Diabetes mellitus, commonly known as diabetes, is a metabolic disorder causing high blood glucose and can lead to serious complications such as cardiovascular disease, stroke, chronic kidney disease, and nerve and vision damage [
Nature has been a generous source of bioactives for medicinal chemistry.
The plant sample was grown and harvested in September 2018 in Hoai Duc province, Vietnam. The plant was identified as
The separation procedure of the ethyl acetate extract.
Compounds isolated from the ethyl acetate extract. (a) EPL1: lupeol, (b) EPL2: friedelan 3-one, (c) EPL3: quassin, (d) EPL4: 17-hydroxy quassin, (e) EPL5: (+)-catechin, (f) EPL6: epicatechin, (g) ELP7: 7-
Swiss mice weighing 18–22 g were provided from the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam, and were humanely treated in accordance with the Ministry of Health Guidelines for the Care and Use of Laboratory Animals. The protocols were approved by the Ethics Committee of VNU Pharmaceutical Chemistry Laboratory (permit number: A 2019–0033). Animals were kept 10 mice per group of a total of 8 groups and divided into two experiments: the high-fat diet-fed (HFD) group with 70 animals (groups 1–7) and the normal control with 10 animals (group NC). The animals were housed in clean metabolic cages placed in a well-ventilated house with an optimum condition (temperature: 25 ± 2°C; photoperiod: 12 h natural light and 12 h dark; and humidity: 45–50%). The cleaning of the cages was done on a daily basis. The HFD group were provided with a high-fat diet consisting of carbohydrate (40%), fat (30%), protein (20%), and other vitamins and minerals (5%). The NC mice were given a standardized diet consisting of protein carbohydrate (53%), protein (20%), and fat (6%). Body weight was recorded weekly. After 8 weeks, HFD mice show a significant increase in body weight (158%–204%) compared with NC mice (78%), as shown in Table
Body weight of mice in the high-fat diet-fed group (HFD) and the normal control group (NC) group before and after 8-week diet.
Group | Week 0 (g) | Week 8 (g) | % increase |
---|---|---|---|
1 | 18.91 | 53.72 | 184.08 |
2 | 19.22 | 52.31 | 172.16 |
3 | 18.73 | 50.24 | 168.23 |
4 | 18.96 | 54.85 | 189.29 |
5 | 19.92 | 51.46 | 158.33 |
6 | 18.15 | 55.24 | 204.35 |
7 | 18.97 | 51.92 | 173.69 |
NC | 18.78 | 33.52 | 78.48 |
Mice whose body weights were over 50 g in the HFD groups (cages 1–6) were injected with STZ (120 mg/kg bw). The high-fat-fed NCF mice (average body weight 33.52 g) were added with physiological saline only. The blood glucose level in both STZ-induced diabetic group and control group was measured by the One Touch Ultra device before the STZ injection and at 48 h, 72 h, 5 days, 7 days, and 10 days after the injection [
Blood glucose level of STZ-induced diabetic group and control group (saline added).
Group time | Control group (saline) (mmol/l) | STZ-induced diabetic group (mmol/l) |
---|---|---|
0 h | 6.4 ± 0.7 | 10.1 ± 0.1 |
48 h | 6.2 ± 0.3 | 16.9 ± 0.8 |
72 h | 5.9 ± 0.6 | 20.2 ± 0.7 |
5 days | 5.8 ± 1.2 | 21.7 ± 0.2 |
7 days | 5.3 ± 1 | 25.8 ± 4.2 |
10 days | 5.7 ± 0.8 | 24.4 ± 0.6 |
Significantly different from blood glucose before STZ injection (
After 10 days of STZ injection, mice with blood glucose levels >18 mmol/L were considered type 2 diabetic [
Treatment and dose for STZ-induced diabetic mice in Cages 1–6.
Cage | Treatment | Dose |
---|---|---|
1 (normal control diabetic) | Saline 0.9% | 10 mL/kg |
2 (positive control) | Glucophage | 70 mg/kg |
3 | EP | 400 mg/kg |
4 | EP3 | 400 mg/kg |
5 | EP4 | 400 mg/kg |
6 | EP6 | 400 mg/kg |
Mice in those cages were given the treatments at exactly 8 a.m. daily. The blood glucose level in each cage was measured before and at 2 h, 4 h, 8 h, 3 days, and 15 days after the oral intake. The results are shown in Figure
Blood glucose level of STZ-induced diabetic mice after treatments.
Blood glucose level of STZ-induced diabetic mice after treatments.
0 h | 2 h | 4 h | 8 h | 3 days | 15 days | % blood glucose decrease | |
---|---|---|---|---|---|---|---|
Cage 1: (NCd) NaCl 0,9% | 26.5 ± 1.1 | 28 ± 1.8 | 28.1 ± 1.3 | 29.3 ± 1.9 | 28.3 ± 0.8 | 31.1 ± 1.1 | 0 |
Cage 2: glucophage | 21.9 ± 1.5 | 23.1 ± 1.8 | 15.5 ± 0.7 | 14.1 ± 3.1 | 9.7 ± 2.2 | 7.8 ± 1.5 | 64.38 |
Cage 3: EP | 23.9 ± 0.9 | 20.1 ± 2.3 | 17.3 ± 5.3 | 14.3 ± 2.1 | 14.2 ± 0.9 | 14.4 ± 0.8 | 39.74 |
Cage 4: EP3 | 27.1 ± 3.2 | 14.5 ± 2.1 | 23.2 ± 2.4 | 15.4 ± 0.3 | 16.5 ± 1.2 | 17.2 ± 4.2 | 36.53 |
Cage 5: EP4 | 23.1 ± 0.9 | 19.6 ± 2.3 | 16.3 ± 5.3 | 13.8 ± 2.1 | 14.0 ± 0.9 | 11.2 ± 0.8 | 51.51 |
Cage 6: EP6 | 28.2 ± 2.3 | 15.1 ± 0.9 | 12.7 ± 1.5 | 10.4 ± 5.3 | 9.8 ± 2.3 | 9.1 ± 1.9 | 67.73 |
Significantly different from blood glucose level of the NC group taken at the same time (
The venous blood of mice was collected from the tail for measuring the blood glucose levels using a glucose monitoring system (One Touch brand). Four groups from obese mice diabetic group treated with EP6 (Cage 6), obese mice diabetic with no treatment (Cage 1), high-fat-fed mice (NCf), and normal mice (NC) were chosen, and their blood was collected and centrifuged at 3,000 rpm for 5 minutes. The separation serum was used for the biochemistry assay. The serum parameters: cholesterol level and triglycerides level, alanine aminotransferase level (ALT), and aspartate transaminase level (AST) were estimated using QuickDetect Total cholesterol/Triglyceride/AST/ALT (Rat) ELISA Kit (Ray Biotech). Consequently, these blood samples were centrifuged at a velocity of 3000 rpm for 15 minutes. Sera were then analyzed with commercially available kits (Sandwich method) according to the manufacturer’s protocol by ELISA apparatus (Photometer 5010 V5+, Robert Riele, Germany). Changes in color were checked at a wavelength of 450 nm. According to manufacture information, both the intra-assay and interassay coefficients of variation were below 12%.
Data are presented as mean
EPL1, lupeol, white crystals, 1H NMR (CDCl3, 500 MHz),
EPL2, friedelan 3-one, white powder, 1H NMR (CDCl3, 500 MHz),
EPL3: quassin, white powder, 1H NMR (CDCl3, 500 MHz),
EPL4: 17-hydroxy quassin, white powder, 1H NMR (CDCl3, 500 MHz),
EPL5: (+)-catechin, white powder, 1H NMR (CD3OD, 500 MHz),
EPL6: epicatechin, white powder, 1H NMR (CD3OD, 500 MHz),
ELP7: 7-
EPL8: 7, 4′-
From Figure
Obese mice have high levels of cholesterol and triglycerides. Based on Table
Comparison of blood glucose level, cholesterol level, triglycerides level, and liver function level (AST, ALT) between diabetic mice treated with EP6 (Cage 6), diabetic mice without any treatment (Cage 1), overweight mice (NCf), and normal mice (NC).
Glucose (mmol/L) | Cholesterol (mmol/L) | Triglycerides (mmol/L) | AST (U/L) | ALT (U/L) | |
---|---|---|---|---|---|
Cage 6 | 11.01 | 2.0 | 1.25 | 23.10 | 26.50 |
Cage 1 | 25.71 | 1.22 | 0.83 | 100.02 | 99.59 |
NCf | 11.50 | 2.91 | 1.99 | 25.91 | 30.81 |
NC | 5.620 | 0.71 | 0.72 | 21.90 | 11.85 |
The STZ administration to damage the pancreas in obese mice led to an increase in their blood sugar levels. The condition accompanied by high consumption of food and water indicates signs of diabetes in mice [
The present research indicates the potential of
The data used to support the findings of this study are included within the article.
The authors declare that there are no conflicts of interest.
This research was funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under Grant no. 104.01-2017.332.