POTENTIAL BIOACTIVITY OF BIOACTIVE FRACTION NANOFORM OF MELINJO (GNETUM GNEMON L.) SEED’S HARD-SHELL EXTRACT AS AN ANTIHYPERURICEMIA

• Main Author: Saraswaty, Vienna
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/51330
• Institution: Institut Teknologi Bandung

Hyperuricemia is a condition characterized with high concentration of blood serum uric acid levels (>5.6 mg/dl for women and 7.0 mg/dL for men). Uncontrolled hyperuricemia condition may result in deposition of uric crystals in joints as well as kidney, hence causing inflammation, gout, and various complications including renal failure, hypertension, and cardiovascular disease. Thus, hyperuricemia seems to be a considerable risk in reduction of the qualiy and life expectancy. Therefore, it is important to control and manage hyperuricemia condition. Melinjo (Gnetum gnemon L.) seed extract has shown a wide pharmacological activity, including antioxidative, anticancer, tyrosinase inhibitor, antibacterial as well as antihyperuricemic. However, the anti-hyperuricemic activity of melinjo seeds extract that is associated with the inhibition of the xanthine oxidase (XO) enzyme has not optimal yet. By-products of the agroindustry are a potential source of bioactive compounds. Therefore, the exploration of the by-product of melinjo seed that is melinjo seed’s hard shell (MSHS) as an antioxidant and antihyperuricemia in association with inhibition of XO enzyme is required. The low concentration of active compounds in natural product extract is due to the mixture of neither active nor inactive compounds in the extract. In addition, in general the active compounds from natural product extract are poorly soluble in water. Due to advancements in technology, inactive compounds can now be separated by a fractionation process. In addition, several studies have shown that the nanotechnology approaches can improve the poor water solubility of drugs then enhancing the efficacy. As an approach, combination of fractionation by macroporous adsorptive resin (MAR) HPD-600 and nanonization of the most active fraction was investigated in this project. To test the hypothesis that the fractionation of MSHS crude ethanol extract (MSHS- CE) and nanonization of MSHS-CE’s most active fraction enhances antioxidant and antiyperuricemia activity, the MSHS-CE, MSHS-CE most active fraction and its nanoform were evaluated. The antioxidant activities were tested on the free iradical scavenging effect by using DPPH and ABTS free radicals and its reducing capacity by the CUPRAC and FRAP methods. Whereas to prove antihyperuricemic activity, the in vitro test of XO enzyme inhibitory activity and in vivo tests in potassium oxonate (PO) induced hyperuricemic rats were observed. The in vivo test was evaluated at dose of 500, 250, and 125 mg/kg body weight (bw). To observe the evolution during nanonization of the most active fraction, a set of parameters which included observations of particle size by a particle size analyzer (PSA), particle morphology by a Scanning Electron Microscope (SEM), the degree of crystallinity by X-Ray diffractometer (XRD), thermal properties by thermal gravimetric analysis (TGA) and digital scanning calorimetry (DSC), changes of functional groups by Fourier-Transform Infrared Spectroscopy (FTIR) as well as the total phenolic content (gallic acid equivalent) was observed. The acute oral toxicity assay with a single fixed dose of 5000 mg/kg bw was observed to evaluate the initial toxic response. Whereas for compound characterization of the most active fraction, an evaluation by using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument was observed. MSHS-CE obviously showed antioxidant and XO inhibitory activity. The MSHS-CE that was produced from old seeds exhibited the highest free radical scavenging activity with a 50% inhibitory concentration (IC50) 160.4 ± 1.5 ppm and 18.7 ± 0.35 ppm for DPPH and ABTS free radicals respectively. Whereas the Cu reducing capacity was 2 ± 0.2 µg (equivalent to ?-tocopherol)/100 µg extract and 89 ± 11.52 µg (equivalent to ascorbic acid)/100 µg extract. Furthermore, the Fe reducing capacity was 217 ± 13.7 µg (equivalent to ?-tocopherol)/100 µg extract. Interestingly, the IC50 value of XO enzyme inhibitory activity was 62 ± 1.29 ppm, showing greater XO inhibitory activity than previous report of melinjo seed extract. The fractionation process by using MAR HPD-600 succesfully eliminated the presence of free sugar and increased the antioxidant activities by 3-7 times higher in comparison with the MSHS-CE. Furthermore, the fraction-50 exhibited the greatest XO inhibitory activity with an IC50 value of 46.24 ± 0.63 ppm. The antihyperuricemia activities of MSHS-CE and fraction-50 were also confirmed in vivo using PO-induced hyperuricemia male wistar rats. A considerable reduction of blood serum uric acid levels was detected when 500 mg/kg bw of fraction-50 was orally administered to PO-induced hyperuricemic rats. The blood serum uric acid levels significantly (p <0.05) decreased to ~46% at minute of 150 (T150), from 4.63 ± 0.34 to 2.68 ± 0.37 mg/dL. The nanonization was successfully carried out for fraction-50. The produced fraction-50 nanoform clearly depicted a change in physical characteristics in the form of smaller particle size (~216.9±2.6 nm, with a polydispersity index of 0.292±0.02), increased surface area as well as an increased degree of amorphicity as suggested by particle size, SEM images, and XRD pattern. The thermal analysis showed that the glass transition temperature of fraction-50 nanoform decreased from 80.8°C to 77.6°C and decomposed more easily at temperature higher than 650°C. The nanosization process of fraction-50 up to 120 minutes of milling did not lead to compound decomposition, which is evidenced from the FTIR spectrum that was completely preserved after the nanonization. In addition, an analysis of the total phenolic content showed an increment from 186.2±17.34 µg (gallic acid equivalent (GAE))/100 mg dried fraction to 240.9±1.62 µg (GAE)/100 mg dried fraction. In addition, the fraction-50 nanoform showed a 2 to 11-fold increment in antioxidant activities (at various method). The in vitro XO enzyme inhibitory activity of the fraction-50 nanoform also increased three-fold as suggested by decreasing 68% of IC50 value from 46.24 ± 0.63 to 14.72 ± 2.70 ppm. Confirmation of anti-hyperuricemic activity in vivo showed that the fraction-50 nanoform at 500 mg/kg bw lowered blood serum uric acid levels more rapidly as compared with fraction-50 (lowered to 1.94±0.44 mg/dL at T60 or 38% lower than hyperuricemic rats at T60). Unexpectedly, the decrease in blood serum uric acid levels was followed by an increase in these levels during at T90-T150. The increment of the blood serum uric acid levels is suspected due to the faster clearance of the bioactive compound as suggested by the higher urinary volume output which was similar with probenecid at 25 mg/kg bw. The acute oral toxicity assay showed that at dose of 5000 mg/kg bw no mortality or toxicity symptoms were observed, indicating that lethal dose (LD)50 of MSHS-CE, fraction-50 and its nanoform are higher than 5000 mg/kg bw, hence relatively safe and practically non toxic. The compounds characterization by using LC-MS/MS instrument showed the presence of flavonoid and phenolic compounds including gnetin C, transresveratrol, gnetol, kaempferol-3-O-rutinoside, Isorhamnetin-3-O-?-rutinoside, gnemonosida A, and gnemonosida D. Among these compounds, gnetin C, transresveratrol and kaempferol-3-O rutinoside are known as XO enzyme inhibitor. While others are natural antioxidant compounds. In summary, melinjo seed’s hard-shell is potential source of antioxidant and antihyperuricemic compounds which are gnetin C, trans-resveratrol, gnetol, kaempferol-3-O-rutinoside, Isorhamnetin-3-O-?-rutinoside, gnemonosida A, and gnemonosida D. Fractionation process by using MAR-HPD600 is potential for separation of inactive or free sugar compounds. By applying top down milling a high energy milling-based nanotechnology for reduction of particle into nanoform, the dissolution rate of the fraction-50 improved hence enhancing its antioxidant and antihyperuricemia efficacy. However, the presence of higher volume of urinary output which was expected as a diuresis action may limit its potential as suggested by its short duration of effect. Hence, we suggest separating diuretic compounds present in the melinjo seed’s hard-shell extract. In addition, optimization of particle size and sustained release formulation are other approaches to prolong its hyperuricemic effect.