| dc.description.abstract | Diabetes is a metabolic disease characterized by a high blood glucose level on fasting, which is above 126 mg/dL. Type-2 diabetes mellitus (T2D) was dominant (90%) among three types of diabetes (type-1 diabetes mellitus, type-2 diabetes mellitus, and gestational diabetes mellitus). Hyperglycemic conditions characterize the T2D traits as the result of abnormalities in insulin secretion and activity. These conditions may trigger the accumulation of free radicals. Therefore, consuming food with antihyperglycemic and antioxidant therapies might be a recommendation for DT2 patients’ treatments. Handling of DT2 sufferers can be done by consuming foods that contain antihyperglycemic and antioxidants, including karamunting (M. malabathricum) leaves which the people of West Kalimantan have often used as herbal tea given to diabetics, which is brewed with hot water. One of the taste described by the community from the brewed water of karamunting leaves (ASDK) is astringency.
Previous studies have proven the antidiabetic activity of karamunting leaves by in vivo testing of ethanol and methanol extracts. Chemical compounds that act as antidiabetic, their application in diabetic animal models, and the sensory profile of the karamunting leaves, especially when consumed in brewing water, have not been reported. This study has the general objectives of identifying bioactive antidiabetic compounds with the metabolomic method, knowing the mechanism of antidiabetic in vivo using diabetic animal models, and evaluating the effect of various forms of brewing karamunting leaves (Melastoma malabathricum) on sensory profiles by analyzing multivariate (sensometric) data and their antidiabetic activity.
The study was divided into three steps as follows: 1) Obtaining an LC-MS/ MS-based fingerprint profile from water extract of karamunting leaves, identifying antidiabetic bioactive compounds (antihyperglycemic and antioxidant) from brewed water of karamantung leaves with a metabolomic approach based on LC-MS/MS and molecular docking methods, and determining the best brewing conditions for karamunting leaves, and; 2) Studying the mechanism of ASDK antidiabetic activity with diabetic animal models; and: 3) Identifying and mapping the ASDK based on their sensory attributes using the sensometric method.
Our study was initiated by analyzing the fingerprint profile based on LC-MS/MS, antioxidant activity (DPPH), and toxicity (brine shrimp lethality test / BSLT) of the water extract of karamunting leaves at various extraction conditions with water temperature (room temperature and hot temperature at room temperature). 90 ± 2 ºC) and different times (15, 30, 45 minutes). Six compounds namely, 4-O-caffeoylquinic acid, quercimeritin, digiprolactone, 3-O-trans-couma-roylquinic acid, norbergenin, and arteamisinine I were identified in the aqueous extract of karamunting leaves. Karamunting leaf water extract with hot water (90 ± 2 ºC) at a low dry leave:water ratio (5:100 w/v) for 15 minutes has high antioxidant activity and low toxicity. Therefore, brewing with hot water and a brewing time of under 15 minutes, i.e., 5 minutes, was used for further experiments. Furthermore, the LC-MS/MS profiles of the ASDK obtained from twelve different brewing conditions (with or without ultrasonication; dry leave:water ratio of 20:100, 7:100, and 3:100 (w/v); brewing times of 5 and 15 minutes) were used as variables in the identification stage of antidiabetic compounds. (antihyperglycemic and antioxi-dant) with a metabolomic approach. The antihyperglycemic activity was carried out in vivo and in vitro. In vitro study used the Oral Glucose Tolerant Test (OGTT) and in vitro analysis by anti-α-amylase and anti-α-glucosidase investigation. Antioxidant activity testing was carried out in vitro (DPPH and FRAP).
Data analysis using principal component analysis (PCA) followed by orthogonal projections to latent structure (OPLS) succeeded in identifying 26 compounds that were positively correlated with antidiabetic activity. Based on the results of the OGTT test, the best brewing conditions were determined from the lowest AUC mg/dL glucose value obtained from the brewing conditions without ultrasonication with a dry leave:water ratio of 7:100 (w/v) for 5 minutes (T0715). The extract from the brewing condition also had high anti α-amylase and antioxidant activity (DPPH radical scavenging activity and potential FRAP), while moderate activity was as anti α-glucosidase. The extract was characterized by a high content of compounds correlated with antidiabetic activity, namely epicatechin, citric acid, and isoquercetin. Confirmation by molecular docking studies showed that epicatechin and citric acid have a non-competitive inhibitory mechanism against α-glucosidase enzymes. The exact mechanism is also shown isoquercetin against α-amylase enzyme. The molecular docking test results show a potential synergism of insulin-mimetic activity between epicatechin and citric acid in lowering blood glucose levels where the two compounds interact with insulin receptors in the kinase-linked receptors (KLRs) domain.
In the second step of the study, an in vivo test was conducted to determine the antidiabetic mechanism with a diabetic animal model, namely rats of the Sprague Dawley strain (10% fructose at libitum for two weeks and induced by streptozotocin 40 mg/kg) which were treated with the ASDK (prepared according to Phase 1 results) 125 mL HED at different doses (1 to 3 times). The antidiabetic mechanism was determined based on the results of the analysis of the pancreas and the profiles of glucose, lipids, amino acid cleavage enzymes, and antioxidant activity of animal blood models. The frequency of administration 3 times/day for 21 days in diabetic model animals, in general, had conditions that were not significantly different based on multivariate analysis (OPLS-DA) with normal animal models (negative control). These conditions include fasting blood glucose levels, HbA1c, cholesterol, triglycerides, low AST and ALT enzyme activity and high HOMA-. Based on this, the brewed water of karamunting leaves, besides being an antihyperglycemic and antioxidant, also shows its potential as an antihyperlipidemic. The insulin-mimetic activity of ASDK was also confirmed at this stage, namely by a decrease in blood glucose levels which was not always followed by high insulin levels.
In the third stage of the study, the sensory profile of the steeping karamunting leaf was conducted using qualitative descriptive analysis (QDA) and multivariate data analysis. The samples used were infusions prepared at high dry leave:water ratio (7:100 w/v) for a short time (5 minutes) and low dry leave:water ratio (3:100 w/v) for a longer time (15 minutes). The brewing methods used were: conventional hot brewing with high (T7) and low dry leave:water ratio (T3); cold brewing with high dry leave:water ratio (C7); hot brewing with a drip bag with high dry leave:water ratio (D7); and hot brewing in tea bags with low dry leave:water ratio (B3). Commercial herbal tea in tea bags with low dry leave:water ratio (K3) was used as a comparison. Karamunting leaf brewing water T7 has a preference score among other brewing methods (the highest score is samples C7, D7, T3, B3) and K3 (lowest score). Sample T7 has sensory harshness attributes, which are positively correlated, and stew and astringency, which are negatively correlated with liking level. Chemical analysis of total phenol (TPC) and total flavonoid (TFC), as well as in vitro antidiabetic activity (antihyperglycemic and antioxidant), were also carried out to study their effect with various brewing methods In addition to sample T7 (the best brewing condition from Stage 1/T0705), other brewing methods also have the potential to be developed with different functional characterizations, but lower and the level of preference is better than T7. The results of OPLS-DA show that the brewing method T3 (code T0315 in Phase 1) and C7 has functional properties as anti α-amylase, B3 as anti α-glucosidase, and D7 as antioxidant (DPPH radical scavenging activity and potential FRAP).
The results of this study indicate that the antidiabetic activity of karamunting leaves can be obtained by conventional brewing, namely 7 g of dried leaves added to 100 mL of hot water (temperature of 90 + 2 oC which can be achieved by boiling water and then allowed to stand for 1 minute), mixed and allowed to stand for 5 minutes, then filtered. This brewed water has a description of harshness and astringency as well as a functional effect as an antidiabetic when consumed 125 mL with a frequency of 3 times / day for 21 days. Another brewing method, using a drip bag, has functional characteristics as an antioxidant, so it can be recommended for non-diabetic conditions. | id |
