|Year : 2023 | Volume
| Issue : 4 | Page : 237-242
Potential use of Balanophora latisepala (V. Tiegh.) Lec. extract for the treatment of diabetes
Nguyen Trong Hong Phuc1, Nguyen Thi Yen Lan2, Nguyen Van Ay3
1 Department of Biology, Can Tho University, Can Tho City, Vietnam
2 Phan Van Tri High School, Can Tho University, Can Tho City, Vietnam
3 College of Agriculture, Can Tho University, Can Tho City, Vietnam
|Date of Submission||01-May-2023|
|Date of Decision||05-Aug-2023|
|Date of Acceptance||08-Aug-2023|
|Date of Web Publication||11-Sep-2023|
Nguyen Van Ay
Campus II, 3/2 Street, Xuan Khanh Ward, Ninh Kieu, Can Tho City
Source of Support: None, Conflict of Interest: None
INTRODUCTION: Balanophora latisepala (BL) is a chlorophyll-free and obligate parasitic plant found across mountainous areas of An Giang, Vietnam. This study was conducted to evaluate the effectiveness of extracts from BL, herbs mentioned in many traditional medicines in the treatment of diabetes, on laboratory mice.
MATERIALS AND METHODS: The effects of BL aqueous, methanol, and ethanol extracts on the blood glucose levels, hematological indices, liver enzymes, and pancreatic tissue structure of normal, glucose-tolerant mice, and diabetic mice treated with streptozotocin (STZ) were assessed. Mice were orally administered extracts at a concentration of 500 mg/kg.
RESULTS AND DISCUSSION: The aqueous and ethanol extracts did not affect the blood glucose levels of healthy and glucose-tolerant mice (P > 0.05). The methanol extract reduced the blood glucose of healthy mice (P < 0.05) but did not affect the blood glucose of glucose-tolerant mice 3 h after administration (P > 0.05). When taking the methanol and ethanol extracts, the diabetic mice experienced a 45.67% and a 40.33% decrease in blood glucose levels after 21 days of research, respectively. Aqueous extract and gliclazide (10 mg/kg) showed no effect on reducing blood glucose levels in diabetic mice over the 21 days of the study. Both methanol and ethanol extracts were effective in regulating liver enzyme levels in diabetic mice. In both untreated and treated diabetic mice, pancreatic islet structures were degraded with the appearance of gaps resulting from β-cell degeneration.
CONCLUSION: This implies that ethanol extracts have potential applications in regulating blood glucose levels.
Keywords: Balanophora latisepala, diabetes, pancreas, streptozotocin
|How to cite this article:|
Hong Phuc NT, Yen Lan NT, Ay NV. Potential use of Balanophora latisepala (V. Tiegh.) Lec. extract for the treatment of diabetes. Indian J Pharmacol 2023;55:237-42
|How to cite this URL:|
Hong Phuc NT, Yen Lan NT, Ay NV. Potential use of Balanophora latisepala (V. Tiegh.) Lec. extract for the treatment of diabetes. Indian J Pharmacol [serial online] 2023 [cited 2023 Oct 3];55:237-42. Available from: https://www.ijp-online.com/text.asp?2023/55/4/237/385497
| » Introduction|| |
Currently, one of the most prevalent diseases, diabetes can lead to numerous other major health issues. A World Health Organization assessment indicates that approximately 8.5% of adults globally have diabetes. Therefore, many studies have been conducted to find medications and herbs that can prevent and treat diabetes.
Balanophora latisepala (BL) (V. Tiegh.) Lec. has no chlorophyll and is an obligate parasite on the roots of the host plants. In the traditional medicine of some mountainous ethnic groups, species of the genus Balanophora is often used as tonics, drugs to strengthen bones, medicines to balance blood sugar, or to treat sexual dysfunction syndromes and intestinal diseases. Furthermore, many species of this genus have been studied and shown to have strong antioxidant properties (Balanophora laxiflora Hemsl.), which can inhibit HIV activity (Balanophora japonica and B. laxiflora). They have also been shown to have hypoglycemic (Balanophora polyandra), analgesic, and anti-inflammatory effects. BL is an endemic species of southern Vietnam. This species has not been studied scientifically, yet it is only used in folk medicine to treat a number of diseases such as pain, lung tonic, and diabetes. The effect, dose, and method of the remedies used depend on the clinical experiences of the physicians without any published information. Therefore, this study is to examine the pharmacological properties and blood glucose regulatory functions of BL. The results from this study will provide more scientific information about the potential functions of BL in the drug development for the treatment of diabetes.
| » Materials and Methods|| |
At Cam Mountain, An Giang, between June and September of 2020, BL was collected at 10°29'08.4”N and 104°58'59.8”E, at an elevation of roughly 200–450 m. The specimens (Blatis082020-AGVN) have been stored at the Plant Laboratory, Department of Biology, School of Education, Can Tho University.
Male Swiss albino mice (30–35 g, aged 7–10 weeks) were provided by the Biotechnology Center of Ho Chi Minh City, Vietnam. The animals were separated into groups with pelleted food and water available, maintained under laboratory conditions, at a temperature of 24°C–28°C, relative humidity of 60%–70%, and 12 h dark/light cycle. Research and manipulation activities on animals were approved by the research ethics board in accordance with document number AWA2021-04/KSP.
Preparation of plant extracts
After being gathered, the BL was cleaned and dehydrated in the incubator at 60°C until it reached a steady weight. The sample was powdered and submitted to decoction (10% weight/volume, powder: water) for 3 h at room temperature. The mixture was then boiled for 1 h. It was then filtered using filter papers and evaporated using a rotary evaporator (SCI100, Scilogex Pro, USA). Following vacuum filtration, the aqueous extract was lyophilized and dissolved in water at suitable quantities for biological tests, as depicted by Félix-Silva et al. Correspondingly, the medicinal powder was soaked in methanol (10% weight/volume, powder: methanol) and ethanol (10% weight/volume, powder: ethanol) for 3 h before being filtered through filtered paper. The solution was then soaked with methanol or ethanol in a ratio of 1:10 for 3 h (three times repeat). A rotary evaporator (SCI100, Scilogex Pro, USA) was used to evaporate the mixture at 50°C once the extract had been combined. The final product was stored at 4°C.
Study of Balanophora latisepala extracts in healthy and oral glucose tolerance mice
Investigation of the effect of BL extract on blood glucose levels of healthy and glucose-tolerant mice orally were carried out according to the methods of Nguyen et al. and Sikarwar and Patil with some modifications., Fifteen male mice were evenly distributed randomly into five treatments. Mice were given distilled water in Group I, gliclazide at 10 mg/kg in Group II, and aqueous, methanol, and ethanol extracts of BL at a concentration of 500 mg/kg mice body weight in groups 3, 4, and 5, respectively. Before conducting the experiment, mice fasted for 12 h and were given water to drink freely. Distilled water, gliclazide, and extracts were inserted directly into the mice's stomach with a curved blunt-tipped needle at a volume of 10 mL/kg. Blood was collected from mouse tails to determine the blood glucose concentrations at 0, 1, and 3 h of the experimental period.
Effects of Balanophora latisepala extracts in streptozotocin-induced diabetic mice
The effect of BL extracts on blood glucose regulation in diabetic mice induced by streptozotocin (STZ) was performed according to Furman and Nguyen et al. with some adjustments.,
Induced diabetes in male mice by injecting STZ (150 mg/kg body weight, mixed with 50 mM sodium citrate buffer, pH = 4.5) into the abdominal cavity of mice. Blood glucose levels were checked 72 h after the STZ injection. After fasting, diabetic mice were defined as having blood glucose levels more than 200 mg/dL that had been used in the study.
Experimental mice were distributed into six treatments (three mice each), including Nor (healthy mice were given distilled water); Diab (diabetic mice were given distilled water); Diab + Gly (diabetic mice treating with 10 mg/kg gliclazide); Diab + Aqu (diabetic mice were given 500 mg/kg of BL aqueous extract); Diab + Met (diabetic mice were given 500 mg/kg of BL methanol extract); and Diab + Eth (diabetic mice were given 500 mg/kg of BL ethanol extract).
Before conducting the experiment, mice were given free water to drink and abstained for 12 h. A single daily dose of 10 mL/kg body weight of distilled water, gliclazide, or extract was given to mice at 8–10 a. m.
The experiment was carried out for 3 weeks. After each week, mice were weighed, and blood glucose was determined. After 21 days, mice were anesthetized, and blood was then sampled from the heart for hematological analysis (red blood cell count [RBC], hemoglobin [HGB], hematocrit [HCT], platelet count, plateletcrit, white blood cell count [WBC], neutrophil count [NEU], lymphocyte count [LYM], and monocyte) by the CELL-DYN Ruby Automated Hematology Analyzer (Abbott, USA) and biochemical testing (aspartate aminotransferase [AST], alanine aminotransferase [ALT], and uric acid) by a Cobas clinical chemistry automatic analyzer (La Roche Ltd., Japan). Laboratory mice were euthanized for pancreatic dissection to perform pancreatic tissue microscopy by tissue fixation and double staining with hematoxylin and eosin Y (H and E).
Using Microsoft Excel (Ver. 2019; Microsoft Inn., USA), experimental data were analyzed and graphed. With IBM SPSS 22 software (SPSS Inn., USA), mean differences were determined through ANOVA analysis and Duncan post hoc test at 95% confidence.
| » Results|| |
Effect of Balanophora latisepala extracts in healthy mice
[Table 1] displays the impact of BL extracts on the blood glucose levels of healthy mice. Mice drinking distilled water and gliclazide had stabilized blood glucose levels after 1 h and 3 h compared with 0 h (P > 0.05). Mice drinking aqueous or methanol extract had an increase in blood glucose levels after 1 h of administration [Table 1].
|Table 1: Effect of Balanophora latisepala extract on healthy mice's blood glucose concentration|
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Effect of Balanophora latisepala extracts on oral glucose tolerance
The glucose concentration in the mouse blood was to increase about 1 h after receiving glucose, and the difference was especially significant in the control and ethanol extract mice (P < 0.05).
The results of investigating the effect of BL extract on blood glucose concentration in healthy mice with oral glucose tolerance showed that most treatments had blood glucose levels return to normal after 3 h of glucose intake [Table 2]. There were two differences in mice's glucose concentrations in the groups drinking distilled water and ethanol extract. Mice had an increase in blood glucose concentration after 1 h of drinking glucose (P < 0.05). Meanwhile, the mice taking gliclazide, aqueous extract, and methanol extract had blood glucose levels kept at normal levels compared to at 0 h (P > 0.05).
|Table 2: Effect of Balanophora latisepala extracts on blood glucose levels of glucose-tolerant healthy mice|
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Effect of Balanophora latisepala extracts on streptozotocin-induced diabetes mice
The weight of control mice increased in 21 days of testing, from 32.55 g to 38.52 g. Diabetic mice treated with gliclazide or extracts (aqueous, methanol, and ethanol) at a dose of 500 mg/kg had an insignificant change in weight (P > 0.05). In contrast, untreated diabetic mice had weight loss during the experiment (P < 0.05).
Effect on blood glucose level
Control mice using distilled water had stable blood glucose levels for 21 days of the experiment. In untreated diabetic mice, blood glucose levels were very high (>500 mg/dL) [Figure 1]. Gliclazide did not show an effect on lowering the blood glucose of diabetic mice, whereas Gliclazide-treated mice had increased blood glucose levels from day 7 to day 21 (P < 0.05).
|Figure 1: Effect of BL extracts on blood glucose in diabetic mice. Mean bars ± standard deviation have different letters in a treatment are significantly different (Duncan test, P < 0.05)|
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Different types of extracts had various effects on the ability to lower blood sugar in diabetic mice. Mice treated with aqueous extract did not reduce blood glucose levels. In this treatment, mice showed signs of ruffled hair, frequent urination, anorexia, paralysis of limbs, a deep coma, and death after 18 days of experimentation. Therefore, the aqueous extract-treated mice were not assessed for their blood glucose levels at day 21. In contrast, the diabetic mice treated with methanol and ethanol extracts exhibited a noticeable reduction in blood glucose compared with others. In mice treated with methanol extract, blood glucose levels decreased by 45.67% after 21 days of treatment, and in the group treated with ethanol extract, they decreased by 40.33% (P < 0.05).
Hematological and biochemical parameters
The test results of hematological indicators [Table 3] after 21 days showed that there was no significant difference between all treatments (P > 0.05). RBC, HCT, HGB, mean red blood cell volume, mean hemoglobin, mean platelet volume, platelet distribution leukocyte count (PDW), WBC, NEU, and LYM in the control treatments, gliclazide, methanol extract, and ethanol extract were all insignificantly different, and they were not different from the biological control (P > 0.05).
|Table 3: Effect of Balanophora latisepala extracts on hematological parameters of diabetic mice|
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Different from hematological indicators, diabetic mice induced with STZ had different biochemical indices of liver enzymes and uric acid compared with controls [Table 4]. The untreated diabetic mice had AST levels 3.16 times higher than controls and ALT levels 5.11 times higher than control mice (P < 0.05). Diabetic mice treated with gliclazide, ethanol, and methanol extracts of BL had unchanged AST and ALT levels compared with the control treatment (P > 0.05). The test results of the uric acid index did not show any difference between treatments (P > 0.05).
|Table 4: Effect of Balanophora latisepala extracts on biochemical indices of diabetic mice|
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Pancreas histopathological examination
The effect of BL extract on histopathological changes of the pancreas in diabetic mice was observed by the H and E staining method [Figure 2]. In normal pancreatic tissues, the Islets of Langerhans More Details had uniformly distributed cells, forming clusters of round or oval cells with well-defined boundaries. The islets of Langerhans were lighter in color than the surrounding acinar cells. Untreated diabetic mice had severely damaged pancreatic structures.
|Figure 2: Mice pancreatic tissue. IS: Islets of pancreas, A: Acinar cells, D: Pancreatic duct|
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| » Discussion|| |
For healthy mice, the presence of carbohydrates and cardiac glycosides in BL extracts might have raised the blood sugar levels of tested mice for about 1 h. The glucose levels then returned to normal after 3 h, which were not significantly different from the control group (P > 0.05). Particularly, for the methanol extract, the blood sugar levels of healthy mice had a significant decrease after 3 h of using the extract (P < 0.05).
Based on the study results shown in [Table 2], the blood sugar levels of these healthy mice were quickly stabilized after 3 h of glucose intake (P > 0.05). There was only one case where glucose tended to be lowered in the gliclazide-treated mice (P < 0.05). Those experimental results showed that the extracts of BL did not physiologically affect blood glucose balance in healthy mice, which was similarly observed in the control mice.
STZ is a very powerful antibiotic, capable of causing enormous harm to laboratory mice. When injecting STZ to induce diabetes in mice, control mice experienced significant weight loss, whereas the trials using extracts had changes that did not differ from controls. Gliclazide did not show glycemic regulation in mice when mice were poisoned by STZ. Meanwhile, BL extracts show certain abilities in regulating blood glucose.
Diabetes is one of the most common causes of liver disease, including abnormal changes in liver enzymes, liver fibrosis, hepatocellular carcinoma, and acute liver failure. Liver enzyme indices, including AST and ALT enzymes, are the main markers used to evaluate liver dysfunction., In this study, untreated STZ-infected mice had increased AST and ALT levels compared with the control mice (P < 0.05). The increase in AST and ALT levels in the serum of diabetic mice is due to the leakage of these enzymes from damaged hepatocytes into the bloodstream. The AST and ALT levels of STZ-infected mice treated with gliclazide and extracts of BL did not increase and were similar to those of the control group (P > 0.05). It has been shown that BL extract has roles in protecting membrane integrity and reducing liver damage caused by chronic hyperglycemia.
It was observed that the pancreatic islets were degenerated, lipolysis occurred, islet cell contours were lost, and acinar cells were swollen. Observing diabetic mice pancreatic tissue treated with gliclazide, methanol, and ethanol extracts, there was a reduction in pancreatic islet degeneration, a marked reduction in lipolysis, and a decrease in swelling of acinar cells. Meanwhile, in diabetic mice treated with aqueous extract, 18-day pancreatic tissue samples tested showed that pancreatic islets still had lipolysis, cells were enlarged, cell membranes were thin, or cells were dissolved. In addition to β-cell destruction, it is well documented that oxidative stress and disturbances of carbohydrate, protein, and lipid metabolism occur in diabetes mellitus.
STZ is a highly toxic antibiotic that stimulates H2O2 generation in β-cells, can induce lipid peroxidation, and depletes antioxidant enzymes. This antibiotic causes structural damage to the tissues of organs, especially the pancreas, including the induction of oxidative reactions, and the formation of nitric acid in the tissues associated with damage to the DNA of cells., The primary causes of damage to B cells are the formation of free radicals and nitric oxide.
The aqueous, ethanol, and methanol extracts of BL all had relatively good antioxidant and free-radical scavenging activities. Therefore, its ability to protect β-cells in pancreatic islets could be related to its free-radical scavenging activity. Moreover, the blood glucose-lowering effect of BL extract could be due to the presence of chemical components present in the plant. Research has indicated that medicinal herbs with phenols, glycosides, and saponins have been proven to have antidiabetic properties. Saponins reduce fasting blood glucose and serum insulin. Terpenoids either stimulate insulin secretion from β-cells or act like insulin. Steroids reduce blood glucose levels and restore insulin in STZ-induced diabetic mice. Flavonoids improve cellular glucose uptake by stimulating the recovery of damaged β-cells. Gum can correct adipose tissue dysfunction in patients with type 2 diabetes and decrease the amount of sugar absorbed from diet to lower blood sugar levels. The above chemical components are all found in BL. However, the composition and content of these compounds in different solvents were different, which could be the reason for the results of blood glucose regulation in diabetic mice by STZ of BL extracts were different.
| » Conclusion|| |
The study on the blood glucose-lowering effect of BL extracts showed that aqueous and ethanol extracts did not affect the blood glucose levels of normal mice or normal glucose-tolerant mice. Diabetic mice had reduced blood glucose levels when treated with methanol and ethanol extracts at a concentration of 500 mg/kg. These extracts also showed their roles in restoring biochemical parameters in diabetic mice. Of the three BL extracts investigated, the ethanol extract had the highest potential for the development of drugs for the treatment of diabetes.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]