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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 65  |  Issue : 3  |  Page : 125-135

Insulin sensitizer and antihyperlipidemic effects of Cajanus cajan (L.) millsp. root in methylglyoxal-induced diabetic rats


1 Department of Beauty Science and Graduate Institute of Beauty Science Technology, Chienkuo Technology University, Changhua, Taiwan
2 Department of Food Science and Biotechnology, Da-Yeh University, Changhua, Taiwan
3 Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taiwan
4 Division of Urology, Department of Surgery, Yuanlin Christian Hospital, Changhua, Taiwan
5 Taitung District Agricultural Research and Extension Station, Council of Agriculture, Taitung, Taiwan
6 Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan

Date of Submission22-Oct-2021
Date of Decision09-Feb-2022
Date of Acceptance30-Mar-2022
Date of Web Publication27-Jun-2022

Correspondence Address:
Prof. Tuzz-Ying Song
No. 168, University Rd., Dacun, Changhua 515006
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjp.cjp_88_21

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  Abstract 


Cajanus cajan (L.) Millsp., known as pigeon pea, is one of the major grain legume crops of the tropical world. It recognizes as an ethnomedicine to possess various functions, such as helping in healing wound and cancer therapy. We investigated whether 95% ethanol extracts from C. cajan root (EECR) protect against methylglyoxal (MGO)-induced insulin resistance (IR) and hyperlipidemia in male Wistar rats and explored its possible mechanisms. The hypoglycemic potential of EECR was evaluated using α-amylase, α-glucosidase activities, and advanced glycation end products (AGEs) formation. For in vivo study, the rats were divided into six groups and orally supplemented with MGO except for Group 1 (controls). Group 2 was supplemented with MGO only, Group 3: MGO + metformin, Group 4: MGO + Low dose-EECR (L-EECR; 10 mg/kg bw), Group 5: MGO + Middle dose-EECR (M-EECR; 50 mg/kg bw), and Group 6: MGO + High dose-EECR (H-EECR; 100 mg/kg bw). EECR possessed good inhibition of α-glucosidase, α-amylase activities, and AGEs formation (IC50 = 0.12, 0.32, and 0.50 mg/mL), respectively. MGO significantly increased serum levels of blood glucose (GLU), glycosylated hemoglobin, homeostasis model assessment of IR, AGEs, lipid biochemical values, and atherogenic index, whereas EECR decreased these levels in a dose-dependent manner. EECR can also act as an insulin sensitizer, which significantly decreased (47%, P < 0.05) the blood GLU levels after intraperitoneal injection of insulin in the insulin tolerance tests. The hypoglycemic and antihyperlipidemic mechanisms of EECR are likely through several possible pathways including the inhibition of carbohydrate-hydrolyzing enzymes (α-glucosidase and α-amylase) and the enhancement of MGO-trapping effects on inhibition of AGEs formation.

Keywords: Advanced glycation end products, antihyperlipidemic, Cajanus cajan (L.) Millsp. root, insulin resistance, insulin sensitizer, methylglyoxal


How to cite this article:
Yang SE, Lin YF, Liao JW, Chen JT, Chen CL, Chen CI, Hsu SL, Song TY. Insulin sensitizer and antihyperlipidemic effects of Cajanus cajan (L.) millsp. root in methylglyoxal-induced diabetic rats. Chin J Physiol 2022;65:125-35

How to cite this URL:
Yang SE, Lin YF, Liao JW, Chen JT, Chen CL, Chen CI, Hsu SL, Song TY. Insulin sensitizer and antihyperlipidemic effects of Cajanus cajan (L.) millsp. root in methylglyoxal-induced diabetic rats. Chin J Physiol [serial online] 2022 [cited 2022 Aug 11];65:125-35. Available from: https://www.cjphysiology.org/text.asp?2022/65/3/125/348363




  Introduction Top


Diabetes mellitus (DM) is a serious threat to human health, with the number of people suffering from diabetes in the world being as high as 135 million to 160 million. In the United States and Canada, for example, diabetes is ranked third in the top ten causes of death, second only to cardiovascular disease and cancer. According to the Ministry of Health and Welfare Statistics, diabetes ranks fourth in Taiwan's top ten causes of death. The type of diabetes in Taiwan is mainly type 2 diabetes mellitus (T2DM), accounting for about 95% of the incidence rate. Therefore, optimal treatment and prevention strategies for T2DM are urgently needed.

T2DM is a heterogeneous disorder that is associated with both β-cell dysfunction and insulin resistance (IR).[1],[2] Many investigators have suggested that IR is the primary abnormality, and that β-cell dysfunction is a subsequent event arising from the cumulative burden of persistent increases in insulin secretion on pancreatic β-cells.[3] It is generally agreed that IR is a key pathogenic factor for T2DM.[4]

Postprandial hyperglycemia (PH) is an early detected symptom in T2DM, which occurs when pancreatic β-cells fail to secrete a sufficient amount of insulin.[5] Evidence suggests that PH induces glucose toxicity and deteriorates β-cell function,[6] which results in an irreversible state of diabetes.[7] PH is related to the amount of and the digestion rate of consumed starch, which is the primary source of blood glucose. Thus, it is a major strategy to reduce or slow dietary carbohydrate digestion and absorption for treating PH,[8] and it can be achieved by inhibiting starch-hydrolyzing enzymes such as α-amylase and α-glucosidase in the small intestine to reduce glucose release.[9]

Recently, synthetic or naturally derived compounds have been shown to inhibit α-amylase and α-glucosidase activities.[10],[11],[12] Phenolic compounds (such as genistein, quercetin, resveratrol, and syringic acid) existed in plants have been found to be potent α-amylase and α-glucosidase inhibitors to regulate carbohydrate metabolism and hyperglycemia.[13] Dietary antioxidants have also been associated with the reduced risk of T2DM by inhibiting peroxidation chain reactions.

Cajanus cajan (L.) Millsp., known as pigeon pea, is one of the major grain legume crops of the tropical world [Figure 1]. Taiwanese aborigines have been eating C. cajan since ancient times, and its roots, seeds, and leaves have been shown to possess various functions, such as suppressing organ swelling, helping wound healing and cancer therapy.[14] The extracts of C. cajan leaves are beneficial medicine against bronchitis, jaundice, cough, diarrhea, abdominal pains, sores, and bladder stones.[15],[16],[17] In addition, C. cajan roots (CCR) have been used as anthelminthic, alexeritics, and expectorant.[18] Zhang et al.[19] demonstrated that the major active compounds responsible for CCR are polyphenols, especially isoflavonoids. Recently, we found that CCR roots have a higher total polyphenol content and total flavonoid content than its leaves and seeds.[20] In addition, the 95% ethanol extracts of CCR (EECR) have the strongest antioxidant, antihyperglycemic (inhibition of α-glucosidase and α-amylase activities), antiglycation, and antibacterial ( Escherichia More Details coli, Staphylococcus aureus, Porphyromonas gingivalis, and Streptococcus mutans) activities than the hot water or 50% ethanol extracts of CCR.[20]
Figure 1: The morphological botany of Cajanus cajan (L.) Millsp.

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Nahar et al.[21] have reported an antidiabetic activity of the methanol extract of CCR in diabetic mice induced by alloxan. Alloxan is known to destruct the β-cells of the pancreas, leading to reduced insulin secretion and increased incidence of diabetes.[22] Because diabetes in mice was induced by alloxan through tail vein injection, the alloxan-induction model cannot reflect the actual condition of T2DM in humans. In contrast, Guo et al.[23] indicated that oral methylglyoxal (MGO) induces IR in Sprague-Dawley rats by increasing oxidative stress and/or advanced glycation end products (AGEs) formation. Thus, in this study, we chose MGO to induce IR in rats to mimic T2DM in humans for the evaluation of the anti-IR potential of the 95% ethanol extracts of CCR (EECR) by determining their starch-hydrolyzing enzymes activities, IR, and hypoglycemic and hypolipidemic activities.


  Materials and Methods Top


Chemicals

CCR (Taitung No. 3) was kindly provided by Dr. Chen-Yi Chen (Taitung District Agricultural Research and Extension Station, Council of Agriculture, Executive Yuan, ROC). p-Nitrophenyl α-D-glucoside (pNPG), acarbose, porcine pancreatic α-amylase, type VI-B (A3176), porcine pancreatic lipase, Type ll (L3216), Folin-Ciocalteu reagent, gallic acid, rutin, Trolox™, fluorescein, 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and MGO were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). The ethanol and acetone solvents were HPLC grade (Fisher Scientific Co.).

Preparation of hot water and ethanol extracts of Cajanus cajan roots

CCR were rinsed with running water to remove soil. The surface of the CCR was sterilized in 95% ethanol for 3 min and then rinsed three times in sterile distilled water. The surface-sterilized CCR was milling to powder. A portion of the CCR powder was extracted with 1:20 ratio (w/v) of 97°C ± 2°C hot water for 3 h or extracted with 1:20 95% ethanol by soaking in the room temperature for 72 h. After filtering through Whatman No. 2 filter paper, the filtrates were concentrated (10-fold) under reduced pressure and lyophilized to obtain the hot water (HWCR) and 95% ethanol extract of CCR (EECR). The extraction yield of HWCR and EECR was 6.7% and 5.0%, respectively.

α-Amylase inhibition assay

The assay was done as illustrated by Shu et al.[24] with some modifications. Briefly, 50 μL of EECR of various concentrations (0.01–1.0 mg/ml) in 0.02 M phosphate buffer (pH 6.9, containing 0.006 M NaCl) was mixed with 25 μL of pancreatic α-amylase solution (0.5 mg/ml) and incubated at 25°C ± 1°C for 10 min. Thereafter, 50 μL of starch solution (0.5% (w/v), 0.02 M phosphate buffer, pH 6.9, containing 0.006 M NaCl) was added and incubated for 10 min at 25°C ± 1°C. Then, the reaction was terminated by adding 100μL of dinitrosalicylic acid (DNS) reagent (12 g of sodium potassium tartrate tetrahydrate in 8.0 mL of 2 M NaOH and 20 mL of 96 mM of 3,5-dinitrosalicylic acid solution) and immediately kept in water at 100°C in a water bath for 10 min. The absorbance of the resultant solution was taken at 540 nm. The uninhibited enzyme was taken as control. Suitable blank was used for all the samples. Acarbose was used as the standard inhibitor of the enzyme. The alfa-amylase inhibitory activity was calculated as percentage inhibition: % inhibition = [(OD540 control- OD540 sample)/(OD540 control- OD540 blank)] ×100%, to obtain different inhibitory activities under various concentrations of EECR.

α-Glucosidase inhibitory assay

The slightly modified method of McCue and Shetty[25] was followed. In brief, in a 96-well microplate, 50 μL rat intestinal α-glucosidase (1.0 U/ml) in phosphate buffer (0.1 M, pH 6.8) for 10 min at 37°C was incubated with 50 μl of EECR of various concentrations (0.01–1.0 mg/ml). The reaction was prompted by addition of 50 μL of 5 mM p-nitrophenyl-α-D-glucopyranoside in a 0.1 M phosphate buffer at pH 6.8. p-Nitrophenol release kinetics were read with a microplate spectrophotometric reader Multiskan MS™ (Labsystems, Minneapolis, MN, USA) for 5 min at intervals of 30 s and absorbance was measured at 405 nm. Acarbose served as the reference standard. The alfa-glucosidase inhibitory activity was calculated as percentage inhibition: % inhibition = [(OD405 control- OD405 sample)/(OD405 control- OD405 blank)] ×100%, to obtain different inhibitory activities under various concentrations of EECR. The minimum inhibitory concentration of the extract IC50 was then estimated from the concentration-dependent graph.

In vitro antiglycation assay

The methodology was based on that of Brownlee et al.[26] BSA (l0 mg/mL) was incubated with glucose (500 mM) in phosphate-buffered saline (PBS) (5 mL total volume, pH 7.4) and extract containing 0.02% sodium azide at 37°C with a final concentrations of BSA (2 mg/mL), glucose (40 mM), EECR (0.25–1.0 mg/mL), or aminoguanidine (AMG; 2.5 mM). All the reagent and samples were sterilized by filtration through 0.2 μm membrane filters. The protein, the sugar, and the prospective inhibitor were included in the mixture simultaneously. AMG was used as an inhibitor positive control. Reactions without any inhibitor were also setup. Each solution was kept in the dark in a capped tube. After 21 days of incubation, fluorescence intensity (excitation wavelength of 330 nm and emission wavelength of 410 nm) was measured for the test solutions. Percent inhibition was calculated as follows:

Inhibition % = [1− (As – Ab)/(Ac-Ab)] ×100

Where As = fluorescence of the incubated mixture with sample, Ac = the fluorescence of the incubated mixture without sample as a positive control, and Ab = the fluorescence of incubated mixture without sample as a blank control.

Animal experimental design

Wistar rats (6–7 weeks old) were used for the experiments. The rats were maintained on a 12:12 h light-dark cycle and provided with food (LabDiet 5001 Rodent Diet; PMI Nutrition International) and water ad libitum throughout the study (a total of 84 days). The rats were acclimatized for 1 week before experimentation and divided into the following six experimental groups using randomization. Each group contained six rats. Group 1: CON (control); Group 2: MGO (1% in water, approximately 500 mg/kg/day; po); Group 3: MGO + MET (10 mg/kg/day; po); Group 4: MGO + L-EECR (10 mg/kg/day; po); Group 5: MGO + M-EECR (10 mg/kg/day; po); and Group 6: MGO + H-EECR (50 mg/kg/day; po). MGO (100 mg/kg bw, po) was administered to the rats by oral gavage on days 1–84. EECR and MET were administered to the rats by oral gavage starting from day 21 till day 84. The body weight, food intake, and water intake were recorded daily, and the blood glucose level was measured every week. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) (intraperitoneal injection, 0.5 U/kg bw) in all rats were evaluated once every 2 weeks. All experimental procedures involving animals were conducted in accordance with the Council of Agriculture, Executive Yuan, guidelines. This experiment was approved by the Institutional Animal Care and Use Committee (IACUC, no. 104001) of the Da-Yeh University.

Serum biochemical tests

Blood was collected in serum separator tubes and centrifuged at ×775 g for 15 min to obtain blood serum. Serum biochemical tests were conducted using an automatic biochemical analyzer (Chiron Diagnostics Corporation, Oberlin, OH, USA), and the test items included blood urea nitrogen (BUN), creatinine (CRE), glucose, albumin, cholesterol, triglyceride (TG), glutamic-oxaloacetate transaminase (GOT), and glutamic-pyruvic transaminase (GPT).

Insulin quantification via enzyme-linked immunosorbent assay

Serum insulin levels were quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's recommendations (EMD Millipore; EZRMI-13K; Darmstadt, Germany) from serum obtained as described above in serum separator tubes (SST™, BD Biosciences) at the time of sacrifice and used to evaluate homeostasis model assessment of IR (HOMA-IR), which was calculated as previously described by Matthews et al.[27] using the following formula:

HOMA-IR = Fasting serum insulin (ng/mL) × fasting blood (mg/dL).

Quantification of methylglyoxal-induced advanced glycation end products

MGO-AGEs were quantified using a competitive ELISA according to the manufacturer's instructions (STA-811, Cell BioLabs, San Diego, CA). This ELISA uses a primary antibody that recognizes the hydroimidazolone (H1) moiety created by the modification of protein residues by MGO. Whole blood taken from the left ventricle before transcardial perfusions was collected in serum separator tubes (SST™, BD Biosciences, Franklin Lakes, NJ) and allowed to clot for 30 min. Clotted blood in SSTs was centrifuged at ×5000 g for 10 min at 4°C and the serum was transferred to fresh microcentrifuge tubes and stored at −80°C until MG-H1 ELISA analysis. Serum samples were diluted 1:2 in 0.1 M PBS to obtain concentrations in the span of the standard curve.

Preparation of liver tissue homogenates

After completion of treatment with EECR (on day 84), the rats were killed by decapitation under CO2 anesthesia. All the following procedures were carried out at 0°C–4°C. Whole liver were homogenized in 1:10 buffer (5.0 mM Tris base, 150 mM NaCl, and 20 mM EDTA, pH 7.5) (w/v). The homogenates were sonicated for 30 s in a disruptor (Bronson Sonic, NY, USA) and centrifuged at ×16,000 g for 10 min. The supernatant was kept at −80°C until use.

Determination of superoxide dismutase and catalase activity in liver tissues

The activity of superoxide dismutase (SOD) was measured by the inhibition of pyrogallol autoxidation procedure of Marklund and Marklund[28] with minor modifications. The assay mixture was transferred to a 3.5 mL cuvette and the rate of increase in the absorbance at 420 nm was recorded for 3 min after an initial lag period of 30 s in a ultraviolet (UV)-visible spectrophotometer. The lag period of 30 s was allowed for steady state of autoxidation of pyrogallol to be attained, which is important for reproducibility of results. One unit of SOD activity was defined as the amount required for inhibiting pyrogallol autoxidation by 50% per min. SOD activity was expressed as units of SOD per μg of protein (U/mg protein).

Determination of GSH/GSSG ratio in liver tissues

GSH/GSSG ratios in liver tissue homogenates were assayed according to the HPLC method of Schofield and Chen[29] with some modification. In brief, after adding 100 μ L of 10% perchloric acid to precipitate the protein of liver homogenates (1.0 mL), the supernatants were treated with iodoacetic acid in the dark at 40°C for 1 h. A volume (0.2 mL) of 3% (v/v) 2,4-dinitrofluorobenzene was added to the reaction mixture and allowed to react at 40°C for 4 h in the dark. After centrifugation (3000 g for 15 min), a portion (25 μL) of the supernatant was injected onto HPLC column. The HPLC system consisted of a Hitachi l-6200 pump, a Hitachi D-2500 Chromato Integrator, a Hitachi L-4200 UV-Vis detector, and a 3-aminopropyl column (200 × 4 mm, 5 μ m). The mobile phase consisted of (A) water/methanol (1:4, v/v) and (B) 2 M sodium acetate, pH 4.6/methanol (36:64, v/v). The 3-aminopropyl column was eluted by a 30-min gradient from 75% (A) to 5% and then isocratically maintained for 15 min at a flow rate of 1.2 mL/min and UV detection at 365 nm.

Determination of malondialdehyde in liver tissues

Lipid peroxidation, as evidenced by the formation of malondialdehyde (MDA), was assayed by the method described by Buege and Aust.[30] Butylated hydroxytoluene (10 μL, 50 mM) was added to the tissue homogenate (1.0 mL) to terminate the peroxidation reaction and then mixed with 1 mL of 7.5% (w/v) cold trichloroacetic acid to precipitate proteins. The supernatant was allowed to react with 1 mL of 0.8% (w/v) thiobarbituric acid in a boiling water bath for 45 min. After cooling, levels of thiobarbituric acid reactive substances were determined spectrofluorimetrically (at 515-nm excitation and 555-nm emission) using 1, 1, 3,3-tetraethoxypropane as the standard and were expressed as pmol per milligram of protein.

Oral glucose tolerance test and insulin tolerance test

OGTT and ITT were performed on day 77 and 83, respectively. In the OGTT, after a 16 h fasting period, the rats were orally administered a GLU solution (2 g/kg). Blood GLU levels were measured using a glucometer after 30, 60, 90, and 120 min of GLU load. In the ITT, following a 4 h fast, the rats were intraperitoneally injected with insulin solution (0.5 U/kg). Blood GLU level was recorded after 30, 60, 90, and 120 min of the insulin injection.

Statistical analysis

The data were presented as the mean ± standard deviation. Statistical differences between groups were evaluated by one-way analysis of variance. SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) was used to perform all the statistical analysis. P ≤ 0.05 was considered statistically significant.


  Results Top


Antistarch-hydrolyzing enzymes activity

The effects of hot water and 95% ethanol extracts (EECR) from CCR on α-glucosidase activity

As shown in [Figure 2], both HWCR and EECR can inhibit the activity of α-glucosidase. At a concentration of 2.0 mg/mL, they can almost completely inhibit the activity of α-glucosidase (>99%), but the inhibitory ability of EECR was significantly better than HWCR (P < 0.05), with IC50 of 0.12 and 0.25 mg/mL, respectively. At low concentrations (<1.0 mg/mL), the ability of EECR to inhibit the activity of α-glucosidase was better than that of the known hypoglycemic agent, acarbose. In addition, the inhibition effect of HWCR was almost equivalent to that of acarbose.
Figure 2: The effects of hot water and EECR on α-glucosidase activity.

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The effects of hot water and EECR on α-amylase activity

[Figure 3] shows that the ability of EECR to inhibit amylase activity significantly increased with the increasing concentration; IC50 was about 0.32 mg/mL. The IC50 of EECR is much lower than that of acarbose (16 μg/mL). In contrast, HWCR had no obvious ability to inhibit amylase activity. There is no significant dose-dependent effect between the concentration of 0.1 and 2.0 mg/mL (P > 0.05); their inhibitory effects were only about 12%–16%.
Figure 3: The effects of hot water and EECR on α-amylase activity.

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Antiglycation activity

The antiglycation ability of EECR is evaluated by its ability to inhibit the formation of AGEs from the conjugation of GLU and BSA, that is, the stronger the ability to inhibit the formation of AGEs, the better the antiglycation effect. As shown in [Figure 4], the higher the concentration of EECR, the better the antiglycation ability. At a concentration of 0.25 mg/mL of EECR, there is about 32.4% of the antiglycation effect. When the concentration was 0.5–1.0 mg/mL of EECR, the antiglycation effect is about 51%–55%, which is comparable to that of 2.5 mM (0.34) aminoguanidine (AMG, 54%) (P > 0.05).
Figure 4: The anti-glycation activity of EECR. Values (means ± standard deviation, n = 3 for the test groups) not sharing the same superscript letter are significantly different (P < 0.05).

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Effect of EECR on body weights of rats treated with methylglyoxal

To investigate whether the EECR have beneficial effects on MGO-induced IR, the rats were orally administered the EECR (10, 50, or 100 mg/kg) for 63 (21–84) days when simultaneously receiving 1% MGO (approximately 500 mg/kg/day; po). As shown in [Table 1], MGO alone significantly decreased body weights of rats starting on day 64 of the experiment until day 84, as compared to the control rats (P < 0.05). However, supplementation with EECR (10–100 mg/kg bw) resulted in substantial recovery of body weights, and no significant differences in body weights were found on days 64–84 in these groups of rats, as compared with the control rats (P > 0.05).
Table 1: Effects of ethanol extracts of cajanus cajan roots on body weight changes in rats treated with methylglyoxal

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EECR on fasting blood glucose level, glycosylated hemoglobin, insulin, homeostasis model assessment of insulin resistance, and advanced glycation end products in methylglyoxal-administered rats

[Table 2] indicates that fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), and insulin of MGO-administrated rats presented a remarkable elevation in comparison to the control group (P < 0.05). At the end of the experiment (on 84 days), H-EECR-treated groups presented a remarkable reduction (P ≤ 0.05) in FBG, HbA1c, and insulin (P ≤ 0.05) compared to the MGO-treated rats. Although compared with the control group, the FBG of M-EECR group was not significantly lower than that of the MGO treatment group, the HbA1c% was significantly lower than that of the MGO treatment group (P < 0.05), and there was no significant difference from the control group (P > 0.05). FBG, HbA1c, and insulin levels induced by MGO could completely inhibit by H-EECR, which are better than the MET group. There was a significant increase in HOMA-IR (P < 0.01) in the MGO group. However, metformin and EECR significantly reduced HOMA-IR (P < 0.05), and EECR showed a significant dose-dependent effect to reduce the increase in HOMA-IR induced by MGO. The AGEs level was indicated as the glycated proteins in the membrane. AGE level was markedly enhanced in the MGO-administered group as compared to the CON group. As expected, administration of the EECR or metformin significantly suppressed the AGEs formation in the MGO-administered rats [Table 2]. These findings suggested that the EECR inhibited MGO-induced AGEs formation in vitro and in vivo.
Table 2: The effects ethanol extracts of cajanus cajan roots on fasting blood glucose, glycosylated hemoglobin, insulin, homeostasis model assessment of insulin resistance and advanced glycation end products in methylglyoxal -administered rats on day 83

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EECR on serum lipid biochemical values in methylglyoxal-administered rats

As shown in [Table 3], total cholesterol (TC), TG, low-density lipoprotein (LDL), and atherogenic index of MGO groups compared with CON group were significantly increased (P < 0.05). In L-EECR (P < 0.05), M-EECR (P < 0.05), and H-EECR (P < 0.01) groups, a certain lowering effect in TC was observed in three doses of treatment. The effect of decreasing TG and LDL in L- and M-EECR groups (P < 0.05) and H-EECR (P < 0.01) was statistically significant compared with the CON group. However, there was no significant difference in serum high-density lipoprotein (HDL) between the MGO-administrated rats and the control group (P > 0.05). Noticeably, the M- and H-EECR treatments substantially decreased the atherogenic index, with the M-EECR treatment resulting in a level (1.59) that is even lower than that (1.79) of the CON group.
Table 3: The effects ethanol extracts of cajanus cajan roots on serum lipid biochemical values and atherogenic index in methylglyoxal-administered rats

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The effects of EECR on biochemical markers of liver (glutamic-oxaloacetate transaminase and glutamic-pyruvic transaminase) and kidney (blood urea nitrogen and creatinine) in rats serum induced by methylglyoxal

The effects of EECR for 63 days on biomarkers of liver injury (GOT and GPT) and kidney injury index (BUN and CRE) in MGO-treated rats are presented in [Table 4]. In the MGO group, only GPT (liver injury index) and BUN (kidney injury index) increased insignificantly compared to the CON group (P < 0.05). However, H-EECR insignificantly reduced the serum GPT induced by MGO (P < 0.05). Although EECR tended to reduce serum BUN, the effect was not significant compared to that of MGO treatment (P > 0.05).
Table 4: The effects ethanol extracts of cajanus cajan roots on biochemical markers of liver (glutamic-oxaloacetate transaminase, glutamic-pyruvic transaminase) and kidney (blood urea nitrogen, creatinine) in rat serum induced by methylglyoxal

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The effects of EECR on antioxidant status of liver in rats induced by methylglyoxal

As presented in [Table 5], liver MDA concentration in the MGO group was significantly increased (P < 0.001) vs. the CON group. Administration of L-, M-, and H-EECR and metformin to MGO-treated rats caused a significant decrease in liver MDA concentration vs. the MGO group (P < 0.05). There was a significant decrease (P < 0.01) in SOD activity and GSH/GSSG ratio in the MGO group vs. CON group. However, L-, M-, and H-EECR and metformin significantly increased SOD activity and GSH/GSSG ratio (P < 0.05) in the MGO-treated rats vs. MGO group.
Table 5: The effects ethanol extracts of cajanus cajan roots on antioxidant status of liver in rats induced by methylglyoxal

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Effects of EECR on glucose tolerance and insulin sensitivity in methylglyoxal-administered rats

To investigate whether the EECR have beneficial effects on MGO-induced IR, the rats were orally administered the EECR (10, 50, or 100 mg/kg) for 63 days when simultaneously receiving 1% MGO (approximately 500 mg/kg/day; po). As shown in [Figure 5]a, except for the treatment of MGO alone, the rats in each group had a maximum blood sugar value of about 122–139 mg/dL at 60 min, and the blood sugar value at 120 min dropped to 88-108 mg/dL; there was no significant difference in blood sugar between the MET and L-, M-, and H-EECR-treated group and the control group (P > 0.05). The blood sugar level of Group 2 (MGO only) rats reached the maximum at 90 min (133 md/dL), and the blood GLU level only dropped to 121 mg/dL at 120 min. Although the blood GLU levels of rats in the MET and L-, M-, and H-EECR-treated groups were lower than those in the MGO alone-treated group, the decrease did not reach a significant level (P > 0.05). In addition, calculation of the blood sugar area under the curve (AUC) revealed that EECR administration did not significantly affect AUC, as compared with MGO treatment alone or the CON group [Figure 5]b.
Figure 5: Effect of EECR on glucose tolerance and insulin sensitivity in methylglyoxal-treated rats. Oral glucose tolerance test and insulin tolerance tests were carried out on day 84. Blood sugar levels (a) and area under the curve (b) of oral glucose tolerance test. Blood glucose levels (c) and area under the curve (d) of insulin tolerance tests. Two groups of rats were supplemented with saline solution (control group) or methylglyoxal solution (methylglyoxal group) without administration of test materials. The other methylglyoxal-treated rats were supplemented with metformin (10 mg/kg/d), low-dose of EECR (L-EECR, 10 mg/kg/day), middle-dose of EECR (M-EECR, 50 mg/kg/day), and high-dose of EECR (H-EECR, 100 mg/kg/day). Values (means ± standard deviation, n = 6 for the test groups) not sharing the same superscript letter are significantly different (P < 0.05).

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In the ITT, insulin sensitivity was markedly reduced after 78 days of MGO treatment, as demonstrated by the higher blood GLU levels in Group 2 rats, as compared with those of CON group after insulin injection (at 30, 90, and 120 min). The reduction of insulin sensitivity was attenuated by the administration of EECR, and the effect of EECR was significant at 10–100 mg/kg of EECR (P < 0.05) [Figure 5]c and [Figure 5]d. Metformin treatment also showed significant hypoglycemic effects in the MGO-administered rats (P < 0.05) [Figure 5]c and [Figure 5]d.


  Discussion Top


Long-term hyperglycemia is known to cause many complications that can endanger life. Therefore, strict control of blood sugar level is the best way to prevent and improve diabetes and its complications. Recently, we found that the EECR has antioxidant, anti-inflammatory, antibacterial, and antiglycation abilities.[20] We also found that EECR contains abundant flavonoids such as genistein, cajanol, daidzein, and Biochanin A.[31],[32] In this study, we found that EECR effectively inhibited MGO-induced: 1) IR (assessed by HOMA-IR) to improve insulin sensitivity, 2) fasting blood sugar and lipids (TG, cholesterol, and LDL) in serum of rats, and 3) liver and kidney damage. Therefore, our study supports the insulin-sensitizing activity of EECR and suggests that the extract might improve other manifestations of the metabolic syndrome.

Hyperglycemia is characterized by a rapid increase in blood GLU levels and is due to quick and continuous hydrolysis of starch by pancreatic α-amylase and absorption of GLU by the intestinal α-glucosidases.[33] One of the therapeutic approaches to decrease PH is to slow down digestion of GLU by the inhibition of these carbohydrate-hydrolyzing enzymes (α-amylase and α-glucosidase) in the digestive tract.[33] Therefore, inhibition of α-amylase and α-glucosidase can significantly decrease PH after a carbohydrate-mixed diet and can be an important strategy in the therapy of DM.[34]

In the present study, the EECR are a mild inhibitor of α-amylase and a strong inhibitor of α-glucosidase, respectively. This is because higher concentration (0.32 mg/mL) of the extract is required to effectively inhibit α-amylase compared to the α-glucosidase, which was obtained at 0.12 mg/mL. This is proved by the respective IC50 generated for the inhibition of the enzymes by EECR. This is in agreement with the notion of a previous report[35] that any medicinal plant that may be used as antidiabetic agents should be a mild inhibitor of α-amylase and a strong inhibitor of α-glucosidase.[35] The reason for the different inhibitory effects of EECR on α-glucosidase and α-amylase is unclear, but similar results have been reported in several studies from different plants, i.e., the hexane extract has, in general, a higher inhibitory effect on α-amylase activity than the extracts of polar solvents (e.g., methanol, acetone, or ethyl acetate).[36],[37]

The inhibitory effects of EECR on α-glucosidase are better than that of the synthetic drugs, acarbose, which strongly inhibits both α-amylase and α-glucosidase, leading to side effects such as abdominal distention, flatulence, meteorism, and sometimes diarrhea due to abnormal bacterial fermentation of undigested carbohydrates in the colon.[20],[38]

MGO is the most important and well-studied precursor of AGEs. This highly reactive dicarbonyl metabolite reacts with arginine or lysine residues of proteins to form AGEs.[39] AGEs accumulate in vivo and activate various signaling pathways (including the mitogen-activated protein kinase, nuclear transcription factor (NF-κB), and signal transducer and transcriptional activator pathways) closely related to the occurrence of various chronic metabolic diseases (diabetes, atherosclerosis, and Alzheimer's disease).[39] AGEs accumulation has been reported to contribute to inflammation and oxidative stress via the ROS overproduction, which is recognized as the major factors in IR.[40] Extracellular AGEs also activate RAGE signaling, leading to oxidative stress, inflammation, and IR. Inhibition of AGEs formation can be considered as a promising therapeutic strategy to diminish glycative stress-induced IR.[41]

Therefore, we investigated whether the EECR can affect AGEs formation induced by BSA-GLU glycation. We evaluated the inhibitory effects of the EECR on AGEs formation in vitro. We found that the formation of AGEs increased with time between 7–21 days of BSA-GLU incubation. The EECR (250–1000 μg/mL) inhibited the AGEs formation in a dose-dependent manner. The ability of 500 ug/mL EECR to inhibit the formation of AGEs is equivalent to 2.5 mM (185 μg/mL) of AMG (54% of inhibition). In vivo, treatment with EECR also significantly suppressed the serum AGEs level in MGO-administered rats.

Insulin induces phosphorylation of tyrosine residues on insulin receptor substrate (IRS) proteins by binding to the insulin receptor. Then, the phosphorylation of IRS results in the activation of Akt, which stimulates the translocation of glucose transporter (GLUT) from the cytosol to the cell membrane, facilitating glucose uptake into cells.[42] Previous studies indicated that treatment with MGO resulted in IR in vitro and in vivo by decreasing IRS phosphorylation and induced insulin-dependent Akt activation and NO generation.[43] In the present study, EECR significantly improved glucose tolerance (reduced FBG and HbA1c) and insulin sensitivity (HOMA-IR) in MGO-administered rats. The result suggests that EECR mediates the inhibition of MGO-induced IRS1/Akt/eNOS pathway. In addition, the reduction of IR (assessed by HOMA-IR) in the EECR group indicates the improvement of insulin sensitivity. Foretz et al.[44] indicated that metformin reduces blood glucose by increasing insulin sensitivity and reducing hepatic gluconeogenesis. We found that the improvement of EECR on IR was even better than that of metformin.

Song et al.[39] indicated that the mechanisms of inhibition of glycative stress by natural compounds (such as polyphenols, polysaccharides, terpenoids, vitamins, and alkaloids) include inhibition of the glycation of proteins, scavenging oxidative free radicals, regulating receptors of AGEs, trapping active dicarbonyl compounds, chelating metal ions, inhibition of aldose reductase, and lowering blood glucose levels.

Wang et al.[45] reported that dietary genistein ameliorates body weight gain, fat deposits, dyslipidemia, hyperglycemia, and fatty liver in the accumulation of MGO and AGEs mice. Genistein inhibits AGEs formation by trapping MGO to form adducts and upregulating the expressions of glyoxalase I and II and aldose reductase in liver and kidney to detoxify MGO. Moreover, it has been shown that genistein is a potent α-glucosidase inhibitor.[46] Biochanin A administration in STZ-induced type 1 diabetes mellitus rats lowers blood glucose levels and decreases angiogenesis and inflammation via suppressing vascular endothelial growth factor, tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β.[47] Cinnamaldehyde ameliorates STZ-induced rat diabetes through modulation of IRS1/PI3K/AKT2 pathway and AGEs/RAGE interaction.[48]

The phosphorylation of ERK, p38, and NF-κB, and the mRNA expression of proinflammatory molecules including TNF-α and IL-1β and iNOS were shown to increase in MGO-administered mice.[49] Recently, our study indicated that EECR mainly activates nuclear factor (NF) erythroid 2related factor 2 (Nrf-2)/antioxidant protein heme oxygenase1 (HO-1) and inhibits NF-κB signaling pathway.[31]

A significant reduction in body weight has been reported in poorly managed diabetes.[50] A similar observation was reported in this study where the MGO-administrated rats show substantial weight reduction compared to CON group. This reduction could be correlated with metabolic derangements associated with poor glucose utilization. Truong et al.[49] indicated that treatment with MGO significantly decreased the insulin-stimulated glucose uptake compared to the cells receiving no MGO treatment. Dare et al.[51] reported that sample treatment reduce weight loss during diabetes could halt some metabolic derangements associated with muscle wasting and loss of adipose tissues.

Metabolic abnormalities within the IR syndrome, such as IR, hyperinsulinemia, hypertension, hypertriglyceridemia and decreased HDL cholesterol, and chronic inflammation commonly parallel the excess of body weight.[52],[53] Thus, we speculated EECR administration to MGO-treated rats improves body weight could be related with EECR can ameliorate MGO-induced IR by increasing insulin sensitivity to maintain metabolic regularity.

EECR treatment recovers the activity of SOD and GSH/GSSG ratio (a useful indicator of oxidative stress in cells and tissues) in the liver tissue of the MGO-administered rats. These findings are consistent with the suggestion that EECR also reduces the MDA level and the markers of hepatic and kidney injury (GPT and BUN) by upregulation of antioxidant enzymes, as the removal of ROS is regulated by enhancing antioxidant enzymes, including SOD, HO-1 and Nrf-2.[31] Thus, we speculate the ameliorative effects of EECR on IR in MGO-induced rats is mainly due to its enhanced antioxidant and anti-inflammation activities.

To our best knowledge, there is no published article on the safe dose of Cajanus cajan root, but we recently reported in a rat study that the NOAEL for EECR is approximately 1 g/kg bw,[54] which, by extrapolation from rats to humans, is equivalent to approximately 972 mg/60 kg person/day.[55] Since the insulin-sensitizing dose of EECR used in this study (50-100 mg/kg bw) is much lower than the estimated safe dose, the future applicability of EECR can be demonstrated.


  Conclusion Top


Overall, the present findings indicate the potential of EECR in the prevention and treatment of IR. [Figure 6] schematically summarizes the effect of EECR existing strategies and suggests several new directions in diminishing glycative stress that may induce IR, including: (1) As AGEs inhibitors and MGO trapping; (2) inhibited the AGE-RAGE signaling pathway; (3) diminished pro-inflammatory cytokines levels (IL-1 β, IL-6 and TNF-α) as well as downregulated NF-κB; (4) ameliorated the oxidative stress by increasing GSH/GSSG ratio and reducing MDA levels; and (5) as a carbohydrate hydrolyzing enzyme inhibitor to decrease PH. In addition, isoflavonoids (such as: Genistein, cajanol, biochanin A) in EECR may play a major role in prevention IR.
Figure 6: Schematic summaries the hypoglycemic and hypolipidemic effects of EECR on methylglyoxal-induced insulin resistance in male Wistar rats. ⊕: activation; ∅: inhibition.

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Acknowledgements

We thank Professor Ming-Ching Cheng of Chung Chou University of Science and Technology kindly provide administrative and technical support. This research was funded by the Ministry of Science and Technology of the Republic of China (ROC), Taiwan, for financially supporting this research under contract No. MOST 106-2320-B-212-002, and partly funded by the Taichung Veterans General Hospital Project, Da-Yeh University Taiwan, R.O.C. under contract No. TCVGH-DYU1068305 and Ministry of Education awards and subsidies to Chienkuo Technology University (CTU-108-RP-BS-001-005-A).

Financial support and sponsorship

This research was funded by the Ministry of Science and Technology of the Republic of China (ROC), Taiwan, for financially supporting this research under contract No. MOST 106-2320-B-212-002, and partly funded by the Taichung Veterans General Hospital Project, Da-Yeh University Taiwan, R.O.C. under contract No. TCVGH-DYU1068305 and Ministry of Education awards and subsidies to Chienkuo Technology University (CTU-108-RP-BS-001-005-A).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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