Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Collagenase was purchased from Roche Diagnostics (Indianapolis, IN, USA).

Persimmon leaves were raised and harvested in Wanju (Jeonbuk, Korea) in June 2013 by Dongsangmyeon Saramdeul Inc. (Jeonbuk, Korea). Persimmon leaves were dried in the shade for one week prior to being powdered and passed through 60-mesh sieves. One volume of persimmon leaf powder was added to 10 volumes of distilled water and extracted at 90–100°C for 3 h. The aqueous phase was filtered and concentrated with a vacuum evaporator (Eyela, Japan). After lyophilization, the powder was stored at −80°C until used. The components of PLE were analyzed by the Development Institute of Traditional Korean Medicine (Jeonnam, Korea) using a high-pressure liquid chromatography workstation (Shimadzu, Japan) (Fig. 1). Analyses were performed on an X-bridged C18 column with a mobile phase gradient of A) 0.1% formic acid and B) acetonitrile over 50 min. Gradient elution was programmed at a flow rate of 0.25 ml/min as follows: 0 min (100%), 10 min (90%), 30 min (40%), 45 min (30%) and 50 min (90%). The injection volume was 20 μl. The column temperature was kept constant at 25°C, and the mobile phase flow rate was 1 ml/min with ultraviolet detection at 265 nm. PLE was standardized to contain 4–7 mg total quercetin 3-O-2′galloylglucoside (C₂₄H₂₄O₁₉) and kaempferol 3-O-2′galloylglucoside (C₂₄H₂₄O₁₈) per 1 g of extract.

Yeast α-glucosidase (0.5 U) dissolved in 0.2 M potassium phosphate buffer (pH 6.8) was mixed with various concentrations of PLE or acarbose. After incubation at 37°C for 15 min, 3 mM p-nitrophenyl-α-D-gluc opyranoside was added. The reaction was further incubated at 37°C for 10 min and then stopped by the addition of 0.1 M Na₂CO₃. The absorption (Abs) of 4-nitrophenol was measured at 405 nm. Reaction mixture without any sample was used as a control, and the mixture without substrate was used as a blank. The percent inhibition of α-glucosidase was calculated as [1-(Abs_sample−Abs_blank)/Abs_control]×100. Measurements were performed in triplicate.

The anti-oxidant activity of each sample extract was assessed by the ability of the extract to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. The extracts, in separate test tubes, were allowed to react with DPPH. DPPH free radical scavenging activity was monitored by measuring the decline in absorbance at 517 nm. Butylated hydroxyanisole was used as the standard compound.

Pathogen-free, male C57BL/6 mice were purchased from Orientbio (Sungnam, Korea). The mice were housed at 20°C with 50% relative humidity, a 12-h light/dark cycle (light from 6:00 am to 6:00 pm) and were provided free access to drinking water. To induce diabetes, eight-week-old male C57BL/6 mice were injected via the tail vein with 100 mg/kg body weight streptozotocin (STZ) dissolved in 0.1 mol/l sodium citrate buffer (pH 4.0). The control mice received citrate buffer alone. PLE (50 or 250 mg/kg body weight) was injected daily for five days via oral gavage prior to administration of STZ. STZ was first administered on day one. On day six, mice were sacrificed by decapitation without anesthesia and trunk blood was collected.

In addition, seven-week-old male C57BL/KsJ-db/db (db/db) mice were purchased from the Jackson Lab (Bar Harbor, ME, USA) and fed a normal chow diet. Starting at eight weeks of age, the point at which the mice become diabetic, the db/db mice were treated with PLE (50 or 250 mg/kg) for eight weeks via oral gavage once daily. Each group was made up of five mice. As a positive control, acarbose (10 mg/kg) was administered instead of PLE. Food consumption and body weight were recorded every week. At the end of the experimental period, an oral glucose tolerance test (OGTT; 1 g/kg body weight) was performed. After a 14 h fast, glucose was administered by oral gavage (2 mg/g). The blood glucose level was subsequently determined from the tail vein at 0, 15, 30, 60 and 120 min following the glucose administration. Animals were sacrificed by decapitation, after which blood samples were collected, and livers were removed and weighed. All of the animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 2011). The protocol of the present study was approved by the Institutional Animal Care and Use Committee of Chonbuk National University (permit no. CBU 2014-00048).

Mice were classified into four groups (1–4) containing five mice each. Groups 1 and 2 received phosphate-buffered saline (PBS) as a negative control or acarbose (3 mg/kg) as a positive control, respectively. Groups 3 and 4 were treated with PLE at two doses (50 and 250 mg/kg). All samples were administered orally to 12-h fasted mice, and 3 g/kg of maltose was administered 5 min thereafter. Blood was collected from the tail vein at 0, 15, 30, 60 and 120 min after loading maltose.

Blood glucose levels were measured by Accu-Chek Aviva glucose monitors (Roche Diagnostics, Indianapolis, IN, USA) and plasma insulin was measured using an ELISA kit (cat. no. EZRMI-13K; Millipore, Bedford, MA, USA). Plasma levels of total cholesterol (TC), triglyceride (TG) and HDL-cholesterol were measured using commercially available kits (cat. no’s. AM202-K, AM157S-K and AM203-K, respectively; Asan Pharmaceutical, Seoul, Korea). For liver TG quantification, liver tissues were homogenized and extracted in chloroform, methanol and DW (2/1/1 ratio).

Tissues were removed and immediately placed in 10% formalin solution, embedded in paraffin and cut into 5-μm sections. Specimens were stained with hematoxylin and eosin (H&E) to identify morphological changes. For immunohistochemical analysis, tissue sections were subjected to a microwave antigen retrieval procedure (1,000 watts for 5 min; CPC-600; Cuisinart, East Windsor, NJ, USA) in 0.01 mol/l sodium citrate buffer. After blocking endogenous peroxidase, the sections were incubated with Protein Block Serum-Free (DAKO, Glostrup, Denmark) to block non-specific staining and then with rabbit anti-insulin antibody (cat. no. sc-9168; 1:100; Santa Cruz Biotechnology, Dallas, TX, USA) for 12 h at 4°C. Peroxidase activity was detected using 3-amino-9-ethyl carbazole. Tissue sections were observed using a light microscope (Eclipse E600 polarizing microscope; Nikon, Tokyo, Japan).

Pancreatic islets were isolated from 12-week-old mice using the collagenase digestion method as previously described (14). Following isolation, islets were cultured overnight in RPMI-1640 supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin in humidified air containing 5% CO₂ at 37°C. Prior to experiments, islets were washed three times in RPMI-1640 and cultured overnight. After the initial culture period, islets were cultured for three days in identical RPMI-1640 containing 5.5 or 30 mmol/l glucose and subsequently washed three times in Krebs-Ringer bicarbonate buffer [25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 115 mmol/l NaCl, 24 mmol/l NaHCO₃, 5 mmol/l KCl, 1 mmol/l MgCl₂, 2.5 mmol/l CaCl₂ and 0.1% bovine serum albumin, pH 7.4] containing 2.8 mmol/l D-glucose. Insulin secretion assays were performed with 2.8 or 16.7 mmol/l glucose and measured using an ELISA kit (cat. no. EZRMI-13K; Millipore, Bedford, MA, USA).

Statistical analysis was performed using analysis of variance and Duncan’s tests on through GraphPad Prism v5.02 (GraphPad Software Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference between values.

The in vitro inhibitory activity of PLE against yeast α-glucosidase is shown in Fig. 2A. PLE inhibited α-glucosidase activity in a dose-dependent manner and therefore should be considered an effective α-glucosidase inhibitor: PLE at a concentration of 100 μg/ml inhibited α-glucosidase activity by 70.5% and was 16.0% less potent than acarbose, which was used as a positive control. The half maximal inhibitory concentration (IC₅₀) value on α-glucosidase activity was 4.7 μg/ml.

The results of the maltose tolerance experiment are presented in Fig. 2B. After the administration of maltose, the blood glucose levels in normal mice increased after 15 min. This elevation was statistically significant compared with the levels at time 0 for each group. At 120 min, the blood glucose levels returned to their basal values. Oral administration of 250 mg/kg PLE inhibited the increases in glucose levels after 30 min, which was statistically significant compared with levels in the corresponding controls at each time-point. The administration of acarbose also significantly decreased the increase of postprandial blood glucose. The areas under the curve (AUC) for glucose were reduced by 15.34% by PLE at 50 mg/kg, 23.63% by PLE at 250 mg/kg and 26.51% by acarbose.

Furthermore, the anti-oxidant properties of PLE were determined using the cell-free DPPH assay. PLE inhibited DPPH activity in a dose-dependent manner (Fig. 3), which confirms the findings of a previous study (15).

Powdered persimmon leaf has previously been reported to have glucose-and lipid-lowering effects in db/db mice (13). First, the present study evaluated the hypoglycemic effects of PLE at concentrations of 50 mg/kg (low-dose) and 250 mg/kg (high-dose) in STZ-induced diabetic mice. As shown in Fig. 4A, a single intravenous injection of STZ induced the increase of fasting blood glucose levels to ~400 mg/dl. However, the fasting glucose levels were significantly reduced to 176 mg/dl in the STZ mice pre-treated with high-dose PLE. The group of mice treated with acarbose (10 mg/kg) showed similar blood glucose levels. Of note, the body weight among the groups did not change over the course of the study (data not shown).

The effect of PLE on the post-prandial increase in blood glucose in STZ-induced diabetic mice was determined via OGTT Consistent with the above results, oral administration of high-dose PLE significantly prevented an increase in plasma glucose levels (Fig. 4B). High-dose PLE and acarbose decreased the AUCs for the postprandial glucose responses by 61.6 and 62.4%, respectively, compared with that in the PBS-treated group (P<0.01). These results indicated that treatment with PLE prevents STZ-induced β-cell damage in mice.

To further evaluate the therapeutic effects of PLE on diabetes, the long-term anti-diabetic effects of PLE in db/db mice were evaluated. Food intake was not significantly influenced by PLE treatment (Fig. 5A); however, the body weight was significantly decreased by high-dose PLE treatment (Fig. 5B). Diabetic db/db mice at 16 weeks of age that were fed normally had hyperglycemia with postprandial blood glucose levels of ~494 mg/dl (Fig. 5C). High-dose PLE decreased postprandial blood glucose levels in db/db mice as effectively as acarbose. The decrease in blood glucose levels by PLE appeared to be dose-dependent, as low doses of PLE (50 mg/kg) revealed no glucose lowering effects, whereas high doses of PLE (250 mg/kg) significantly decreases glucose levels (P< 0.01).

An OGTT was performed to determine the effects of PLE on glucose tolerance after eight weeks of PLE treatment (Fig. 5D). The AUC for glucose response of high-dose PLE-treated db/db mice was significantly lower than that of the (db/db) mice. By contrast, oral administration of low-dose PLE did not show any improvement in glucose tolerance.

Plasma insulin levels between db/db mice that received PBS and those that had been administered PLE were also compared. The serum insulin levels in high-dose PLE-treated mice tended to be higher than those in PBS-treated mice (Fig. 6A). In addition, as shown in Fig. 6B and C, high-dose PLE significantly lowered plasma TG (160.5±15.9 mg/dl; P<0.01) and TC levels (209.2±74.5 mg/dl; P<0.05) compared with those in the control mice (208.9±32.4 and 263.9±19.3 mg/dl, respectively). Plasma HDL-cholesterol levels were not significantly different among groups (Fig. 6D). Overall, these results suggested that PLE has glucose- and lipid-lowering effects in db/db mice.

As diabetes can trigger hepatic steatosis, livers were assessed for the extent of fat accumulation. The results indicated that PLE supplementation was associated with less steatotic livers (data not shown). Examination of H&E-stained sections demonstrated marked macrovesicular steatosis in db/db mice, and the degree of hepatic steatosis was markedly alleviated by PLE (Fig. 7A). Liver TG and liver weight were concordant with the histological findings (Fig. 7B and C). Treatment of db/db mice with high-dose PLE resulted in significant decreases in hepatic TG contents (85.3±12.5 mg/100 mg vs. 54.2±15.8 mg/100 mg; P<0.01) and liver weight (3.8±0.8 vs. 2.7±0.6 g; P=0.088) as compared with that in the control db/db mice.

As PLE administration increased plasma insulin levels (Fig. 6A), pancreata were examined using H&E staining. Pancreatic islets of diabetic db/db mice exhibited degeneration and poorly defined margins (Fig. 8A). Immunostaining with an insulin antibody showed weak insulin staining. By contrast, pancreatic islets of high-dose PLE-treated mice had a round shape and high insulin immunoreactivity, suggesting the protection of β-cells by PLE.

To determine the protective effects of PLE on β-cells, the present study examined whether it was effective in protecting pancreatic islets from glucotoxicity. Islets from mice were isolated and incubated under normal glucose (5.5 mM) or high-glucose (30 mM) culture conditions. Basal and glucose-stimulated insulin secretion was assessed after three days of culture (Fig. 8B). Results showed that the amount of glucose-stimulated insulin secretion was 6.65±0.48 ng/islet/h in normal glucose-cultured islets and 4.80±1.48 ng/islet/h in high-glucose-cultured islets. Following pre-treatment with PLE, however, the insulin secretion under high-glucose conditions was significantly restored to a level closer to the control value. Basal insulin release among the groups was similar. In conclusion, in vitro and in vivo results of the present study suggested that PLE exhibits protective effects on β-cells.