Antidiabetic Activity of Extract Combination of Orthosiphon aristatus and Oryza sativa L. var glutinosa

Traditionally and scientifically, research has shown that Orthosiphon aristatus and Oryza sativa L. var. glutinosa have antidiabetic activity. The combination of two medicinal plants can increase their biological activity. This study aimed to determine the antidiabetic activity of O. aristatus and O. sativa L. var. glutinosa on single and combined extracts. Phytochemical screening of the single extract was done qualitatively. The α-glucosidase inhibitory method was used as an antidiabetic activity. The results showed that every extract contained alkaloids, steroids/triterpenoids, flavonoids, tannins, quinones, and coumarins. A single extract of O. sativa L. var glutinosa, O. aristatus, and their combinations (1:1, 1:2, and 2:1) had an α-glucosidase enzyme inhibitory activity with an IC50 value of 67.82, 80.93, 73.81, 88.72, and 61.51 μg/ml, respectively. The combination shows that the ratio of 1:1 was nearly additive, 1:2 was slight to moderate antagonism, and 2:1 was moderate to slight synergism. The combination of 96% ethanol extract of O. sativa L. var. glutinosa and O. aristatus in a ratio of 2:1 was the most effective in increasing its inhibitory activity.


INTRODUCTION
Diabetes mellitus (DM) is a condition in which an increase in blood sugar levels (hyperglycemia) that caused by a decrease in insulin secretion and activity 1 . It is because of the damage in the metabolic system, especially carbohydrates, fats, and proteins. Diabetes mellitus is one of five diseases with a high number of cases and death rates worldwide. The recent report estimates that diabetes mellitus attacked more than 463 million people worldwide and around 4.2 million of them died in 2019 2 . According to the Indonesian Health Ministry report, about 16.5 million Indonesians aged over 15 years were diagnosed with diabetes in 2018. In diabetic patients, the production of free radicals will be higher due to the auto-oxidation process of glucose 3 .
Although diabetes mellitus is a chronic disease that does not cause immediate death, it can be fatal if the curing management is not proper. Management of diabetes mellitus requires drug therapy and non-drug therapy.
Acarbose, a class of α-glucosidase enzyme inhibitors, is one of the drug therapies used in diabetic patients 4 . The α-glucosidase enzyme is an enzyme that plays a role in the breakdown of carbohydrates into glucose in the digestive tract to control glucose absorption 5 11 . All the extracts were weighed using analytical balance to determine the yield.

Phytochemical screening
Qualitative phytochemical screening was performed based on the standard procedures 12 .

Antidiabetic activity assay
A total of 1 mg of an α-glucosidase enzyme from S. The inhibitory activity of α-glucosidase was calculated based on the absorption values at the wavelength of 400 nm using a UV-Vis spectrophotometer 13 . The IC50 value, whereas the extract concentration can inhibit 50% of the α-glucosidase enzyme activity, was calculated based on the linear regression equation 14 .

Extract combination analysis
The inhibitory activity of the α-glucosidase enzyme from the combined extract was calculated using the combination index 15 between BKH and KK through the following Formula 1.

Plant extraction
The percentage of yields was obtained from the ratio of the extract and the number of herbal substances. In this study, the yield of KK extract was higher than BKH extract. The solvent used for extraction is more efficient in attracting the compound in the KK than BKH extract. A high yield value can indicate the number of bioactive compounds contained. Likewise, the higher the yield percentage, the more bioactive compounds contained in 17 . Therefore, we can assume that more bioactive components in KK compare to BKH extract. Table I showed that the 96% ethanol extract weight of BKH and KK were 7.29 and 21.56 g, and the yields were 7.27% and 21.52%, respectively.

Antidiabetic activity assay
The α-glucosidase enzyme inhibitory activity from the single extract of BKH and KK and their combination (1:1, 1:2, and 2:1) were presented in Tables III to V. In this study, KK's 96% ethanol extract had an inhibitory activity of the α-glucosidase enzyme with an IC50 value of 80.93 µg/mL. This value was better than in the research conducted by Juliani et al. 9 , which reported that the IC50 value of O. aristatus butanol extract was 154.07 µg/mL. In  In carbohydrate metabolism, carbohydrates entering the digestive tract would be digested into simpler sugars and then absorbed by the small intestine. The α-glucosidase is an enzyme commonly used for in vitro antidiabetic activity assay. It is commonly found in the small intestine and converts disaccharides into monosaccharide carbohydrates 33 . Therefore, inhibition of the αglucosidase enzyme activity will reduce the breakdown of disaccharides into glucose which eventually reduces the blood glucose levels 34 . In the in vitro study, the αglucosidase enzyme hydrolyzes the substrate ρnitrophenyl-α-D-glucopyranoside to become yellow ρnitrophenyl and glucose 35 .

Extract combination analysis
The effectiveness of the combination 96% ethanol extract BKH and KK on α-glucosidase enzyme inhibition, with the Combination Index (CI) value as the analysis parameter, were presented in Table VI. The use of a combination of extracts with a low ratio is a simple initial step to determine the effect of each extract on efficacy and toxicity. This is essential as initial data to determine which extract influences the efficacy and toxicity when the extract is combined so that the ratio of the combination of extracts to be used can be estimated 36, 37 .

DATA AVAILABILITY
None.