Antihyperglycemic, antihyperlipidemic and antioxidant effects of standard ethanol extract of Bombax ceiba leaves in high-fat-diet- and streptozotocin-induced Type 2 diabetic rats

Chinese Journal of Natural Medicines 2017, 15(3): 01680177

Chinese Journal of Natural Medicines

Antihyperglycemic, antihyperlipidemic and antioxidant effects of standard ethanol extract of Bombax ceiba leaves in high-fat-diet- and streptozotocin-induced Type 2 diabetic rats XU Guang-Kai 1, QIN Xiao-Ying 1∆, WANG Guo-Kai 2, XIE Guo-Yong 1, LI Xu-Sen 1, SUN Chen-Yu 1, LIU Bao-Lin 3*, QIN Min-Jian 1* 1

State Key Laboratory of Natural Medicines, Department of Resources Science of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 210009, China; 2 Anhui Key Laboratory of Modernized Chinese Materia Medica, Anhui University of Traditional Chinese Medicine, Hefei 230012, China; 3 Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China Available online 20 Mar., 2017

[ABSTRACT] The present study aimed at exploring the therapeutic potential of standard extract of Bombax ceiba L. leaves (BCE) in type 2 diabetic mellitus (T2DM). Oral administration of BCE at doses of 70, 140, and 280 mg·kg−1, to the normal rats and the high-fat-diet- and streptozotocin-induced T2DM rats were carried out. Effects of BCE on blood glucose, body weight, and a range of serum biochemical parameters were tested, and histopathological observation of pancreatic tissues was also performed. HPLC-ESI-Q/TOF-MS/MS analysis indicated that the chemical composition of BCE mainly contained mangiferin, isoorientin, vitexin, isomangiferin, isovitexin, quercetin hexoside, 2'-trans-O-cumaroyl mangiferin, and nigricanside. BCE caused a significant decrease in the concentrations of fasting blood glucose, glycosylated hemoglobin, total cholesterol, triglyceride, low density lipoprotein-cholesterol, serum insulin, and malondialdehyde, and increases in oral glucose tolerance, high density lipoprotein-cholesterol, and superoxide dismutase in the T2DM model rats. Moreover, considerable pancreatic β-cells protection effect and stimulation of insulin secretion from the remaining pancreatic β-cells could be observed after BCE treatment. The results indicated that BCE exhibited an excellent hypoglycemic activity, and alleviated dyslipidemia which is associated with T2DM. Antioxidant activity and protecting pancreatic β-cells are the possible mechanisms involved in anti-diabetic activity of BCE. [KEY WORDS] Bombax ceiba ; Type 2 diabetic mellitus; Antihyperglycemic; Antihyperlipidemic; Antioxidant

[CLC Number] R965

[Document code] A

[Article ID] 2095-6975(2017)03-0168-10

Introduction Diabetes mellitus, an extensive chronic metabolic disease, is characterized by hyperglycemia and carbohydrate, protein, and fat metabolism disturbances. Persistent hyperglycemia often induces complications affecting patient’s visual, nervous, renal, and other systems. There are many issues to be addressed to cure diabetes, in addition to glycemic control. Currently available synthetic oral antihyperglycaemic agents [Received on] 15-Apr.-2016 [*Corresponding author] Tel: 86-25-86185127, Fax: 86-25-86185292, E-mail: [email protected] (LIU Bao-Lin); Tel: 86-25-86185130, Fax: 86-25-85301528, E-mail: [email protected] (QIN Min-Jian). ∆ Co-first author These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

have not shown to alter the progressive β cell failure and may be associated with an increased risk of unwanted effects after prolonged use. As traditional herbal medicines may have multiple effects on diabetes with greater tolerability and fewer side effects, there is an increasing need to search for more natural therapeutic agents from natural plants. Bombax ceiba L. (Bombacaceae), also known as “Hero Tree” or “Panzhihua” in China, is widely distributed and cultivated in temperate Asia, tropical Asia, Africa, and Australia [1]. The plant contains health-promoting phytopharmaceuticals such as phenolics, flavonoids, sesquiterpenoids, steroids, naphthoquinones, and neolignans [2-6], possessing medicinal properties and a strong ethnobotanical background [7]. It has been extensively used in southern China, India, and rural area of northern Pakistan as a famous folk medicine

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in the treatment of a range of diseases, including edema, hepatotoxicity [3, 8-10], ulcer [11], pyrexia [6], hypertension [2, 12], and diabetic mellitus [13-14]. In addition, the flower, leave, stem bark, and root of Bombax ceiba are all shown to have antimicrobial and antioxidant activities [13, 15-17]. Through bioassay screening for beneficial biological agents for diabetes from ethnomedicines, we found that ethanol extract of Bombax ceiba leaves (BCE) possessed antihyperglycemic effects. Therefore, in the present study, standard BCE was prepared and high-performance liquid chromatography-electrospray ionization quadrupole timeof-flight tandem mass spectrometry (HPLC-ESI-Q/TOFMS/MS) analysis method was used to reveal the phytopharmaceuticals of BCE. It was assumed that herbal medicines can only be effective as an alternative to oral hypoglycaemic agents in type-2 diabetes mellitus (T2DM) when pancreatic islets are not totally destroyed. Therefore, a series of systematic pharmacological experiments of BCE on high-fatdiet (HFD) and streptozotocin (STZ) induced T2DM rats were carried out. The results from the present study could provide scientific evidences for the utilizing of Bombax ceiba leaves to treat T2DM.

Materials and Methods Chemicals and reagents The kits for measurement of superoxide dismutase (SOD), fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoproteincholesterol (LDL-C), and malondialdehyde (MDA) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). The ELISA kit for insulin detection was purchased from R&D Systems (Minneapolis, MN, USA). Streptozotocin was purchased from Sigma Co. (St Louis, MO, USA). The standard antidiabetic drug glimepiride was obtained from Wanbang Pharmaceutical GmbH (Xuzhou, Jiangsu, China). Mangiferin (purity > 98%) was purchased from Guangrun Pharmaceutical Technology Co. Ltd. (Nanjing, China). Rutin (purity > 92.6%) was purchased from National Institutes for Food and Drug Control (Beijing, China). All solvents used in the present study were of analytical reagent grade. Column chromatography was carried out with macroreticular resin AB-8 (Chemical Plant of Baoen, Hebei, China). Plant materials and preparation of BCE The leaves of Bombax ceiba L. were collected from Ledong, Hainan, China, in August, 2013, and identified by Prof. QIN Min-Jian (China Pharmaceutical University, Nanjing, China). A voucher specimen (No. BM-201301) was deposited in the Herbarium of Medicinal Plants of China Pharmaceutical University, Nanjing, China. Air-dried Bombax ceiba leaves (2.5 kg) were refluxed with 80% ethanol at the ratio of 1 : 10 (solvent and sample ratio, V/W) for 2 h. The extracted solutions were combined, filtered, and then concentrated by a rotary evaporator under

reduced pressure to remove the ethanol solvent. The powdered extract was then dissolved with deionized water at appropriate concentrations, adsorbed to macroporous resin column, and then eluted with distilled water and 20%, 65%, and 95% ethanol successively. The 65% fraction was concentrated using a vacuum evaporator, and vacuum dried at room temperature, to obtain the standard Bombax ceiba leaves ethanol powder extract (BCE, yield 12.81%, W/W). Phytochemical profiling The HPLC-ESI-Q/TOF-MS/MS analysis was conducted using an Agilent Technologies Series 1290 Infinity HPLC instrument (Agilent, Waldbronn, Germany) coupled with an Agilent 6530 Q-TOF mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with an automatic degasser, an autosampler and a column compartment. Chromatographic separation was performed at 30 °C on a C18 column (210 mm × 4.6 mm, 5 μm; Hanbang Company, China) at a flow rate of 1.0 mL·min−1, and the injection volume was 10 μL. The mobile phase was consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B) using a gradient elution as follows: 0−15 min, 14% B; 15−20 min, 14%−17% B; 20−45min, and 17%−18% B. The mass spectra were acquired across the range of m/z 150−950 in a negative mode. The operating parameters of mass spectrometer were as follows: drying gas (N2) flow rate, 10 L·min−1; drying gas temperature, 320 °C; nebulizer, 35 psig; capillary voltage, 3 000 V; fragment voltage 120 V; skimmer voltage, 60 V, and Oct RFV, 750 V. The collision energy was set at 15 V. All the MS data were controlled by MassHunter software B.02.00 ChemStation (Agilent Technologies, Santa Clara, CA, USA). Determination of total phenolic compounds The determination of total phenolic compounds in BCE was accomplished using the method described in Chinese Pharmacopoeia [18]. 2-mL extract appropriately diluted was mixed with 2 mL AlCl3 (0.1 mol·L−1) and 3 mL of KAc (1 mol·L−1), andthe mixture was adjusted to 10 mL with 60% ethanol and let it rest for 30 min. Its absorbance (A) was measured at 421 nm, and 60% ethanol was used as a blank control. Rutin was used as a reference standard and the content of total phenolics was expressed as rutin equivalents (RE, μg·mg−1 extract) through the calibration curve with rutin. HPLC-DAD analysis High performance liquid chromatography (HPLC) analysis was conducted on an Agilent Series 1260 LC instrument (Agilent Technologies, Santa Clara, CA, USA) equipped with an on-line degasser, a quaternary pump, a diode-array detector (DAD), a thermostated column compartment, and an auto-sampler. The analytes separation was performed on a C18 analysis column (210 mm × 4.6 mm, 5 μm; Hanbang Company, China). The column temperature was maintained at 30 °C. The mobile phase was consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B), and the gradient program was optimized as follows: 10%−16% B at 0−1 min, 16%−18% B at 1−11 min and

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18%−90% B at 11−15 min. Sample injection volume was 5 μL and the flow rate was maintained at 1 mL·min−1. The elutes were detected at 258 nm. The standard stock solution of 200 μg·mL−1 of mangiferin was prepared in methanol and diluted to obtain standard solutions containing 160, 120, 100, 80, 60, 40, and 20 μg·mL−1 of mangiferin. The calibration curve was constructed by plotting area under the peak versus corresponding concentration of mangiferin. The sample solution was prepared as follow: BCE (10 mg), was milled by 60 meshes, and extracted with 50 mL of methanol in a round-bottom flask at 75 °C for 30 min. After reflux extraction, the sample was centrifuged at 1 000 × g for 10 min to collect the supernatant, which was then diluted to 100 mL with the extraction solution and filtered through a 0.22-μm microfiltration membrane before HPLC analysis. Animals Male Sprague Dawley (SD) rats were purchased from the Aiermaite Experimental Animal Corporation (Suzhou, China). The animals were housed in standard polypropylene cages and maintained under controlled room temperature (22 ± 2 ºC) and humidity (55% ± 5%) with 12 h light/12 h dark cycle. All the rats were provided with commercially available rat normal pellet diet (Qinglongshan Experimental Animal Co., Nanjing, China) and water ad libitum, prior to the dietary manipulation. All procedures were approved by the Ethics Committee for the Use of Experimental Animals of China Pharmaceutical University, Nanjing, China. HFD-fed and STZ-induced T2DM rats c The rats were fed with normal pellet diet for two weeks and those weighing 200–240 g were allocated into two dietary regimens by feeding either normal pellet diet or HFD (consisting of 18% fat, 20% carbohydrate, 3% egg and 59% basic diet (W/W) [19]). After six weeks of dietary manipulation, the group of HFD-fed rats were injected intraperitoneally (i.p.) with low dose of STZ (35 mg·kg−1 suspended in 0.1 mol·L−1 citrate buffer at pH 4.4), while the respective control rats were given vehicle citrate buffer (pH 4.4) only in a dose volume of 1 mL·kg−1, i.p.. The fasting blood glucose (FBG) was measured five days after the vehicle or STZ injection. The rats with the FBG of more than 11.1 mmol·L−1 [20] were used for further study. The rats were feed continually on their respective diets until the end of the experiment. Acute oral toxicity testing Acute oral toxicity of BCE was tested on male SD rats, according to OECD (Organization of Economic Cooperation and Development) Guideline 423. Two groups of four rats each were used for the study. Group I served as control and received distilled water. Group II received single oral dose of BCE (2 000 mg·kg−1). The animals were observed for gross behavioral, neurological, autonomic, and toxic effects at short intervals of time for 24 h and then daily for two weeks. Food consumption was monitored daily and body weights were recorded weekly. On Day 14, the animals were sacrificed and all the organs were removed for gross pathological

examination. BCE treatments In this experiment, a total of 60 male SD rats (10 normal; 50 diabetic rats induced by HFD and STZ) were divided into six groups of 10 rats in each. Group I: normal control rats administered with 0.5% CMC-Na daily (NC); Group II: diabetic control rats administered with 0.5% CMC-Na daily (DC); Group III: diabetic rats administered with a low dose of BCE (70 mg·kg−1) (DC + L); Group IV: diabetic rats administered with a middle dose of BCE (140 mg·kg−1) (DC + M); Group V: diabetic rats administered with a high dose of BCE (280 mg·kg−1) (DC + H); Group VI: diabetic rats administered with the reference drug glimepiride (5 mg·kg−1) (DC + GL). All test BCE samples and positive control drug were dissolved with 0.5% sodium carboxymethylcellulose (CMCNa) and were administered orally via a standard orogastric cannula once a day for three weeks. The normal control and diabetic control rats were administrated with the same volume of vehicle. The FBG levels were determined every 7 days after STZ injection. Other biochemical parameters were determined on Day 21 after the animals were fasted overnight and sacrificed by decapitation. Clinical observations Mental activity, fur condition, water and food intake, urine output and survival of the rats were observed every day. Body weight and food intake of the rats were determined every week. Oral glucose tolerance test (OGTT) The OGTT was performed on overnight fasted rats at the end of the treatments. Glucose (2 g·kg−1, i.g.) was administered and blood samples were collected at 0 (just before glucose ingestion), 30, 60, 90, and 120 min after the ingestion of glucose. Serum glucose levels of these samples were immediately determined with glucose kit based on the glucose oxidase method [21]. The results were expressed as integrated area under the curve for glucose (AUCglucose), which was calculated by the trapezoid rule. Blood sampling and analysis of biochemical metabolic parameters On Day 21, the rats were overnight fasted and the blood samples from eye pit were collected and centrifuged at 3 000 g for 15 min at 4 ºC and the serum was separated and stored at −80 ºC until analysis. Fasting serum insulin (FINS), TC, TG, HDL-C, LDL-C and HbA1c were measured using commercial assay kits, according to the manufacturer’s instructions. The contents of MDA and the activity of SOD were determined with commercially available kits, according to the manufacturer’s instructions. Histopathological studies On Day 21, when the animals were sacrificed, and the

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pancreas tissues were removed and stored in 10% formalin after washing with normal saline. The tissues were embedded in paraffin and sectioned with 4-μm thickness (Leica, Wetzlar, Germany), and then stained with hematoxylin eosin for microscopic assessment (Olympus DX45, Tokyo, Japan). For the quantitative analysis of pancreatic islets, the number of pancreatic islets was counted under a microscope (100 ×). by two investigators blinded to the experimental groups. The area of islets was measured under a microscope (200 ×) through the computer image analysis system (DP2-BSW) which Olympus microscope equipped with. Five vision fields were counted and measured, and an average of the serial three slices was taken from each individual rat. Statistical analysis All the values are expressed as mean ± SD (standard deviation). The significance of differences between the

experimental groups and the control groups was determined by Student’s-test. Differences among multiple groups were analyzed by one-way analysis of variance (ANOVA). The differences were considered statistically significant when P < 0.05.

Results Chemical Characterization of BCE HPLC-ESI-Q/TOF-MS/MS analysis was carried out in a negative ion mode and the total ion chromatograms of the BCE are shown in Fig. 1A, and the UV and MS data are listed in Table 1. According to these results, BCE was mainly composed of eight flavonoids, namely mangiferin, isoorientin, vitexin, isomangiferin, isovitexin, quercetin hexoside, 2'-trans-O-cumaroyl mangiferin, and nigricanside. Fig. 1B shows the product ions scan spectra and the molecular formula of these analytes.

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Fig. 1 The total ion chromatograms of BCE (A) and the product ion mass spectra of detected compounds (B). Compound 1: mangiferin; compound 2: isoorientin; compound 3: vitexin; compound 4: isomangiferin; compound 5: isovitexin; compound 6: quercetin hexoside; compound 7: 2'-trans-O-cumaroyl mangiferin; and compound 8: nigricanside Table 1 Characterization of chemical constituents of ethanol extract of Bombax ceiba L. leaves by HPLC-ESI-Q/TOF-MS/MS Compound tR/min 1

14.058

UV(nm)

Quasi-molecular (Error, × 10−6)

Molecular formula

m/z Calculd.

MS/MS fragments

258; 318

421.077 2 (−1.08)

C19H18O11

421.077 6

403, 331, 301, 285, 271, 259

Mangiferin

31 32

Proposed compound References

2

21.133

256; 350

447.094 2 (−2.00)

C21H20O11

447.093 3

357, 339, 327, 311, 297, 285

Isoorientin

3

27.546

258; 322

431.098 7 (−0.77)

C21H20O10

431.098 4

341, 323, 311, 283, 269

Vitexin

33

4

28.993

258; 318

421.077 9 (−0.61)

C19H18O11

421.077 6

403, 331, 301, 285, 272, 258

Isomangiferin

31

5

30.502

256; 322

431.098 5 (−0.36)

C21H20O10

431.098 4

413, 341, 323, 311, 283, 269

Isovitexin

6

33.294

256; 366

463.089 (−1.6)

C21H20O12

463.088 2

300, 271, 255

7

37.518

256; 318

541.098 6 (0.36)

C26H22O13

541.098 8

8

42.322

256; 318

421.078 2 (−1.39)

C19H18O11

421.077 6

Mangiferin and total phenolic compounds content in BCE In order to comprehend the components of active fraction, the total phenolic compounds content in BCE was analyzed. The results showed that the total phenolic compounds were expressed as rutin equivalents 81.08% (W/W) in the BCE. The HPLC-DAD analysis method was applied to determine the contents of mangiferin in BCE. The amount of mangiferin in BCE was determined to be 65.2% (W/W). No acute oral toxicity observed in rats following BCE treatment In the acute toxicity testing, BCE treated animals did not show any change in their behavioral patterns. There were no significant differences in the body weight and food consumption when compared to the vehicle treated group. Also, no gross pathological changes were seen. Thus, it was concluded that BCE was safety at dose of 2 000 mg·kg−1. Improved general features of experimental rats following BCE treatment Compared to the non-diabetic control, the diabetic rats showed tarnished fur and significantly decreased body weight (Fig. 2) with symptoms, i.e., polydipsia, polyuria and thickened urine smell. The treatment with BCE improved these general features. Chronic treatment with BCE or glimepiride prevented the body weight loss, polydipsia, and also showed a certain downward trend in food intake (Table 2).

Quercetin hexoside 2'-Trans-O-Cumaroy 421, 403, 331, 301, 283 lmangiferin 331, 313, 301, 285, 271, 259 Nigricanside

34 35 36 37

Fig. 2 Effects of BCE treatment on body weight in HFD and STZ induced diabetic rats (n = 10, mean ± SD). STZ: HFD fed rats injecting STZ on the sixth week after dietary manipulated; ig 1, 2 and 3 was the week after T2DM rat model was induced successfully

Improved glucose tolerance in diabetic rats following BCE treatment As shown in Fig. 3, intra gastric administration of glucose (2 g·kg−1) did not cause significant change in blood glucose level in normal control rats (from 7.90 ± 0.57 to 10.77 ± 0.86 mmol·L−1, n ≥ 6), while the diabetic rats (from 30.44 ± 2.92 to 46.15 ± 3.35 mmol·L−1, n ≥ 6) exhibited severe and significant impairment in glucose tolerance. Blood glucose reached the highest level at 30 min and then showed a certain downward trend. After treatment with the BCE for 21

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Table 2 Effects of BCE treatment on food intake in HFD and STZ induced diabetic rats (g) Before administration (week)

Group NC

After administration (week)

1

2

3

4

5

6

1

2

3

4

222 0

226 2

228 0

231 0

234 0

234 6

233 4

234 0

235 2

234 6

DC

113 4

118 2

126 0

135 0

142 8

145 8

148 2

153 6

156 0

155 4

DC + GL

104 4

107 4

111 0

118 2

123 0

127 2

129 0

125 4

122 4

120 6 149 4

DC + L

118 8

123 0

131 4

140 4

147 0

153 6

156 0

154 2

150 6

DC + M

111 0

114 6

121 2

126 6

129 0

136 8

138 6

133 2

131 4

130 2

DC + H

111 6

115 2

120 6

127 2

130 2

138 0

140 4

133 8

132 6

132 0

when treated with BCE middle and high doses (14.82 ± 1.32 and 13.46 ± 0.92 mmol·L−1, respectively) in diabetic rats versus the diabetic control group (16.67 ± 1.61 mmol·L−1, n ≥ 6) from the first week. On Day 21, the effects of BCE were more obvious; even low-dose BCE treated diabetic rats were found to have a significant decrease in serum levels of glucose (P < 0.05). The effect of GL on FBG level appeared a similar pattern. Effects of BCE on SOD, MDA and insulin levels As shown in Table 4, HOMA-IR was tested to evaluate the insulin resistance in the diabetic rats. The serum insulin level in the diabetic control rats (88.72 ± 1.26 mIU·L−1) abnormally raised and subsequently triggered insulin resistance. The serum insulin levels of high dose of BCE (25.78 ± 0.83 mIU·L−1) and positive controls groups (29.18 ± 0.75 mIU·L−1) were notably lower than that of the diabetic control group (Table 4). After the treatment with BCE or glimepiride, the activity

Fig. 3 Effects of BCE on OGTT in HFD and STZ induced diabetic rats (n ≥ 6, mean ± SD)

days, the rats in tested groups exhibited significant (P < 0.05) reduction in blood glucose level in 2 h, indicating that BCE can improve the glucose tolerance of diabetic rats. Effects of BCE on fasting blood glucose (FBG) levels The FBG level is the most important and obvious indicator for detection of diabetes. As shown in Table 3, the FBG level was found to be significantly (P < 0.05) decreased

of total SOD was markedly increased, compared with the diabetic control mice (P < 0.05, Table 4). The serum MDA contents were lower in the BCE treated groups than that in the diabetic control group (P < 0.05).

Table 3 Serum levels of FBG in different groups (mmol·L−1, n ≥ 6, mean ± SD) Days

NC

DC

DC + L

DC + M

DC + H

DC + GL

0

6.72 ± 0.36

15.72 ± 1.26#

16.17 ± 1.73

16.31 ± 1.91

15.78 ± 0.83

16.18 ± 0.75

7

6.66 ± 0.44

16.67 ± 1.61#

15.71 ± 1.25

14.82 ± 1.32*

13.46 ± 0.92*

8.24 ± 1.13*

14

6.34 ± 0.91

16.99 ± 1.53#

14.33 ± 1.31*

13.53 ± 1.12*

12.98 ± 1.85*

8.53 ± 0.96*

6.57 ± 0.63

#

*

*

*

9.21 ± 1.10*

21 *

17.01 ± 0.47

13.41 ± 0.37

12.89 ± 0.76

11.88 ± 1.47

#

P < 0.05 vs diabetic control group; P < 0.05 vs normal control group

Table 4 Serum levels of several biochemical parameters in different groups (n ≥ 6, mean ± SD) NC

DC

DC + L

DC + M

DC + H

DC + GL

Fins (mIU·L−1)

19.72 ± 0.36

88.72 ± 1.26#

42.17 ± 1.73*

36.31 ± 1.91*

25.78 ± 0.83*

29.18 ± 0.75*

HOMA-IR

3.75 ± 1.85

30.20 ± 18.58#

11.71 ± 9.25*

9.46 ± 2.32*

5.99 ± 3.21*

6.75 ± 2.40*

*

*

*

−1

SOD (mmol·mL )

210.63 ± 2.51

168.44 ± 4.50

#

177.33 ± 6.31

MDA(mmol·L−1) 4.72 ± 0.23 8.07 ± 0.53# 7.41 ± 0.37 P < 0.05 vs diabetic control group; #P < 0.05 vs normal control group

189.99 ± 9.12

6.89 ± 0.76*

194.60 ± 7.20

6.63 ± 0.22*

192.37 ± 13.67* 5.61 ± 0.41*

*

Effects of BCE on lipid profile As shown in Fig. 4, the increased serum levels of TG, TC, LDL-C were found to be significant (P < 0.05), and the suppression ratios were 15.7%, 59.2% and 48.3%,

respectively; whereas HDL-C was found to be significantly (P < 0.05) decreased when treated with high dose BCE in the diabetic rats versus the diabetic control group. The effect of GL on lipid profile level appeared a similar pattern.

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Effects of BCE on β-cells The results of BCE in histopathologic examination are shown in Fig. 5 and Table 5. These results provided the histological evidence of severe pancreatic damage in conjunction with the blood biochemical results. As shown in Fig. 5, compared with normal control rat (Fig. 5A), the diabetic control rats showed decreased number and size (0.65 ± 0.23, n = 6) of pancreatic islets, ambiguity of their verges, vacuolation, and invasion of connective tissues (Fig. 5B). The diabetic control rats showed apparent islet atrophy: smaller islet in size and irregular shrinkage shape, and smaller parenchymal cells accompanied with interstitial fibrosis (arrow head). Although low dose (70 mg·kg−1) BCE treatment showed little improvement of islet cell atrophy (Fig. 5C), the middle and

Fig. 4 Effects of BCE treatment on lipid profile in HFD and STZ induced diabetic rats (n ≥ 6, mean ± SD). *P < 0.05 vs diabetic control group; #P < 0.05 vs normal control group

Fig. 5 Effects of BCE on the islet morphology of the HFD and STZ induced diabetic rats. Arrow: islets and parenchymal cells. A: normal control, B: diabetic control, C: diabetic rats with 70 mg·kg−1 extract, D: diabetic rats with 140 mg·kg−1 extract, E: diabetic rats with 280 mg·kg−1 extract, F: diabetic rats with glimepiride (Scale, 300 ) Table 5 Effects of BCE on the number and area of the islets (n = 6, mean ± SD) NC

DC

DC + L #

Average number of islets (100 ×) 1.23 ± 0.50 0.65 ± 0.23 Average area of the islet (μm2, 20 246 6 029# 100 ×) * P < 0.05 vs diabetic control group; #P < 0.05 vs normal control group

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DC + M

0.41 ± 0.37

1.29 ± 0.58

7 173

14 806*

DC + H *

1.48 ± 0.76 9 572*

DC + GL *

1.05 ± 0.60* 8 728*

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high doses of BCE treatments (140 mg·kg−1, Fig. 5D and 280 mg·kg−1, Fig. 5E) remarkably protected the islet cell atrophy and stimulated parenchymal cells hypertrophy with abundant and basophilic cytoplasm, and the interstitial fibrosis was not obvious. The reference drug glimepiride treatment group could also relieve islet atrophy and fibrosis to some extent (Fig. 5F). On Day 21, when the animals were sacrificed, the pancreas tissues were removed for microscopic assessment. Compared with the diabetic control group, the number of islets was significantly increased by 227.6% and 198.4% in the BCE (H) and BCE (M) group respectively, and the area of the islet was increased by 245.5% and 158.7% respectively. The GL treated group increased 161.5% and 144.7% for the number and area of the islet, respectively.

Discussion The HFD and STZ induced diabetic rats were characterized by hyperglycemia, compensatory hyperinsulinaemia, elevated triglycerides and hypertriglyceridaemia which were similar to the features of the later stage of type 2 diabetes [20, 22]. Streptozocin is a highly selective cytotoxic agent to pancreatic islet β-cells. When fed HFD, the rats’ β-cells are under the strain of compensatory hyperinsulinaemia and become more susceptible to the diabetogenic effect of STZ [19]. Hyperglycemia is the most important feature of diabetes mellitus [23]. The significant reduction in the blood glucose level of HFD and STZ induced diabetic rats following administration of BCE indicated that Bombax ceiba L. leaves possess hypoglycemic activity. This may be attributed to the phytochemical constituents of the extract such as mangiferin, vitexin, isovitexin, and other phenolic compounds. Similar reductions in the FBG level of the diabetic rats have been reported following administration of other medicinal plant extracts [2, 4, 24]. The observed general features of the experimental diabetic rats included increased food consumption intake and reduced body weight, which were similar to the symptoms of patients with diabetes. These symptoms of diabetic rats were markedly ameliorated after administration with glimepiride or BCE for three weeks, which may be attributed to the ability of BCE to control hyperglycemia and improve glucose homeostasis. Moreover, the reversal of STZ-mediated reduction in blood insulin by BCE could be due to the presence of mangiferin and isovitexin which have been reported to significantly stimulate insulin secretion in hyperglycemic rats [4, 25]. These HFD and STZ induced diabetic rats also showed dyslipidemia as evidenced from the elevated levels of TC, LDLc, VLDLc, and TG with concomitant decreased HDLc, as in human type 2 diabetes [26]. It has been found that mangiferin upregulates proteins pivotal for mitochondrial bioenergetics and downregulates proteins controlling de novo lipogenesis [27]. This provides a molecular basis supporting the capability of BCE to regulate diabetic complications through

improving dyslipidemia. It is well known that diabetes is correlated closely with oxidative stress, resulting in an increased ROS production or a reduction in the antioxidant defense system [28]. SOD is an endogenous antioxidase which can protect the cells from wound by eliminating the oxygen radicals. Lipid peroxidation can produce MDA, which is an index of metabolic level of free radicals. In the present study, , the SOD activity of diabetic rats treated with glimepiride or BCE for three weeks was remarkably higher than that of hyperglycemic rats. Whereas, MDA content was significantly lower than that in the diabetic rats. According to the results of HPLC-ESI/MS analysis, BCE was mainly composed of phenolic constituents, including flavonoids, which possess putative antioxidant activity. These data indicated an obvious antioxidant effect of BCE on the diabetic rats, which might be one of the possible reasons for its antidiabetic effect. It is well acknowledged that STZ enters the β-cells and causes DNA oxidative damage. Moreover, STZ also liberates toxic levels of nitric oxide that inhibits aconitase activity and contributes to DNA damage. As a result, β-cells undergo the destruction by cell necrosis [29-30]. Such damage was protected by BCE treatment, which may result from the capability of the extract to enhance antioxidant activities. The extensive damage to the islets of Langerhans and reduced dimension of islets in the diabetic rats were restored by glimepiride to normal cellular population size of islets with hyperplasia. Similarly, regeneration of islet cells and mild expansions were observed in diabetic rats treated with BCE at various doses. Therefore, the possible mechanism by which the BCE brings about its hypoglycemic effect may be increasing the insulin level because of its protective effect on pancreatic β-cells and stimulation of insulin secretion from the remaining pancreatic β-cells.

Conclusions In summary, BCE is a relatively non-toxic natural product extracted from Bombax ceiba leaves and attenuatessymptoms of T2DM. The anti-diabetic activity of BCE may be attributed to the improvement in regulation of glucose and lipid metabolism and antioxidant activities and to increased pancreatic β-cells function in type 2 diabetic mice. Further in-depth studies should be carried out to investigate the exact mechanisms for these pharmacological actions. The results of HPLC-ESI-Q/TOF-MS/MS analysis indicate that the chemical composition of BCE mainly contain eight flavonoids and among them, the amount of mangiferin is dominant, which may contribute to the principal pharmacological actions of BCE.

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Cite this article as: XU Guang-Kai, QIN Xiao-Ying, WANG Guo-Kai, XIE Guo-Yong, LI Xu-Sen, SUN Chen-Yu, LIU Bao-Lin, QIN Min-Jian. Antihyperglycemic, antihyperlipidemic and antioxidant effects of standard ethanol extract of Bombax ceiba leaves on high-fat diet and streptozotocin induced Type 2 diabetic rats [J]. Chin J Nat Med, 2017, 15(3): 168-177.

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