Chemical composition and hypoglycaemic effect of polyphenol extracts from Litchi chinensis seeds

Journal of Functional Foods 22 (2016) 313–324

Available online at www.sciencedirect.com

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Chemical composition and hypoglycaemic effect of polyphenol extracts from Litchi chinensis seeds Shuli Man a,b,*, Jiang Ma a,b, Chunxia Wang a, Yu Li a, Wenyuan Gao c, Fuping Lu a,b,* a

Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China b Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China c School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China

A R T I C L E

I N F O

A B S T R A C T

Article history:

The hypoglycaemic effects of Litchi chinensis Sonn. seeds extract (LSE) on type 2 diabetic (T2D)

Received 13 September 2015

rats and the active constituents from LSE were investigated. As a result, 21 compounds in-

Received in revised form 15 January

cluding 3,5-dihydroxybenzoic acid, 3,4-dihydroxybenzaldehyde, procyanidin D, cianidanol,

2016

cinnamtannin B1, procyanidin A1, scopoletin, rutin, phlorizin and epicatechin–epicatechin–

Accepted 18 January 2016

catechin were identified by UPLC-Q/TOF-MS. The content of polyphenols reached 43.37 ± 2.34 g

Available online

of cianidanol/100 g. After one-month treatment, LSE improved quality of life of streptozotocin (STZ)/high fat diet induced T2D rats and protected against pancreas, liver and kidney tissues

Keywords:

damage through the improvement of glucose tolerance and insulin resistance. In addition,

Litchi seeds

LSE influenced lipid metabolism and increased mRNA levels of Bax and NF-κB, thereby in-

Diabetes

hibiting apoptosis-induced hepatic damage and inflammation to protect the body against

Lipid metabolism

diabetic exacerbation. For the first time, this study delineated Litchi seeds’ anti-diabetic pros-

Inflammation

pect of treatment for type 2 diabetes.

Apoptosis

1.

Introduction

Litchi (Litchi chinensis Sonn.) as a common fruit has been widely planted in Southeast Asia. Because of its attractive colour and delicious taste, litchi has been widely accepted by consum-

© 2016 Elsevier Ltd. All rights reserved.

ers. Generally speaking, litchi seeds contain many nutrient components such as polyphenols and saponins (Queiroz, de Abreu, dos Santos, & Simao, 2015). However, they are usually discarded as fruit waste upon consumption and processing. It has been reported that the seeds possess many biological activities including antioxidant (Wang, Lou, Ma, & Liu, 2011),

* Corresponding authors. Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China. Tel.: +86 022 60601265; fax: +86 022 60601265. E-mail addresses: [email protected] (F. Lu); [email protected] (S. Man). Abbreviations: ACO, Acyl-CoA oxidase; AUC, area under curve; DGAT, diacyl glycerol acyl transferase; ELISA, enzyme-linked immunosorbent assay; FAS, fatty acid synthetase; FBG, fasting blood glucose; FSI, fasting serum insulin; HDL-C, high density lipoprotein cholesterol; HFD, high fat diet; HPLC, high performance liquid chromatography; ISI, insulin sensitivity index; IRI, insulin resistance index; LDH, lactate dehydrogenase; LDL-C, low density lipoprotein cholesterol; LSE, Litchi seeds extract; OGTT, oral glucose tolerance tests; RT-PCR, reverse transcription polymerase chain reaction; STZ, streptozotocin; T-CHO, total cholesterol; T2D, type 2 diabetes; TG, total triacylglycerol; UPLC/ DAD-ESI-Q/TOF-MS, Ultra-performance liquid chromatography/diode-array detection electrospray ionization quadrupole time-of-flight mass spectrometry http://dx.doi.org/10.1016/j.jff.2016.01.032 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

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Journal of Functional Foods 22 (2016) 313–324

anti-obesity (Qi, Huang, Huang, Wang, & Wei, 2015), inhibition of α-glucosidase (Ren, Xu, Gao, Ma, & Gao, 2013), antiviral (Xu, Xie, Wang, & Wei, 2010), and antityrosinase activities (Prasad et al., 2009). As a consequence, a very limited amount of research has determined litchi seeds’ anti-diabetic potential for the treatment of type 2 diabetes (T2D) and their constituents were rarely investigated. The prevalence of T2D, which has been characterized clinically by progressive impaired insulin secretion, resistance of peripheral tissues to the effect of insulin and augmented hepatic glucose production, has alarmingly increased (Bergman, Stefanovski, & Kim, 2014). Impaired insulin secretion disturbs both glucose and lipid metabolism (Qi et al., 2015). Streptozotocin (STZ) as a well-known diabetogenic agent was used at a low dose in combination with high fat diet (HFD) for efficient induction of T2D and widely applied in understanding the pathogenesis of T2D and therapeutic studies in recent years. This study chemically and pharmacologically profiled litchi seeds’ anti-diabetic capacities. The major compounds were identified by UPLC-Q/TOF-MS for the first time. Meanwhile, the concentration of major nutrients was evaluated. This study could help better utilize litchi seeds that are usually discarded as fruit waste in the food industry.

2.

Materials and methods

2.1.

Material

Litchi (L. chinensis Sonn.) seeds were purchased from a local herbal pharmacy in Yunnan. All seeds were harvested at their commercial ripening stage. All the samples were individually washed, dried and stored at 4 °C.

2.2.

Extraction procedure

The dried litchi seeds (1 kg) were crushed with a hammer and then extracted with ethanol/water (95:5, 6:4, 3:7, v/v; 3 L, 2 times) for 2 h at 80 °C. In each case, the solutions were combined, transferred to a rotary evaporator and concentrated under vacuum at 55 °C. Then the concentrated solution was applied to a D101 macroporous resin (Nankai Hecheng S&T, Tianjin, China). The elution was initiated with 30% ethanol and increased with a gradient to 90% ethanol. The fractions of litchi seed extract (LSE) collected in the 30–75% ethanol range were combined and concentrated in vacuo to dryness at 55 °C.

2.3.

Determination of total polyphenols

Total polyphenol content of samples was determined using Folin–Ciocalteu method. Briefly, the aliquot (50 µL) of each sample (dissolved in methanol, 1 mg/mL) was mixed with 850 µL of dd H2O and 50 µL of Folin–Ciocalteu reagent. Subsequently, 150 µL of 10% Na2CO3 solution was added to the mixture and placed in the dark at ambient temperature for 20 min. The absorbance of the reaction mixture was read at 560 nm against deionized water as a blank on a Multimode Reader (Tecan Infinite 200 PRO, Männedorf, Switzerland). Cianidanol was chosen as a standard. Using a seven point standard curve (0.005–

0.035 mg/mL), the content of total polyphenols was determined in triplicate, respectively. The regression equation was y = 12.04x + 0.0465, R2 = 0.9931 (y was the absorption of the mixture; x was the total polyphenol concentration of samples (%)). The data were expressed as milligram Cianidanol equivalents/100 mg lyophilized powder.

2.4.

UPLC-Q/TOF-MS analysis

One milligram of LSE was dissolved in 1 mL of 50% acetonitrile and filtered through 0.22 µm microporous membrane (Xinjinghua Co., Shanghai, China) before use. An aliquot of 4 µL of the filtrate was injected into the Ultra-performance liquid chromatography (UPLC) equipment for analysis. Chromatographic analysis was performed on a Waters Series ultra-fast HPLC system (Waters Co., USA) equipped with a quaternary pump, micro-degasser, an auto-plate-sampler, a thermostatically controlled column apartment, coupled with diode-array detection (DAD) and electrospray ionization (ESI) ion source. Chromatographic separation was performed at 30 °C on a Waters Acquity UPLC TM BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters, Massachusetts, USA). The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The linear gradient was programmed: 5–60% B at 0–10 min, 60– 90% B at 10–20 min, 90–100% B at 20–21 min, isocratic 100% B at 21–22 min and 100–5% B at 22–23 min. The re-equilibration time of gradient elution was 5 min. The flow rate was 0.4 mL/ min with the scan range from 200 to 400 nm. The monitoring UV wavelength was set at 280 nm.

2.5.

HPLC analysis of main compounds

Twenty milligrams of LSE were dissolved in 1.0 mL of 50% acetonitrile and filtered through 0.22 µm microporous membrane before use. The analytical conditions were an Agilent ZORBAX SB-C18 column (250 × 4.6 mm i.d., 5 µm; Agilent, Palo Alto, CA, USA) and a solvent system composed of acetonitrile (A) and 0.1% formic acid in water (B) (condition: 10–15% A from 0 to 10 min; isocratic 15% A from 10 to 15 min; 15–20% A from 15 to 20 min; 20–25% A from 20 to 30 min; 25–30% A from 30 to 35 min; 30–50% A from 35 to 40 min; 50–95% A from 40 to 45 min; and kept at 95% A from 45 to 55 min) at a flow rate of 1 mL/min. The injection volume was 10 µL, and the detection was at 280 nm. Seven concentrations (1.6, 3.2, 8, 25, 62.5, 125 and 250 µg/mL) of 3,5-dihydroxybenzoic acid, 3,4dihydroxybenzaldehyde, procyanidin D, cianidanol, scopoletin, rutin, and phlorizin dissolved in MeOH were injected into HPLC. The linear regression equation for each calibration curve was established by plotting the concentration of standard compound injected against the peak area.

2.6.

Experimental animals

Eighteen male Sprague-Dawley rats weighing 100~130 g were obtained from the Laboratory Animal Center of Academy of Military Medical Sciences (Beijing, China quality certification number: SCXK (Jun) 2012-0004). This animal study was approved by the Institutional Animal Care and Use Committee of China, and institutional guidelines for animal welfare and experimental conduct were followed.

Journal of Functional Foods 22 (2016) 313–324

315

Fig. 1 – Scheme of the study design describing three groups of animals treated over ten weeks with intravenous STZ and oral HFD (Model group), intravenous STZ and oral HFD + LSE (LSE group), and intravenous saline solution and oral vehicle (Normal group).

After one week of acclimatization, the rats were randomly allocated into 3 groups: normal control group (Normal), untreated diabetes (Model) and diabetes treated with LSE (LSE) (Fig. 1). In the first 4 weeks, rats in model and LSE groups were given a high fat diet (HFD) alone containing 53.7% basal chow, 20% saccharose, 15% lard, 10% egg yolk powder, 1.1% cholesterol and 0.2% sodium cholate, which were purchased from the Experimental Center of Aoyide in Tianjin, whereas the normal group received regular chow only. At the beginning of the 5th week, rats fed with HFD received streptozotocin (STZ) (30 mg/ kg, i.p., dissolved in 0.1 M cold citrate buffer, pH 4.5) once or twice to initiate diabetes. Three days after STZ injection, rats with fasting blood glucose level > 11.1 mM, measured by a glucometer, were recognized as diabetic. Then the LSE-treated rats were administrated orally 30 mg of LSE in 10 mL of 0.9% sodium chloride per kilogram of body weight every day for six weeks. The model group received appropriate vehicles. The normal group received equivalent volumes of the abovementioned vehicles. During the experiment, the body weight of each rat was measured every week. Food consumption, water consumption, urine volume, density and pH were monitored every other day during the course of the study. Fasting blood samples were collected into heparinized tubes from each rat by the puncture of the retro-orbital sinus every other week starting at the 5th week. Blood was immediately processed for serum by centrifugation at 3000 g for 10 min at 4 °C. To avoid haemolysis phenomenon, serum samples were separated and kept in aliquots at −20 °C for biochemical assays. After the end of the collection, all the rats were sacrificed. Autopsies were harvested. Portions of each tissue were fixed in 10% formalin (pH 7.4) for histology, and snap frozen in liquid nitrogen for molecular biology test.

2.7.

rate (%) was calculated by the following formula: (FBG value before oral administration of LSE − FBG value after oral administration of LSE)/FBG value before oral administration of LSE × 100%) (Li et al., 2013). The oral glucose tolerance test (OGTT) was performed by orally administering gavage using a 10% (w/v) solution of glucose (1 g/kg). Blood samples were drawn from the lateral tail vein immediately before and 30, 60, and 120 min after bolus glucose loading, and tested by One Touch Ultra glucose monitor. The area under the concentration-by-time curve (AUC) of glucose during the oral glucose tolerance test was calculated by the trapezoidal method (Diz Chaves, Spuch Calvar, Perez Tilve, & Mallo Ferrer, 2003).

2.9.

Serum insulin was measured by immunoenzymatic ELISA kit (Lufeng, Shanghai, China). The results were calculated by correlative formula (Li et al., 2013). The data were calculated according to the following formula: ISI = Ln (1/(FBG × FSI)), IRI = (FBG × FSI)/22.5.

2.10.

Histological evaluation

For the histopathological examination, portions of liver, kidney and pancreas tissues were fixed in 10% formalin, and after proper dehydration, the tissues were embedded in paraffin wax. Five-micrometre-thick sections were prepared and stained with haematoxylin and eosin. Every organ was randomly cut into 6 histological sections. Histopathology examination was completed using Nikon eclipse TE2000-U Microscope and performed by a pathologist who was unaware of whether tissues were treated.

Biochemical assays 2.11.

Lactate dehydrogenase (LDH), total triacylglycerol (TG), total cholesterol (T-CHO), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured by spectrophotometry, using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.8.

Index of insulin resistance

Blood glucose test

Fasting blood glucose (FBG) was tested every week after STZ injection by a glucometer (Shanghai, China). Hypoglycaemic

Reverse transcription PCR

The mRNA abundances of Bax, Acyl-CoA oxidase (ACO), NFκB, fatty acid synthetase (FAS), diacylglycerol O-acyltransferase 2 (DGAT2), and GAPDH were measured by PCR according to a previous report (Man et al., 2015). Briefly, total RNA extraction from liver tissue by Trizol reagent was reversely transcribed into cDNA using MMLV RT according to the manufacturer’s instruction. PCR was performed in a volume of 25 µL with a 1 µL aliquot of cDNA. The products were stained with ethidium bromide and detected under an ultraviolet lamp. The densi-

316

Journal of Functional Foods 22 (2016) 313–324

tometry of each band was analysed using professional image analysis software, and the ratio of the target gene against the GAPDH internal standard was calculated.

2.12.

Statistical analysis

SPSS 17.0 for Windows (SPSS Inc.) was used to analyse the data. Data were expressed as the means ± standard error (SE). The statistically significant differences observed between these groups were determined by the single factor-test (ANOVAtailed). Differences were considered statistically significant if p-values were less than 0.05.

3.3.

The effects of LSE on FBG, OGTT, IRI and ISI are shown in Fig. 4. As shown in FBG test, the blood glucose in diabetic rats was higher than in healthy ones. Compared with the model group, LSE intervention group effectively caused a marked reduction of blood glucose in diabetic rats, whose hypoglycaemic rate reached 27.88%, but still higher than that in the normal group (Fig. 4A). At the same time, the increasing blood glucose induced the elevation of urinary glucose (Fig. 4C), which due to hyperglycaemia evoked cellular glucose overload in the kidneys (Vanhorebeek et al., 2009).

3.4.

3.

Results

3.1.

Chemical composition of LSE

3.1.1. UPLC-Q/TOF-MS identification of main active compounds Using a sensitive and specific UPLC coupled with DAD and ESIquadrupole time-of-flight mass spectrometry (Q/TOF-MS) assay and according to the references reported previously (Queiroz et al., 2015), 21 compounds, including 3,5-dihydroxybenzoic acid, 3,4-dihydroxybenzaldehyde, procyanidin D, cianidanol, cinnamtannin B1, procyanidin A1, scopoletin, rutin, phlorizin and epicatechin–epicatechin–catechin were identified in LSE (Fig. 2 and Table 1).

3.1.2.

Blood samples were collected immediately before and during 120 min after glucose loading. OGTT showed the appearance of the glucose peak at 30 min after glucose administration. The AUC of glucose was significantly elevated in the diabetic group compared with the normal one (Fig. 4B). LSE decreased the AUC of glucose compared with the model group.

3.5.

Effects of treatment on insulin levels

The insulin metabolism was also assessed in rats through performing analyses of ISI and IRI. The serum insulin level in model group was abnormally raised and subsequently triggered insulin resistance. Compared with the model group, the serum level of IRI in LSE group was notably lowered, which in turn increased ISI progressively (Fig. 4D).

3.6.

Effects of treatment on organ weight

Organ weight data are shown in Table 3. Especial for thymus, liver and kidney, thymus was significantly decreased in HFD/ STZ induced diabetic rats, while liver and kidney were remarkably elevated. However, LSE suppressed this trend to protect liver and kidney tissues. No statistically significant variations were detected in any other organs.

HPLC analysis of main active compounds

To analyse the concentration of major nutrients in LSE, seven compounds were used as polyphenol standards. Furthermore, optimization of extraction and chromatographic conditions were obtained to separate the ingredients of LSE with linear gradient elution of acetonitrile: 0.1% formic acid in water system. As Table 2 indicates, all calibration curves showed good linear regressions (R2 ≥ 0.99).

3.2.

Effects of treatment on glucose tolerance levels

Determination of total polyphenols

Previous chemical studies revealed that polyphenols were the major components in litchi seeds (Queiroz et al., 2015). According to the above UPLC-Q/TOF-MS identification, several kinds of catechin-rich compounds were identified. Therefore, according to the Folin–Ciocalteu method and cianidanol standard indication, the content of polyphenols was determined as 43.37 ± 2.34 g of cianidanol/100 g.

3.1.3.

Effects of treatment on blood glucose levels

General observations of LSE on diabetic rats

STZ-induced diabetic rats exhibited the conditions of dispiritedness and emaciation, accompanied with typical symptoms of hyperphagia, polydipsia, polyuria and loss of weight. The urinary output of the STZ-diabetic group was significantly increased at all times. Meanwhile, the body weight decreased gradually after STZ treatment. After a 2-week administration of LSE, the average water consumption and urine volume of the LSE-treated rats were gradually decreased compared with the model group (Fig. 3).

3.7.

Effects of treatment on histopathologic examination

The pathological examination of pancreatic, kidney and liver sections using HE-staining is displayed in Fig. 5. Pancreatic tissues exhibited the islet in the normal group with the complete structure, uniform arrangement and numerous pancreatic β-cells (Fig. 5A1). In contrast, pathology of islet in the model group showed damage with loosening or deforming. The reduction of pancreatic β-cells was obvious (Fig. 5A3). As shown in the LSE group, the number of pancreatic β-cells and the cellular morphology of islet had a certain degree of recovery (Fig. 5A2). Renal histological examination revealed partial contraction of renal tubule and slight congestion of glomerular tufts in the untreated diabetic groups (Fig. 5B3). Treatment with LSE improved renal morphology (Fig. 5B2). Serum urea nitrogen concentration also indicated that LSE protected STZ induced renal injury (from 5.01 ± 0.91 mM (model) to 3.90 ± 0.28 mM (LSE)). Normal level was 2.81 ± 0.17 mM.

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317

Fig. 2 – UPLC-Q/TOF-MS chromatograms and structures of main compounds identified in LSE. (A) The chemical structures of the main compounds have been identified by the NCBI PubChem Compound database. (B) UPLC-Q/TOF-MS analysis of LSE. The number represented the compound was consistent with the No. in Table 1.

318

Table 1 – Accurate mass measurement for the molecular ions of parent compounds in LSE.

tR, retention time. Data in brackets mean ppm error. c Ara, arabinosyl; Glc, glucopyranosyl; Rha, rhamnopyranosyl. Shading represents the compounds containing (epi)catechin. The underline indicated the speculative structure between two ion fragments. b

Journal of Functional Foods 22 (2016) 313–324

a

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Table 2 – Linearity of calibration curve for seven standards and their content in LSE (n = 3). No.

Standards

1 2

3,5-Dihydroxy-benzoic acid 3,4-Dihydroxybenzaldehyde Procyanidin D Cianidanol Scopoletin Rutin Phlorizin

3 4 9 10 15 a b

tRa (min)

Regression equationb

R2

Linear range (ng)

Content in LSE (mg/g)

6.8 9.9

Y = 1.070x + 32.602 Y = 2.473x + 22.981

0.9986 0.9999

16–625 16–625

1.02 0.94

12.2 13.4 22.8 23.0 31.1

Y = 1.028x − 28.995 Y = 0.598x + 36.651 Y = 1.080x + 19.324 Y = 0.519x + 22.295 Y = 2.184x + 76.803

0.9987 0.9977 0.9940 0.9986 0.9973

16–625 32–1250 32–1250 32–1250 16–625

2.87 6.31 7.84 9.65 1.33

tR, Retention time. Y, Peak area; x, amount injected (ng).

Morphological changes of liver were also observed. Steatosis and severe cytoplasmic vacuoles were found in the liver of the diabetic group (Fig. 5C3). LSE ameliorated these changes (Fig. 5C2).

In the present study, mRNA levels of Bax, NF-kB, FAS and DGAT2 in non-treated diabetic rats were significantly increased compared with the normal ones and the LSE group (p < 0.05). However, expression of ACO was decreased in the model group (Fig. 6).

3.8. Biochemical analysis especially for plasma lipid in diabetic rats As revealed in Fig. 6, the serum levels of TG, T-CHO and LDH in diabetic rats were visibly elevated, whereas the ratio of HDL-C to LDL-C decreased as compared with the normal group. Instead, LSE lessened these trends in comparison with the nontreated diabetic rats.

3.9. mRNA levels of lipid metabolism relative genes in diabetic rats’ liver The liver as an insulin-dependent tissue played a pivotal role in glucose and lipid metabolism (Li, Ji, Zhong, Lin, & Lv, 2015).

4.

Discussion

Litchi seed is regarded as an abundant and under-utilized subtropical commodity. Previous research focused on litchi seeds containing a great amount of polyphenols and saponins (Queiroz et al., 2015), which exhibited strong inhibitory activity on α-glucosidase (Ren et al., 2011, 2013) and a significant rise in glucose consumption activity (Lv et al., 2014). In the present study, we used macropores to purify these compounds and evaluate their antidiabetic activity in STZ/HFD rats.

Fig. 3 – Effect of LSE on general health condition of the diabetic rat. (A) Body weight changes; (B) The average food consumption; (C) The average water consumption; (D) Urine volume change. Different letters mean significant differences between two groups (p < 0.05).

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Fig. 4 – Blood glucose and insulin responses in STZ-treated rats. (A) FBG detection. (B) Oral glucose tolerance test (OGTT). 1, Data on the 9th week. 2, Data on the 10th week. (C) Urinary glucose measurement. (D1) IRI and (D2) ISI evaluation of insulin tolerance. Different letters mean significant differences between two groups (p < 0.05).

First of all, using a sensitive and specific UPLC coupled with DAD and ESI-Q/TOF-MS assay and according to the references, several kinds of compounds were identified in LSE (Table 1). Secondly, based on the Folin–Ciocalteu method, the content of polyphenols was determined and reached 43.37 ± 2.34 g of cianidanol/100 g. As previously reported, catechin-rich agents have several therapeutic uses in the prevention of obesity and down-regulation of blood glucose in T2D patients (Nagao et al., 2009). Especially for cianidanol, it prevents T1D by modulating immune function and thereby preserving islet mass (Fu, Yuskavage, & Liu, 2013). Procyanidin D protects against the damage of diabetic pancreas through

anti-inflammatory effects (Yin et al., 2015). In addition, phlorizin reduces acute renal toxicity (Brouwers et al., 2013) and hepatic damage in diabetic mice (Lu et al., 2012). Scopoletin (Chang et al., 2015), rutin (Dhanya et al., 2014), and 3,5dihydroxybenzoic acid (Scazzocchio et al., 2015) act as antiglycation and anti-diabetic agents in T2D rats. Therefore, HPLC analysis was used to determine the content of these typical polyphenol compounds (Table 2). It was possible that all these compounds played the hypoglycaemic roles in LSE preventing T2D progression. STZ-induced diabetes has been described as a useful experimental model to study the activity of hypoglycaemic agents

Table 3 – Absolute organ weights influenced by LSE (g). Group

Heart

Lung

Brain

Pancreas

Spleen

Thymus

Liver

Kidney

Normal LSE Model

1.4 ± 0.1 1.3 ± 0.0 1.4 ± 0.0

1.7 ± 0.1 1.5 ± 0.0 1.6 ± 0.1

2.0 ± 0.0 2.1 ± 0.0 2.0 ± 0.0

0.82 ± 0.10 0.80 ± 0.10 0.62 ± 0.05

0.70 ± 0.04 0.64 ± 0.04 0.64 ± 0.04

0.44 ± 0.02a 0.20 ± 0.02b 0.18 ± 0.01b

16 ± 1a 17 ± 0ab 19 ± 1b

2.7 ± 0.1a 3.0 ± 0.1a 3.4 ± 0.1b

Different letters mean significant differences between two groups (p < 0.05).

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Fig. 5 – Representative sections (HE staining, ×100) of rat tissues. A, pancreatic tissue; B, kidney; C, liver tissues. 1, normal; 2, LSE; 3, model groups. Arrows represent islet with pancreatic beta cells in A1–3. Contraction of renal tubule and slight congestion of glomerular tufts were shown by arrows in B2–3. Arrows indicate cytoplasmic vacuoles in C2–3. There were also some binucleated cells in C2–3.

(Dominguez, Yorek, & Grant, 2015). In the present study, LSE improved quality of life of STZ/HFD induced diabetic rats through regulating symptoms of hyperphagia, polydipsia and polyuria (Fig. 3). Organ weight and histopathologic examination indicated that pancreas, livers and kidneys suffered different levels of damage in STZ/HFD exposed rats. LSE alleviated this injury. Blood glucose levels responded to the diabetes directly. Yet a randomized detection of glucose did not give clear hypoglycaemic effects of the agents. OGTT was considered as the most common and more sensitive test for short-term evaluation, compared to FBG or HbA1c (Farhan et al., 2012). In this research, oral administration of LSE to diabetic rats remarkably reduced the blood glucose concentrations without causing hypoglycaemia (Fig. 4). OGTT indicated that glucose tolerance AUC of the model group was greater than that of LSEtreated diabetic rats. Diabetes was caused by impaired insulin secretion and resistance of peripheral tissues to the glucose utilization of insulin (Bergman et al., 2014). Insulin resistance assay exhibited that the low level of ISI and high level of IRI in model rats denoted perturbations in β-cell function, whereas LSE rats alleviated these trends, indicating that treatment improved β-cell function and insulin resistance, thereby helping relieve diabetes syndrome through reversing the injured islet in diabetic rats and mean blood glucose levels. Hyperglycaemia evoked cellular glucose overload in the kidneys, which was associated with mitochondrial dysfunction and renal injury (Vanhorebeek et al., 2009). LSE decreased the levels of urinary glucose, which might be the reason why it induced the earlier recovery from renal injury (Chiu et al.,

2014) and protected kidney mass and morphology (Fig. 5B) in diabetic rat. Meanwhile, insulin resistance was assumed to disturb both glucose and lipid metabolism (Fizelova et al., 2015). Liver as an insulin-dependent tissue played a pivotal role in this pathway (Li et al., 2015). Morphological examination indicated that steatosis and severe cytoplasmic vacuoles happened in the liver tissues (Fig. 5C3). Furthermore, it was observed the phenomenon of atherogenic dyslipidaemia, such as increasing TG, T-CHO and LDH levels and decreasing HDL-C/LDL-C ratio in diabetic rats. As we know, increased levels of triacylglycerols were associated with increased risk of diabetes, whereas increased levels of large HDL particles protected diabetes (Fizelova et al., 2015). LSE decreased levels of TG and T-CHO in comparison with the model group and increased ratio of HDL-C/LDL-C to protect diabetes. Meanwhile, serum LDH was used as a marker for the evaluation of disease severity in the early stage of toxic reaction (Yun et al., 2008). LSE treatment decreased the levels of LDH to protect the body from damage (Fig. 6). For the above disturbance of glucose and lipid metabolism, lipogenesis enzyme FAS (Abe et al., 2009), fatty-acid β-oxidation enzyme ACO (Xu, Zhu, Kim, Yamahara, & Li, 2009) and TG synthesis enzyme TGAT (Srivastava, 2009) also participated in the liver metabolism. In this research, LSE significantly reduced mRNA expression of DGAT2 and FAS compared with the model group to decrease hepatic lipid accumulation (Wang et al., 2013). Previous research revealed that oxidative stress, inflammation, and apoptosis took part in the development of diabetic hepatic complications (Pal, Sinha, & Sil, 2014). NF-κB as an inflammation-related factor and Bax as a pro-apoptotic protein both induced hepatic injury (Park et al., 2011). LSE

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Fig. 6 – Effect of LSE on T-CHO, TG, LDH, HDL-C/LDH-C, and mRNA expression of Bax, ACO, NF-kB, FAS and DGAT2. Different letters mean significant differences between two groups (p < 0.05).

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decreased mRNA levels of NF-κB and Bax, which indicated that LSE could prevent apoptosis-induced hepatic damage and improve inflammation, thereby preventing diabetic exacerbation. In conclusion, this study demonstrated that LSE contained several kinds of catechin-rich compounds and pinocembrin glycosides, which played key hypoglycaemic roles in LSE preventing T2D progression. After one-month of treatment, LSE improved quality of life of STZ/HFD induced diabetic rats and protected against pancreas, liver and kidney damage via improvement of glucose tolerance and insulin resistance. In addition, LSE ameliorated glucose and lipid metabolism and inhibited the apoptosis-induced hepatic damage and inflammation to protect the body against diabetic exacerbation. All these results indicated that LSE postponed pancreas, liver and renal damage, and had a protective effect on STZ-induced T2D rats. Therefore, LST may serve as a potential agent for the prevention of early stage T2D.

Conflict of interest We declared that there were no conflicts of interest in this paper.

Acknowledgements This work was supported by grant 81202952 from the National Natural Science Foundation of China and 2015M570229 from the Postdoctoral Science Foundation of China.

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