parts of the fruit- seed, pulp and whole fruit were investigated for their antioxidant potential to find out the part of the fruit exhibiting the highest activity. Methanol, ethanol and water extracts of seed, pulp and whole fruit (oven-dried and fresh) were assessed for their abilities to inhibit oxidized LDL- induced foam cell formation, ox-LDL induced apoptosis and nitric oxide formation after LPS stimulation of RAW 264.7 macrophages and their activities were compared to the each other. Amongst the solvent extracts of fresh fruit parts, water extract of pulp showed the highest activity. However the activity of the fresh pulp water extract was less than that of oven-dried pulp water and methanol extracts. It was seen that of all the extracts of oven-dried and fresh fruit parts tested, oven-dried pulp methanol and water extracts showed the highest foam cell inhibition activity, ox-LDL induced apoptosis inhibition activity and LPS-induced nitric oxide scavenging activity in RAW 264.7 macrophages. The activities of oven-dried pulp methanol and water extracts were found to be equal. Hence for the remaining investigations, the pulp methanol and water extracts of oven-dried E. officinalis fruits were used. Next, different phytochemicals namely phenolics, carotenoids, tannins, alkaloids and ascorbic acid were isolated from the pulp of E. officinalis fruit and each of these phytochemicals were subjected to LDL oxidation inhibition assay to select the phytochemical showing the highest activity for further analysis. It was seen that the phenolic compound rich extract showed the highest percentage of inhibition (100% at 5h) of LDL oxidation. High Performance Liquid chromatography (HPLC) analysis of phenolic compounds revealed the presence of 7 fractions in the water extract (phenolic compound rich extract) which were identified by HPLC standards and mass spectrometry and their identities were confirmed by nuclear magnetic resonance imaging (NMR) analysis. Each of these fractions were isolated and subjected to LDL oxidation inhibition assay. They were also assessed for their abilities to prevent ox-LDL induced proliferation in RAW 264.7 macrophage and A7r5 vascular smooth muscle cell lines, ox-LDL induced foam cell formation in RAW 264.7 macrophage cell line, LPS stimulated free radical formation assays in RAW 264.7 macrophage and A7r5 vascular smooth muscle cell lines and ox-LDL induced apoptosis in RAW 264.7 macrophages. It was seen that, out of the 7 fractions identified, 4 fractions showed very high activities. These fractions were fraction numbers 2, 5, 6 and 7 with the highest activity shown by fraction 7 (myricetin), followed by fraction 6 (caffeic acid), fraction 2 (4-hydroxybenzoic acid) and fraction 5 (coumaric acid). In all these assays, it was seen that the individual fractions showed lesser activities compared to those of the crude methanol and water extracts. Hence combinations of these fractions were studied in macrophage and smooth muscle cell lines to find out the combination giving the highest activity. In the activity studies using combinations of fractions, the IC50 of each of fractions obtained in different assays were used to make combinations in the respective assays. The effect of E. officinalis methanol and water extracts on different anti-atherosclerotic and anti-inflammatory parameters in RAW 264.7 macrophage cell lines and A7r5 vascular smooth muscle cell lines were also studied. In macrophages, methanol and water extracts at 50μg/ml showed the highest degree of inhibition in ox-LDL induced proliferation in macrophage and vascular smooth muscle cell lines. 50μg/ml of methanol and water extracts also showed the highest level of inhibition in platelet derived growth factor induced A7r5 vascular smooth muscle cell migration, ox-LDL induced foam cell formation and enhancement of cholesterol efflux and ox-LDL induced apoptosis in RAW 264.7 macrophages. 50μg/ml of methanol and water extracts enhanced migration of macrophages in the presence of ox-LDL. This degree of activity shown by 50μg/ml crude extracts was shown by the combination of fraction numbers 2, 6 and 7 corresponding to 4-hydroxybenzoic acid, caffeic acid and myricetin respectively. The crude methanol and water extracts at 50μg/ml and combination of fraction numbers 2,6 and 7 showed similar inhibition in foam cell formation in macrophages by producing a similar degree of inhibition of scavenger receptor gene expression like CD36, lectin-like oxidized LDL receptor (LOX-1) and scavenger receptor A-1 and upregulation of cholesterol efflux pathway genes like Liver X Receptor- alpha (LXR-α), ATP- binding cassette transporter A1(ABCA1), ATP- binding cassette transporter G1 (ABCG1) in macrophages. Crude methanol and water extracts at 50μg/ml showed the highest level of inhibition of inflammation in both macrophage and vascular smooth muscle cell lines by inhibiting cytokine release, free radical production under Lipopolysaccharide stimulation and also enhanced the antioxidant enzyme activities of these cell lines. This same level of inhibition was also shown by the combination of fraction numbers 2, 6 and 7. Western blotting analysis showed that the extracts and the fraction combination 2, 6, 7 brought about these effects in macrophages and vascular smooth muscle cells by inhibiting the activation and phosphorylation of epidermal growth factor receptor (EGFR), Akt, P38 MAP kinase and reduced inflammation by preventing the activation of nuclear factor (NF)-κB. After investigating the anti-atherosclerotic effects of the E. officinalis fruit, the effects of different processing methods on the bioactivities of the fruit pulp were studied, culminating in food product formulation. The first method of processing studied was yeast fermentation of the fruit to produce wine. For this, three varieties of E. officinalis fruits- cultivated variety (common E. officinalis used in the previous experiments), wild variety and Mysore variety also called Phyllanthus acidus were taken and each of them were processed by fermentation separately to make three varieties of wines. Then assessment of lipopolysaccharide induced nitric oxide scavenging activities and ox-LDL induced foam cell inhibition abilities of these three wines in comparison with their juices was done in RAW 265.7 macrophage. It was found that wine made from the cultivated variety had the highest activity in macrophage cell line system. HPLC analysis of phenolic compounds showed that some phenolic compounds were preserved and some increased during the course of fermentation. The second product was prepared by the processing of E. officinalis fruit by osmodehydration using fructooligosaccharides (FOS) (a prebiotic). Here, the best temperature causing maximum infusion of FOS into the fruits was selected and then the antioxidant activity, LDL oxidation and foam cell inhibition abilities of FOS osmodehydrated fruits were compared to those of untreated E. officinalis fruits. It was found that osmodehydrated fruits showed the same activities as those of untreated fruits. Phytochemical analysis and HPLC profiling of phenolic compounds showed that the processing method of osmodehydration preserved the phenolic compounds present in the original fruit. Finally, preparation of muffins from osmotically dehydrated FOS infused E. officinalis fruits was done by powdering the FOS infused fruits and mixing it with wheat flour in different proportions. It was found that the best sensory and texture profile was shown by 10% FOS- E. officinalis incorporated muffins compared to control muffins (without addition of FOS- E. officinalis).

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	anti-atherosclerotic effect, Emblica officinalis, processing methods
Subjects:	600 Technology > 08 Food technology > 24 Fruits > 03 Citrus fruits 600 Technology > 08 Food technology > 32 Antioxidants
Divisions:	Plant Cell Biotechnology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	19 May 2016 07:44
Last Modified:	19 May 2016 07:44
URI:	http://ir.cftri.res.in/id/eprint/12179

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