PHARMACOGNOSTIC STUDY OF KABAU (ARCHIDENDRON BUBALINUM (JACK) NIELSEN.) AND ANALYSIS OF ANTIDIABETIC POTENTIAL COMPOUNDS

• Main Author: Riasari, Hesti
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/62225
• Institution: Institut Teknologi Bandung

Kabau is an endemic plant of Indonesia, especially in the island of Sumatra. Kabau belongs to the Fabaceae family which has three sub-family namely Faboideae, Caesalpinoideae, Mimosoideae. Kabau belongs to the sub-family Mimosoideae, genus Archidendron, with the species name Archidendron bubalinum (Jack) Nielsen. From a chemotaxonomic perspective, it is estimated that there is a similar chemical structure with jengkol which allows for the similarity of pharmacological effects, therefore members of the Archidendron can be developed as antidiabetic drugs. One type of the Archidendron species that has been tested for antidiabetic activity is jengkol (Archidendron jiringa), so it can be used as a reference for the same test. In general, this study included macroscopic and microscopic characteristics, analysis of odor compounds using GC-MS, total phenol content, total flavonoid content, antioxidant activity, in vitro and in vivo antidiabetic activity testing and analysis of compounds that have the potential to have antidiabetic activity. Macroscopic and microscopic characterization was carried out on fresh and dry kabau seeds by observing their shape, size, smell and taste, and making a 100 µm thick incision on fresh kabau seeds. In addition to fresh kabau seed plant samples, microscopic sample preparations were also carried out on simplicia powder and histochemical testing with the addition of specific reagents using a light microscope equipped with a digital camera and analysis using the S-Viewer program. Morphological analysis, both macroscopic and microscopic on kabau seeds, was to identify the type of Archidendron bubalinum in its morphoanatomical form and distribution as well as accumulation of primary and secondary metabolites. The morpho-anatomical character of kabau seeds is cylindrical in a neatly arranged arrangement consisting of five to six seeds in each pod, which smells like jengkol with a slightly bitter sweet taste and a soft texture. Kabau seeds have an average length of 2 cm, a diameter of 1.5 cm and have cotyledons (seed germination). Microscopically, kabau seeds consist of 3 layers, namely sarcotesta, and endotesta containing amyloplasts with starch and oil sacs. Histochemical analysis of kabau seeds contains phenolic compounds, tannins, alkaloids, terpenoids, proteins/amino acids and starch which are distributed differently in the kabau seeds, making it easier to identify compounds that contribute to the activity of kabau seeds. The next stage of research was the extraction of kabau seeds by continuous extraction using a Soxhlet device, using solvents with gradient polarity, namely nhexane, ethyl acetate and 96% ethanol. The type of solvent used affects the active compounds that are also extracted. Polar solvents will attract polar compounds, non-polar solvents will attract non-polar compounds and semi-polar solvents will attract semi-polar compounds. The yield obtained from the extraction with nhexane solvent was 0.76%, ethyl acetate 0.83% and ethanol 96% as much as 10.66 %. The difference of the type of solvent affects the extracted metabolites so that the yield of the resulting extract was different. The n-hexane extract organoleptically has a characteristic odor of kabau seeds which was very dominant, so that the n-hexane extract was analyzed for odor compounds using GC-MS. The results of GC-MS show various compounds of saturated fatty acids, unsaturated fatty acids and compounds thought to give odor to kabau seeds, namely lentionin and methyl imidosulfur difluoride in which both compounds contain sulfur functional groups. In the next step, the three extracts were tested for total phenol content, total flavonoid content, antioxidant activity and antidiabetic activity in vitro and in vivo. The results of the highest total phenol and flavonoid levels in the ethanol extract, and highly correlated with antioxidant activity with an IC50 of 41.18 µg/mL. The next stage of the study was to carry out antidiabetic activity using in vitro methods by testing the inhibitory activity of the a -glucosidase enzyme. The results showed that the most active extract inhibiting the a-glucosidase enzyme with the lowest IC50 value was n-hexane extract. The results of GC-MS on the nhexane extract showed that there were saturated and unsaturated fatty acids as well as compounds containing sulfur that gave kabau a distinctive odor. Unsaturated fatty acids inhibit the activity of the a-glucosidase enzyme better than saturated fatty acids, the more double bonds in fatty acids, the stronger the enzyme inhibitory activity and the inhibitory potential depends on the length of the carbon chain and the position of the double bond in the acid molecule. For extracts that are active in inhibiting -glucosidase enzymes, the compound separation process was carried out by gradient elution column chromatography method using a combination of n-heptane, n-hexane and ethyl acetate solvents using silica gel 60 as a stationary phase. During the separation process produces vial no. 61 -91 with white powder and then monitored by TLC, vial No. 73 showed a spot pattern of pure compound with a weight of 183.2 mg and a characteristic odor, which was then named VKH isolate. VKH isolate was monitored for purity using 3 developer TLC and 2 dimensional TLC, the result was that VKH isolates were pure. Furthermore, VKH isolates was analyzed using FTIR spectroscopy, 1H-NMR (500MHz) and 13C-NMR (125 MHz), confirming that VKH isolate was oleic acid. Oleic acid compounds were tested for antidiabetic activity in vitro. Based on the results of the inhibition of a-glucosidase enzyme, it was shown that oleic acid gave a better % inhibition compared to n-hexane extract In the next research stage, in vivo antidiabetic activity testing of n-hexane, ethyl acetate and ethanol extracts was carried out using the glucose tolerance test method, measuring glucose levels in alloxan-induced animals and measuring MDA and SOD levels. The results of the glucose tolerance test showed a significant effect of reducing glucose levels by ethyl acetate extract at a dose of 750 mg/kgbw and ethanol at a dose of 250 mg/kgbw. These results indicated that kabau seed extract can be used as an alternative for antidiabetic treatment and as an antioxidant. This was supported by the results showing that the measured SOD levels were higher in the group given the extract than normal, and close to the levels in the group given ascorbic acid. The next research stage is the fractionation process of the ethanol extract using the solid liquid method, where 15 grams of ethanol extract is dissolved with 150 ml of ethyl acetate, sonicated for 30 minutes then stirred, the filtrate is taken then the extract is added back to ethyl acetate and repeated three times. Furthermore, the extract was dissolved with 150 ml of ethyl acetate - methanol (1:1), sonicated for 30 minutes then stirred, the filtrate was taken then the extract was added back to ethyl acetate - methanol (1:1) and carried out triplo, the last step the extract was added 100% methanol and tripled. then the compound monitoring test was carried out on TLC and guided activity by in vivo testing using the glucose tolerance test method, the test material showed antihyperglycemic activity in hyperglycemic-induced animals, the smaller the AUC value, the lower the amount of glucose in the blood, when compared to the treatment group, the lower the AUC value. The higher the antidiabetic potential. All test materials showed the same potential atihyperglycemic activity assessed from the AUC0-150 values which were not significantly different from each other, but when viewed from the decrease in AUC, the methanol fraction had the potential to lower glucose levels better than the other fractions. The next stage of research was subfractionation to obtain compounds that have anti-diabetic potential in the methanol fraction, subfractionation was carried out using medium pressure liquid chromatography (mplc) with a sample weight of 1g with a silica stationary phase of 12 mg with a flow rate of 15 ml/minute, a pressure of 300psi and the mobile phase using chloroform – methanol - formic acid (4 : 6: 1), obtained 117 subfractions and monitored using TLC with silica gel F254 as a stationary phase and ethyl acetate - methanol - formic acid - water as a mobile phase (6 : 2 : 1 : 1 ), the results of mplc on vial 41 showed a single spot which was later called M41LM isolate in the form of a sticky yellow liquid as much as 73.9 mg. Furthermore, testing the purity of the M41LM isolate using 3 developer TLC and 2-dimensional TLC showed that the isolate was still not pure because there were still other spot patterns when sprayed with H2SO4 , to determine the secondary metabolite group in M41LM isolates, sprayed the stains from TLC with spray reagent. Lieberman Buchard, the stains give a red color which indicates that the isolate was a terpenoid group. Furthermore, M41LM isolate was monitored by densitometry to see the spectrodensitogram pattern with the result showed that the purity of the isolate was 77.75%. Characterization of M41LM isolates using 1 H-NMR (500MHz) and 13 C-NMR (125 MHz) obtained predictions of structural candidate from M41LM isolates of compounds diterpene group with sugar substitution.