Anti-hyperglycemic Activity and Inhibition of Advanced Glycation End Products by Lonicera japonica Thunb. in Streptozotocin-induced Diabetic Rats

Abstract

We investigated whether L. japonica Thunb. extract (LTE) has anti-hyperglycemic activity and/or inhibits the formation of advanced glycation end products (AGEs) in streptozotocin (STZ)-induced diabetic rats. LTE was examined for free-radical scavenging activity and α-glucosidase inhibitory effects, LTE strongly exhibited both activities, ED₅₀ was 3.08 mg/mL, and IC₅₀ was 1.348 mg/mL, respectively. The concentration of myo-inositol, which evaluates the insulin-mimetic activities of LTE was 2.24 g/100 g. These and other results suggest that LTE corrects intracellular myo-inositol deficiencies and improve insulin sensitivity. After STZ-induced diabetic rats freely ingested LTE for 12 weeks, glycoalbumin and blood glucose levels were reduced while serum biochemical profiles were improved. When tested in rat lens crystallins, 0.25% LTE inhibited N^ε-(carboxymethyl) lysine formation by 17.7% when compared with the untreated groups. These results suggest that LTE ameliorates diabetes-induced abnormalities by an anti-hyperglycemic activities and inhibitory effects on the formation of AGEs.

Introduction

Metabolic syndromes have become a hot topic as they are diseases brought on by lifestyle. Diabetes mellitus, high blood pressure, and hyperlipidemia are common lifestyle-related diseases. In general, type 2 diabetes (T2D) develops in adults due to lack of exercise, overeating, and drinking in excess resulting in reduced insulin secretion and sensitivity. On the other hand, type 1 diabetes (T1D) is due to the destruction of β-cells in the pancreas that produce insulin. The incidence of T1D has been increasing at a rate of approximately 3% per year since the 1960s (Pitkäniemi et al., 2004). Diabetes causes nerve damage, retinopathy, and nephropathia, as well as angiopathy, infection disease, cataracts, and cutaneous diseases (Dyck et al., 1993). In particular, cataracts develop due to the glycation and oxidation of crystallin proteins in the eye lens (Ahmed, 2005). Diabetic retinopathy is a collective term for all disorders of the retina caused by diabetes. The majority of type 1 diabetic patients will develop retinopathy over a 15–20-year period (Hazin et al., 2001). The advanced glycation end products (AGEs) related with diabetic conditions suggests that AGEs may be responsible for some retinal pathologies (Stitt, 2003). The detection of N^ε-(carboxymethyl) lysine (CML), one of the main antigenic structures of AGEs, is widely used to demonstrate the contribution of the CMLs in the etiology of diabetic complications (Koito et al., 2004).

Oral anti-diabetes drugs have been used in diabetic care; however, side effects such as abdominal distension, increase of flatulence, diarrhea, anemia, hypoglycemia symptom, and hepatic damage all may occur (Cicero et al., 2004). With regards to safety, prevention and suppression of diabetes using diet is very effective. The antidiabetic effects of resveratrol (Silan, 2008), chlorogenic acid (Taguchi et al., 2014), morin (Iizuka et al., 2011), black and green tea (Vinson and Zhangm, 2005), and genistein (Hanamura et al., 2006) also been reported. In addition, herbal medicine research is attracting attention (Jia et al., 2003). Although herbal medicines take time to be effective, their efficacy has been demonstrated. Furthermore, the side effects of herbal medicines appear to be significantly reduced when compared to antidiabetic drugs (Rao et al., 2010).

Lonicera japonica Thunb., (Japanese Honeysuckle) has been widely used in the food industry because of its health benefits, such as anti-inflammatory, antibacterial, antiviral, antioxidative, and hepatoprotective activities (Shang et al., 2011a). Usually, L. japonica Thunb is extracted with hot water before drinking. Many compounds, such as chlorogenic acid (Shang et al., 2011a), luteolin (Yip et al., 2006), and iridoids (Choi et al., 2007), have been isolated and identified from L. japonica Thunb. These compounds are known to aid in lifestyle-related disease prevention by suppressing inflammation and oxidative stress (Choi et al., 2007). Myo-inositol was isolated from ethanol extracts of Lonicera bournei Hemsl flower buds, which has the same honeysuckle genus as L. japonica Thunb. (Song et al., 2006; Han et al., 2015). Myo-inositol is a cyclitol naturally contained in food derived from plants (e.g., cereals and fruits) or animals (e.g., meat). Several inositol isomers and derivatives display insulin-mimetic activities (Loewus and Murthy, 2000). Abnormalities in the metabolism of inositol are associated with insulin resistance (Croze and Soulage, 2013). In addition, several authors have suggested that diabetes is a condition of inositol depletion (Greene et al., 1975; Salway et al., 1978; Scioscia et al., 2007). It is expected that T1D may be improved by supplementing myo-inositol.

In recent years, the antidiabetic effects of L. japonica in T2D rats have been reported (Xiang et al., 2001). Therefore, L. japonica is also expected to be effective for T1D, and we investigated the inhibitory effect of L. japonica Thunb. extract (LTE) on hyperglycemia in T1D rats. The present study first examines the antioxidative and α-glucosidase inhibitory activities of LTE. Following, the chlorogenic acid and luteolin content in the LTE was determined. In addition, the levels of myo-inositol, which had yet been determined in LTE, were measured. Blood glucose levels and serum biochemical profiles were also observed during LTE treatment. Finally, because it has been reported that streptozotocin (STZ)-induced diabetic rats had significantly increased CML concentrations in the lens crystallins (Karachalias et al., 2003), the inhibitory effects of CML formation in STZ-induced diabetic rats was investigated.

Materials and Methods

Extract from the dried flower bud of L. Japonica    Dried flower buds of the L. Japonica Thunb. (Jiangsu Kanion Pharmaceutical Co., Ltd., Jiangsu Lianyungang, China, 1.5 kg) were added to a 20-fold volume of distilled water (30 L) and extracted by hot-water at 90 °C for more than 1 h. The solution was filtered and the filtrate was concentrated by using a vacuum evaporator (until a solid content of 35% was obtained) and sterilized via boiling at 80 °C for 30 min. After cooling on ice for 20 min, the resultant thickened liquid was dried with a spray drier (Yamato pulvis spray dryer model GB-22, Yamato Scientific) to obtain a crude dried L. japonica Thunb. extract (LTE) (Fig. 1). Two extractions yielded 919.1 g of dried extract powder, a 30.64% recovery.

Fig. 1.

Before and after drying flower buds of Lonicera japonica Thunb. (a) The flower bud of L. japonica Thunb.; (b) The dried flower bud of L. Japonica Thunb.; (c) The crude dried L. japonica Thunb. extract.

HPLC analysis for the determination of chlorogenic acid and luteolin in LTE    A Cosmosil 5C18-MS-II (5 m, 4.6 mm I.D.×250 mm, NacaleiTesque) column was used for chromatographic separation. The mobile phase consisted of a 0.1 M phosphate buffer (pH 2.5) containing 0.1 mM EDTA (solvent A) and acetonitrile (solvent B). The gradient program was as follows: 20% B for 10 min, 10% B to 25% B for 20 min, 25% B to 40% B for 20 min. The flow rate was 1.0 mL/min for a total run time of 60 min. The column temperature was maintained at 30 °C. The injection volume in each experiment was 5 µL and the detection wavelength was 350 nm. The sample was spiked with chlorogenic acid or luteolin at approximately the same concentration as the sample components. If the peak in the chromatogram became larger, then the peak was identified as chlorogenic acid or luteolin. The calibration curves were constructed using four different concentrations (5, 25, 50, and 125 µg/mL) of each standard (chlorogenic acid and luteolin) by plotting the concentration of the standard against the peak area. Unknown concentrations of chlorogenic acid and luteolin were quantified by relating each respective peak area to the regression line.

Microbiological and HPLC analysis for the determination of myo-inositol in LTE    Myo-inositol content of LTE was analyzed according to a microbiological assay method using Saccharomyces cerevisiae (S. cerevisiae) ATCC 9080 (Duliński et al., 2011). Myo-inositol was extracted from the samples by acid hydrolysis by 18% (v/v) HCl. Yeast was grown in standard inositol solutions to generate a standard curve. Control and sample extracts were measured for turbidity at 600 nm, allowing us to calculate the myo-inositol concentration in our samples from the standard curve.

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay    LTE was dissolved in Milli Q water and methanol (1: 1) and ascorbic acid (0.1, 0.2, 0.25, and 0.5 mg/mL). The test solution (0.1 mL) was mixed with 2.4 mL of 0.1 M acetic buffer and 1.5 mL of a 0.016% DPPH/ethanol solution. After standing for 10 min, 5.0 mL of xylene was added and shaken for 15 sec. The solution was centrifuged (1 000 × g) for 5 min. The upper layer was collected and its absorbance was measured with a UV spectrophotometer in 510 nm. Ascorbic acid was used as a standard for our DPPH radical scavenging assays.

Radical scavenging activity (%)=100-{(a-b)/a}×100

a: the absorption without samples, b: the absorption with samples

α-Glucosidase inhibitory assay    α-Glucosidase activity was measured using a glucoamylase/α-glucosidase assay kit (Kikkoman, Chiba, Japan) according to the manufacturer's instructions. One unit of glucoamylase and α-glucosidase activity was defined as the amount of enzyme that catalyzed the formation of 1 µmol p-nitrophenol (PNP) per minute from PNP-β-d-maltoside and PNP-α-d-glucoside at 37 °C, respectively. α-Glucosidase (12.5 units; from Bacillus stearothermophilus; SIGMA) was dissolved in 5 mL of Milli Q water (2.5 units/mL). This solution was mixed with methanol (LTE 0 mg/mL) or LTE (0.1, 0.5, and 2.0 mg/mL) (1:1 ratio). After pre-incubating with 2.0 mL of substrate solution at 37 °Cfor 5 min, 100 µL of the sample solution was added and incubated at 37 °C for 10 min. The reaction was terminated by the addition of 1.0 mL stop solution. α-Glucosidase activity was determined spectrophotometrically at 400 nm. α-Glucosidase activity was assessed by the following formula:

α-Glucosidase activity (%)=(E2s–E2b)×0.171

E2s: absorbance of test sample; E2b: absorbance of blank (substrate solution).

α-Glucosidase inhibitory activity was assessed by the following formula:

α-Glucosidase inhibitory activity (%)=100×(control–sample)/control

Control: α-Glucosidase activity at 2.5 units/mL without LTE (100%); sample: α-glucosidase activity with LTE.

Animals    Five-week-old male Wistar rats were purchased from Japan SLC Inc. (Hamamatsu, Japan). All animals were maintained in a temperature-controlled room (temperature 23 °C ± 1 °C, humidity 55% ± 5%) on a 12 h light/dark cycle and were acclimatized to the laboratory environment for 1 week before the experiment. Rats were randomly divided 25 animals into 5 groups as follows: normal rat group (water), diabetes mellitus (DM) rat + water group (control group), DM rat + LTE 0.05% group, DM rat + LTE 0.1% group, and DM rat + LTE 0.25% group. DM was induced by injection of streptozotocin (STZ, 40 mg/kg B.W.) dissolved in 0.05 M citric sodium buffer (pH4.5) at the caudal vein. A 10% glucose solution was given for 12 h after injection of STZ, and pathogenesis of DM was confirmed by measurement of blood glucose using free-style freedom Kissei sensor (Kissei pharmaceutical co., Tokyo, Japan) one week after treatment. Rats treated with LTE (0.05%, 0.1%, and 0.25%, dissolved in water) were given free intake for 12 weeks. After the 12 weeks' experimental period, the rats were denied access to food and water overnight and then sacrificed by rapid neck disarticulation. Subsequently, eyes were collected for CML measurements in the lens. After sacrifice, body weight, liver, and kidney weights were measured. A blood sample was obtained from the ventral aorta. To separate the blood plasma, blood samples were collected into blood collection tubes containing heparin lithium (Capiject; Terumo, Tokyo, Japan) and then centrifuged (1 600 × g, 10 min, 4 °C). The intake of LTE powder (mg/g body weight) was determined from the amount of water consumed. All experimental procedures were in accordance with the guidelines of the University of Shizuoka, Japan, based on those of the American Association for Laboratory Animal Science (Permit Number: 145056 and 145075).

Biochemical analysis of the plasma samples    Biochemical analysis was performed for blood plasma samples. The plasma parameters, including blood glucose, glycoalbumin, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglyceride, uric acid, blood urea nitrogen, creatinine, acetoacetate, and 3-hydroxybutyrate and arterial blood ketone body ratio (acetoacetate/3-hydroxybutyrate) were analyzed by the SRL Corporation (Tokyo, Japan).

Measurement of CML level in lens crystalline    Lens crystallins were removed from rat eyes after sacrifice and homogenized with 0.02 M phosphate buffered saline. The suspension was centrifuged (4 °C, 8 000 × g, 30 min) and supernatant was stored for test samples at −80 °C. The protein levels in the supernatant were measured by BCA protein assay. Then, 1 µg/well (10 µg/mL) supernatant was applied by coating buffer (Na₂CO₃ and NaHCO₃ dissolved in Milli Q water, pH9.7). The CML levels in test sample were measured by ELISA (Moriyama et al., 2010). Each well of a 96-well microtiter plate was filled with 100 µL of test sample (10 µg/mL protein) in coating buffer and incubated for 12 h at 4 °C. After incubation, the wells were washed three times in the washing buffer, and blocked with 200 µL of blocking buffer (0.5% gelatin in coating buffer) and incubated for 1 h at room temperature. The wells were washed three times in the washing buffer and then incubated with 100 µL of anti-AGEs-BSA antibody (0.1 µg/mL; Clone No.6D12) (Trans Genic Inc.) for 1 h at room temperature. The wells were washed three times in the washing buffer and incubated with 100 µL of HRP-conjugated anti-mouse IgG antibody (1:5 000 in washing buffer; Seikagaku Co. Tokyo, Japan) for 1 h at room temperature. The wells were then washed four times in washing buffer and reacted with substrate solution (O-phenylenediamine tablet in 10 mL substrate buffer containing 6 µL 30% hydrogen peroxide) for visualization. After 3.5 min, the reaction was stopped by the addition of 1 M sulfuric acid, and the absorbance at 492 nm was measured using a microplate reader.

Statistical analysis    Each experiment was conducted at least three times. The results shown in all figures correspond to the means ± SD. The results were analyzed using one-way ANOVA, followed by the Dunnett's test using Microsoft Excel 2016 (Microsoft, Redmond, WA, USA). The Tukey-Kramer test was used to compare differences between groups using Microsoft Excel 2016 (Microsoft). A p value of <0.05 or less was considered significant in this study.

Results

Chlorogenic acid, luteolin and myo-inositol content of LTE    The HPLC chromatogram of LTE, standards of chlorogenic acid and luteolin are shown in Fig. 2. The content of chlorogenic acid and luteolin in our LTE samples was 15.5 and 4.45 mg/g, respectively. The content of myo-inositol in our LTE samples measured by microbiological assay method was 22.4 mg/g.

Fig. 2.

(a) The HPLC chromatogram of Lonicerajaponica Thunb. Extract; (b) HPLC chromatogram of standards of chlorogenic acid and luteolin.

DPPH radical scavenging assay    Results for the DPPH radical scavenging activity of LTE is shown in Fig. 3. The DPPH radical scavenging activity of LTE increased in a dose dependent manner at sample concentrations between 0.1 and 5.0 mg/mL (0.1 mg/mL, 4.2%; 0.5 mg/mL, 12.0%; 1.0 mg/mL, 25.5%; 5.0 mg/mL, 78.8%). Furthermore, we compared LTE DPPH radical scavenging activity with ascorbic acid to determine its antioxidant potential. The ED₅₀ for ascorbic acid was 0.268 mg/mL. This concentration is equivalent to 3.08 mg/mL of LTE (Fig. 3 (b)).

Fig. 3.

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of Lonicera japonica Thunb. extract (LTE). (a) Ascorbic acid standard curve of DPPH radical scavenging assay, (b) ED₅₀ values for LTE. Values represent the mean ± SD (n = 3).

α-Glucosidase inhibitory assay    α-Glucosidase activity at 2.5 units/mL α-glucosidase was assessed at 100%, and α-glucosidase inhibitory activity was measured using the formula given in Materials and Methods. The calibration curve for the inhibitory activity of α-glucosidase was shown in Fig. 4 (a). At concentrations of 100, 500, and 2 000 µg/mL of LTE, the inhibitory effect of α-glucosidase reached 1.14, 22.63 and 73.29%, respectively. LTE inhibited α-glucosidase activity in a dose dependent manner, and the IC₅₀ value was determined to be 1.348 mg/mL (Fig. 4 (b)).

Fig. 4.

α-Glucosidase inhibitory activity of Lonicera japonica Thunb. Extract (LTE). (a) Calibration curve for the inhibitory activity of α-glucosidase, (b) ID50 values for LTE. Values represent the mean ± SD (n = 3).

Water intake, body and tissue weights    The water intake was monitored periodically by weighing the leftovers for each cage. The LTE groups tended to have more water intake than the DM group. Water intake was not changed in the control DM group but changed in all three LTE groups treated for 100 days (Fig. 5). Body weight of the treated LTE groups was greater than the control DM group (Fig. 6 (a), Table 1). The daily LTE intake per body weight did not change during the test period except day 91–100 of the LTE 0.25% group. The average LTE intake in the LTE 0.05%, 0.1%, and 0.25% groups per 1 g body weight was approximately 0.30, 0.65, and 1.54 mg, respectively. In the LTE 0.25% group, LTE intake per body weight increased when days 1–10 and 91–100 were compared (Fig. 6 (b)). In the LTE 0.05% and 0.1% groups, there were no changes in LTE intake per body weight until 100 days. The body weight and kidney weight were significantly different in DM + LTE 0.05% group and 0.25% group when compared to control DM group (Table 1).

Fig. 5.

Daily water intake of Wistar rats treated for 100 days with Lonicera japonica Thunb. extracts (LTE). The graph represents average daily water (± SEM) intake every 10 days. Values represent the mean ± SD. Means with the same alphabet are not significantly different from each other (Tukey-Kramer test, represents p < 0.05 compared to each control).

Fig. 6.

Changes in body weight and daily intake of Lonicera japonica Thunb. extract (LTE). (a) Longitudinal changes in body weight of Wistar rats treated for 100 days with LTE. The body weight data are shown as means ± SD. Statistical analysis was performed with Dunnett's test. * represents p < 0.05 compared to the first day of the trial, ** represents p < 0.01 compared to the first day of the trial. (b) Changes in the daily intake of LTE (mg/g body weight). The intake of LTE (mg/g body weight) was determined from the amount of water consumed. The daily intake data are shown as means ± SD. Statistical analysis was performed with Dunnett's test at each group. * represents p < 0.05 compared with 1–10 days.

Table 1. Effect of Lonicera japonica Thunb. extract (LTE) effects on the body and organ weights of Wistar rats.

	Normal	Control DM	DM +LTE 0.05%	DM +LTE 0.1%	DM +LTE 0.25%
Body weight (g)	355.8 ± 17.2a	148.4 ± 14.0b	190.6 ± 55.4b	181.6 ± 18.0b	177.8 ± 19.5b
Liver weight (g/kg b.w.)	25.7 ± 0.8a	52.6 ± 3.1b	51.0 ± 8.3b	48.9 ± 3.9b	50.5 ± 5.0b
Kidney weight (g/kg b.w.)	5.5 ± 0.3a	16.5 ± 1.3b	14.4 ± 3.3b	14.2 ± 1.9b	13.1 ± 1.3b

Normal, normal rat + water group; DM, diabetes mellitus (DM) rat + water group. DM + LTE, diabetes mellitus (DM) rat + LTE group. Means denoted by the same letter are not significantly different from each other (Tukey-Kramer test, p < 0.05).

Biochemical analysis of the plasma samples    The effects of oral administration of LTE on blood glucose, glycoalbumin, total cholesterol, HDL cholesterol, LDL-cholesterol, triglyceride, uric acid, blood urea nitrogen, creatinine, acetoacetate, 3-hydroxybutyrate, and arterial blood ketone body ratios are presented in Table 2. The DM-induced biochemical changes were less in the DM + LTE 0.25% group than in the control group. There was a significant (p < 0.05) increase in blood glucose, glycoalbumin, total cholesterol, LDL-cholesterol, triglyceride, urea nitrogen levels and ketone bodies ratio in STZ-induced control DM rats compared with the normal group. The levels of glycoalbumin decreased in LTE 0.05%, 0.1%, and 0.25%-treated rats (Table 2). The control DM group blood glucose levels increased by 3.2 fold (429 ± 42.8 mg/dL) when compared to control. In contrast, rats treated with LTE (0.05%, 0.1%, and 0.25%) had reduced blood glucose (88.6%, 85.5%, and 79.5%, respectively) when compared with the control DM group (Table 2). In addition, total cholesterol, LDL-cholesterol (+ LTE 0.05%, 0.1%, and 0.25%), and creatinine (+ LTE 0.25%) levels were lower in the DM + LTE groups than in the control DM group. Triglyceride, uric acid, blood urea nitrogen, acetoacetic acid, 3-hydroxybutyric acid levels and ketone body ratios tended to decrease between DM + LTE 0.25% groups when compared controls (Table 2).

Table 2. Effects of Lonicera japonica Thunb. extract (LTE) on serum biochemical markers in Wistar rats.

	Normal	Control DM	DM +LTE 0.05%	DM +LTE 0.1%	DM +LTE 0.25%
Blood glucose (mg/dL)	124.0 ± 17.4a	429.0 ± 38.3b	379.6 ± 24.6bc	366.6 ± 24.9bc	341.4 ± 21.6c
Glycoalbumin (%)	9.1 ± 1.9a	41.4 ± 2.1b	33.1 ± 3.2b	32.5 ± 1.3b	33.0 ± 2.6b
T-CHO (mg/dL)	34.8 ± 3.9a	126.0 ± 49.4b	62.8 ± 21.4a	65.3 ± 17.8a	59.8 ± 27.1a
HDL-CHO (mg/dL)	20.8 ± 1.1	35.6 ± 6.7	37.2 ± 23.6	34.5 ± 6.6	35.0 ± 3.5
LDL-CHO (mg/dL)	4.4 ± 0.9a	75.2 ± 32.5b	27.6 ± 7.0a	24.0 ± 15.2a	22.5 ± 13.4a
TG (mg/dL)	135.2 ± 19.8a	566.0 ± 342.6b	291.2 ± 159.0ab	394.0 ± 111.7ab	275.2 ± 39.7ab
Uric acid (mg/dL)	1.1 ± 0.3	1.4 ± 0.3	1.4 ± 0.5	1.1 ± 0.2	0.9 ± 0.3
BUN (mg/dL)	20.6 ± 2.0a	70.5 ± 10.3b	54.9 ± 13.8b	59.6 ± 8.5b	56.2 ± 4.3b
Creatinine (mg/dL)	0.35 ± 0.02a	0.29 ± 0.08ab	0.25 ± 0.04bc	0.22 ± 0.02bc	0.18 ± 0.01c

Normal, normal rat + water group; Control DM, diabetes mellitus (DM) rat + water group; DM + LTE, diabetes mellitus (DM) rat + LTE group; T-CHO, total cholesterol (mg/dL); HDL-CHO, high-density lipoprotein cholesterol (mg/dL); LDL-CHO, low-density lipoprotein cholesterol (mg/dL); TG, triglyceride (mg/dL); BUN, blood urea nitrogen (mg/dL); AcAc, acetoacetate (µmol/L); 3-OHB, 3-hydroxybutyrate (µmol/L). Means denoted by the same letter are not significantly different from each other (Tukey-Kramer test, p < 0.05).

Measurement of CML level in lens crystalline    A non-competitive ELISA method was used to investigate the effect of LTE on CML formation in lens crystalline. The CML levels in the lens crystalline of the control DM group were significantly higher than in the normal group (Fig. 7). LTE at concentrations of 0.1% and 0.25% inhibited CML formation by 8.1% and 17.7% compared with control DM group. There was a significant (p < 0.05) decrease in CML formation in the lens crystalline of STZ-induced DM + LTE 0.25% groups compared with the control DM rats (Fig. 7).

Fig. 7.

The effect of Lonicera japonica Thunb. extract (LTE) on N^ε-(carboxymethyl) lysin (CML) formation as measured by ELISA. Normal, normal rat + water group; DM, diabetes mellitus (DM) rat + water group, DM + LTE, diabetes mellitus (DM) rat + LTE group. The bar graph depicts the inhibition effects of LTE on CML levels determined in the presence of different concentrations (0.05%, 0.10%, and 0.25%) of LTE. Values represent the mean ± SD (n=3). Means with the same alphabet are not significantly different from each other (Tukey-Kramer test, p < 0.05).

Discussion

This study clearly demonstrates that LTE have antioxidative and α-glucosidase inhibitory activities. Chen et al. reported that the ED₅₀ value for antioxidant activity of LTE was 0.45 mg/mL (Chen et al., 2013). Our studies (ED₅₀ value; 3.08 mg/mL) showed lower antioxidant activity than those reported by Chen et al. We suggest that the increased ED₅₀ in our study is due to lower total phenolic content because our LTE is a water extract. Shang et al. reported that the content of chlorogenic acid and luteolin in L. japonica should be ∼ 1.5 and 0.1%, respectively (Shang et al., 2011). Our results show a similar content of chlorogenic acid (1.55%) and luteolin in LTE (0.45%). Therefore, phenolic compounds in addition to chlorogenic acid and luteolin in L. japonica have antioxidative activity.

α-Glucosidase (EC 3.2.1.20) is a key enzyme, which catalyzes the final step during the digestive process of carbohydrates. Therefore, α-glucosidase inhibitors delay the release of d-glucose and disaccharides from oligosaccharides and dietary carbohydrates, leading to delayed glucose absorption. Indeed, the reduction of postprandial plasma glucose suppresses postprandial hyperglycemia (Lebovitz, 1997). Zhanga et al. reported that methanol extracts from L. japonica flower buds inhibit rat intestinal α-glucosidase activity. At a concentration of 2 mg/mL, rat intestinal maltase inhibition reached 66%, but only 13% inhibition for rat sucrase (Zhang et al., 2013). Luteolin (0.5 mg/mL) inhibited α-glucosidase by 36%, suggesting that it was able to effectively suppress postprandial hyperglycemia in non-insulin dependent diabetes patients (Kim et al., 2000). Our results suggest that luteolin in LTE is related to α-glucosidase inhibitory activity. It is necessary to clarify other components within LTE may also inhibit α-glucosidase activity.

Intracellular myo-inositol is decreased and Na-K-ATPase activity is reduced in hyperglycemic states that cause nerve conduction disorders (Nishizuka, 1983). It has also been reported that the efficacy of dietary myo-inositol supplementation improves motor nerve conduction velocity in STZ-diabetic rats (Greene et al., 1982). Because myo-inositol displays insulin-mimetic activities, abnormalities in inositol metabolism are likely associated with insulin resistance (Loewus and Murthy, 2000). Therefore, the content of myo-inositol in LTE was examined. In this study, the concentration of myo-inositol in LTE was 2.24 g/100 g dry weight. Several animal and human studies have demonstrated a beneficial effect of a myo-inositol supplementation for diabetic nerve disorders. Green et al. demonstrated that a 1% (w/w) myo-inositol supplemented diet (vs. 0.011% or 0.069% free myo-inositol in normal diets) restored neuronal myo-inositol intracellular levels in STZ-diabetic rat models (Greene et al., 1982). Salway et al. reported that myo-inositol, 500 mg twice a day, given to seven diabetic patients for two weeks, improved diabetic neuropathy (Salway et al., 1978).

In this study, the average myo-inositol intake in the LTE 0.05%, 0.1%, and 0.25% groups per kg body weight were approximately 6.72, 14.56, and 34.50 mg, respectively. It has also been reported that oral administration of myo-inositol (667 mg/kg) for 3 weeks inhibits the diabetes-induced neurological disorder (Mayer and Tomlinson, 1983). The intake of myo-inositol in this study was less than 667 mg/kg. However, the experimental period reported by Mayer and Tomlinson (1983) was 3 weeks, whereas the experimental period in the present study was 100 days (about 14 weeks); hence, it is possible that the improvement of STZ-induced DM was obtained with a small amount of myo-inositol.

When STZ-diabetic rats were treated with a free intake of LTE (0.05%, 0.1%, and 0.25%) for 12 weeks, a reduction of blood glucose levels and improvement of serum biochemical profiles were observed (Table 2). In this study glycoalbumin, an estimate of short-term (2 weeks) circulating plasma glucose levels, tended to reduce in DM rat + LTE 0.1% and 0.25% groups (Table 2). Our data suggests that plasma glucose levels decrease in response to LTE treatment. There are several reports that hyperglycemia in STZ-induced DM is accompanied by increases in serum cholesterol and triglyceride levels (Choi et al., 1991; Platel et al., 1993; Sharma et al., 1997). Uric acid is a mediator of diabetic kidney injury. Dyslipidemia retards urate clearance, causing elevated serum uric acid level (Estevez et al., 1990). Also, the ketone body synthesis pathway is induced during insulin dependent diabetes (Cook et al., 2017). Ketone bodies refer to three molecules, acetoacetate, 3-hydroxybutyrate, and acetone (Laffel, 1999). It is presumed that the excess triglycerides and free fatty acids accumulate in the liver because fatty liver occurred in control group. In contrast, total cholesterol, LDL-cholesterol, triglyceride, uric acid, urea nitrogen, creatinine levels and ketone body ratios were lower in the DM rat + LTE groups. In addition, the kidney weight was significantly different in DM + 0.25% group compared to control DM group (Table 1). Renal hypertrophy is an early feature of diabetes, and it may predispose the kidney to the eventual development of parenchymal dysfunction (Choi et al., 1991; Sharma et al., 1997). These results suggest that STZ-treated rats feeding on LTE showed amelioration of renal dysfunction and hyperuricemia due to decreased renal hypertrophy. Cytochrome P-4502E1 (CYP2E1) is known to metabolize ketone bodies, ethanol, and fatty acids induced by diabetes (Woodcroft et al., 2002). Further studies are required to confirm and further examine the mechanisms that decrease ketone bodies.

The most common AGE generated by oxidation reactions and glycation is CML (Fujiwara et al., 2011). Previous reports indicate that the ability to inhibit AGE formation is linked to the plant extract antioxidant properties; which scavenge free-radicals formed during the Maillard glycation reaction (Boo et al., 2012). Tzeng et al. reported that ethanol extracts of L. japonica flowers downregulated the protein expression of p38 mitogen-activated protein kinase in the kidney and inhibited the progression of diabetic nephropathy in diabetic rats (Tzeng et al., 2014). Ghanem et al. also reported on the potential association between diabetic retinopathy and levels of CML. CML may indeed be a suitable biochemical marker for glycoxidation based on our study and others, and is associated with the progression of diabetic retinopathy (Ghanem et al., 2011). Our results suggest that LTE inhibits the progression of diabetic retinopathy due to LTE inhibited AGE formation in lens crystalline. The total LTE intake for 100 days was 0.91 (DM + LTE 0.05% group), 1.94 (DM + LTE 0.1% group), and 4.61 (DM + LTE 0.25% group) mg/g body weight (Fig. 6 (B)). A correlation was observed between the inhibition rate of CML formation and the LTE intake (LTE mg/g body weight) of each group (r = 0.896). LTE anti-glycation activity may be due to its antioxidant properties and/or phenolic content.

Many herb and plant extracts have been shown to have hypoglycemic and hypolipidemic properties (Jia et al., 2003; Cicero et al., 2004; Vinson et al., 2005; Hanamura et al., 2006; Silan et al., 2008; Rao et al., 2010; Iizuka et al., 2011; Taguchi et al., 2014). Our studies demonstrate that the feeding of LTE to STZ-induced DM rats results in the improvement of blood glucose levels, serum biochemical profiles (Table 2), and the inhibition of AGE formation (Fig. 7). Components such as chlorogenic acid, contained in L. japonica Thunb., also lower fasting blood glucose levels during late diabetes in vivo (Jin et al., 2015). Chlorogenic acid also inhibits AGEs formation with an IC₅₀ value of 148.32 µM and is more effective than aminoguanidine, a well-known AGE inhibitor (IC₅₀; 807.67 µM) (Tzeng et al., 2014). In addition, luteolin significantly improves blood glucose levels (Kim et al., 2011) and inhibits AGEs formation (85.4% inhibition at 100 µg/mL) (Zang et al., 2016). Therefore, we suggest that the chlorogenic acid and luteolin in LTE is related to its anti-hyperglycemic activity and inhibition of AGE formation.

In this study, treatment of rats with LTE (0.05%, 0.1%, and 0.25%) reduces blood glucose levels significantly compared with the control. Formation of intracellular AGEs is faster in response to hyperglycemia (Shinohara et al., 1998). Blood glucose levels have been suggested to be the most important factor for AGE formation in vivo under diabetic conditions (Kim et al., 2011). Therefore, suppression of AGE formation may be a more feasible therapeutic method for preventing diabetic complications. AGE formation in the retina during diabetes is linked to microvascular dysfunction, which causes diabetic complications such as diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy (Grzegorczyk-Karolak et al., 2016). Further studies are needed to elucidate whether LTE inhibits microvascular dysfunction.

Acknowledgments    The authors would like to thank Enago (www.enago.jp) for the English language review.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations:

LTE

Lonicera japonica Thunb. extract

AGEs

advanced glycation end products

STZ

streptozotocin

CML

N^ε-(carboxymethyl) lysin

T2D

type 2 diabetes

T1D

type 1 diabetes

DPPH

2,2-diphenyl-1-picrylhydrazyl

PNP

p-nitrophenol

DM

diabetes mellitus

HDL

high-density lipoprotein

LDL

low-density lipoprotein

PBS

phosphate buffered saline.

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