|Year : 2020 | Volume
| Issue : 1 | Page : 47-53
Insignificant antidiabetic activities of ethanolic extracts of seeds of Archidendron pauciflorum
Sundarasekar Jeevandran1, Sahgal Geethaa2, Wong Jin Yi3, See Wei Yuan3, Subramani Parasuraman4, Sundram Karupiah3
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University; Department of Biotechnology, Faculty of Applied Science, AIMST University, Kedah, Malaysia
2 Department of Pharmaceutical Technology, Faculty of Pharmacy, AIMST University, Kedah, Malaysia
3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, Kedah, Malaysia
4 Department of Pharmacology, Faculty of Pharmacy, AIMST University, Bedong, Kedah, Malaysia
|Date of Submission||01-May-2020|
|Date of Decision||14-Jun-2020|
|Date of Acceptance||18-Jun-2020|
|Date of Web Publication||20-Jul-2020|
Dr. Sundram Karupiah
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, Semeling, Bedong, Kedah
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Archidendron pauciflorum (Fabaceae), commonly recognised as Dogfruit or Jering (Malaysia), is indigenous to Southeast Asia. The pounded leaves and bark of A. pauciflorum are used to treat gum pains, toothache, chest pains, skin ailments, and wounds. The antidiabetic effect of A. pauciflorum is not clear; hence, the present study is planned to evaluate the antidiabetic activities of ethanol extract of seeds of A. pauciflorum (ESAP) using both in vitro and in vivo models. Materials and Methods: Seeds of A. pauciflorum were sequentially extracted with hexane, chloroform, and ethanol, and the ethanolic extract was used in this study due to its various phytochemical constituents. Inhibitory activity of α-amylase and α-glucosidase (in vitro) was measured using the spectrophotometric method. For in vivo study, rats were used to induce diabetes mellitus (induced by streptozotocin) and ESAP was administered at the dose levels of 125, 250, and 500 mg/kg once daily through oral gavage for 21 consecutive days, and the antidiabetic effect of ESAP was compared with glibenclamide. Changes in glucose level and body weight were measured at regular intervals. Results: In vitro inhibition studies demonstrated that the ESAP had a moderate inhibitory activity for α-amylase and α-glucosidase. The rats treated with ESAP showed a significant decrease in their body weight at 21 days, whereas the glibenclamide treated rats showed a significant increase in their body weight. The standard drug glibenclamide was observed to significantly lower the blood glucose level, whereas the extract treated group did not show a significant change in levels of blood glucose when compared with control. Conclusion: The ESAP did not show any antidiabetic effect on diabetic rats.
Keywords: Glibenclamide, glucose, streptozotocin, α-amylase, α-glucosidase
|How to cite this article:|
Jeevandran S, Geethaa S, Yi WJ, Yuan SW, Parasuraman S, Karupiah S. Insignificant antidiabetic activities of ethanolic extracts of seeds of Archidendron pauciflorum. J Pharm Negative Results 2020;11:47-53
|How to cite this URL:|
Jeevandran S, Geethaa S, Yi WJ, Yuan SW, Parasuraman S, Karupiah S. Insignificant antidiabetic activities of ethanolic extracts of seeds of Archidendron pauciflorum. J Pharm Negative Results [serial online] 2020 [cited 2020 Aug 11];11:47-53. Available from: http://www.pnrjournal.com/text.asp?2020/11/1/47/290198
| Introduction|| |
Diabetes mellitus (DM) is a syndrome characterized by high blood glucose levels that result from defects in the body's ability to produce and/or use insulin. Both type 1 and type 2 diabetes comprise abnormalities of insulin action, which includes insulin insensitivity and resistance. It is a group of heterogeneous, hormonal, and metabolic disorders characterized by hyperglycemia and glucosuria, with disturbances in carbohydrate, fat, and protein metabolism resulting from defects in insulin secretion, insulin action, or both. DM is a major killer with about 422 millions of people worldwide in 2014 and also is considered as a major risk factor for cardiovascular disorders such as peripheral artery disease, cerebral stroke, and ischemic heart disease. It is also related to lasting complications such as angiopathy, neuropathy, nephropathy, and retinopathy.,
Lately, diabetes has been observed as the most challenging disease faced by medical professionals. Their growing occurrence places a huge burden on the society and the public health sector. Globally, the estimated number of DM patients is increasing, and it is predicted that more than 578 million people will be affected by DM in 2030. This scenario stresses the importance of better treatment and management for all diabetes-related chronic disorders.,
The actual therapies of DM are quite effective but often restricted because of their ways of application, side effects, loss of the efficacy after long-term use, cost, and unavailability in rural areas where they are mostly needed. Some severe side effects such as diarrhea, lactic acidosis, and liver problems had also been observed when using several hypoglycemic agents for the treatment. Therefore, constant research and development to find new potent and comfortable classes of blood glucose-lowering compounds with minimal side effects is imperative.
Recently, there has been a resurgence of interest in the study of plant materials as a source of the potential medicinal substance. These medicinal plants have continued to contribute significantly in developing the state-of-the-art drugs by providing lead compounds., Therefore, natural bioactive compounds for diabetes have attained greater importance in recent times. Since bioactives from plant sources are safe and easily available, potential Archidendron pauciflorum (A. pauciflorum) seeds with high phytochemical constituents were decided to be used in this investigation.
A. pauciflorum (family: Fabaceae) which is commonly known as Jering in Malaysia, is indigenous to Southeast Asia and also considered to be a local delicacy in that region. The tree grows up to 25 m in height and produces dark purple-colored pods containing three to eight round and flat beans per pod. The beans are eaten raw, fried, boiled, or roasted; sometimes, they are served as a dessert. People in this region consume parts of this plant because of its therapeutic value which includes blood purification, antidiabetic agent, overcoming dysentery, treating toothache, gum pains, chest pains, and skin ailments.
Furthermore, A. pauciflorum seeds are being used as folklore medicine in many Southeast Asian countries to treat various ailments. The seeds are cost-effective, affordable, and easy to obtain, which continues to be an important area of research. The antidiabetic activity of seeds of A. pauciflorum is not clear; hence, the present study is planned to evaluate the antidiabetic activities of ethanol extract of seeds of A. pauciflorum (ESAP) using bothin vitro models andin vivo models.
| Materials and Methods|| |
A. pauciflorum seeds were purchased from the local market in Singkir, Kedah. Authentication of the plant was done, and a voucher specimen of the plant was prepared and submitted to the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, for future reference (Herbarium voucher specimen with accession number: AIMST/FOP/19).
Healthy, adult Sprague Dawley (SD) rats (180 ± 20 g) were used for the study. The rats were obtained from the Central Animal House, AIMST University, Malaysia. The rats were housed in a large, spacious poly acrylic cage under an ambient room temperature (±25°C), 45%–55% humidity, and 12 h of light and dark cycle. A minimum of 5 days of acclimatization period was allowed before the animals were used in the experiment. The animals were fed with water and rat pellets ad libitum. Prior permission was obtained from the AIMST University Human and Animal Ethics Committee to carry out the animal experiment (AUAEC/FOP/2018/10).
The seeds were carefully removed from its pods and washed with running tap water to remove any dirt prior to the drying process. The seeds later were spread evenly and dried at 40°C for a week to remove the moisture content. The samples were powdered using a heavy-duty blender (Warring, America, 380V). The powdered seeds were extracted by means of three solvent sequential extraction method using hexane, chloroform, and ethanol, respectively. The extracts were filtered through double-layered muslin cloth, and filtrates were collected and concentrated in a rotary evaporator (RII0, Buchi, Switzerland) at 40 °C. The concentrated extracts were dried in an oven at 40°C for 3 days to obtain a consistent weight prior to storage at −20°C. The percentage extract yield was estimated as dry weight/dry material weight ×100.
In vitro antidiabetic activity
Alpha-amylase inhibition assay
Solution of 0.20 mM phosphate buffer (pH 6.9) containing 0.5 mg/mL of α-amylase was prepared. Five hundred microliters of ESAP extract (100, 200, 400, 800, and 1000 μg/mL, respectively) was added into 500 μl of the prepared phosphate buffer. The samples were incubated at 25°C for 10 min. The same procedure was followed for the standard drugs at different concentration (100, 200, 400, 800, and 1000 μg/mL, respectively). After that, a solution of 0.02 M sodium phosphate buffer (pH 6.9) containing 1% of starch solution was prepared. Five hundred microlitres of this solution was added into each of the test tubes containing test samples and standard drugs. These reaction mixtures were then incubated for 10 min at 25°C. One milliliter of 3,5-dinitrosalicylic acid color reagent was added into each test tubes to stop the reactions. Following that, for 5 min, the test tubes were incubated in a boiling water batch before allowing it to cool to room temperature. Distilled water (10 mL) was added to the reaction mixture to dilute it before measuring its absorbance at 540 nm. Control represents 100% enzyme activity and was conducted in a similar way by replacing extract with vehicle.
At 50% inhibitory concentration (IC50) estimation of the enzymatic action of the sample was dictated by the assay carried out with various concentrations of the samples. The IC50 value was identified from the plotted graph of percentage inhibition against inhibitor concentration. The assay was computed by nonlinear regression analysis from the main inhibitory values. The percentage of inhibition was calculated using the following formula:
% Inhibition = (Absorbancecontrol– Absorbanceextract)/Absorbancecontrol× 100
The experimental value is expressed as the mean ± standard deviation (SD), and all experiments were carried in triplicates (n = 3). The IC50 values, the concentration required to inhibit the α-amylase activity by 50%, were computed.
Alpha-glucosidase inhibition assay
A solution of starch substrate (2% w/v maltose or sucrose), 1 mL of 0.2 M Tris buffer (pH 8.0), and ESAP at various concentrations (100, 200, 400, 800, and 1000 μg/mL, respectively) were incubated at 37°C for 5 min. One milliliter of an α-glucosidase enzyme (1 U/mL) was added to each test tubes, followed by incubation at 37°C for 5 min to initiate the reactions. To stop the reaction, the reaction mixture was heated in boiling water bath for 2 min. Using the glucose peroxidase method, the amount of glucose produced was measured.
Calculation of 50% inhibitory concentration values
Using the percentage scavenging activities at five different concentrations (100, 200, 400, 800, and 1000 μg/mL) of the extract, the concentration of ESAP required to scavenge 50% of the radicals (IC50) was calculated. Percentage inhibition (I %) was calculated by:
I % = (Ac-As)/Ac × 100,
where Ac is the absorbance of the control and As is the absorbance of the sample.
Acute oral toxicity studies
Acute oral toxicity of the fractionated extract of ESAP was carried out as per the guidelines set by the Organization for Economic Co-operation and Development, revised draft guidelines 423 using female SD rats. The step-by-step procedure required by the principle dictates the use of least number of animals in each step, in order to obtain adequate information on the acute toxicity of the substance being tested so that proper classification can be made. For the experiment, three animals per dose of healthy SD rats were used. All the rats were fasted overnight before being fed with the ESAP in increasing dose levels (5, 50, 300, and 2000 mg/kg body weight, respectively). After the dosing, behavioral, autonomic profiles, and neurological changes in the rats were closely monitored for a period of 24 h. After this period, for 2 times/day, behavioral, autonomic profiles, neurological changes, and mortality were observed in the animals.,
Antidiabetic activity(in vivo)of ethanolic extract of seeds of Archidendron pauciflorum
Healthy, adult, male SD rats were used in the study. Induction of diabetes was done based on the method published by Petchi et al. Briefly, DM was induced in overnight-fasted rats by a single intraperitoneal injection of freshly prepared 55 mg/kg body weight streptozotocin (STZ) in normal saline. After 24 h of DM induction, the rats were given 5% w/v of glucose solution (2 mL/kg body weight) to prevent hypoglycemic mortality. DM was confirmed after 48 h of induction by measuring fasting blood glucose levels using a tail vein blood sample. Rats with fasting blood glucose of more than 200 mg/dL were considered as diabetics and used for further experiment.
Diabetic animals were randomly divided into five groups (Group II–VI) as follows:
- Group 1: Normal control
- Group II: Diabetic control
- Group III: Diabetic animals treated with glibenclamide (20 mg/kg)
- Group IV: Diabetic animals treated with ESAP (125 mg/kg)
- Group V: Diabetic animals treated with ESAP (250 mg/kg)
- Group VI: Diabetic animals treated with ESAP (500 mg/kg).
Group I (normal control) and Group II (diabetic control) animals have received 0.5% w/v carboxymethylcellulose (CMC). Animals in Group III were treated with glibenclamide (20 mg/kg BW), and animals in Group IV–VI were treated with ESAP at various doses (125, 250, and 500 mg/kg of BW, respectively). The doses of ESAP were selected based on the results obtained from toxicology study. Continuously, for 21 days, both the standard and ESAP (suspended in 0.5% w/v CMC) were administered once daily through an oral gavage. On the 7th and 14th days of the experiment, few drops of venous blood were collected for the estimation of blood glucose (whole blood) using a glucometer (Roche, Switzerland). Throughout the study, experimental animals' body weight variations were monitored at regular intervals. On the 21st day, using retro-orbital plexus puncture method, blood samples were collected from all the experimental animals and the serum was separated and used for biochemical analysis.
At the end of the study, blood sample were collected from each of the experimental animals through retro-orbital plexus puncture under mild diethyl ether anesthesia for biochemical analysis. The blood sample was collected in a tube coated with sodium ethylenediaminetetraacetic acid. The collected blood sample was centrifuged at 3000 rpm and plasma was collected and stored at −20°C until further analysis. The plasma sample was used to determine total protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, total bilirubin, urea, creatinine, total serum cholesterol, serum triglyceride (TG), and high-density lipoprotein (HDL) using Reflotron Plus biochemical analyzer (Roche Diagnostics, Germany) with the help of commercially available Reflotron strips.
The level of low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) was calculated mathematically. The LDL was calculated using the Friedewald formula: Total cholesterol − HDL − TG/5, and the VLDL level was calculated using the formula: LDL/5.
All the data were expressed as mean ± standard error of mean (SEM); the statistical analysis was carried out using one-way analysis of variance (ANOVA), followed by Tukey's test post hoc test method, and values of P < 0.05 were considered as statistically significant.
| Results|| |
The powdered seeds of A. pauciflorum were extracted sequentially by different solvents, hexane, chloroform, and ethanol, respectively, in increasing polarity by maceration method. The percentage of yield of hexane, chloroform, and ethanolic extract of seeds of A. pauciflorum was 15.8, 9.3, and 8.4%, respectively.
In vitro antidiabetic activity
Alpha-amylase inhibition assay
Alpha-amylase inhibitory studies demonstrated that the ESAP had moderate inhibitory activity [Table 1]. IC50 value for acarbose and ESAP was found to be 64.48 ± 10.65 μg/mL and 686.80 ± 16.55 μg/mL, respectively.
|Table 1: Percentage inhibition of α-amylase by ethanol extract of Archidendron pauciflorum|
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Alpha-glucosidase inhibition assay
Alpha-glucosidase inhibition activity on ESAP revealed moderate activity [Table 2]. IC50 value for acarbose is 61.82 ± 9.26 μg/mL, whereas for the ESAP, it is 472.66 ± 15.35 μg/mL.
|Table: 2: Percentage inhibition of α-glucosidase by ethanol extract of Archidendron pauciflorum|
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Acute oral toxicity studies
Acute toxicity study did not show any toxic signs and mortality up to 2000 mg/kg given as a single oral administration. Hence, the dose levels at 125, 250, and 500 mg/kg were selected for antidiabetic study.
In vivo antidiabetic activity
Effects on body weights
The diabetic animals and the diabetic animals administered with ESAP at various dose levels (125 and 500 mg/kg) showed a significant decrease in the body weight when compared to the normal control, whereas the diabetic animals treated with glibenclamide prevented the diabetic-induced body weight reduction. The effect of ESAP on body weight is shown in [Figure 1].
|Figure 1: Effect of ethanolic extracts of Archidendron pauciflorum on body weight. All the values are mean ± standard error of mean (n = 6). *P < 0.05 when compared with normal control|
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Effects on serum glucose level
Throughout the study, the diabetic animals and the diabetic animals administered with ESAP at different dose levels (125, 250, and 500 mg/kg) showed a significant increase in the blood glucose levels when compared to the normal control, whereas glibenclamide prevented the STZ-induced increases in the levels of glucose from week 3 onward when compared to the normal control [Table 3].
|Table 3: Effect of ethanolic extract of A. pauciflorum on glucose level in rats|
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At the end of the study, there was a significant increase in the levels of AST, creatinine, and urea in the diabetic animals and the diabetic animals administered with ESAP, compared to the normal control [Table 4]. ESAP also increases the levels of total bilirubin when compared to the normal control. The diabetic animals treated with glibenclamide showed a significant increase in the levels of AST and urea. In lipid analysis, diabetic animals showed a significant increase in the levels of total cholesterol, triglyceride, LDL, and VLDL and a significant decrease in the level of HDL, when compared to the normal control [Table 5], whereas glibenclamide and ESAP at the dose levels of 125, 250, and 500 mg/kg prevented STZ-induced abnormalities in lipid profile [Table 5].
|Table 4: Effects of ethanolic extracts of A. pauciflorum on biochemical parameter in rats|
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|Table 5: Effect of ethanolic extracts of A. Pauciflorum on serum lipid profile in rats|
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| Discussion|| |
The plant-based diets and medicines have recently been reviewed and have gained importance for the control of type 2 DM., They are being used directly or indirectly for the preparation of many modern drugs. Although A. pauciflorum have been used for a long time as traditional or folklore medicine, it has not gained much recognition as medicine and one of the factors being lack of systematic scientific research and supportive animal or clinical trials. In the present study, antidiabetic effect of ESAP was investigated using bothin vitro andin vivo models. Plants are known to possess the broad range of phytochemicals varying from nonpolar to polar. Therefore, the plant materials were sequentially extracted (hexane, chloroform, and ethanol, respectively) to ensure complete extraction of all nonpolar as well as polar components and thereby inclusion of all components in the screening study. Initial phytochemical studies (unpublished) show that various types of phytochemical constituents are present in the ethanolic extract of the plant, and hence, the ESAP was used in this study. Normally plants are identified to produce a large assortment of alpha-amylase and glucosidase inhibitors that provide protection against insects and microbial pathogens., Thus, ESAP extract was analyzed for α-amylase and α-glucosidase inhibitory activities. The α-glucosidase inhibition significantly decreases postprandial hyperglycemia (PPHG) in the treatment of T2DM patients. Amylases and glucosidases are the important enzymes of dietary carbohydrate, and inhibitors of these enzymes may be efficient in impeding glucose absorption to suppress PPHG. In T2DM, one of the reasons for hyperglycemia is triggered by excessive hepatic glycogenolysis and gluconeogenesis, which results in decreased utilization of glucose by tissues. The glucosidase inhibitors work by blocking the α-1,6-glucosidase enzyme in the liver which is supposed to hydrolyze glycogen, and hence, it reduces the glycogenolytic rate which increases the accumulation of glycogen storage in the liver., As a short-term effect, the current blood glucose level in diabetic patient is decreased, and alternately, the hemoglobin A1c level is reduced, as a long-term effect.
Bunawan et al. reported that A. pauciflorum extract shows good phytochemical properties such as phenolics. Rich phenolic compounds in plants are responsible for its potent α-amylase activity. These phenolics can bind to the reactive sites of the enzymes to alter its catalytic activity. The mechanism of inhibition for α-amylase is suggested to happen by direct blockage of the active center at several subsites of the enzyme.In vitroα-amylase and α-glucosidase inhibitory studies demonstrated that the ESAP has moderate inhibitory activities [Table 1] and [Table 2] compared to the standard that had been used. The percentage inhibition at 100, 200, 400, 800, and 1000 μg/mL concentrations of samples showed a concentration-dependent percentage of inhibition. Phytochemical studies (unpublished) show the presence of tannins and glycosides in the ethanolic extract of the plant. Lately, tannin has been reported as nonspecific inhibitors for numerous hydrolytic enzymes such as lipases, α-glucosidases, α-amylases, and invertase. The glycosides from the plant extract can act as a substrate for the α-glucosidase enzyme and may be accountable for the inhibitory activity. The enzyme amylase works by degrading starch by cleaving its glycosidic bonds. The ESAP contains glycosides with glycosidic bonds which can act as substrates for amylase. By following the logic of α-amylase inhibition mechanism, the starch in the body will not be cleaved to form a disaccharide. Therefore, it will be helpful for the action of glucosidase by not providing substrates for the conversion of disaccharides into monosaccharides (i.e., glucose), and the level of blood glucose level can be controlled.
Bioflavonoids are documented for their multidirectional biological activities, including their antidiabetic efficacy., Dietary flavonoids exercise their antidiabetic effect by affecting various cellular signaling pathways in the pancreas. Flavonoids exert their effect by affecting β-cell mass and function as well as energy metabolism and insulin sensitivity in peripheral tissue., In the present study, STZ is used to induce DM. STZ is commonly used for the induction of DM in rodents that works by inhibiting insulin secretion and causes insulin-dependent DM. It is a glucosamine–nitrosourea originated from Streptomyces achromogenes (Gram-positive bacterium), and it is utilized for the treatment of pancreatic beta-cell carcinoma and to stimulate DM in rodents. STZ triggers hyperglycemia following 2 h of injection, hypoglycemia in 6 h, and ultimately hyperglycemia by decreasing the insulin levels through the inhibition/destruction of pancreatic beta-cell function. STZ totally destroys beta-cells by amassing in pancreatic beta-cells via the low-affinity glucose transporter GLUT2 in the plasma membrane and by production of reactive oxygen species. In the present study, diabetes is characterized by low-glycemic intensity, which was induced to the rats by an STZ injection (dose of 55 mg/kg BW) intraperitoneally. In this study, increased muscle wasting and tissue proteins may be the underlying reason for the observed decrease in the body weight of the diabetic control rats. Clinical studies show that frequent urination and over conversion of glycogen to glucose can result in weight loss.,
Higher concentrations of liver transaminases such as ALT and AST are considered biomarkers of hepatocellular damage, correlated with fatty liver disease and hyperglycemia in diabetes. ESAP-treated animals did not reduce the levels of AST, suggesting that the plant extract did not improve the STZ-induced hepatic damage in diabetic rats.
Renal damage and liver disease related to insulin resistance in diabetic control animals can be predicted by the hike in the concentrations of renal markers and liver enzymes, respectively. In this study, rats treated with glibenclamide and plant extract prevented any abnormalities in biochemical parameter. There are also abnormalities in lipid profile by the diabetic control animals. Concentrations of total cholesterol, LDL, and VLDL are found to be increased, whereas the concentration of HDL is lower than the normal control animals. Research blames exogenous fat loading, enhancement of intestinal CoA-dependent esterification, and an abnormal increase in small intestinal acyl coenzyme A: cholesterol acyltransferase activity for this. The results from the study [Table 4] and [Table 5] also indicate that ESAP can reduce the levels of serum urea, serum creatinine, and total cholesterol significantly.
| Conclusion|| |
In vitro α-amylase and α-glucosidase inhibitory studies demonstrated that the ESAP had moderate inhibitory activity. The ESAP showed insignificant activity in controlling the body weights of the rats compared to the standard drug being used. All three concentrations of the ethanol extract of seeds of A. pauciflorum (125 mg/kg, 250 mg/kg, and 500 mg/kg BW) were unable to reduce the blood glucose level significantly compared to glibenclamide.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]