STUDY OF ACTIVITY AND ACTIVE COMPOUNDS IN TEMPUYUNG (SONCHUS ARVENSIS (LINN.)) LEAVES AS ANTIHYPERTENSIVE AGENT

• Main Author: Suryani
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/57159
• Institution: Institut Teknologi Bandung

Tempuyung (Sonchus arvensis Linn.) has been used empirically to lower blood pressure and have a diuretic effect. Diuretic is one of the action mechanisms of antihypertensive drugs. The purpose of this study was to examine the pharmacological activity of the bioactive compounds of tempuyung leaves as an antihypertensive agent. The study was carried out through several mechanisms such as antioxidants, ?-blockers, angiotensin-converting enzyme (ACE) inhibitors, and angiotensin receptor blockers (ARB) conducted in vitro, in vivo, and ex vivo study. The investigation was started by tempuyung leaves extraction using maceration methods with ethanol 96% as a solvent. The yield of tempuyung leaves extract was 7.79%. Fractionation of tempuyung leaves extract was performed by liquid-liquid extraction method using n-hexane, ethyl acetate, and water, respectively. Aqueous fraction, ethyl acetate fraction, and n-hexane fraction were obtained from this method with the yield of 28.01, 8.72, and 42.74%, respectively. Phytochemical screening and thin-layer chromatography (TLC) screening revealed that all three solvents could separate compounds in tempuyung leaves extract based on polarity; one of them was the presence of flavonoid. Aqueous fraction and ethyl acetate fraction showed the presence of flavonoids, but not detected on n-hexane fraction. The TLC and high-performance liquid chromatography (HPLC) analysis were demonstrated that the flavonoid compounds found in extract and ethyl acetate fraction were luteolin, luteolin-7-O-glycosides, apigenin, apigenin-7-O-glycosides, kaempferol, rutin, and quercetin. The concentration of luteolin as the primary component in tempuyung leaves was 6.44 ± 0.61 ?g/mg. Total flavonoid content in tempuyung leaves extract, and ethyl acetate fraction was 26.08 ± 3.03 ?g/mg and 36.49 ± 2.08 ?g/mg, respectively, they counted as equality with luteolin. The antioxidant activity in tempuyung leaves extracts and their three fractions was measured with the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method. The examination of antioxidant activity of aqueous fraction and ethyl acetate fraction showed the values varied from 971.34 ± 12.33 to 226.77 ± 6.91 µg/mL. In comparison, the tempuyung leaves extract (IC50 > 250 µg/mL) and n-hexane fraction (IC50 > 1000 µg/mL) were the lowest and the highest antioxidant activity compared to other fractions in this study. Ethyl acetate fraction has greater antioxidant activity than other fractions although it is significantly lower for vitamin C. The ACE inhibition activity of the extract and fractions was evaluated as the liberation of hippuric acid (HA) from hippuryl-L-histidyl-L-leucine (HHL). The study revealed that tempuyung leaves (100 ?g/mL) inhibited ACE by 66.19 ± 1.98%, while aqueous fraction by 28.62 ± 13.95%, n-hexane fraction by 69.36 ± 0.60%, and ethyl acetate fraction by 78.59 ± 7.01%. The ethyl acetate subfractions (50 ?g/mL) showed that subfraction numbers 4-9 had an ACE inhibitor activity of more than 50%. TLC analysis demonstrated that these subfractions had luteolin, apigenin, quercetin, and kaempferol content. Tempuyung leaves activity as a ?receptor antagonist was conducted in male Wistar rats using epinephrine-induced acute hypertension method followed by a relaxation activity test on the heart muscle of norepinephrine-induced frogs. Moreover, Mitogen signaling pathway inhibitors Activated Protein Kinase (MAPK) in HEK 269 cells overexpression ?receptors was also analyzed against isoproterenolinduced Extracellular signal Regulated Kinase (ERK) phosphorylation. Tempuyung leaves extract (100 mg /kg BW) was able to inhibit epinephrine 0.25 mg/kg BW in raising blood pressure (p<0.05). Ethyl acetate fraction (16 mg/kg BW) exhibited antihypertensive activity (p<0.05), with systolic and diastolic blood pressure inhibition of 58.56 and 68.24%. Contraction of frog heart muscles induced by norepinephrine decreased with the administration of ethyl acetate fraction (27.7%). Bioactive compounds (luteolin, apigenin and quercetin) in ethyl acetate fraction might suppress blood pressure because of ?receptor activation through adrenalin stimulation and prevent the increase in heart muscle contractions. Thus, these bioactive compounds might be potential as ?receptor antagonists by inhibiting the phosphorylation of ERK1/2 of MAPK signaling pathways which plays a role in contraction process and cell movement. In vitro activity of bioactive compounds of tempuyung leaves as an ARB was performed by a cellular mechanism in CHO-AT1r cells and vascular smooth muscle cells (VSMC) A7R5 with angiotensin II inductors (Ang II). Ang II stimulates MAPK signaling pathways through ERK1/2 phosphorylation, which plays a role in cell contraction, proliferation, differentiation, and cell migration. Tempuyung leaves extract (100 µg/mL) significantly inhibited ERK1/2 phosphorylation (p<0.01) while ethyl acetate fraction 100 ?g/mL exhibited higher activity than tempuyung leaves extract and equivalent to olmesartan. Luteolin, apigenin, kaempferol, and rutin (5- 40 ?g/mL) found in tempuyung leaves could inhibit phosphorylation ERK1/2. Inhibition of MAPK signaling pathways occurs because luteolin, apigenin, kaempferol, and rutin can inhibit phosphorylation tyrosine kinase protein, and luteolin inhibit Ang II-induced the internalization of AT1r receptors into the cytoplasm. Microscopic immunofluorescence analysis revealed that AT1r receptors found on the surface of cell membranes indicated the mechanism of action of bioactive compounds of tempuyung leaves. Upstream signal analysis of the cytoplasm suggested that inhibition of ERK1/2 phosphorylation reduced its translocation into the nucleus. Moreover, the downstream signal analysis demonstrated this by expressing the CREB transcription factor and low c-fos (p<0.01). Inhibition of MAPK signaling pathways decreased transcription factors' expression, resulting in reduced cell growth and migration. Luteolin (5-10 ?g/mL), apigenin (20 ?g/mL), and kaempferol (20 ?g/mL) was found to inhibit Ang II, thus significantly reducing the proliferation and migration rate of VSMC cells (p<0.01). Luteolin suppressed CREB and c-fos expression up to 3-fold by reducing cell proliferation (59.85%) and VSMC cell migration (82.58%). Apigenin and kaempferol showed lower activity compared to luteolin. Control of proliferation and migration of vascular smooth muscle cells can prevent vascular remodeling. The rise in blood pressure can be triggered by vascular remodeling. Ethyl acetate fraction had significant antioxidant activity, ACE inhibitors, ? receptor antagonist, and angiotensin II receptor antagonist compared to aqueous fraction and n-hexane fraction. The highest flavonoid content in the ethyl acetate fraction might contribute to the pharmacological effects produced by this fraction. Luteolin, apigenin, and kaempferol found in ethyl acetate fraction revealed to have pharmacological effects as antihypertensives. Luteolin is a biomarker compound in tempuyung leaves and has the highest activity compared to other flavonoid compounds in tempuyung leaves. The information that can be provided from this study is the complex phytochemical content contained in a plant makes tempuyung leaves has the potential as an antihypertensive with more than one of mechanisms of action including antioxidants, ACE inhibitors, ?-blocker, and ARB. Ethyl acetate fraction is the selected fraction that can be developed into an herbal medicine to help hypertension therapy derived from tempuyung leaves because it contains bioactive compounds as antihypertensives which produce the best activity in the pharmacological tests.