Macrophylloflavone: A New Biflavonoid from Garcinia macrophylla Mart. (Clusiaceae) for Antibacterial, Antioxidant, and Anti-Type 2 Diabetes Mellitus Activities

Investigations of antibacterial, antioxidant, and anti-type 2 diabetes mellitus activities have been carried out on Garcinia macrophylla Mart. plant extract fractions. An isolate from a fraction of ethyl acetate extract was characterized with spectroscopic data. A new biflavonoid compound was found to have a skeleton of 5,7,4′,5″,7″,3‴,4‴-heptahydroxyflavanone[3-6″] flavones which was named macrophylloflavone (1). The compound was evaluated for its antibacterial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 with cephazolin as a positive control, antioxidant assay against 2,2 diphenyl-1-picrylhydrazyl (DPPH) with ascorbic acid as the positive control, and anti-type 2 diabetes mellitus treatment with metformin as a positive control. The biflavonoid compound exhibited a good inhibition for bacteria and free radical DPPH. Furthermore, biflavonoid compound treatment on the diabetic rats suggested its ability to decrease the blood glucose level. This study provided evidence that the plant has antibacterial, antioxidant, and antidiabetic properties.


Introduction
Degenerative diseases contribute the most number of world's mortalities and morbidities. According to many studies, the main factor causing the degenerative diseases is free radicals, which are actively produced through human body metabolic process [1][2][3][4]. Diabetes mellitus is an example of degenerative diseases which is infamous for the death of 1.6 million people worldwide in 2016. e disease is believed to be the main cause due to a lack of insulin secretion by the pancreas or metabolic disorders when blood glucose levels are higher (hyperglycemia) than normal levels [5]. Recent studies also found that people with the disease are more susceptible to dermatologic infections, such as staphylococcal follicular skin infections, erysipelas, and cellulitis. is is made worse by the fact that the human body is vulnerable to pathogenic bacteria, even when we carry out our daily routine [6,7]. It leads to a situation where patients take many kinds of medicines. To deal with the bacterial disease problems, many people rely on antibiotics. However, the rise of antibiotic-resistant bacteria has grown a concern to find the alternative for antibiotics [8]. In addition, synthetic drugs are often used for maintenance-free radical and diabetes mellitus, not to mention the fact that synthetic drug intake can exert multiple adverse effects on health [9,10].
As an alternative, this study proposed the use of ethnomedicinal plants which have antibacterial, antioxidant, and antidiabetic activities [11,12]. Plant-based medicines are famous for their low toxicity, minimal side effects, and high availability. e plants have potential medicinal uses 2.2. Sample Collection. G. macrophylla stem bark sample was collected from Gayo Lues Regency, Aceh Province, Indonesia (4°3′ 10,56″ N 97°24′ 18,15″ E), in September 2016. It was identified by a botanist of Research Center for Biology at Indonesian Institute of Sciences (LIPI), Bogor, Indonesia. e specimen voucher was labeled as 5/Medang Kandis. In Indonesia, the plant is called Kandis Gajah [22].

Extraction, Isolation, and Elucidation.
e sample was chopped and dried. 2.5 kg of the dried sample was extracted with maceration using methanol 96%. All filtrates obtained were concentrated with the rotary evaporator. e methanol extract (212.6 g) was sequentially partitioned with 96% nhexane (500 mL) solvent and 96% ethyl acetate (1,500 mL) solvent in a separating funnel. e solvent of the fraction was evaporated to obtain the n-hexane fraction (10.5 g), the ethyl acetate fraction (101.4 g), and the methanol fraction (100.4 g), respectively.
An ethyl acetate extract fraction (50 g) was isolated with column chromatography (v � 5 cm). Isolation was conducted using silica gel G 60 F (500 g) as stationary phase and dichloromethane-ethyl acetate as mobile phase, by the increase of polarity. e addition of polarity in the chromatography process was maintained until dichloromethane-ethyl acetate ratio reached 0:100 (v/v). Afterward, the purification to yield pure isolate was performed with rechromatography, preparative TLC, and recrystallization. A total of 60 mg of the pure isolate was elucidated by 1D-NMR (1H and 13C), 2D-NMR (HSQC and HMBC), GC-MS, FTIR, and UV-Vis spectrophotometers. e final product of this process was a biflavonoid compound 1.

Antioxidant Assay.
A free radical scavenging experiment was accomplished by modifying the DPPH reduction method [23]. is experiment was carried out using 99.9% methanol as a blank. DPPH 0.1 mM of 11 mL was prepared (0.0005 grams of DPPH crystal in 11 mL of 99.9% methanol solvent). A negative control was formulated by adding 3 mL of 99.9% methanol solvent to 1 mL of DPPH 0.1 mM. A positive control (ascorbic acid) and tested compound (compound 1) were prepared in stock beforehand with concentrations of 100 ppm (0.001 grams in 10 mL of 99.9% methanol solvent), respectively. e positive control and the tested compound stock solutions were diluted to varying doses of 2, 4, 6, 8, and 10 (ppm), respectively. A total of 1 mL of 0.1 mM DPPH solution was pipetted and added to 3 mL variation of the positive control and the tested compound doses, respectively. All solutions were incubated under conditions at room temperature in a dark room for 30 minutes, and the process of measuring absorbance was continued at a wavelength of 517 nm with a spectrophotometer. All absorbance values were calculated for percent inhibition and IC 50 .

Antibacterial Assay.
A subculture method was performed to inoculate each strain of bacteria [24]. A LB medium of 20 mL was prepared for each bacterium. A bacterium was taken from agar media stock and added to each LB medium. e bacterium in LB media was regenerated overnight at 37°C. e fresh bacterium was suspended in 10 mL NaCl 0.85% until an absorbance reached 0.5-0.8 (in order to afford 1 × 10 8 CFU/mL) on OD 600 by a spectrophotometer [25]. e antibacterial activities were carried out by the disc diffusion method [26]. 20 mL of MHA solution was poured into a Petri dish glass (size of 100 × 15 mm). Furthermore, fresh E. coli and S. aureus were smeared over agar media of the Petri dish. e paper discs containing the compound 1 2 e Scientific World Journal (doses of 30 μg/mL, 60 μg/mL, and 120 μg/mL), the positive control of cephazolin, and the negative control of DMSO were placed on the top of the growth media. Incubation was carried out at 37°C for 24 hours. e inhibition zones were measured by a caliper in millimeters (mm) as an indication of antibacterial activity.
2.6. Antidiabetic Assay. Rattus norvegicus rats (150-250 g) aged 2.5-3.0 months in healthy conditions were divided into 5 groups with 5 rats in each group. All rats were adapted for 7 days by giving pellets as food and distilled water as drinks in the cage [27]. Each rat for the experiment was given a group code, and its blood sugar level was checked initially after 7 days of adaptation (referred to as pretreatment). e group code was confirmed as follows: the negative control group was coded with B0, the positive control group was given the metformin drug coded with B1, and the groups were given the compound 1 with varying doses of 6, 7, and 8 (μg/kg body weight) given their respective codes B2, B3, and B4. Furthermore, a glucose monohydrate was injected with a dose of 150 mg/kg body weight in all rats [28]. After 7 days of the glucose monohydrate administration, blood glucose levels were measured again in these rats (referred to as diabetic blood). A diabetic rat was given oral treatment in each group once a day, namely, group B0 (given the distilled water), B1 (given the metformin), B2 (given the compound 1 at dose of 6 μg/kg body weight), B3 (given the compound 1 at dose of 7 μg/kg body weight), and B4 (given the compound 1 at dose of 8 μg/kg body weight). e blood glucose levels were checked every 7 days until they became normal again (referred to as posttreatment). Selected rats from each group at the end of the treatment were sacrificed for taking kidney, pancreatic, and liver organs in order to do histopathological analysis.

Histopathological
Studies. e kidney, pancreas, and liver organs in the treatment rats (various group codes as B0, B1, B2, B3, and B4) and normal rats without the treatment (blank control with code A) were targeted for histopathological examination. e organs were soaked for 7 days into a 10% NBF before histopathology [29]. Histopathology was carried out by staining organs that have been sliced with hematoxylin-eosin dyes and placed on glass preparation. e glass preparation organs were observed using a biological microscope to determine cell performance in the kidneys, pancreas, and liver of rats.

Statistical Analysis.
e obtained data were handled through Microsoft Excel 2010 software (Microsoft Corp., Redmond, WA). All data were presented as means ± standard deviation in the assay. e IC 50 value in the antioxidant assay was calculated based on linear regression analysis of plot concentration variation (μg/mL) to inhibition (%). Two-way ANOVA was used for the study of antidiabetic assay using GraphPad software Prism 7 (GraphPad Software, Inc., San Diego, California, USA). Matching between groups was carried out by Tukey's post hoc examination. p values <0.05 were considered significantly different. e one-way ANOVA for the study of histopathological data was run by R 3.6.2 software.

Structure Elucidation.
e substance 1 isolated from the ethyl acetate fraction of the G. macrophylla stem barks possessed yellow solid appearance. UV spectrum gave the λ max of MeOH at 260.0 nm and 274.0 nm, ascribed to the presence of π-π * electron excitation, which was typical for chromophore moiety with conjugated double bonding systems -C�C-C�C-on the aromatic rings. Meanwhile, the λ max at 331.2 nm and 355.0 nm indicated the n-π * electron excitation (i.e., presence of heteroatom or conjugated carbonyl -C�C-C�O).
e FTIR spectrum of compound 1 which was subjected directly to the GC-MS instrument exhibited the specific absorption signals corresponding to particular functional groups at respective wavenumber; they were 3252-3387 cm   e two-dimensional correlation of the HMBC and the COSY cross-peaks is displayed in Figure 1.
e signals at δ C-4″ 183.83 ppm and δ C-4 197.91 ppm were a unique attribute for carbonyl carbon at a dimeric flavonoid compound. Moreover, the signals at δ C-2 82.72 ppm, δ C-3 50.84 ppm, δ C-6 97.43 ppm, δ C-8 96.42 ppm, δ C-3″ 103.37 ppm, δ C-6″ 102.02 ppm, and δ C-8″ 99.83 ppm were a unique feature of the flavanone[3-6″] flavone skeleton pattern. It was clearly confirmed that the δ C-6″ appeared in the downfield region and the δ C-8″ existed in the upfield region. Referring to the δ C-6″ and δ C-8″ values of compounds 2, 3, and 4, the δ C-6″ value always appeared in the upfield area compared to the δ C-8″ value which exists in the downfield area where the linkage occurs in the flavanone [3-8″] flavone skeleton pattern [30,[39][40][41][42]44]. Linkage performance between C-3 in flavanone moiety and C-6″ in flavone moiety could also be observed through the HMBC relationship of compound 1 which was compared with the HMBC of compound 3 as shown in Figure 2. e overall assignment based on spectroscopic data of compound 1, by comparing data to the model compounds, demonstrated that macrophylloflavone (1) is a new biflavonoid compound with linkage flavanone[3-6″] flavone system.

Antibacterial Activity.
e classification of antimicrobial strength such as weak, moderate, and strong is indicated by the inhibition zones of <12, 12-20, and >20 mm, respectively [46]. Based on the results, the biflavonoid compound 1 was found to have strong antimicrobial activity against both the E. coli and the S. aureus bacteria. e inhibition zones in various concentrations were found to be around 16.65 ± 0.43 to 20.29 ± 0.28 mm (mean ± SD) for E. coli (30 μg/mL, 60 μg/mL, and 120 μg/mL). Meanwhile, for S. aureus, the inhibition zone was around 15.54 ± 0.39 to 23.16 ± 0.32 mm (mean ± SD). e antibacterial properties of flavonoids were based on their ability to inhibit nucleic acid synthesis, cytoplasmic membrane function, energy metabolism, and porins in cell membranes [47].

Antioxidant Activity.
e antioxidant performance of the compound 1 against DPPH free radicals gave the IC 50 values of 3.69 ± 0.29 μg/mL (mean ± SD). e strength of an antioxidant's activity is based on the IC 50 value criteria; i.e., the IC 50 value <10 μg/mL indicates very strong activity, the value 10-50 μg/mL suggests strong activity, the value 50-100 μg/mL implies moderate activity, the value 100-250 μg/mL reveals weak activity, and the value >250 μg/ mL signifies no activity [48]. In this research, the IC 50 value of compound 1 was found to be <10 μg/mL indicating a very strong antioxidant activity. Almost all flavonoid compounds are antioxidants. It is ascribed to their possession of hydroxyl groups in the flavonoids [49]. e compound 1 acted as an antioxidant agent donating its hydrogen atoms from the 4 e Scientific World Journal substituent content of the hydroxyl groups towards DPPH free radical, allowing the reduction of the DPPH radical. e reduction of DPPH radicals can be observed through the color change, from purple (DPPH) to yellow (1,1-diphenyl-2-picrylhydrazine) [50].

Anti-Type 2 Diabetes Mellitus
Activity. e compound 1 has the ability to reduce the blood glucose levels in rats with diabetes ( Figure 3). Several studies have reported that flavonoids contribute to the prevention of diabetes mellitus by regenerating the formation of β-cells pancreas which acts to produce insulin. e formation allows the decrease of glucose by converting it into energy, thus maintaining the blood glucose levels under a normal condition [51]. e results of the group treatment in decreasing the blood glucose levels are presented in Table 3 and Figure 3. e data were analyzed with two-way ANOVA to interpret the significance of the interaction effect at F (8, 40) � 146.2, p < 0.0001. e main effect between treatment (doses of compound 1) and control (negative and positive controls) referred as F (2, 40) � 2985, p < 0.0001, produces a significantly different value in blood glucose levels at the posttreatment (mean ± SD), namely, negative control (412.20 ± 7.76), positive control (98.00 ± 1.67), dose 6 μg/kg body weight (171.00 ± 3.81), dose 7 μg/kg body weight (138.00 ± 1.87), and a dose of 8 μg/kg body weight (108.40 ± 3.21). According to the American Diabetes Association, blood glucose levels >180 mg/dL indicate hyperglycemia, while the levels <100 mg/dL indicate hypoglycemia [52]. erefore, it could be assumed that blood glucose levels given by the treatment group were found to be within the normal range. e blood glucose levels in the positive control group, however, indicate the risk of hypoglycemia. e blood glucose levels produced the main effect at F ratio of F (4, 20) � 239, p < 0.0001 generating the significant force. e analysis of Tukey's post hoc test showed that the positive control has a significant different effect of compound 1 on the dose of 6 μg/kg body weight (p < 0.0001), 7 μg/kg body weight (p � 0.0049), and 8 μg/kg body weight (p � 0.0055). is means that all variations of compound 1 dosage have antidiabetic activity better than the positive control, but a very significant different result in reducing blood glucose in diabetic rats was shown by compound 1 at a dose of 6 μg/kg body weight. In other words, there were variations in the high and low doses of compound 1 have no effect in decreasing the blood glucose of diabetic rats.
Decreasing of blood glucose levels in diabetic rats was caused by the activity of flavonoid dimers in compound 1, i.e., naringenin and luteolin. Flavonoid compounds have double bonds, carbonyl groups, and hydroxy groups which are very important in the antidiabetic activity [53]. Naringenin and luteolin, in particular, have been reported to have the ability to overcome the hyperglycemia by inhibiting intestinal α-glucosidase activity, reducing oxidative stress, increasing antioxidant enzymes, and stimulating insulin secretion [54,55]. e results of the decrease in blood sugar levels obtained after administration of compound 1 conclusively proved that the macrophylloflavone (1) has antitype 2 diabetes property.       8 e Scientific World Journal e histopathological data of kidney tubular cells, pancreatic β-cells, and liver hepatocytes cell required the normal rat cells (given code A), as a benchmark in the observations. e data in Table 4 have shown changes in the number of kidney tubular cells, pancreatic β-cells, and liver hepatocytes cells.
ese were ascribed to the presence of necrosis in cells by glucose monohydrate (alloxan). Alloxan can cause significant damage to these cells by triggering the formation of free radicals that lead to diabetes in rats [56]. e administration of the compound 1 (on doses of B2, B3, and B4) reduced the necrosis in kidney tubular cells (Table 4). It was evident that the compound 1 could repair the tubular cell damage in the kidney. e number of necrotic cells in the kidney tubules in the treatment group with 8 μg/kg body weight dose (B4) revealed the similarities with the normal rat kidney tubules (A) (p < 0.05). e kidneys contribute to glucose homeostasis through the processes of gluconeogenesis, glucose filtration, glucose reabsorption, and glucose consumption [57]. Kidney gluconeogenesis is more sensitive to insulin. e condition of diabetes results in an increase in kidney glucose production that is not proportional to the amount of insulin, hence the consequent reduction in glucose absorption in the proximal tubule by the insulin-independent process [58]. Excessive glucose and oxidative stress in the kidneys cause damage to tubular cells. e antioxidant properties of the compound 1 have produced a positive effect in preventing damage to kidney tubular cells so that the appearance of tubular cells is closer to the appearance of tubules under normal conditions ( Figure 4).
An increase in the number of pancreatic β-cells has occurred in the administration of the compound 1 at various doses (B2, B3, and B4) ( Table 4). Administration of the compound 1 with a dose of 8 μg/kg body weight (B4) has to afford no difference in the number of normal pancreatic β-cells (A) (p < 0.05). Application of the compound 1 was observed to increase the formation of β-cells in the pancreas ( Figure 5). is was possible due to the compound 1 ability to neutralize free radicals that inhibit the formation of β-cells in the pancreas. Flavonoid compounds in the case of  (1) Macrophylloflavone (2)     A: the blank control, B0: the negative control, B1: the positive control, B2: the compound 1 given at a dose of 6 μg/kg body weight, B3: the compound 1 given at a dose of 7 μg/kg body weight, B4: the compound 1 given at a dose of 8 μg/kg body weight. Different letters showed significant differences between the groups (p < 0.05). 10 e Scientific World Journal diabetes were used for maintaining the function of β-cell pancreatic and increasing the reformation of β-cells in the pancreas [59]. e liver hepatocyte cell damage decreased after giving the compound 1 with various doses (B2, B3, and B4) ( Table 4). e compound 1 with dose of 7 μg/kg body weight (B3) and dose of 8 μg/kg body weight (B4) resulted in a number of liver hepatocyte necrosis cells same as the number of normal hepatocyte necrosis cells (A) (p < 0.05). e compound 1 was shown not to damage liver hepatocyte e Scientific World Journal 11 cells (not toxic) and regenerate liver hepatocyte cells in the normal range ( Figure 6). Antioxidant agents based on liver pathology are administrated to repair or prevent various liver diseases which are commonly caused by oxidative stress disorders [60].

Conclusions
e macrophylloflavone (1) as new biflavonoid compound with linkage 3-flavanone-6″-flavone has been successfully isolated from the ethyl acetate fraction from the stem bark of G. macrophylla plant. e in vitro evaluation of 1 suggested strong antibacterial and also antioxidant activities. More importantly, the in vivo assay exhibited the ability of 1 to decrease blood glucose levels in the diabetic rats to the normal level.

Data Availability
e data generated to support the findings during this study are included within this article.

Ethical Approval
Ethical clearance approval for using animals in this study was granted by the Veterinary Ethics Committee of

Conflicts of Interest
e authors declare that they have no conflicts of interest.