INTRODUCTION
Diabetes mellitus (DM) is a group of metabolic disorders characterized by high blood glucose levels (hyperglycemia) and abnormalities in carbohydrate, fat, and protein metabolism. Due to disturbances or defects in insulin secretion, insulin sensitivity, or both.[ 1 ] Diabetes mellitus is also known as the silent killer because sufferers are often unaware and when it is realized, complications have occurred.
According to the 2019 International Diabetes Federation data, it is estimated that there are 223 million women aged 20–79 years living with diabetes. By 2045, the number is expected to increase to 343 million people with diabetes aged 20–79 years.[ 2 ] According to the World Health Agency, the World Health Organization (WHO) predicts an increase in the number of people with diabetes mellitus, which is one of the global health threats.
Treatment of diabetes can be done by several mechanisms such as suppressing hepatic glucose production (biguanides), stimulating insulin secretion (sulfonylureas and glinides), delaying the digestion and absorption of intestinal carbohydrates to maintain postprandial glucose levels (α-glucosidase and -amylase inhibitors), increasing receptor sensitivity. insulin and peripheral glucose uptake (thiazolidinedione and metformin) or insulin.[ 1 ] Various efforts to prevent and treat it continue to be made because most oral antidiabetic drugs provide unwanted side effects such as bloating, diarrhea, and stomach cramps, thus experts conduct research including developing various types of treatment. One of the treatments for diabetes is to find sources of treatment using natural ingredients that are relatively safe, including using plants as medicines.[ 3 ]
One of the plants that can be used to lower blood sugar levels in the community is binahong leaves (Anredera cordifolia Ten.Steenis). Binahong is a vine plant that comes from the Basellaceae family. People usually use the leaves of binahong as a medicine for skin wounds and post-operative wounds, ulcers, hypertension, inflammation, and gout.[ 4–7 ] Binahong leaves contain secondary metabolites including saponins, alkaloids, flavonoids, phenols, triterpenoids, and sterols.[ 8 ]
In addition to binahong leaves, there are stems of brotowali, which can also be used to lower blood sugar levels. Brotowali is a medicinal plant belonging to the Menispermaceae family. Brotowali is also a vine plant that lives in the tropics and is evenly distributed in Indonesian forests. Brotowali is known to contain alkaloids, flavonoids, saponins, and tannins.[ 9 , 10 ]
Furthermore, the cherry leaf is also one of the Indonesian plant known as kersen, which contains several secondary metabolites including flavonoids, triterpenoids, tannins, saponins, and glycosides. Some research on this plant showed antidiabetic activity, gout, hypertension, cough laxative, flu, headache, antiseptic, antioxidant, and anti-inflammatory activities.[ 11 ]
Based on empirical information and several studies support that binahong leaf extract (Anredera cordifolia Ten.Steenis), brotowali stem (Tinospora crispa L.), and cherry leaves (Muntingia calabura L.) can lower blood glucose levels with various active doses.
In this study, we examined the activity of the three extracts and their combination in therapeutic use for the treatment of diabetes mellitus with in vitro and in vivo methods of lowering blood sugar.
MATERIALS AND METHODS
The study was conducted in several stages, including preparation of materials, extraction, in vitro alfa glucosidase inhibitory assay, deficiency insulin assay, and histology pancreas.
Chemicals and reagents
The materials used in this experiment were glucosidase enzymes, acarbose, dilute hydrochloric acid LP, 96% ethanol, 2% HCl, NaOH, dimethyl sulfoxide, p-nitrophenyl-α-D-glucopyranoside, Na2 CO3 , Aqua Dest, 0.1 M phosphate buffer solution (pH 6.8).
Plants
Binahong leaves (Anredera cordifolia Ten.Steenis), brotowali stems (Tinospora crispa L.), and cherry leaves (Muntingia calabura L.) [Figure 1 ] were planted in the local garden in Bandung City, West Java, Indonesia. This plant was identified and confirmed by Herbarium Jatinangor, Biology Department of Padjadjaran University, Indonesia.
Figure 1: Anredera cordifolia Ten. Steenis (a) Muntingia calabura L. (b) Tinospora crispa L. (c)
Sample preparation
Binahong leaves (Anredera cordifolia Ten.Steenis), brotowali stems (Tinospora crispa L.), and cherry leaves (Muntingia calabura L.) were washed, cleaned in running tap water, cut into small pieces, and dried to constant weight using an oven at 40°C. The sample was pounded into small size using a grinder machine; it was stored in an air-tight container until when needed.
Extraction
Binahong leaf powder, cherry leaf powder, and brotowali stem powder that has been sifted were weighed as much as 200 g each, then extracted using 96% ethanol. Powder of each simplicia as much as 200 g was macerated with 2000 mL of ethanol 96% ratio (1:10) for 3 × 24 h, then filtered. The macerate was then concentrated with a rotary evaporator and weighed so that the extract yield was known.
Experimental animal
White Wistar rats aged 2–3 months and weighing 150–250 g (University of Padjajaran) were used in this study. Before animals were tested, they were adapted to new cage environments including feeding. Animal experiments were conducted according to the Commission of the Ethics of Health Research Faculty of Medicine, the University of Padjajaran Bandung (613/UN6.KEP/EC/2021).
Insulin deficiency
This method was carried out curatively using Swiss Webster mice as test animals. An animal model of insulin resistance was formed using the induction of Alloxan 55–60 mg/kg BW orally for 14 days with the parameter observed as the value of blood sugar levels. The acclimatized mice fasted for 6–8 h, and then their initial blood glucose levels were measured and then randomly divided into six groups, namely the positive control group, the negative control group, the Glibenclamide group 65 mg/Kg BB, sample extract 75, 150, and 300 mg/kg BW.
After that, alloxan was used to induce mice in all groups except the negative group (−). After 3 days of induction, therapy was given for 14 days orally. On day 3rd (t0 ), 7th (t7 ), and 14th (t14 ). days the parameters blood glucose levels were measured.[ 12 , 13 ]
Alfa glucosidase inhibitory activity assay
The α-glucosidase enzyme plays a role in the process of converting carbohydrates into glucose; therefore, if there is inhibition of the activity of α-glucosidase it can lower blood sugar. Enzyme inhibitory activity was tested using 50 μL of phosphate buffer solution, added 25 μL of test sample/acarbose solution, added 25 μL of 0.1 M phosphate buffer (pH 6.8) containing glucosidase solution (0.6 U/mL) incubated in 96-well plates at 37°C for 15 min. After pre-incubation, 25 μL of 15 mM p-nitrophenyl-α-D-glucopyranoside (pNPG) in phosphate buffer (pH 6.8) was added to each well and incubated at 37°C for 15 min. Then, the reaction was stopped by adding 150 μL of 0.2 M NaCO3 to each well, and the absorbance reading was recorded at a wavelength of 405 nm with a microplate reader.
RESULTS AND DISCUSSION
In this study, we tested alfa glucosidase inhibitory activity and insulin resistance from Binahong leaf, cherry leaf, and brotowali stem extracts and their combinations.
The powdered simplicia of binahong leaves, cherry leaves, and brotowali stems were each weighed as much as 200 g and then extracted by maceration with a ratio of 1:10. The principle of maceration is that the liquid will penetrate the cell wall and enter the cell. The extraction method chosen was maceration because it is easy and uses simple tools and can avoid damage to compounds that are not heat resistant. Maceration was carried out for 3 × 24 h using 96% ethanol as solvent. Liquid extracts then were concentrated by evaporation of the solvent until the extract became thick. Binahong leaves, cherry leaves, and brotowali stem thick extract, respectively, were obtained at 16.705 g, 31.78 g, and 27.82 g. Table 1 shows the extract yield.
Table 1: Extract rendemen
Insulin deficiency
This test was carried out curatively where the test animals were induced first and then treated. The comparison drug used was glibenclamide with its mechanism of increasing insulin release from the pancreas. In this method, alloxan was used at a dose of 55–60 mg/kg BW to destroy the pancreatic cells in the insulin-deficient animal model.[ 12 ] Alloxan was administered intravenously to the tails of mice in all groups except the negative control group (−). After 3 days of alloxan induction, fasting blood sugar levels were checked in mice using the Easy Touch® glucometer. Animals used for research included animals with blood glucose levels reaching 200 mg/dL (t0). The test animals to be studied were divided into several groups, namely group I as a negative control, group II as a positive control, group III as a comparison using glibenclamide 0.65 mg/Kg BW, and group IV–VI combination extracts. For treatment, it was given orally for 14 days. During therapy, fasting blood glucose levels will be checked on days 3(t0), 7(t7), 11(t11), and 14(t14). Blood sampling was carried out by piercing the venous blood flow using a lancet on the tail of the mice.
In vivo antidiabetic test results method of insulin deficiency are shown in Table 2 . The results showed a decrease in levels of blood glucose that varied enough to give a fairly large standard deviation. In general, the whole test group showed a decrease in glucose levels in blood on the 14th day compared to the condition beginning, except in combination 3. Based on the control group’s positive data, the test animal still showed hyperglycemia until the end of day 14; so, this alloxan diabetes method is suitable for use as an animal model for testing antidiabetic activity.
Table 2: Insulin deficiency
In the results of the test using the insulin deficiency method, ethanol extract of cherry leaves, binahong leaves, and stem brotowali had good antidiabetic activity, which showed a decrease in blood glucose in mice on 14th day as well as the administration of glibenclamide as a standard. Animals that have been tested for 19 days were sacrificed, two from each group, then the pancreas was isolated and Gomori staining was performed using victoria blue dye and floxin.[ 13 ] Gomori staining results are shown in Figure 2 .
Figure 2: Pancreas histology image. Negative control (a) Positive control (b) Standard (c) Combination1 (d) Combination 2 (e) Combination 3 (f)
Parameters observed to assess the success of therapy can be seen from the average area islets of Langerhans, the number of alpha cells, and the number of beta cells in the islets of Langerhans in units of the same area [Figure 2 ]. Alpha cells have a portion of about 20%, whereas beta cells have a portion of about 75% of the islets of Langerhans. Alpha cells produce the hormone glucagon, whereas beta cells produce insulin. There is improvement in therapy indicated by the high number of beta cells, on the contrary, the high number of alpha cells will aggravate the condition of diabetes mellitus. The results of histologic observations can be seen in the following image:
Inhibitory α glucosidase activity
The inhibitory activity of the alpha-glucosidase enzyme was tested using the acarbose standard. It aims to compare the IC50 value of acarbose with the test sample. Acarbose was chosen as a comparison because it is an antidiabetic drug that works by inhibiting glucosidase. In addition, in terms of structure, acarbose has a similar structure to the substrate p-nitrophenyl-α-D-glucopyranoside (p-NPG).[ 14 ]
This research was conducted at pH 6.8 and a temperature of 37°C, adjusted to the optimum reaction temperature enzymatic. Reduction of enzyme activity can be caused by storage factors after production, which do not meet the criteria listed on the enzyme label, such as exposed sunlight, storage temperature too high, and other things that can trigger loss of enzyme activity. Enzyme activity determined in international units, namely the number of enzymes that catalyze the formation of 1.0 mol D-glucose from p-nitrophenyl-α-D glucosidase at pH 6.8 and 37°C for 1 min.
Incubation was carried out in two stages, first incubation for 20 min to give time for the test solution to reach a temperature of 37°C, and the second stage of incubation for 20 min, which is incubation for enzymatic reactions. Then, the reaction was stopped by adding natrium carbonate. The product of the reaction between glucosidase and p-nitrophenyl-α-D-glucosidase is p-nitrophenyl, which is yellow; thus, this product can be measured with a microreader at 425 nm wavelength. To correct the absorption generated, the measurement was carried out by blank absorption by replacing the position enzymes with buffers. It is intended to see the absorption given by the test sample without an enzymatic reaction. The magnitude of the inhibition of enzyme activity was seen from the decrease in p-nitrophenyl, which formed when compared to the activity of early enzymes. The magnitude of the inhibitory activity of each concentration range is seen from the percentage of the resulting inhibition; the greater the percentage of inhibition produced, the more inhibited many enzyme activities.[ 15 , 16 ]
In this test, sample absorbance and blank absorbance were measured. The treatment for measuring the absorbance of the sample and blank was the same, using sample control and blank control as a correction factor for the sample and blank absorbance values because the color of the extract can also provide absorption at that wavelength.[ 15 ] The absorbance of p-nitrophenol formed was measured at a wavelength of 405 nm.
The enzyme inhibitory activity test was carried out on the ethanol extract of binahong leaves, cherry leaves, and brotowali stems and their combinations. Each extract was weighed as much as 10 mg and added DMSO (dimethyl sulfoxide), which serves to help dissolve the extract as much as 100 L in the micropipette and made up to 10 mL with phosphate buffer pH 6.8 to obtain a concentration of 1000 g/mL. Then, the dilution was carried out with phosphate buffer pH 6.8 to obtain a concentration of 400 g/mL; 200 g/mL; 100 g/mL; 50 g/mL; 25 g/mL. The results of the glucosidase activity inhibition test from the sample and acarbose are shown in Figure 3 .
Figure 3: Inhibition α glucosidase activity extract
After obtaining the IC50 value of each extract [Table 3 ], further testing was carried out on the combination of extracts, where the concentration of each extract in the combination was 15 g/mL (equivalent to an average of half the IC50 value of each extract).
Table 3: Inhibitory α-glucosidase activity
From the combination of extracts result, it was found that the combination of extracts of cherry: binahong 1:1, 1:2, and 2:1, binahong: brotowali 1:1, 1:2, and 2:1 can inhibit alpha-glucosidase enzymes successively in 85.11; 88.58; 79.48; 85.29; 83.49, and 91.29% [Figure 4 ].
Figure 4: Inhibition α glucosidase activity combination extract
The best combination in inhibiting the alpha-glucosidase enzyme is a combination of extracts binahong: brotowali 2:1 with the highest inhibition of the alpha-glucosidase enzyme at 91.29%.
CONCLUSION
The combination of binahong leaves (Anredera cordifolia Ten.Steenis) and cherry leaves (Muntingia calabura L. and the combination of brotowali stems (Tinospora crispa (L.) and binahong leaves showed in vivo antidiabetic activity with insulin deficiency method. The combination of these extracts was able to reduce blood sugar levels until the observation on day 14. Meanwhile, the combination of brotowali stems and cherry leaves could not reduce blood sugar levels in in vitro tests.
In vitro testing by inhibiting alpha-glucosidase enzymes, both binahong leaves extract, brotowali stems, and cherry leaves were able to inhibit alpha-glucosidase enzymes at IC50 , respectively, that is, 35.07 ± 2.35; 29.42 ± 1.40; and 26.63 ± 1.15 µg/mL. The best combination of extracts by in vitro and in vivo methods was shown in the combination of binahong leaf and brotowal stem extract binahong leaves, brotowali stems (2:1). This research needs to be continued with making the preparations containing binahong leaf-brotowali stem extract and binahong leaves-brotowali stems extract (2:1).
Financial support and sponsorship
Bhakti Kencana University Research Funding.
Conflicts of interest
There are no conflicts of interest.
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