ANTIHYPERURICEMIC ACTIVITY OF FOUR SPECIES OF ANNONACEAE AND ISOLATION OF CHEMICAL CONTENT OF SELECTED PLANT [STELECHOCARPUS BURAHOL (BLUME) HOOK. F. THOMSON

• Main Author: Sunarni, Titik
• Format: Dissertations
• Language: Indonesia
• Subjects: Farmakologi dan terapeutik
• Online Access: https://digilib.itb.ac.id/gdl/view/33145
• Institution: Institut Teknologi Bandung

Hyperuricemia is one of the risk factor for gouty, even correlated with renal dysfunction, cardiovascular disease, diabetic and metabolic syndrome. Hyperuricemia can cause precipitation of uric acid in various tissues especially in the joint that will initiate inflammation and tissue damage. Hyperuricemia can be treated with antihyperuricemic agent, xanthin oxidase (XO) inhibitor and uricosuric as allopurinol and probenecid. The usage of allopurinol and probenecid may accompanied with side effects that can be fatal such as Stevens-Johnson syndrome. Several plants of Annonaceae have been used traditionally to treat hyperuricemia, such as kepel [Stelechocarpus burahol (Blume) Hook. f. Thomson], soursop (Annona muricata L.), sugar apple (Annona squamosa L.) and mulwo (Annona reticulata L.). However, this traditional used is mostly not supported by scientific data on efficacy and safety, so that study is aim to evaluate antihyperuricemic properties of four species of Annonaceae plant followed by isolation of chemical constituent of selected Annonaceae plants Extraction was performed by maceration using 96% ethanol. Antihyperuricemic activity of extract was conducted in vivo using hyperuricemic rat model induced by oxonic potassium and in vitro through xanthine oxidase inhibitory activity. The extract showed highest activity to decrease uric acid serum level was further fractionated by liquid-liquid extraction (LLE) with n-hexane, ethyl acetate and water. Ethyl acetate fraction was subfractionated by vacuum liquid chromatography (VLC) using silica gel H as stationary phase and eluted with a gradient of a combination of n-hexane-ethyl acetate-methanol as mobile phase. An active subfraction was then further subfractionated by classical column chromatography using silica gel 60 as stationary phase and eluted with isocratically solvent of chloroform-methanol (8:2). An active subfraction of classical column chromatography was purified by preparative paper chromatography, using 10% acetic acid as mobile phase and followed by further separated using preparative thin layer chromatography (preparative TLC) with chloroform-methanol (8:2) as mobile phase. The active extract, fraction and subfraction were also tested for their uricosuric activity. iv Four extracts of Annonaceae plants had antihyperuricemic activity at a dose of 75 mg/kg with a highest decreased in uric acid level (57%) given by ethanolic extract of kepel leaves. On XO activity test, all of the four extracts showed very weak effect (IC50 >200 mg/mL) except only mulwo extract with IC50 171.73 ± 17.17 ????g/mL. N-hexane and ethyl acetate fractions of kepel extract obtained through liquid-liquid extraction method had equivalent activities in decreasing uric acid level i.e., by 32 and 28% decreased in uric acid level, respectively. Nhexane fraction had very weak effect on XO activity (IC50 >200 ????g/mL), while ethyl acetate and water fractions had no effect on XO activity. Subfractionation by vacuum liquid chromatography yielded seven subfractions i.e. E.1 - E.7 and only E.3, E.4 and E.5 subfractions had antihyperuricemic activity with a percentage of decreasing in uric acid levels 43, 46 and 33%, respectively. The two subfractions E.3 and E.4 inhibited XO activity with IC50 >200 ????g/mL. Subfractionation of E.3 and E.4 by classical column chromatography yielded three and four subfractions (E.3.1 –E.3.3 and E.4.1 –E.4.4), however only E.3.2 and E.4.3 subfractions decreased uric acid level by 29 and 38 %. The two subfraction (E.3.2 and E.4.3) inhibited XO activity with IC50 128.39 ± 20,21 and >200 ????g/mL, respectively). TLC of E.3.2 subfraction showed 6 spots, while E.4.3 produced only 3 spots. Under uricosuric activity test, the kepel extract, ethyl acetate fractions, E.4 and E.4.3 subfractions increased the urine excretion of uric acid. This data indicated that those substances have uricosuric effect. Purification of subfraction E.4.3 done by preparative paper chromatography produced three band i.e., named as E.4.3.1 - E.4.3.3. Further separation of E.4.3.2 by preparative TLC produced E.4.3.2.2. Purity of E.4.2.2 by single development TLC and spectrophotodensitometry showed one spot, name as isolate R. Characterization and identification by ultraviolet-visible spectrophotodensitometry, 1 H-NMR, 13 C-NMR, HSQC dan HMBC spectrometry suggested that isolate was kaempferol-3-O-rhamnoside (C21H20O10). Mass spectrum showed molecular ion [M+H + ] at 433.38, while the molecular weight of kaempferol-3-Orhamnoside (C21H20O10) is 432.38 g.mol -1 . Further analyses with 1 H-NMR and 13 C-NMR spectra showed that the isolate R were comparable to structure of kaempferol-3-O-rhamnoside isolated from Sirraitia grasvenorri. Both substances had the similar chemical shift pattern. It might be concluded that isolate was kaempferol-3-O-rhamnoside. Based on the results it can be concluded that among the four species of Annonaceae tested, ethanolic extract of kepel leaves demonstrated the strongest in vivo antihyperurisemic activity than soursop, sugar apple and mulwo leaves extracts. The fourth extract had no effect on XO activity. The kepel leaves extract, ethyl acetate fraction, E.4 and E.4.3 subfraction showed antihyperurisemic and uricosuric activities, however they had no effect on XO activity. The isolate isolated from antihyperuricemic active subfraction of kepel leaves ethanol extract was identified as kaempferol 3-O-rhamnoside.