In-vitro studies on antioxidant and antidiabetic potential of Sesoot ( Garcinia picrorrhiza Miq.) fruit ethanolic extract from Indonesia

Diabetes mellitus (DM) is a disease that can be identified by high levels of blood glucose. Garcinia plants have been widely used for many traditional medicines as antioxidant, anticancer, antidiabetics, and antiinflammation. The antioxidant and antidiabetic activities of ( Garcinia picrorrhiza Miq.) or sesoot fruit extract were evaluated in this study and compared with xanthone. The antioxidant and antidiabetic of ethanolic ripe sesoot ( G. picrorrhiza Miq.) fruit extract (GpKar) was evaluated by 2,2-Azinobis 3-ethyl benzothiazoline 6-sulfonic acid (ABTS •+ ) reducing activity, α-Glucosidase, β-Glucosidase, and α-amylase inhibitor activity. GpKar showed higher ABTS •+ -reducing activity (IC 50 = 49.30 µg/mL) than xanthone (IC 50 = 404.30 µg/mL). GpKar showed IC 50 = 109.32 µg/mL for α-glucosidase inhibitory activity, while xanthones had a better activity (IC 50 = 33.97 µg/mL). GpKar also showed lower α-amylase inhibitory activity and β-Glucosidase (IC 50 = 126.01 and 9432.09 µg/mL) compared to xanthone (IC 50 = 44.32 and 405.03 µg/mL, respectively). The compounds of GpKar are proven to have antioxidant and antidiabetic activities. Therefore, it will be industrially relevant to develop a natural medicine for decreasing DM risk, thus evaluating the antioxidant and antidiabetic effect of G. picrorrhiza by a pre-clinic study is needed.


INTRODUCTION
In 2019, there are about 463 millions people around the world have been diagnosed with diabetes. Moreover, the International Diabetes Federation (IDF) said that diabetes cases are anticipated to grow higher up to 700 millions by 2040 (Pasupuleti et al., 2020). Diabetes is a disease stimulated by high levels of blood glucose (hyperglycemia). Hyperglycemia is a condition in which insulin secretion and/or insulin action experiences some defects (American Diabetes Association, 2013). Diabetes is divided into two types and Type 2 diabetes mellitus (T2DM) can lead to various illnesses and premature death (Pasupuleti et al., 2020). Defect of insulin action and production leads to long-term health problems. In addition to insulin, defects of carbohydrates, lipids, proteins metabolisms also play a role in health problems (American Diabetes Association, 2013). α-amylase and α-glucosidase are enzymes that stimulate the starch breakdown which is the main source of increased blood sugar levels. Blood glucose levels can be decreased by inhibiting the enzymes and it is an essential strategy in diabetes (Lordan et al., 2013;Widowati et al., 2018).
Diabetes can cause activation of several pathological diseases, chronic hyperglycemia, and free radical generation (Yaribeygi et al., 2019). Free radicals can be neutralized by electron donation from antioxidants. Catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD) are some antioxidants that counter the deleterious effect of free radicals. These antioxidants are produced in the body or provided through the diet. Furthermore, traditional medicinal plants also have antioxidants (Kumar et al., 2021). Antioxidant plays a protective role in diabetic pathologies and cardiovascular disease.
Garcinia is one of the Clusiaceae family which is native to Asia, Australia, and tropical regions. Garcinia fruit is a nutrient-rich source, minerals, vitamins, and dietary fiber. In several studies, Garcinia plants have been widely used as a traditional natural medicine for antioxidant, anticancer, antidiabetic, and antiinflammation, due to their good source of bioactive compounds (Aisha et al., 2013;Murthy et al., 2019). G. picrrorhiza Miq. grows in tropical to temperate climates and in the original region known as sesoot (Utami et al., 2017). It contains bioactive compounds including xanthones, benzophenones, hydroxycitric acid, and anthocyanins (Espirito Santo et al., 2020). Xanthones are one of the compounds highly found in natural sources, including plants, fungi, ferns, and lichens. Xanthones could be found in several plant families, including Garcinia. Their pharmacological properties have raised a great interest (Negi et al., 2013) and xanthones themselves are polyphenolic compounds that have been widely used in the treatment of DM (Putri, 2015).
Based on the previous study, the extract of G. picrorrhiza Miq. has antioxidant activity and anticholinesterase activity which is potentially used to decrease Alzheimer's disease progression (Utami et al., 2017). Another study related to the Garcinias reported that extract of G. mangostana L. has antidiabetic activity by decreasing cholesterol levels and lipidic peroxidation (Husen et al, 2017); however, the effect of G. picrorrhiza Miq has not been much reported. The genus Garcinia has more than fifty species. However, G. picrorrhiza Miq. is one of the species which is rarely studied. Based on these facts, more comprehensive studies are needed to investigate another bioactivity on G. picrorrhiza Miq. extract. The potential of ripe fruit G. picrorrhiza Miq. for DM can be investigated by evaluating the antioxidant activity with the ABTS •+ method, while the antidiabetic activity was evaluated by α-glucosidase, α-amylase, and β-Glucosidase assay.

Method Preparation of G. picrorrhiza extract
Ripe fruits of G. picrorrhiza Miq. (1330 g) were ground and immersed in ethanol 70% for 24 hours. The extraction was carried out twice. This procedure was redone until the colorless filtrate was obtained. The collected filtrates were subsequently evaporated at 40˚C using a rotatory evaporator, and the extract (GpKar) was kept at -20 ℃ (Prahastuti et al., 2020;Rusmana et al., 2017;Widowati et al., 2016Widowati et al., , 2018.
Formula 1 was used to calculate α-glucosidase assay as below: (1) Where C = absorbance value of the negative control S = absorbance value of the sample
A microplate reader was used to calculate the absorbance at 405 nm while the inhibition of β-Glucosidase was evaluated according to formula 2: (2) Where C = absorbance value of the negative control S = absorbance value of the sample α-amylase inhibitory activity assay The assay of α-amylase inhibitory activity was done according to the breakdown of the substrate to generate a colored product and the absorbance was measured over a certain period (Utami et al., 2019;Widowati et al., 2015Widowati et al., , 2018. For this assay, dimethyl sulfoxide (DMSO) was used as the blank, 30 µL of GpKar,xanthone (20.83,41.67,83.33,166.67,333.33,and 666.67 µg/mL) and 10 µL of αamylase enzyme 250 units/g (Sigma Aldrich 7595) were put into the well. The reaction mixture was then incubated for 10 minutes at 37˚C. For the treatment group, 40 µL starch solution was added after incubation while for the control group 40 µL phosphate buffers were supplemented. The reaction mixture was again kept for another 15 minutes at 37˚C. The 100 µL acidic iodine solution (except black) was also added to terminate the enzymatic reaction after incubation. Using a microplate reader, the absorbance was then measured at 565 nm (Utami et al., 2019;Widowati et al., 2018). Formula 3 was used to calculate α-amylase assay, seen as below: (3) Where C = absorbance value of the negative control S = absorbance value of the sample

Data Analysis
All data measurements were done three times. Data are shown as means ± standard deviation. In this study, the statistical analysis was performed using the SPSS Statistics 20.0 computer program. The analysis used the One Way ANOVA, followed by Tukey's HSD Post Hoc Test. The significance value was determined with a p-value of ≤ 0.05.

RESULT AND DISCUSSION
Sesoot fruit is one of those natives to Asia and Africa which contains many bioactive compounds including xanthones, flavonoids, triterpenoids, and benzophenones (Gutierrez-Orozco and Failla, 2013). Xanthones are polyphenol groups commonly found in mangosteen. Xanthones contain α-mangostin, γ-mangostin, and another minor compound (Mohammad et al., 2019). Xanthones from Garcinia fruit were known to exhibit pharmacologic properties such as antioxidant (Gondokesumo et al., 2019), antidiabetic (Ewenighi et al., 2015), and anti-inflammatory (Mohan et al., 2018). The ABTS •+ -reducing activity of GpKar and xanthone is shown in Figure. 1, and the IC 50 of samples in ABTS •+ -reducing activity are shown in Table 1.  Based on Figure 1, both GpKar and xanthone showed an ABTS •+ reducing activity percentage which depended on the concentration-manner. The higher concentration of the sample shows the higher ABTS •+ reducing activity. Therefore, as seen in Table 1, GpKar was shown to have a lower IC 50 value at 49.30 µg/mL than xanthone at 404.30 µg/mL. The IC 50 is the the sample concentration to collect 50% of the free radical. The lower the IC 50 value indicates the higher antioxidant activity, and GpKar was categorized highly-active antioxidant (Marjoni and Zulfisa, 2017).
This result conforms with the previous study where ABTS •+ antioxidant activity of methanolic extract G. talbotii from India had an IC 50 value of 40.09 µg/mL (Kureshi et al., 2020) which was in line with its ABTS •+ -reducing activity. Meanwhile, our previous study revealed that the H 2 O 2 inhibitory scavenging activity of GpKar had a higher activity than xanthone (Utami et al., 2017). The high antioxidant activity of GpKar is probably caused by secondary metabolites. The major secondary metabolites in mangosteen are xanthones and more than 68 xanthones derivatives are isolated. The αmangosteen and γ-mangosteen are the major xanthone derivates that show powerful antioxidant activity (Aizat et al., 2019;Tjahjani and Widowati, 2013).
Plant extracts are commonly used to treat various diseases including diabetes because of their effectiveness, safety, and abundant sources (Taher et al., 2016). The α-glucosidase inhibitors delay the digestion as well as the complex carbohydrates absorption to keep blood glucose at lower levels (Nisha, 2017). The αglucosidase and β-Glucosidase inhibitors are promising therapeutic agents, such as for diabetes (Kumar et al., 2011), breast cancer (Zhou et al., 2017), antitumor (Li et al., 2018), hepatitis B, and human immunodeficiency virus (HIV) (Abid et al., 2016). The result of the αglucosidase inhibitory activity assay of GpKar and xanthone can be seen in Figure 2. Table 2 illustrates the results of the α-Glucosidase, β-Glucosidase, and α-amylase inhibitory activity by the IC 50 value.  Based on Figure 2, both GpKar and xanthone showed α-glucosidase inhibitory activity depending on concentration-manner. Based on Table 2, GpKar showed higher IC 50 value (109.32 µg/mL) compared to xanthone (33.97 µg/mL); xanthone was categorized highly active (Marjoni and Zulfisa, 2017). Based on Figure 3, both GpKar and xanthone showed β-Glucosidase inhibitory activity depending on concentration-manner. Based on Table 2, GpKar showed a higher IC 50 value (126.01 µg/mL) than xanthone (44.32 µg/mL) in inhibition of β-Glucosidase. This result indicates that GpKar has a lower β-Glucosidase inhibitory activity than xanthone. Xanthones are categorized as highly active in β-Glucosidase activity based on these results (Marjoni and Zulfisa, 2017). Xanthones from mangosteen have been proven to suppress glucose absorption and potential compounds for in vitro αglucosidase inhibition (Ryu et al., 2011). The extract of G. mangostana pericarp demonstrated a mild lowering effect on glucose level in normoglycemic animals, which was comparable with glibenclamide (standard drug) (Taher et al., 2016). The antioxidant compounds such as epicatechin and xanthones may play a role in this effect. Therefore, G. mangostana extract is also a strong candidate for DM management (Taher et al., 2016). Based on Figure 4, the α-amylase inhibitory activity of GpKar and xanthone showed a concentration dependent-manner. Table 2 shows that the IC 50 value of GpKar was higher (9432.09 µg/mL) than xanthone (405.03 µg/mL). Xanthone was categorized weak in α-amylase inhibitory activity and GpKar was categorized inactive (Marjoni and Zulfisa, 2017).
There was a report stated that extract of G. mangostana can deaccelerate the absorption of glucose by inhibiting the carbohydrate hydrolyzing enzymes like α-amylase (Manaharan et al., 2012). The result in this present study has been in line with the previous study. Inhibition of amylase and glucosidase enzymes is proven to lower the postprandial elevation of blood glucose after a mixed carbohydrate diet (Thamizharasan and Umamaheswa, 2016). Meanwhile, G. kola possesses hypoglycemic properties and significantly lowered blood glucose in alloxan-induced diabetic rats (Ewenighi et al., 2015).
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are included to free radicals. Unstable electrons in free radicals react with lipids, proteins, and/or DNA resulting in various damages. Oxidative damage happens when free radicals and antioxidant agents are imbalanced (Ayepola et al., 2014). However, oxidative stress affects various diseases such as diabetes, vascular and neural disorders (Pasupuleti et al., 2020). Various mechanisms of oxidative stress play a role in diabetes, including excess oxygen radicals, glycated protein formation, and the glycation of antioxidant enzymes. This will limit the ability of antioxidants to prevent free radicals (Pasupuleti et al., 2020). The previous study showed that various group compounds such as phenolic, flavonoids, anthocyanins, and carotenoids could have a contribution to the antioxidant activity in the extracts of G. parvifolia fruit (Hassan et al., 2013). Moreover, Hassan stated that the sum up phenolic content strongly correlated with the activity of antioxidant (Hassan et al., 2013). The quality of antioxidant properties relates to various factors, and the major factors that affect the activities are the maturation stage, extraction solvent, and materials preparations (Suttirak and Manurakchinakorn, 2014).

CONCLUSION
The extract of sesoot fruit has highly active antioxidants while xanthone has weak antioxidant properties. The extract of sesoot has moderate α and β-Glucosidase inhibition and inactive α-amylase inhibition properties. On the other hand, xanthone has highly active α and β-Glucosidase inhibition and weak α-amylase inhibition properties. Further research to develop its activity in antidiabetic prevention by pre-clinic studies is needed.