Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Research Paper
Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats Dan Li a,b, Cheng Peng a,b,n, Xiaofang Xie a,b,n, Yu Mao c, Min Li a,b, Zhixing Cao a,b, Danqing Fan a a
Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China State Key Laboratory Breeding Base of Systematic Research Development and Utilization of Chinese Medicine Resources Co-Founded by Sichuan Province and MOST, Chengdu 611137, PR China c Southwest University for Nationalities, Chengdu 610041, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 4 July 2013 Received in revised form 13 January 2014 Accepted 14 February 2014
Ethnopharmacological relevance: The leaf of Malus toringoides (Rehd.) Hughes is a traditional folk medicine in Tibet, China, which is called “E Se” in Tibetan language. This original plant grows on snow mountains at an attitude of 3000 to 3700 m. It is primarily used to treat hypertension, hyperlipidemia, hyperglycemia, indigestion and other diseases. This study aimed to evaluate the antidiabetic effect of flavonoids extracted from E Se (ESF) and to explore the potential mechanism in streptozotocin (STZ) or alloxan (ALX) induced diabetic mice and STZ-induced diabetic rats. Materials and methods: 72 h after the establishment of a diabetic model, STZ or ALX induced diabetic mice and STZ-induced diabetic rats were treated daily with ESF at doses of 45, 90, 180 mg/kg and 37.5, 75, 150 mg/kg, respectively. Both mice and rats were fasted for 5 h before administration and the blood glucose (BG) levels were tested 1 h after treatment. Body weight was determined every other day. For STZ-induced rats, glycosylated hemoglobin (Hb1Ac), serum insulin and c-peptide, hepatic glycogen, superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in liver were assessed on the fourth day after BG level detection. Results: Compared with the model group, the general behavior of mice treated with ESF (90, 180 mg/kg) and rats treated with ESF (75, 150 mg/kg) became better and BG levels were significantly reduced (P o0.05). Significant decrease (P o0.05) in Hb1Ac level was observed in ESF-treated rats compared with diabetic rats. Significant increase (Po 0.05 ) in serum insulin and c-peptide were detected in ESF-treated rats. The treatment also significantly (P o 0.05) elevated SOD activity and reduced MDA level in the liver of diabetic rats. Besides, ESF 150 mg/kg had a trend of rising hepatic glycogen content of diabetic rats. Conclusions: The findings of this study suggest that flavonoids from the Malus toringoides (Rehd.) Hughes leaves may possess an antidiabetic activity in animals with established diabetes. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Tibetan folk medicine Malus toringoides (Rehd.) Hughes Flavonoids Hypoglycemic Antioxidant Chemical compounds studied in this article: Phlorizin (PubChem CID:6702)
1. Introduction Diabetes mellitus (DM) is a multifactorial disease caused by a metabolic disorder, characterized by chronic hyperglycemia syndrome, and is associated with co-morbidities such as kidney failure, nerve damage, hyperlipidemia, hypertension, and cardiovascular
Abbreviations: ALX, Alloxan; BG, Blood glucose; DM, Diabetes mellitus; ESF, The flavonoids of E Se; Hb1Ac, Glycosylated hemoglobin; Hb, Hemoglobin; HPLC, High performance liquid chromatography; MDA, Malondialdehyde; SOD, Superoxide dismutase; STZ, Streptozotocin n Corresponding authors at: Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China. Tel./fax: þ 86 28 61800018. E-mail address: [email protected] (C. Peng).
diseases. DM has become a major public health problem with rapidly increasing incidence. Epidemiological studies showed that this disease affected more than 340 million people worldwide and became the third chronic non-communicable disease after cardiovascular diseases and cancer (Rubio et al., 2008; Fradkin, 2012). At present, a number of hypoglycemic agents including insulin and oral drugs such as sulphonylureas, biguanides and glycosidase inhibitors are popularly used in clinics to keep blood glucose in normal level. Unexpectedly, these agents produced serious side effects in the clinical application, such as weight gain, gastrointestinal disturbances, edema, hypoglycemia and insulin resistance (Vasconcelos et al., 2011). Therefore, it is important to discover alternative therapies that may have less or no side effects (Wu et al., 2011). Medicinal plants used by folk medicinal healers are successfully used in many countries to control
http://dx.doi.org/10.1016/j.jep.2014.02.026 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
D. Li et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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diabetes, and have become the most important sources for seeking safe, specific and effective hypoglycemic agents (Hamza et al., 2010; Ibeh and Ezeaja, 2011). Moreover, many hypoglycemic components have been obtained from the medicinal plants, mainly including flavonoids, alkaloids, polysaccharides, saponins, terpenoids and unsaturated fatty acids (Wang et al., 2008). E Se is a kind of common Tibetan folk medicine in China. It is obtained from the leaf of Malus toringoides (Rehd.) Hughes or Malus tiansitoria (Batal.) Schneid., growing on the snow mountains at an attitude of 3000 to 3700 m. The earliest record about E Se is found in the ‘Tibetan Medicine the crystal mirror Materia Medica’ (pinyin: zang yao jing jing ben cao) and ‘Tibetan and Chinese Dictionary’ (pinyin: zang han da ci dian). In Chinese folk medicine, it has been used in the treatment of hypertension, indigestion, liver injury, hyperlipidemia and hyperglycemia (Mao et al., 2009a; 2009b). It is recently reported that E Se contains a special kind of flavonoid named E Se flavonoid (ESF) (Wang et al., 2011) whose bioactivity is not clear. Therefore, the main purpose of this study is to evaluate the antidiabetic effect of ESF on diabetic animals.
2. Materials and methods
analysis using a Shimadzu system (LC-20AT). Chromatographic separation was performed by using a Scienhome Kromosil C18 column 250 4.6 mm2 at 30 1C. A gradient elution was performed by the proportion of solvent A (0.1% phosphate acid) and solvent B (acetonitrile) from 0 to 20% of B (0–30 min). The ESF and the standard were dissolved in methanol and filtered through a 0.45 μm membrane (Millipore, USA) prior to injection of 10 μl. 2.4. Animals Adult, Kunming mice of either sex weighing 22–26 g and SD rats of either sex weighing 180–220 g were obtained from Chengdu University of Traditional Chinese Medicine. All animals were housed as five rats or ten mice per cage and were fed with standard diet and were allowed free access to water, maintained at a constant temperature (227 2 1C) and 45–55% relative humidity with a 12 h light–dark cycle. All the experimental procedures and protocols involving animals were reviewed by the Institutional Animal Ethical Committee of Chengdu University of Traditional Chinese Medicine.
2.1. Chemicals
2.5. Hypoglycemic activity of ESF on diabetic mice
STZ and ALX were purchased from Sigma-Aldrich (St. Louis, USA) and stored at 20 1C and 2–4 1C, respectively. Metformin was purchased from Bristol-Myers Squibb (Shanghai, China). Hb1Ac, hepatic glycogen, coomassie brilliant blue, superoxide dismutase (SOD) and malondialdehyde (MDA) assay kits were obtained from Nanjing Jiancheng Bioengineer Company (Nanjing, China). Serum insulin and c-peptide ELISA kits were produced by R&D Systems (USA). All other reagents used were of analytical grade.
2.5.1. Establishment of diabetic mice Mice were acclimatized to the laboratory for 1 week prior to the experiment. The STZ-induced diabetic mice and ALX-induced diabetic mice were respectively established according to the methods reported by Masumoto et al. (2009) and Ibeh and Ezeaja (2011) with a little modification. 0.1 M citrate buffer was the carrier used for STZ and saline solution was used for ALX. After fasted for 14 h, the mice body weights were measured and then the mice were randomly divided into group I (n ¼12), group II (n ¼12), group III (n ¼70) and group IV (n ¼70), and intraperitoneally injected with 0.1 M citrate buffer (10 ml/kg b.w., pH 4.5), saline solution(10 ml/kg b.w.), STZ (150 mg/kg b.w.) and ALX (200 mg/kg b.w.), respectively. Then all the mice were fed with standard diet and were allowed free access to water. After 72 h, all the mice were fasted for 5 h and BG levels were monitored from the tip of the tail vein with an autoanalyzer (ACCU-CHEK Active, German) glucose kit. The mice in group I and group II with BG under 9.0 mmol/L were considered to be normal in blood glucose and chosen as SMC (STZ: normal mice) and AMC (ALX: normal mice). Meanwhile, mice in group III and group IV with BG level between 15.0 mmol/L and 33.3 mmol/L and accompanied with manifestations of polydipsia, polyuria and polyphagia were considered to be diabetic mice accepting following experiments.
2.2. Plant material and preparation of the ESF Leaves of Malus toringoides (Rehd.) Hughes were collected in Ganzi Tibetan Autonomous Prefecture in May 2011 and identified by Professor Min Li, a pharmacognosist from the Department of Science of Identification of Chinese Materia Medica, School of Pharmacy, Chengdu University of Traditional Chinese Medicine (Chengdu, China). A voucher specimen of Malus toringoides (Rehd.) Hughes was deposited with the number 20110522002 in the Herbarium of the Department of Pharmacology, School of Pharmacy, Chengdu University of Traditional Chinese Medicine (Chengdu, China). The dried leaves (500 g) were pulverized to prepare the extract. After refluxing extraction with 5 times the amount of 70% (v/v) aqueous ethanol and repeating it twice for 1 h each time, the extract was filtered and then the solvent was extracted with an amount of petroleum ether for ten times. The petroleum ether extractions were discarded; the remainder was purified on a polyamide column and initially eluted with 2000 ml water. A second elution with 2000 ml 90% (v/v) aqueous ethanol was performed to extract flavonoids. The extraction of 90% (v/v) aqueous ethanol was concentrated under reduced pressure by using a rotary evaporator. The concentrates were heat-dried at 50 1C and then stored in a refrigerator at 4 1C for further use. The yield obtained was 1.13%. 2.3. Plant material and high performance liquid chromatography (HPLC) analysis of ESF ESF in this study was provided by associate professor Mao Yu (Southwest University for Nationalities, Sichuan, China) and was extracted from the leaves of Malus toringoides (Rehd.) Hughes. The main component phloridzin was quantified by means of LC-DAD
2.5.2. Treatment and BG detection on mice For STZ-induced diabetic mice, immediately after BG detection, 50 STZ-induced diabetic mice in group III were randomly divided into five groups according to BG levels and body weight and were treated as follows: SMDC (STZ-induced diabetic control mice, distilled water, 10 ml/kg b.w.), SMM (STZ-induced diabetic micemetformin 400 mg/kg b.w.), SMDT45 (STZ-induced diabetic miceESF 45 mg/kg b.w.), SMDT90 (STZ-induced diabetic mice-ESF 90 mg/kg b.w.), and SMDT180 (STZ-induced diabetic mice-ESF 180 mg/kg b.w.). SMC acting as the normal group was treated with distilled water (10 ml/kg b.w.). For ALX-induced diabetic mice, 50 ALX-induced diabetic mice were also randomly divided into five groups as: group AMDC (ALX-induced diabetic control mice), AMM (ALX-induced diabetic mice), AMDT45, AMDT90, and AMDT180 administered with distilled water (10 ml/kg b.w.), metformin (400 mg/kg b.w.) and ESF (45, 90, and 180 mg/kg b.w.), respectively. AMC acting as the normal group was treated with
Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
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distilled water (10 ml/kg b.w.). Each group had 10 mice and was treated for 4 days. BG levels were tested 1 h after treatment with the drugs. Meanwhile, body weight was measured 30 min before administration every other day. 2.6. Antidiabetic effect of ESF on diabetic rats 2.6.1. Establishment of diabetic rats After adapting to the new surroundings, all rats were fasted for 14 h and were divided into group V (n ¼10) and group VI (n ¼ 70). Conducted with the method described by Liu et al. (2011) and preexamination, rats in group VI were intraperitoneally injected with STZ (80 mg/kg b.w). Rats in group V were injected with 0.1 M citrate buffer (10 ml/kg b.w., pH 4.5). Three days after injection, all rats were fasted for 5 h and the BG level was measured by using an autoanalyzer (Accu-Chek Active, German) glucose kit. The rats in group V with BG level under 9.0 mmol/L were considered to be normal in blood glucose and represented as RC (normal rats). The rats of group VI with BG between 15.0 mmol/L and 33.3 mmol/L, as well as with polydipsia, polyuria, and polyphagia were considered to be diabetic and were used in further experiments. 2.6.2. Treatment and BG detection on rats After the successfully induced experimental diabetes, the rats were divided into six groups (n ¼8). RC (normal rats) and RDC (diabetic control rats) received distilled water (10 ml/kg b.w.); RDT37.5, RDT75, and RDT150 received the ESF at the dose of 37.5, 75, and 150 mg/kg b.w., respectively while RM (rats metformim) served as positive control and received the metformin (333.3 mg/kg b.w.). Metformin and ESF were dissolved in distilled water and were administered by gastric gavage for 4 consecutive days. All rats were fasted for 5 h before administration. Blood samples were collected by tail snipping and the BG levels were determined with an autoanalyzer (Accu-Chek Active, German) glucose kit at 1 h after administration. The measurement method of body weight was the same used for mice.
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2.7. Statistical analysis All data were expressed as mean 7SD, tested for normality using SPSS for Windows, version 17.0. One-way analysis of variance (ANOVA) was used for statistical analyses. A P-value of less than 0.05 was considered to be statistically significant.
3. Results 3.1. The analysis of HPLC on ESF The high performance liquid chromatography (HPLC) analysis of ESF is displayed in Fig. 1. The content of phloridzin for HPLC was 79.8%.
3.2. Effect of ESF on BG level and body weight of diabetic mice Before injection of STZ or ALX, the general state of health and the BG of all experimental mice were not different. At the end of the experiment, the healthy states of the ESF-treated groups were better than the SMDC and AMDC groups. As can be observed in Figs. 2 and 3, the dose of 150 mg/kg STZ or the dose of 200 mg/kg ALX is sufficient to significantly (Po0.01) increase the BG of mice, as compared with SMC and AMC groups. The diabetic mice displayed obviously lower BG levels at 1 h after the administration of the ESF or metformin when compared with the SMDC and AMDC. Tables 1 and 2 indicate that the body weight of SMDC and AMDC decreases significantly (Po0.01) compared with the SMC and AMC groups. The treatment of ESF showed a tendency to increase the body weight; however, there was no change statistically.
3.3. Effect of ESF on BG level and body weight of diabetic rats 2.6.3. Biochemical analyses At the end of treatment, blood was collected by femoral artery puncture from anesthetized rats after the BG level detection. The blood was collected with or without heparin sodium for plasma and serum separation, respectively. Once blood had been collected, thymus, spleen and liver were immediately removed, individually weighed and expressed in absolute and relative terms (g and g/100 g b.w., respectively). Then the livers were immediately perfused in 0.9% NaCl injectable solution, blotted with blotting paper and were stored frozen for further use. Exactly 0.4 g of the liver was homogenized in 3.6 ml of cool 0.9% NaCl injectable solution and centrifuged at 3000 rpm for 10 min. The supernatant was decanted into clean tubes and was used for coomassie brilliant blue (this was used for measuring protein concentration of liver and to calculate the value of SOD and MDA), SOD and MDA assays. The hepatic glycogen was determined using the colorimetric method. Insulin and c-peptide concentrations were measured from serum samples by using the Rat ELISA Kit. Hb1Ac and hemoglobin were assayed as per the manufacturer's instruction: (a) 2 ml heparin-anticoagulant whole blood was centrifuged at 1000 rpm for 10 min and was washed twice with saline solution as the above method; (b)1 ml of precipitated red blood cells were homogenized in 1.5 ml of freshly prepared distilled water; this was denoted as liquid A; (c)10 μl of liquid A was homogenized with 2.5 ml of reagent 4 (Nanjing Jiancheng, China) and tubes were maintained in room temperature for 10 min. The absorbance of the mixture was measured at 540 nm and the content of Hb¼S540 nm 0.3677; the content of Hb1Ac¼[(Smeasuring tube Sblank tube)/Hb] 10.
The BG level in the RC group remained unchanged during the experiment. Fig. 4 shows that the BG levels of diabetic rats are significantly higher (Po 0.01) than those of normal rats (RC). The ESF at a dose of 75, 150 mg/kg could obviously reduce (P o0.05) the BG level of diabetic rats after 1 h of the administration for 4 days compared with RDC group. After injection of 80 mg/kg STZ, the body weight of diabetic rats decreased significantly (P o0.01) compared to RC group (Table 3). However, neither the metformin nor the ESF could reverse the loss of body weight.
Fig. 1. High performance liquid chromatography (HPLC) analysis of ESF: phlorizin. Column: Scienhome Kromosil C18 column 250 4.6 mm2 at 30 1C. Mobile phase: gradient elution of solvent A (0.1% phosphate acid) and solvent B (acetonitrile) from 0 to 20% of B, 0–30 min. Flow rate: 0.8 ml/min.
Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
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Fig. 2. Effect of ESF on blood glucose (mmol/L) of STZ-induced mice. The results are expressed as mean 7SD (n¼ 10/group). a – Po 0.01 vs. SMC group, b – Po 0.05 vs. SMDC group and c – P o0.01 vs. SMDC group.
Fig. 4. Effect of ESF on blood glucose (mmol/L) of STZ-induced rats. The results are expressed as mean 7 SD (n¼ 8/group). a – P o0.01 vs. RC group, b – Po 0.05 vs. RDC group and c – Po 0.01 vs. RDC group.
Table 3 Effect of ESF on body weight (g) of STZ-induced rats. Groups
RC RDC RM RDT37.5 RDT75 RDT150
Body weight (g) Before modeling
1 day
3 days
4 days
174.88 7 9.09 171.88 7 9.80 174.137 9.60 176.007 9.43 172.75 7 9.81 177.50 7 10.70
194.36 713.70 155.38 78.91a 160.00 710.31 155.88 77.28 160.38 712.07 164.25 711.17
202.63 719.73 156.75 710.42a 160.88 714.66 157.50 79.13 162.25 713.59 163.25 714.44
203.25 7 22.29 157.75 7 8.61a 160.137 16.73 158.50 7 10.58 165.38 7 14.74 166.757 15.79
The results are expressed as mean 7 SD (n¼ 8/group). a
Fig. 3. Effect of ESF on blood glucose (mmol/L) of ALX-induced mice. The results are expressed as mean 7 SD (n¼10/group). a – P o0.01 vs. AMC group, b – P o0.05 vs. AMDC group and c – P o0.01 vs. AMDC group.
Table 1 Effect of ESF on body weight (g) of STZ-induced mice. Groups
SMC SMDC SMM SMDT45 SMDT90 SMDT180
Body weight (g) Before modeling
1 day
3 days
22.20 70.90 21.85 71.15 22.66 70.81 22.41 70.69 22.62 71.02 22.6771.02
27.62 7 1.77 20.65 7 1.35a 20.067 2.44 21.43 7 1.50 21.167 2.84 21.147 2.93
29.077 1.96 19.05 7 2.16a 18.93 7 2.22 20.32 7 1.85 19.137 1.72 20.50 7 3.03
The results are expressed as mean 7SD (n¼ 10/group). a
P o0.01, vs. SMC group.
Table 2 Effect of ESF on body weight (g) of ALX-induced mice. Groups
AMC AMDC AMM AMDT45 AMDT90 AMDT180
Body weight (g) Before modeling
1 day
3 days
22.23 7 0.86 22.727 0.80 22.377 1.00 22.487 1.37 22.34 7 1.35 22.357 0.98
27.917 1.90 22.05 7 2.16a 22.92 7 3.02 22.517 2.55 23.007 2.64 23.107 2.11
29.46 7 2.22 20.94 7 2.84a 23.54 7 4.00 21.187 3.65 22.89 7 3.11 22.047 3.05
The results are expressed as mean 7SD (n¼ 10/group). a
P o 0.01, vs. AMC group.
P o0.01, vs. RC group.
3.4. Effect of ESF on liver, spleen, and thymus mass of diabetic rats The effects of ESF on masses of liver, spleen and thymus are listed in Table 4. The group RDC showed a significant (P o0.05) decrease in liver, spleen, thymus mass and thymus relative mass compared with group RC, except for the relative mass of liver and spleen. The treatment of ESF had no evident effect on these tissue masses and relative masses in STZ-induced diabetic rats. 3.5. Effects of ESF on hepatic glycogen, SOD activity and MDA level in liver Table 5 shows that there is a slight reduction in liver glycogen level in STZ-induced diabetic rats. The ESF (150 mg/kg) treatment attenuated this reduction in hepatic glycogen content in STZinduced diabetic rats; however, there is no significant change statistically. The SOD activity and MDA level in liver of different groups are measured at the end of the treatment to assess the antioxidant ability of ESF, and the results are listed in Table 5. An obvious (P o0.01) decrease in SOD activity and increase in MDA level in diabetic rats are observed compared with RC group. The high dose of ESF increased SOD activity and decreased MDA level significantly (P o0.01) in liver compared with RDC group. 3.6. Effects of ESF on hemoglobin, Hb1Ac, serum insulin and c-peptide levels of diabetic rats As shown in Table 6, the hemoglobin content of the RDC group is significantly (P o0.01) decreased and the Hb1Ac is significantly (P o0.05) increased compared with the RC group. The treatment with different doses of ESF and metformin significantly (P o0.05) elevated the hemoglobin content when compared with the RDC
Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
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Table 4 Effects of ESF on liver, spleen, and thymus mass of diabetic rats. Parameters
RC
RDC
RM
RDT37.5
RDT75
RDT150
Liver (g) (g/100 g) Spleen (g) (g/100 g) Thymus (g) (g/100 g)
8.017 1.37 3.94 7 0.53 1.089 7 0.310 0.5377 0.141 0.420 7 0.140 0.2077 0.067
6.64 70.68b 4.21 70.31 0.816 70.184a 0.518 70.116 0.200 70.058b 0.127 70.037a
6.90 70.66 4.32 70.30 0.937 70.233 0.586 70.135 0.189 70.090 0.118 70.054
6.57 70.92 4.13 70.35 0.782 70.286 0.488 70.161 0.159 70.051 0.099 70.028
6.90 7 0.62 4.187 0.24 0.805 7 0.220 0.4877 0.133 0.2157 0.059 0.1337 0.044
6.497 0.98 3.89 7 0.41 0.954 7 0.311 0.5667 0.163 0.2577 0.195 0.149 7 0.100
The values are expressed as mean 7 SD (n¼ 8/group). a b
P o 0.05, vs. RC group. P o0.01, vs. RC group.
Table 5 Effects of ESF on hepatic glycogen, SOD activity and MDA level in liver. Groups
Hepatic glycogen (mg/g liver mass)
SOD (U/mgprot)
MDA (nmol/mgprot)
RC RDC RM RDT37.5 RDT75 RDT150
9.1917 2.440 7.320 7 1.001 8.3277 0.697 7.6137 2.117 7.6697 1.013 8.6447 0.687
55.77711.43 40.41 713.58a 49.84 714.62 45.02 710.08 52.48 716.61 59.48 716.66c
57.99 710.01 115.11 726.74a 69.21 714.67c 111.11 725.86 91.38 720.97b 57.81 713.84c
The values are expressed as mean 7 SD (n¼ 8/group). a b c
P o 0.01, vs. RC group. P o0.05, vs. RDC group. P o 0.01, vs. RDC group.
group. The high dose of ESF and metformin also markedly (P o0.05) reduced the Hb1Ac content. The serum insulin and c-peptide levels of different experimental groups are shown in Table 6. The STZ-induced diabetic rats exhibited a significant (Po 0.01) decrease in serum insulin and c-peptide (P o0.05) compared with the RC group. The significant (P o0.05) increase in c-peptide levels of ESF and metformin treated diabetic rats that toward normal level. The administration of ESF in diabetic rats caused a significant (P o0.05) increase in serum insulin when compared with RDC group and RM group.
4. Discussion Herbal medication has been used for treating various ailments by a large number of people in the world (Orhan et al., 2013). China has a remarkable biological diversity and a long history of using traditional plants for treating ailments. For many years, these plants have been successfully used as an important source of medicine for the treatment of variety of diseases and the research on these plants' products has increased. ‘E Se’ is a name called in Tibetan language, its plant names are Malus toringoides (Rehd.) Hughes and Malus tiansitoria (Batal.) Schneid. It is mainly used for the treatment of indisgestion, hypertension and hyperlipidemia (Wang et al., 2011). ESF extracted from Malus toringoides (Rehd.) Hughes is a special kind of flavonoid with a core moiety of phloridzin, which has a high hypoglycemic activity (Zhao et al., 2004; Masumoto et al., 2009; Najafian et al., 2012). In addition, some studies demonstrated that flavonoids have remarkable antidiabetic activity, which can reduce the high blood glucose level, inhibit α-glucosidase and protect pancreas by its antioxidant and antihyperlipidaemic activity (Adaramoye, 2012; Salib et al., 2013; Zheng et al., 2013); higher dietary flavonoids could reduce the incidence of diabetes (Jacques et al., 2013; van Dam et al., 2013). The present study was therefore designed to evaluate the antidiabetic effect of ESF against experimental diabetic animals.
ALX and STZ are two β-cell poisons, and selectively damage islet β-cell of a variety of animals that induce experimental diabetes (Szkudelski, 2001; Gai et al., 2004). The clinical symptoms of experimental animals are similar to type 1 DM, so they are classic and widely used models of diabetes. This study aimed to investigate the hypoglycemic activity of ESF in ALX or STZ induced diabetic mice, and further investigate the possible antidiabetic mechanism in STZ induced diabetic rats. The hypoglycemic activity test of ESF in diabetic mice produced an evident (P o 0.05) decrease in BG level compared with model group, and the doses at 90 and 180 mg/kg of ESF were relatively stable in hypoglycemic effect. However, ESF and metformin could not reverse the loss of body weight in diabetic mice. The treatment of ESF displayed a significant (P o 0.05) decrease in BG level in STZ-induced rats compared with positive and model groups. A loss in body weight in STZ-induced diabetic rats as well as type 1 diabetic patient has been reported previously (Bwititi et al., 2001; McDermott et al., 2003). However, the data of the present study indicates that oral administration of ESF cannot reverse the weight loss in diabetic mice and diabetic rats. The increase in hepatic glycogen can be brought about by a decrease in glycogenolysis and/or an increase in glycogenesis. There was a slight reduction in hepatic glycogen level in STZinduced diabetic rats. The dose of 150 mg/kg ESF could attenuate this reduction in glycogen content in STZ-induced diabetic rats. This result indicated that ESF might inhibit glycogenolysis and/or stimulate glycogenesis in STZ-induced diabetic rats to reduce BG level, which was consistent with the previous report (Gupta et al., 2012). On the other hand, the major compound of ESF is phlorizin, which could inhibit glucose transport in both the small intestine and the kidney (Chan et al., 2012); therefore, the ESF may reduce the renal reabsorption of glucose to lower blood glucose level. We can investigate it in the further study. Serum insulin and c-peptide levels are important for the evaluation of pancreatic islet β-cell. STZ-induced diabetic rats exhibited a significant (Po 0.05) decrease in serum insulin and c-peptide compared with the normal group. Treatment of ESF in diabetic rats showed a significant (P o0.05) increase in serum insulin and c-peptide. This might contribute to the decrease in BG level in diabetic rats. Therefore, ESF might improve the impaired pancreas, possibly due to pancreatic islet β-cell recovery and/or regeneration, which is consistent with the previous report (Gilbert and Liu, 2013). During DM, the excess glucose present in the blood reacts with hemoglobin to form HbA1c; the decrease in total hemoglobin level in diabetic rats is primarily due to the increase in HbA1c formation (Nain et al., 2012). Administration of ESF to diabetic rats reduced the Hb1Ac and increased the level of hemoglobin in diabetic rats. The amount of Hb1Ac increase is directly proportional to the BG level. So, the treatment of ESF could decrease the BG level in STZdiabetic rats.
Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
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Table 6 Effects of ESF on hemoglobin, Hb1Ac, serum insulin and c-peptide of diabetic rats. Groups
Hemoglobin (mg/mL)
Hb1Ac (mmol/mol Hb)
c-peptide (pg/mL)
Serum insulin (mIU/L)
RC RDC RM RDT37.5 RDT75 RDT150
45.32 7 7.25 35.397 4.79a 41.50 7 2.05c 40.63 7 5.26c 41.047 4.07c 44.357 5.88d
31.56 7 0.81 35.607 2.36 a 31.687 1.23c 33.767 1.32 33.007 1.26 31.90 7 0.95c
2058.1827 224.342 1575.9047 161.852b 1820.1187 141.015c 1833.0707 182.518c 1893.1537 256.803d 2022.1727 280.222d
3.848 70.345 3.469 70.207b 3.50770.117 3.738 70.230c 3.763 70.269c,e 3.845 70.254d,f
The values are expressed as mean 7 SD (n¼ 8/group). a
P o0.05, vs. RC group. Po 0.01, vs. RC group. c Po 0.05, vs. RDC group. d Po 0.01, vs. RDC group. e P o 0.05, vs. RM group. f P o 0.05, vs. RM group. b
It was reported that there was a strong relation existing between hyperglycemia, oxidative stress, cellular and endothelial dysfunction (Orhan et al., 2010). Increasing evidence in experimental and clinical studies indicate that oxidative stress plays an important role in DM development and pathogenesis of diabetic complications (Keter and Mutiso, 2012). Streptozotocin and hyperglycemia are two main pathogenic factors for the oxidative stress increasing the imbalance in ROS generation and elimination/ neutralization (Atangwho et al., 2012). The resulting excess ROS initially induces antioxidant enzymes, particularly in the liver, the tissue with the most abundant antioxidant defense enzymes (Tatsuki et al., 1997). SOD is a major scavenging enzyme by removing free radicals and protects from free oxygen radicals by catalyzing the elimination of superoxide radical, which damage the membrane and biological structures (Nain et al., 2012). In the present study, the treatment with ESF showed a significant increase in SOD activity in the liver of diabetic rats. On the other hand, the lipid peroxidation is one of the characteristic features of DM (Nain et al., 2012). The measurement of MDA is used as an index of lipid peroxidation and it helps to assess the extent of tissue damage. Several studies have reported an increase in MDA in plasma, liver and kidney in experimental diabetes mellitus (Deng et al., 2012; Orhan et al., 2012). The results of the present study show that ESF significantly decreases MDA level and reduces the risk of tissue damage. The results of this study clearly indicate that ESF contain a free radical scavenging activity, which can attenuate pathological alteration caused by the presence of superoxide radicals, which is consistent with the previous report (Zheng et al., 2013).
5. Conclusion This is the first report on the antidiabetic effect of ESF on streptozotocin (STZ) or alloxan (ALX) induced diabetic mice and STZ-induced diabetic rats. The results of this present study clearly indicate that the flavonoids of Malus toringoides (Rehd.) Hughes (ESF) exhibit hypoglycemic activity in experimental diabetic animals. The ESF exhibited improvement in parameters like FBG, Hb1Ac, hepatic glycogen, SOD activity and MDA level in liver. This antidiabetic activity may be related to the improvement of impaired pancreatic islet β-cell and antioxidant ability. Therefore, the leaf of Malus toringoides (Rehd.) Hughes is a good candidate as an alternative and/or complementary medicine in the treatment of DM. However, further investigations are needed to identify the lead molecule and to explore the exact mechanism of action for the antidiabetic effect.
Acknowledgment This study was financially supported by the Research on Key Technology and Product Development of Chinese and Tibetan medicine (Grant no. 2013SI0114). The authors would like to thank the associate Professor Mao Yu (Southwest University for Nationalities, Sichuan, China) for providing the ESF. The authors also acknowledge the State Key Laboratory Breeding Base of Systematic Research Development and Utilization of Chinese Medicine Resources Co-Founded by Sichuan Province and MOST for providing laboratories and instruments.
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Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
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Research Paper
Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats Dan Li a,b, Cheng Peng a,b,n, Xiaofang Xie a,b,n, Yu Mao c, Min Li a,b, Zhixing Cao a,b, Danqing Fan a a
Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China State Key Laboratory Breeding Base of Systematic Research Development and Utilization of Chinese Medicine Resources Co-Founded by Sichuan Province and MOST, Chengdu 611137, PR China c Southwest University for Nationalities, Chengdu 610041, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 4 July 2013 Received in revised form 13 January 2014 Accepted 14 February 2014
Ethnopharmacological relevance: The leaf of Malus toringoides (Rehd.) Hughes is a traditional folk medicine in Tibet, China, which is called “E Se” in Tibetan language. This original plant grows on snow mountains at an attitude of 3000 to 3700 m. It is primarily used to treat hypertension, hyperlipidemia, hyperglycemia, indigestion and other diseases. This study aimed to evaluate the antidiabetic effect of flavonoids extracted from E Se (ESF) and to explore the potential mechanism in streptozotocin (STZ) or alloxan (ALX) induced diabetic mice and STZ-induced diabetic rats. Materials and methods: 72 h after the establishment of a diabetic model, STZ or ALX induced diabetic mice and STZ-induced diabetic rats were treated daily with ESF at doses of 45, 90, 180 mg/kg and 37.5, 75, 150 mg/kg, respectively. Both mice and rats were fasted for 5 h before administration and the blood glucose (BG) levels were tested 1 h after treatment. Body weight was determined every other day. For STZ-induced rats, glycosylated hemoglobin (Hb1Ac), serum insulin and c-peptide, hepatic glycogen, superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in liver were assessed on the fourth day after BG level detection. Results: Compared with the model group, the general behavior of mice treated with ESF (90, 180 mg/kg) and rats treated with ESF (75, 150 mg/kg) became better and BG levels were significantly reduced (P o0.05). Significant decrease (P o0.05) in Hb1Ac level was observed in ESF-treated rats compared with diabetic rats. Significant increase (Po 0.05 ) in serum insulin and c-peptide were detected in ESF-treated rats. The treatment also significantly (P o 0.05) elevated SOD activity and reduced MDA level in the liver of diabetic rats. Besides, ESF 150 mg/kg had a trend of rising hepatic glycogen content of diabetic rats. Conclusions: The findings of this study suggest that flavonoids from the Malus toringoides (Rehd.) Hughes leaves may possess an antidiabetic activity in animals with established diabetes. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Tibetan folk medicine Malus toringoides (Rehd.) Hughes Flavonoids Hypoglycemic Antioxidant Chemical compounds studied in this article: Phlorizin (PubChem CID:6702)
1. Introduction Diabetes mellitus (DM) is a multifactorial disease caused by a metabolic disorder, characterized by chronic hyperglycemia syndrome, and is associated with co-morbidities such as kidney failure, nerve damage, hyperlipidemia, hypertension, and cardiovascular
Abbreviations: ALX, Alloxan; BG, Blood glucose; DM, Diabetes mellitus; ESF, The flavonoids of E Se; Hb1Ac, Glycosylated hemoglobin; Hb, Hemoglobin; HPLC, High performance liquid chromatography; MDA, Malondialdehyde; SOD, Superoxide dismutase; STZ, Streptozotocin n Corresponding authors at: Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China. Tel./fax: þ 86 28 61800018. E-mail address: [email protected] (C. Peng).
diseases. DM has become a major public health problem with rapidly increasing incidence. Epidemiological studies showed that this disease affected more than 340 million people worldwide and became the third chronic non-communicable disease after cardiovascular diseases and cancer (Rubio et al., 2008; Fradkin, 2012). At present, a number of hypoglycemic agents including insulin and oral drugs such as sulphonylureas, biguanides and glycosidase inhibitors are popularly used in clinics to keep blood glucose in normal level. Unexpectedly, these agents produced serious side effects in the clinical application, such as weight gain, gastrointestinal disturbances, edema, hypoglycemia and insulin resistance (Vasconcelos et al., 2011). Therefore, it is important to discover alternative therapies that may have less or no side effects (Wu et al., 2011). Medicinal plants used by folk medicinal healers are successfully used in many countries to control
http://dx.doi.org/10.1016/j.jep.2014.02.026 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
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diabetes, and have become the most important sources for seeking safe, specific and effective hypoglycemic agents (Hamza et al., 2010; Ibeh and Ezeaja, 2011). Moreover, many hypoglycemic components have been obtained from the medicinal plants, mainly including flavonoids, alkaloids, polysaccharides, saponins, terpenoids and unsaturated fatty acids (Wang et al., 2008). E Se is a kind of common Tibetan folk medicine in China. It is obtained from the leaf of Malus toringoides (Rehd.) Hughes or Malus tiansitoria (Batal.) Schneid., growing on the snow mountains at an attitude of 3000 to 3700 m. The earliest record about E Se is found in the ‘Tibetan Medicine the crystal mirror Materia Medica’ (pinyin: zang yao jing jing ben cao) and ‘Tibetan and Chinese Dictionary’ (pinyin: zang han da ci dian). In Chinese folk medicine, it has been used in the treatment of hypertension, indigestion, liver injury, hyperlipidemia and hyperglycemia (Mao et al., 2009a; 2009b). It is recently reported that E Se contains a special kind of flavonoid named E Se flavonoid (ESF) (Wang et al., 2011) whose bioactivity is not clear. Therefore, the main purpose of this study is to evaluate the antidiabetic effect of ESF on diabetic animals.
2. Materials and methods
analysis using a Shimadzu system (LC-20AT). Chromatographic separation was performed by using a Scienhome Kromosil C18 column 250 4.6 mm2 at 30 1C. A gradient elution was performed by the proportion of solvent A (0.1% phosphate acid) and solvent B (acetonitrile) from 0 to 20% of B (0–30 min). The ESF and the standard were dissolved in methanol and filtered through a 0.45 μm membrane (Millipore, USA) prior to injection of 10 μl. 2.4. Animals Adult, Kunming mice of either sex weighing 22–26 g and SD rats of either sex weighing 180–220 g were obtained from Chengdu University of Traditional Chinese Medicine. All animals were housed as five rats or ten mice per cage and were fed with standard diet and were allowed free access to water, maintained at a constant temperature (227 2 1C) and 45–55% relative humidity with a 12 h light–dark cycle. All the experimental procedures and protocols involving animals were reviewed by the Institutional Animal Ethical Committee of Chengdu University of Traditional Chinese Medicine.
2.1. Chemicals
2.5. Hypoglycemic activity of ESF on diabetic mice
STZ and ALX were purchased from Sigma-Aldrich (St. Louis, USA) and stored at 20 1C and 2–4 1C, respectively. Metformin was purchased from Bristol-Myers Squibb (Shanghai, China). Hb1Ac, hepatic glycogen, coomassie brilliant blue, superoxide dismutase (SOD) and malondialdehyde (MDA) assay kits were obtained from Nanjing Jiancheng Bioengineer Company (Nanjing, China). Serum insulin and c-peptide ELISA kits were produced by R&D Systems (USA). All other reagents used were of analytical grade.
2.5.1. Establishment of diabetic mice Mice were acclimatized to the laboratory for 1 week prior to the experiment. The STZ-induced diabetic mice and ALX-induced diabetic mice were respectively established according to the methods reported by Masumoto et al. (2009) and Ibeh and Ezeaja (2011) with a little modification. 0.1 M citrate buffer was the carrier used for STZ and saline solution was used for ALX. After fasted for 14 h, the mice body weights were measured and then the mice were randomly divided into group I (n ¼12), group II (n ¼12), group III (n ¼70) and group IV (n ¼70), and intraperitoneally injected with 0.1 M citrate buffer (10 ml/kg b.w., pH 4.5), saline solution(10 ml/kg b.w.), STZ (150 mg/kg b.w.) and ALX (200 mg/kg b.w.), respectively. Then all the mice were fed with standard diet and were allowed free access to water. After 72 h, all the mice were fasted for 5 h and BG levels were monitored from the tip of the tail vein with an autoanalyzer (ACCU-CHEK Active, German) glucose kit. The mice in group I and group II with BG under 9.0 mmol/L were considered to be normal in blood glucose and chosen as SMC (STZ: normal mice) and AMC (ALX: normal mice). Meanwhile, mice in group III and group IV with BG level between 15.0 mmol/L and 33.3 mmol/L and accompanied with manifestations of polydipsia, polyuria and polyphagia were considered to be diabetic mice accepting following experiments.
2.2. Plant material and preparation of the ESF Leaves of Malus toringoides (Rehd.) Hughes were collected in Ganzi Tibetan Autonomous Prefecture in May 2011 and identified by Professor Min Li, a pharmacognosist from the Department of Science of Identification of Chinese Materia Medica, School of Pharmacy, Chengdu University of Traditional Chinese Medicine (Chengdu, China). A voucher specimen of Malus toringoides (Rehd.) Hughes was deposited with the number 20110522002 in the Herbarium of the Department of Pharmacology, School of Pharmacy, Chengdu University of Traditional Chinese Medicine (Chengdu, China). The dried leaves (500 g) were pulverized to prepare the extract. After refluxing extraction with 5 times the amount of 70% (v/v) aqueous ethanol and repeating it twice for 1 h each time, the extract was filtered and then the solvent was extracted with an amount of petroleum ether for ten times. The petroleum ether extractions were discarded; the remainder was purified on a polyamide column and initially eluted with 2000 ml water. A second elution with 2000 ml 90% (v/v) aqueous ethanol was performed to extract flavonoids. The extraction of 90% (v/v) aqueous ethanol was concentrated under reduced pressure by using a rotary evaporator. The concentrates were heat-dried at 50 1C and then stored in a refrigerator at 4 1C for further use. The yield obtained was 1.13%. 2.3. Plant material and high performance liquid chromatography (HPLC) analysis of ESF ESF in this study was provided by associate professor Mao Yu (Southwest University for Nationalities, Sichuan, China) and was extracted from the leaves of Malus toringoides (Rehd.) Hughes. The main component phloridzin was quantified by means of LC-DAD
2.5.2. Treatment and BG detection on mice For STZ-induced diabetic mice, immediately after BG detection, 50 STZ-induced diabetic mice in group III were randomly divided into five groups according to BG levels and body weight and were treated as follows: SMDC (STZ-induced diabetic control mice, distilled water, 10 ml/kg b.w.), SMM (STZ-induced diabetic micemetformin 400 mg/kg b.w.), SMDT45 (STZ-induced diabetic miceESF 45 mg/kg b.w.), SMDT90 (STZ-induced diabetic mice-ESF 90 mg/kg b.w.), and SMDT180 (STZ-induced diabetic mice-ESF 180 mg/kg b.w.). SMC acting as the normal group was treated with distilled water (10 ml/kg b.w.). For ALX-induced diabetic mice, 50 ALX-induced diabetic mice were also randomly divided into five groups as: group AMDC (ALX-induced diabetic control mice), AMM (ALX-induced diabetic mice), AMDT45, AMDT90, and AMDT180 administered with distilled water (10 ml/kg b.w.), metformin (400 mg/kg b.w.) and ESF (45, 90, and 180 mg/kg b.w.), respectively. AMC acting as the normal group was treated with
Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
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distilled water (10 ml/kg b.w.). Each group had 10 mice and was treated for 4 days. BG levels were tested 1 h after treatment with the drugs. Meanwhile, body weight was measured 30 min before administration every other day. 2.6. Antidiabetic effect of ESF on diabetic rats 2.6.1. Establishment of diabetic rats After adapting to the new surroundings, all rats were fasted for 14 h and were divided into group V (n ¼10) and group VI (n ¼ 70). Conducted with the method described by Liu et al. (2011) and preexamination, rats in group VI were intraperitoneally injected with STZ (80 mg/kg b.w). Rats in group V were injected with 0.1 M citrate buffer (10 ml/kg b.w., pH 4.5). Three days after injection, all rats were fasted for 5 h and the BG level was measured by using an autoanalyzer (Accu-Chek Active, German) glucose kit. The rats in group V with BG level under 9.0 mmol/L were considered to be normal in blood glucose and represented as RC (normal rats). The rats of group VI with BG between 15.0 mmol/L and 33.3 mmol/L, as well as with polydipsia, polyuria, and polyphagia were considered to be diabetic and were used in further experiments. 2.6.2. Treatment and BG detection on rats After the successfully induced experimental diabetes, the rats were divided into six groups (n ¼8). RC (normal rats) and RDC (diabetic control rats) received distilled water (10 ml/kg b.w.); RDT37.5, RDT75, and RDT150 received the ESF at the dose of 37.5, 75, and 150 mg/kg b.w., respectively while RM (rats metformim) served as positive control and received the metformin (333.3 mg/kg b.w.). Metformin and ESF were dissolved in distilled water and were administered by gastric gavage for 4 consecutive days. All rats were fasted for 5 h before administration. Blood samples were collected by tail snipping and the BG levels were determined with an autoanalyzer (Accu-Chek Active, German) glucose kit at 1 h after administration. The measurement method of body weight was the same used for mice.
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2.7. Statistical analysis All data were expressed as mean 7SD, tested for normality using SPSS for Windows, version 17.0. One-way analysis of variance (ANOVA) was used for statistical analyses. A P-value of less than 0.05 was considered to be statistically significant.
3. Results 3.1. The analysis of HPLC on ESF The high performance liquid chromatography (HPLC) analysis of ESF is displayed in Fig. 1. The content of phloridzin for HPLC was 79.8%.
3.2. Effect of ESF on BG level and body weight of diabetic mice Before injection of STZ or ALX, the general state of health and the BG of all experimental mice were not different. At the end of the experiment, the healthy states of the ESF-treated groups were better than the SMDC and AMDC groups. As can be observed in Figs. 2 and 3, the dose of 150 mg/kg STZ or the dose of 200 mg/kg ALX is sufficient to significantly (Po0.01) increase the BG of mice, as compared with SMC and AMC groups. The diabetic mice displayed obviously lower BG levels at 1 h after the administration of the ESF or metformin when compared with the SMDC and AMDC. Tables 1 and 2 indicate that the body weight of SMDC and AMDC decreases significantly (Po0.01) compared with the SMC and AMC groups. The treatment of ESF showed a tendency to increase the body weight; however, there was no change statistically.
3.3. Effect of ESF on BG level and body weight of diabetic rats 2.6.3. Biochemical analyses At the end of treatment, blood was collected by femoral artery puncture from anesthetized rats after the BG level detection. The blood was collected with or without heparin sodium for plasma and serum separation, respectively. Once blood had been collected, thymus, spleen and liver were immediately removed, individually weighed and expressed in absolute and relative terms (g and g/100 g b.w., respectively). Then the livers were immediately perfused in 0.9% NaCl injectable solution, blotted with blotting paper and were stored frozen for further use. Exactly 0.4 g of the liver was homogenized in 3.6 ml of cool 0.9% NaCl injectable solution and centrifuged at 3000 rpm for 10 min. The supernatant was decanted into clean tubes and was used for coomassie brilliant blue (this was used for measuring protein concentration of liver and to calculate the value of SOD and MDA), SOD and MDA assays. The hepatic glycogen was determined using the colorimetric method. Insulin and c-peptide concentrations were measured from serum samples by using the Rat ELISA Kit. Hb1Ac and hemoglobin were assayed as per the manufacturer's instruction: (a) 2 ml heparin-anticoagulant whole blood was centrifuged at 1000 rpm for 10 min and was washed twice with saline solution as the above method; (b)1 ml of precipitated red blood cells were homogenized in 1.5 ml of freshly prepared distilled water; this was denoted as liquid A; (c)10 μl of liquid A was homogenized with 2.5 ml of reagent 4 (Nanjing Jiancheng, China) and tubes were maintained in room temperature for 10 min. The absorbance of the mixture was measured at 540 nm and the content of Hb¼S540 nm 0.3677; the content of Hb1Ac¼[(Smeasuring tube Sblank tube)/Hb] 10.
The BG level in the RC group remained unchanged during the experiment. Fig. 4 shows that the BG levels of diabetic rats are significantly higher (Po 0.01) than those of normal rats (RC). The ESF at a dose of 75, 150 mg/kg could obviously reduce (P o0.05) the BG level of diabetic rats after 1 h of the administration for 4 days compared with RDC group. After injection of 80 mg/kg STZ, the body weight of diabetic rats decreased significantly (P o0.01) compared to RC group (Table 3). However, neither the metformin nor the ESF could reverse the loss of body weight.
Fig. 1. High performance liquid chromatography (HPLC) analysis of ESF: phlorizin. Column: Scienhome Kromosil C18 column 250 4.6 mm2 at 30 1C. Mobile phase: gradient elution of solvent A (0.1% phosphate acid) and solvent B (acetonitrile) from 0 to 20% of B, 0–30 min. Flow rate: 0.8 ml/min.
Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
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Fig. 2. Effect of ESF on blood glucose (mmol/L) of STZ-induced mice. The results are expressed as mean 7SD (n¼ 10/group). a – Po 0.01 vs. SMC group, b – Po 0.05 vs. SMDC group and c – P o0.01 vs. SMDC group.
Fig. 4. Effect of ESF on blood glucose (mmol/L) of STZ-induced rats. The results are expressed as mean 7 SD (n¼ 8/group). a – P o0.01 vs. RC group, b – Po 0.05 vs. RDC group and c – Po 0.01 vs. RDC group.
Table 3 Effect of ESF on body weight (g) of STZ-induced rats. Groups
RC RDC RM RDT37.5 RDT75 RDT150
Body weight (g) Before modeling
1 day
3 days
4 days
174.88 7 9.09 171.88 7 9.80 174.137 9.60 176.007 9.43 172.75 7 9.81 177.50 7 10.70
194.36 713.70 155.38 78.91a 160.00 710.31 155.88 77.28 160.38 712.07 164.25 711.17
202.63 719.73 156.75 710.42a 160.88 714.66 157.50 79.13 162.25 713.59 163.25 714.44
203.25 7 22.29 157.75 7 8.61a 160.137 16.73 158.50 7 10.58 165.38 7 14.74 166.757 15.79
The results are expressed as mean 7 SD (n¼ 8/group). a
Fig. 3. Effect of ESF on blood glucose (mmol/L) of ALX-induced mice. The results are expressed as mean 7 SD (n¼10/group). a – P o0.01 vs. AMC group, b – P o0.05 vs. AMDC group and c – P o0.01 vs. AMDC group.
Table 1 Effect of ESF on body weight (g) of STZ-induced mice. Groups
SMC SMDC SMM SMDT45 SMDT90 SMDT180
Body weight (g) Before modeling
1 day
3 days
22.20 70.90 21.85 71.15 22.66 70.81 22.41 70.69 22.62 71.02 22.6771.02
27.62 7 1.77 20.65 7 1.35a 20.067 2.44 21.43 7 1.50 21.167 2.84 21.147 2.93
29.077 1.96 19.05 7 2.16a 18.93 7 2.22 20.32 7 1.85 19.137 1.72 20.50 7 3.03
The results are expressed as mean 7SD (n¼ 10/group). a
P o0.01, vs. SMC group.
Table 2 Effect of ESF on body weight (g) of ALX-induced mice. Groups
AMC AMDC AMM AMDT45 AMDT90 AMDT180
Body weight (g) Before modeling
1 day
3 days
22.23 7 0.86 22.727 0.80 22.377 1.00 22.487 1.37 22.34 7 1.35 22.357 0.98
27.917 1.90 22.05 7 2.16a 22.92 7 3.02 22.517 2.55 23.007 2.64 23.107 2.11
29.46 7 2.22 20.94 7 2.84a 23.54 7 4.00 21.187 3.65 22.89 7 3.11 22.047 3.05
The results are expressed as mean 7SD (n¼ 10/group). a
P o 0.01, vs. AMC group.
P o0.01, vs. RC group.
3.4. Effect of ESF on liver, spleen, and thymus mass of diabetic rats The effects of ESF on masses of liver, spleen and thymus are listed in Table 4. The group RDC showed a significant (P o0.05) decrease in liver, spleen, thymus mass and thymus relative mass compared with group RC, except for the relative mass of liver and spleen. The treatment of ESF had no evident effect on these tissue masses and relative masses in STZ-induced diabetic rats. 3.5. Effects of ESF on hepatic glycogen, SOD activity and MDA level in liver Table 5 shows that there is a slight reduction in liver glycogen level in STZ-induced diabetic rats. The ESF (150 mg/kg) treatment attenuated this reduction in hepatic glycogen content in STZinduced diabetic rats; however, there is no significant change statistically. The SOD activity and MDA level in liver of different groups are measured at the end of the treatment to assess the antioxidant ability of ESF, and the results are listed in Table 5. An obvious (P o0.01) decrease in SOD activity and increase in MDA level in diabetic rats are observed compared with RC group. The high dose of ESF increased SOD activity and decreased MDA level significantly (P o0.01) in liver compared with RDC group. 3.6. Effects of ESF on hemoglobin, Hb1Ac, serum insulin and c-peptide levels of diabetic rats As shown in Table 6, the hemoglobin content of the RDC group is significantly (P o0.01) decreased and the Hb1Ac is significantly (P o0.05) increased compared with the RC group. The treatment with different doses of ESF and metformin significantly (P o0.05) elevated the hemoglobin content when compared with the RDC
Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
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Table 4 Effects of ESF on liver, spleen, and thymus mass of diabetic rats. Parameters
RC
RDC
RM
RDT37.5
RDT75
RDT150
Liver (g) (g/100 g) Spleen (g) (g/100 g) Thymus (g) (g/100 g)
8.017 1.37 3.94 7 0.53 1.089 7 0.310 0.5377 0.141 0.420 7 0.140 0.2077 0.067
6.64 70.68b 4.21 70.31 0.816 70.184a 0.518 70.116 0.200 70.058b 0.127 70.037a
6.90 70.66 4.32 70.30 0.937 70.233 0.586 70.135 0.189 70.090 0.118 70.054
6.57 70.92 4.13 70.35 0.782 70.286 0.488 70.161 0.159 70.051 0.099 70.028
6.90 7 0.62 4.187 0.24 0.805 7 0.220 0.4877 0.133 0.2157 0.059 0.1337 0.044
6.497 0.98 3.89 7 0.41 0.954 7 0.311 0.5667 0.163 0.2577 0.195 0.149 7 0.100
The values are expressed as mean 7 SD (n¼ 8/group). a b
P o 0.05, vs. RC group. P o0.01, vs. RC group.
Table 5 Effects of ESF on hepatic glycogen, SOD activity and MDA level in liver. Groups
Hepatic glycogen (mg/g liver mass)
SOD (U/mgprot)
MDA (nmol/mgprot)
RC RDC RM RDT37.5 RDT75 RDT150
9.1917 2.440 7.320 7 1.001 8.3277 0.697 7.6137 2.117 7.6697 1.013 8.6447 0.687
55.77711.43 40.41 713.58a 49.84 714.62 45.02 710.08 52.48 716.61 59.48 716.66c
57.99 710.01 115.11 726.74a 69.21 714.67c 111.11 725.86 91.38 720.97b 57.81 713.84c
The values are expressed as mean 7 SD (n¼ 8/group). a b c
P o 0.01, vs. RC group. P o0.05, vs. RDC group. P o 0.01, vs. RDC group.
group. The high dose of ESF and metformin also markedly (P o0.05) reduced the Hb1Ac content. The serum insulin and c-peptide levels of different experimental groups are shown in Table 6. The STZ-induced diabetic rats exhibited a significant (Po 0.01) decrease in serum insulin and c-peptide (P o0.05) compared with the RC group. The significant (P o0.05) increase in c-peptide levels of ESF and metformin treated diabetic rats that toward normal level. The administration of ESF in diabetic rats caused a significant (P o0.05) increase in serum insulin when compared with RDC group and RM group.
4. Discussion Herbal medication has been used for treating various ailments by a large number of people in the world (Orhan et al., 2013). China has a remarkable biological diversity and a long history of using traditional plants for treating ailments. For many years, these plants have been successfully used as an important source of medicine for the treatment of variety of diseases and the research on these plants' products has increased. ‘E Se’ is a name called in Tibetan language, its plant names are Malus toringoides (Rehd.) Hughes and Malus tiansitoria (Batal.) Schneid. It is mainly used for the treatment of indisgestion, hypertension and hyperlipidemia (Wang et al., 2011). ESF extracted from Malus toringoides (Rehd.) Hughes is a special kind of flavonoid with a core moiety of phloridzin, which has a high hypoglycemic activity (Zhao et al., 2004; Masumoto et al., 2009; Najafian et al., 2012). In addition, some studies demonstrated that flavonoids have remarkable antidiabetic activity, which can reduce the high blood glucose level, inhibit α-glucosidase and protect pancreas by its antioxidant and antihyperlipidaemic activity (Adaramoye, 2012; Salib et al., 2013; Zheng et al., 2013); higher dietary flavonoids could reduce the incidence of diabetes (Jacques et al., 2013; van Dam et al., 2013). The present study was therefore designed to evaluate the antidiabetic effect of ESF against experimental diabetic animals.
ALX and STZ are two β-cell poisons, and selectively damage islet β-cell of a variety of animals that induce experimental diabetes (Szkudelski, 2001; Gai et al., 2004). The clinical symptoms of experimental animals are similar to type 1 DM, so they are classic and widely used models of diabetes. This study aimed to investigate the hypoglycemic activity of ESF in ALX or STZ induced diabetic mice, and further investigate the possible antidiabetic mechanism in STZ induced diabetic rats. The hypoglycemic activity test of ESF in diabetic mice produced an evident (P o 0.05) decrease in BG level compared with model group, and the doses at 90 and 180 mg/kg of ESF were relatively stable in hypoglycemic effect. However, ESF and metformin could not reverse the loss of body weight in diabetic mice. The treatment of ESF displayed a significant (P o 0.05) decrease in BG level in STZ-induced rats compared with positive and model groups. A loss in body weight in STZ-induced diabetic rats as well as type 1 diabetic patient has been reported previously (Bwititi et al., 2001; McDermott et al., 2003). However, the data of the present study indicates that oral administration of ESF cannot reverse the weight loss in diabetic mice and diabetic rats. The increase in hepatic glycogen can be brought about by a decrease in glycogenolysis and/or an increase in glycogenesis. There was a slight reduction in hepatic glycogen level in STZinduced diabetic rats. The dose of 150 mg/kg ESF could attenuate this reduction in glycogen content in STZ-induced diabetic rats. This result indicated that ESF might inhibit glycogenolysis and/or stimulate glycogenesis in STZ-induced diabetic rats to reduce BG level, which was consistent with the previous report (Gupta et al., 2012). On the other hand, the major compound of ESF is phlorizin, which could inhibit glucose transport in both the small intestine and the kidney (Chan et al., 2012); therefore, the ESF may reduce the renal reabsorption of glucose to lower blood glucose level. We can investigate it in the further study. Serum insulin and c-peptide levels are important for the evaluation of pancreatic islet β-cell. STZ-induced diabetic rats exhibited a significant (Po 0.05) decrease in serum insulin and c-peptide compared with the normal group. Treatment of ESF in diabetic rats showed a significant (P o0.05) increase in serum insulin and c-peptide. This might contribute to the decrease in BG level in diabetic rats. Therefore, ESF might improve the impaired pancreas, possibly due to pancreatic islet β-cell recovery and/or regeneration, which is consistent with the previous report (Gilbert and Liu, 2013). During DM, the excess glucose present in the blood reacts with hemoglobin to form HbA1c; the decrease in total hemoglobin level in diabetic rats is primarily due to the increase in HbA1c formation (Nain et al., 2012). Administration of ESF to diabetic rats reduced the Hb1Ac and increased the level of hemoglobin in diabetic rats. The amount of Hb1Ac increase is directly proportional to the BG level. So, the treatment of ESF could decrease the BG level in STZdiabetic rats.
Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
D. Li et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Table 6 Effects of ESF on hemoglobin, Hb1Ac, serum insulin and c-peptide of diabetic rats. Groups
Hemoglobin (mg/mL)
Hb1Ac (mmol/mol Hb)
c-peptide (pg/mL)
Serum insulin (mIU/L)
RC RDC RM RDT37.5 RDT75 RDT150
45.32 7 7.25 35.397 4.79a 41.50 7 2.05c 40.63 7 5.26c 41.047 4.07c 44.357 5.88d
31.56 7 0.81 35.607 2.36 a 31.687 1.23c 33.767 1.32 33.007 1.26 31.90 7 0.95c
2058.1827 224.342 1575.9047 161.852b 1820.1187 141.015c 1833.0707 182.518c 1893.1537 256.803d 2022.1727 280.222d
3.848 70.345 3.469 70.207b 3.50770.117 3.738 70.230c 3.763 70.269c,e 3.845 70.254d,f
The values are expressed as mean 7 SD (n¼ 8/group). a
P o0.05, vs. RC group. Po 0.01, vs. RC group. c Po 0.05, vs. RDC group. d Po 0.01, vs. RDC group. e P o 0.05, vs. RM group. f P o 0.05, vs. RM group. b
It was reported that there was a strong relation existing between hyperglycemia, oxidative stress, cellular and endothelial dysfunction (Orhan et al., 2010). Increasing evidence in experimental and clinical studies indicate that oxidative stress plays an important role in DM development and pathogenesis of diabetic complications (Keter and Mutiso, 2012). Streptozotocin and hyperglycemia are two main pathogenic factors for the oxidative stress increasing the imbalance in ROS generation and elimination/ neutralization (Atangwho et al., 2012). The resulting excess ROS initially induces antioxidant enzymes, particularly in the liver, the tissue with the most abundant antioxidant defense enzymes (Tatsuki et al., 1997). SOD is a major scavenging enzyme by removing free radicals and protects from free oxygen radicals by catalyzing the elimination of superoxide radical, which damage the membrane and biological structures (Nain et al., 2012). In the present study, the treatment with ESF showed a significant increase in SOD activity in the liver of diabetic rats. On the other hand, the lipid peroxidation is one of the characteristic features of DM (Nain et al., 2012). The measurement of MDA is used as an index of lipid peroxidation and it helps to assess the extent of tissue damage. Several studies have reported an increase in MDA in plasma, liver and kidney in experimental diabetes mellitus (Deng et al., 2012; Orhan et al., 2012). The results of the present study show that ESF significantly decreases MDA level and reduces the risk of tissue damage. The results of this study clearly indicate that ESF contain a free radical scavenging activity, which can attenuate pathological alteration caused by the presence of superoxide radicals, which is consistent with the previous report (Zheng et al., 2013).
5. Conclusion This is the first report on the antidiabetic effect of ESF on streptozotocin (STZ) or alloxan (ALX) induced diabetic mice and STZ-induced diabetic rats. The results of this present study clearly indicate that the flavonoids of Malus toringoides (Rehd.) Hughes (ESF) exhibit hypoglycemic activity in experimental diabetic animals. The ESF exhibited improvement in parameters like FBG, Hb1Ac, hepatic glycogen, SOD activity and MDA level in liver. This antidiabetic activity may be related to the improvement of impaired pancreatic islet β-cell and antioxidant ability. Therefore, the leaf of Malus toringoides (Rehd.) Hughes is a good candidate as an alternative and/or complementary medicine in the treatment of DM. However, further investigations are needed to identify the lead molecule and to explore the exact mechanism of action for the antidiabetic effect.
Acknowledgment This study was financially supported by the Research on Key Technology and Product Development of Chinese and Tibetan medicine (Grant no. 2013SI0114). The authors would like to thank the associate Professor Mao Yu (Southwest University for Nationalities, Sichuan, China) for providing the ESF. The authors also acknowledge the State Key Laboratory Breeding Base of Systematic Research Development and Utilization of Chinese Medicine Resources Co-Founded by Sichuan Province and MOST for providing laboratories and instruments.
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Please cite this article as: Li, D., et al., Antidiabetic effect of flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.026i
