BIOACTIVE FRACTION FROM RED GINGER RHIZOME (ZINGIBER OFFICINALE ROSCOE VAR. SUNTI VAL) AS ANTI-ATHEROSCLEROTIC AGENT CANDIDATES AND ITS MECHANISM OF ACTIONS

• Main Author: Wakhidatun Suciyati, Siti
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/71079
• Institution: Institut Teknologi Bandung

Atherosclerosis is an inflammatory disease of the arteries, one of which is caused by changes in fat metabolism. Atherosclerosis is a major cause of cardiovascular disease, especially ischemia of the heart and cerebrovascular disease. Statin therapy is the only conventional therapy for the treatment of atherosclerosis that is commonly used. However, in the long term side effects of this class of drugs cannot be avoided, such as rhabdomyolysis. Red ginger was chosen as the main candidate to be developed in this study because its easily obtainable, affordable, and has been used empirically for generations by the community in treating diseases. The ethanol extract of red ginger rhizome was shown to have antihuperlipidemic effects. Red ginger rhizome infusion also significantly reduced LDL levels in humans. Based on these considerations, this research was aimed to determine the active fraction of red ginger rhizome that showed anti-atherosclerotic activity and study its mechanisms of action. Study of the content of red ginger rhizome begins with continuous extraction using n-hexane, ethyl acetate, and ethanol as solvents. The results of the phytochemical screening showed the presence of alkaloids and steroids/triterpenoids in the simplicia and extracts extracted from red ginger rhizome. These terpenoid compounds evaporate easily, giving red ginger its distinctive aroma. Flavonoid compounds were detected in ethanol and ethyl acetate extracts with red ginger having the highest total flavonoid and phenolic content compared to other ginger varieties. The antioxidant test results using the DPPH method showed the ethanol extract had the highest inhibitory capacity compared to the ethyl acetate extract and n-hexane extract with IC50 respectively 11.57 ± 0.67 ?g/mL; 28.38 ± 4.76 ?g/mL; and 20.20 ± 3.05 ?g/mL. While the results of the antioxidant test using the NO radical scavenging method showed that n-hexane extract from red ginger rhizome had the highest activity when compared to other extracts with IC50 respectively for ethanol extract, ethyl acetate extract, and n-hexane extract were 84.01 ± 2.81 ?g/mL; 262.99 ± 3.58 ?g/mL; and 150.39 ± 7.32 ?g/mL. Cardiomyocyte treated with extracts extracted from red ginger showed a decreasing trend in the number of living cardiomyocytes as the concentration of the extract was given to cardiomyocytes after 24 hours of incubation. The highest decrease up to 20% was observed at a concentration of 10 ?g/mL of ethanol extract, whereas at a lower concentration the extract did not give a significant change in viability. Cardiomyocytes were then used in the I?B-? phosphorylation inhibition test to determine the presence of anti-inflammatory activity. The n-hexane extract and ethanol extract extracted from red ginger rhizome showed a statistically significant decrease in I?B-? phosphorylation (p<0.05). Compared to the positive control, the n-hexane and ethanol extracts reduced I?B-? phosphorylation by 78.9% and 53.8%. This result is inferior compared to the comparator BAY11-7082 that provided maximum inhibition. Referring to the test results above, the n-hexane extract gave the best results when compared to other red ginger extracts hence it was chosen as the main candidate for an anti-atherosclerotic agent. N-hexane extract also showed a dose-dependent inhibition of I?B-? phosphorylation. Twelve fractions were collected from the n-hexane extract of red ginger rhizome that was fractionated using a vacuum liquid chromatography method with a stationary phase of 60H silica gel and a mobile phase of n-hexane-ethyl acetate at a different gradient of concentrations. The fraction that has highest average weight was the F3(8:2) fraction at 2.15 g. The TLC profile of the red ginger rhizome fraction showed the presence of 6-gingerol in the F6 (5:5) and F7 (4:6) fractions. Viability of cardiomyocytes against fractions of n-hexane extract of red ginger rhizome and 6-gingerol showed a trend of decreasing cell number as the concentration increased. At the same concentration of fractions, fractions F6 and F7 had the highest inhibition of I?B-? phosphorylation of all the fractions obtained. Both fractions were then determined for their effect on decreasing IL-1? and IL-6 mRNA expression. A significant reduction (p<0.05) of IL-1? and IL-6 mRNA expression was shown by the F6 fraction when compared to the positive control, BAY11-7082, 6-gingerol, and F7 fraction. From the results of lipoxygenase inhibitory activity, F6 fraction showed the same lipoxygenase inhibitory activity as zileuton although its potency was still lower than quercetin with the IC50 of zileuton, quercetin, 6-gingerol, n-hexane extract, F6 fraction, and F7 fraction respectively were 43.37 ± 3.27 ?g/mL; 20.96 ± 2.24 ?g/mL; 36.39 ± 2.32 ?g/mL; 69.97 ± 3.05 ?g/mL; 36.40 ± 0.76 ?g/mL; and 52.27 ± 2.39 ?g/mL. The F6 fraction decreased TNF-? production significantly in macrophage cells compared to n-hexane extract or F7 fraction with its levels respectively are 0.79 ± 0.12 pg/mL; 2.28 ± 0.13 pg/mL, and 3.41 ± 0.13 pg/mL. The F6 fraction also decreased TNF-? production significantly in macrophage cells compared to n-hexane extract or F7 fraction with its levels respectively were 7.91 ± 0.001 pg/mL; 14.11 ± 0.007 pg/mL, and 12.6 ± 0.004 pg/mL. Similar results were observed in the lipid oxidation inhibition test. F6 fraction act as a potent inhibitor (IC50: 45.97 ± 5.14 ?g/mL) compared to n-hexane extract and the F7 fraction (IC50: 72.34 ± 3.74 ?g/mL and 66.74 ± 5.77 ?g/mL). F6 fraction has a lower concentration of inhibition compared to 6-gingerol (IC50: 59.40 ± 3.47 ?g/mL), although it was still inferior compared to ascorbic acid (IC50: 11.74 ± 2.08 ?g /mL). Determination of 6-gingerol content showed F6 fraction had a high content of 6-gingerol (194.99 ± 1.43 mg/g extract) compared to the F7 fraction (77.19 ± 1.87 mg/g extract) and the n- hexane (129.77 ± 4.22 mg/g extract). Based on the results of GC MS measurements of the F6 fraction, the anti-atherosclerotic effect of the F6 fraction was better than the n-hexane extract and its fractions also 6-gingerol strongly suspected due to the synergistic effect of the compounds zingeron, 6-paradol, 6-gingerol, 6-shogaol, and 10-shogaol. The information collected from this study is red ginger rhizome has potential as an anti-atherosclerosis with a mechanism of action including as an antioxidant, I?B-? phosphorylation inhibitor in the NF-?B signal transduction pathway, lipoxygenase inhibitor, supressed proinflammatory cytokines production, and prevented oxLDL formations. The fraction of the n-hexane extract (F6) is selected to be developed as preventive or complementary medicine to treat atherosclerosis because of its potential activity in pharmacological studies that have been carried out.