6J diabetic mice.

Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 Contents lists available at ScienceDirect 2 3 4 5 6 journal homepage: www.elsevier.com/locate/jep 7 8 9 Research Paper 10 11 12 13 14 15 16 Jun-fei Gu a,b,1, Zhi-yin Zheng a,c,1, Jia-rui Yuan a,b, Bing-jie Zhao a,b, Chun-fei Wang a,d, 17 18 Q1 Li Zhang a,c, Qing-yu Xu e, Guo-wen Yin e, Liang Feng a,n, Xiao-bin Jia a,b,c,nn 19 a Key Laboratory of Delivery Systems of Chinese Meteria Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing 210028, Jiangsu, China 20 b College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China 21 c College of Pharmacy, Jiangsu University, Zhenjiang 212013, Jiangsu, China d 22 College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230038, China e Department of Intervention, Cancer Hospital of Jiangsu Province, Nanjing 210009, Jiangsu, China 23 24 25 26 art ic l e i nf o a b s t r a c t 27 Article history: Ethnopharmacological relevance: Fresh Portulaca oleracea L. (family: Portulacaceae; POL) has been used as 28 Received 12 March 2014 a folk medicine for the treatment of diabetes mellitus for a long time. More bioactive components with 29 Received in revised form higher activity could be retained in fresh medicinal herbs compared to the dried ones. The present study 30 16 November 2014 was conducted to compare different antidiabetic activity between fresh and dried POL, including 31 Accepted 1 December 2014 hypoglycemic and antioxidant activities both in vivo and in vitro. Furthermore, in order to explore 32 which components were responsible for the antidiabetic activity, the difference on chemical components 33 Keywords: between fresh and dried POL was analyzed and compared. 34 Portulaca oleracea L Materials and methods: Insulin-resistant HepG2 cells induced by insulin were used to evaluate the 35 Fresh and dried herbs promoting effect of the fresh and dried POL on glucose utilization in vitro. Streptozotocin (STZ)-induced Hypoglycemic 36 C57BL/6J diabetic mice were used to compare the differences on hypoglycemic and antioxidant activities Antioxidant 37 of fresh and dried POL, including the fasting blood glucose, glucose tolerance, serum insulin level, Components difference 38 malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in vivo. UPLC/Q-TOF–MS method Diabetes mellitus was performed to analyze the difference of antidiabetic components between fresh and dried POL. 39 Results: Compared with the dried POL extract, the fresh POL extract significantly increased the 40 consumption of extracellular glucose in insulin-resistant HepG2 cells (Po 0.05). In STZ-induced C57BL/ 41 6J diabetic mice, both fresh and dried extracts decreased markedly the fasting blood glucose (FBG) levels, 42 and improved significantly oral glucose tolerance test (OGTT), as well as enhanced significantly insulin 43 secretion and antioxidative activities (P o0.05; P o0.01). Furthermore, the fresh extract showed stronger 44 antidiabetic activity (P o0.05). The UPLC/Q-TOF–MS analysis results also revealed that the relative 45 contents of polyphenols and alkaloids in the fresh herbs were more abundant than those in the 46 dried POL. 47 Conclusion: Our results indicated that both fresh and dried POL possessed antidiabetic activities, besides 48 stronger activity was observed in the fresh herb. These findings provided evidence for the application 49 and development of fresh POL in the treatment of diabetes mellitus. & 2014 Elsevier Ireland Ltd. All rights reserved. 50 51 52 53 54 55 56 57 Abbreviations: POL, Portulaca oleracea L; STZ, streptozotocin; MDA, malondialdehyde; SOD, superoxide dismutase; FBG, fasting blood glucose; OGTT, oral glucose tolerance test; NA, noradrenaline; DA, dopamine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FBS, fetal bovine serum; GC, glucose consumption; ELISA, 58 enzyme linked immunosorbent assay; UPLC/Q-TOF–MS, ultraperformance liquid chromatography/quadrupole-time-of-flight mass spectrometry 59 n Q4 Correspondence to: 100# Shizi Street, Hongshan Road, Nanjing 210028, China. Tel.: þ86 25 85637809. 60 nn Corresponding author at: Key Laboratory of Delivery Systems of Chinese Meteria Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing 210028, Jiangsu, 61 China. Tel./fax: þ 86 25 85637809. E-mail addresses: [email protected] (L. Feng), [email protected] (X.-b. Jia). 62 1 They contributed equally to this work. 63 64 http://dx.doi.org/10.1016/j.jep.2014.12.002 65 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved. 66

Journal of Ethnopharmacology

Comparison on hypoglycemic and antioxidant activities of the fresh and dried Portulaca oleracea L. in insulin-resistant HepG2 cells and streptozotocin-induced C57BL/6J diabetic mice

Please cite this article as: Gu, J.-f., et al., Comparison on hypoglycemic and antioxidant activities of the fresh and dried Portulaca oleracea L. in insulin-resistant HepG2 cells and.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.002i

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J.-f. Gu et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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1. Introduction Fresh herbs, a special application form of traditional Chinese medicinal herbs, are commonly used as a folk remedy to treat a variety of diseases in China (Jia et al., 2011). Accumulating evidence suggest that fresh herbs have stronger efficacy than dried form and may be optimal for the prevention and treatment of some diseases (Henning et al., 2011; Liang et al., 1999). It has been well shown that much higher content of components with bioactivity are retained in the fresh herbs than the dried ones (Tang et al., 2012; Sanchez-Medina et al., 2007). Some unstable components, such as volatile, flavor (Díaz-Maroto et al., 2003) and total phenolic (Henning et al., 2011), may be destroyed by drying (Díaz-Maroto et al., 2003). Therefore, in order to improve the beneficial use of fresh herbs, the activity and components difference need to be compared in the treatment of some diseases. Ethnopharmacological surveys indicate that more than 1200 plants are used in traditional medicine for their alleged hypoglycemic activity (Rucha et al., 2010; Aslan et al., 2010). Among these plants, Portulaca oleracea L. (POL), known as “vegetable for long life” in Chinese folklore, is widely used not only as an edible plant, but also as a traditional Chinese herbal medicine for hypoglycemic. Studies have shown that POL has a variety of bioactivities, such as antibacterial (Zhang et al., 2002), analgesic, anti-inflammatory (Chan et al., 2000), skeletal muscle relaxant and wound-healing (Rashed et al., 2003). POL may be beneficial for type-2 diabetes mellitus patients as an adjunctive and alternative therapy (El-Sayed, 2011a, 2011b). Fresh POL contains abundant catecholamines, phenolic acids and flavonoids, such as noradrenaline (NA), dopamine (DA), caffeic acid, ferulic acid and luteolin, which were considered as the major bioactive components (Ziegler et al., 2012; Strack et al., 2003; Xiang et al., 2005). Research showed that its catecholamines were the effective components for regulating the immune system and preventing diabetes (Del et al., 2011). Moreover, phenolic acids and flavonoids have also been reported to effectively ameliorate diabetes and its complications due to their powerful antioxidant activity (Okezie et al., 2007). However, the antidiabetic acitivities including hypoglycemic and antioxidant activities and the component difference between fresh and dried POL remain unclear. The aim of this study was to compare the difference between fresh and dried POL on the antidiabetic activity and relevant components variance, and provide evidence for further application of fresh POL on diabetes mellitus treatment.

2. Materials and methods 2.1. Reagents Roswell Park Memorial Institute-1640 (RPMI-1640) was obtained from Nanjing Keygen biotech CO., LTD (Jiangsu, China); fetal bovine serum (FBS), trypsinase penicillin and streptomycin were obtained from GIBCO Life Technologies (GIBCO, USA); 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Amresco (SoIon, OH, USA); blood glucose, MDA, SOD, glycogen kits and the insulin ELISA kit were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China); streptozotoin (STZ) was offered by Sigma Chemical Co. (St. Louis, MO, USA); metformin was obtained from Shanghai Medicinal Company (Shanghai, China); Other chemicals were all reagent grade. 2.2. Plant materials Fresh POL was collected from Pukou, Jiangsu Province, China in July, 2011. Its botanical identity was confirmed by professor

De-kang Wu from Nanjing University of Chinese Medicine. The voucher specimen (no. JTCM-20110811-001) was deposited at Key Laboratory of New Drug Delivery Systems of Chinese Meteria Medica, Jiangsu Provincial Academy of Chinese Medicine. 2.3. Preparation of plant extracts Harvested fresh POL (5 kg) was washed by 10 L distilled water to remove impurities. The washed herbs were air-dried and juiced. The herb juice was filtrated. The residues were macerated with 1000 mL distilled water for 24 h until exhaustion, followed by filtration. The filtrate was combined and concentrated in a rotary evaporator at 40 1C under reduced pressure. The concentrated filtrate was subjected to freeze-drying. The yellowish green powder was obtained (35.03 g) and called as “Fresh extract” in this study. The extract yield (w/w) of fresh POL was 0.70%. Harvested fresh POL (5 kg) was dried under open-air and powdered. The powder was macerated first with 1000 mL distilled water for 24 h at room temperature and then refluxed for three times (1.5 h/time). The extraction was filtrated to remove the impurities. The filtered extracts were concentrated in a rotary evaporator 40 1C under reduced pressure and then subjected to freeze-drying. The resulted yellowish green powder (33.75 g, yield 0.68%, w/w) was referred to as “Dried extract” in this study. Finally, these extracts were reconstituted in double distilled water and filtered with 0.22 μm micropore film, then stored at 4 1C. 2.4. Cell culture The HepG2 cell line was obtained from the Cell Bank of the Institute of Cell Biology (Shanghai, China). Then cells were cultured in RPMI-1640 containing 10% FBS with penicillin (100 U/mL)/ streptomycin (100 μg/mL) in a humidified incubator (5% CO2) at 37 1C. The medium was renewed every day. 2.5. Glucose consumption HepG2 cells were seeded into 96-well plates in RPMI-1640 supplemented with 10% FBS and penicillin (100 U/mL)/streptomycin (100 μg/mL), cultured in a humidified incubator (5% CO2) at 37 1C for 24 h. The insulin-resistant cell model was induced according to the previous reference method (Miyagawa et al., 2010). In brief, HepG2 cells were incubated with a fresh medium containing 1% FBS and 1 10 6 mol/L bovine insulin for 24 h. Cells were treated with 10 9 mol/L insulin and fresh or dried POL extract (0.25, 0.5, and 1.0 mg/mL, respectively) or metformin (0.086 mg/mL) in RPMI-1640 medium containing 1% FBS. Medium was used as a blank control. After incubation for 24 h, the glucose concentrations in cell supernatant were determined at 505 nm wavelength by the glucose oxidase method. The glucose consumption (GC) was calculated by measuring the glucose concentrations of blank wells and subtracting the remaining glucose in cell plated wells. GC (mmol/L) ¼[extracellular glucose concentrations of blank wells (mmol/L) extracellular remaining glucose in cell plated wells (mmol/L)] (Lu et al., 2011). 2.6. MTT assay To access the cytotoxicity of POL extracts on insulin-responsive cell viability, MTT assay was used to detect the cell viability after the treatment of POL extracts at different concentrations. The prepared cell suspension was seeded into 96-well plates and incubated at 37 1C for 24 h. Then the cells were starved for 12 h. These cells were treated with POL extracts at different concentrations for 24 h. Then the supernatant was discarded and 20 μL MTT (5 mg/mL) was added into each well. Then the cells were


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maintained at 37 1C for 4 h and the formed crystal was dissolved with 100 μL dimethyl sulfoxide (DMSO). The 96-well plate was shaken in a shaker for 15 min. The optical density (OD) values of samples were read at 485 nm with a microplate reader. MTT assay was performed to evaluate the effect of POL extracts on cell viability after the glucose consumption experiment (Zheng et al., 2011a, 2011b). MTT (5 mg/mL) of 20 μL was added to each well and then maintained for 4 h at 37 1C. DMSO of 100 μL was added to dissolve the crystal. The 96-well plate was shaken in a shaker for 15 min. The optical density (OD) value was measured in a microplate reader at 485 nm wavelength. The result of MTT was used to normalize the glucose utilization results. Glucose consumption (GC) due to the cell proliferation can be deducted by calculating the ratio of the GC and MTT (GC/MTT) (Yin et al., 2002). 2.7. Animal treatment Fifty healthy male C57BL/6J mice, weighed 20 72 g, were obtained from Shanghai SLAC Laboratory Animal Center. Mice were kept at an environmental controlled room (temperature: 20 72 1C, humidity: 60 75%) and housed in plastic cages with a 12:12-h light/dark cycle. The mice in all groups were fed with standard rat chow and tap water ad libitum. Before the experiment, the mice were fasted overnight, and then injected with a single intraperitoneal dose of STZ (35 mg/kg) (Comin et al., 2010) which was freshly dissolved in sodium citrate buffer (pH ¼ 4.5). The same dose of sodium citrate buffer was injected for normal control. The mice which fasting plasma glucose level above 16.8 mmol/L after being injected STZ for 72 h were selected for further experiment. All procedures were carried out in accordance with the Chinese legislation on the use and care of laboratory animals. The suitable mice were treated by oral administration with fresh extract (Group FrL, 200 mg/kg; Group FrH, 400 mg/kg) and dried extract (Group DrL, 200 mg/kg; Group DrH, 400 mg/kg) for 3 weeks (Chen et al., 2012). Metformin hydrochloride (250 mg/kg) was chosen as a positive control (Zheng et al., 2011a, 2011b) while sodium citrate buffer for blank contol. 2.8. Determination of the fasting blood glucose levels Fasting blood glucose concentration was determined using a Glucometer-elite commercial test (GT-1810, Arkray International Trading Co., Ltd. Shanghai, China) according to the glucose oxidase method (Ajmi et al., 2009). Blood samples were collected on 0, 7, 14 and 21 days from the tip of tail after overnight fast. The samples were used for the determination of the fasting blood glucose levels. The optical density (OD) values of samples were determined at 505 nm by SPECTRA MAX 190 microplate reader (Molecular Devices, USA).

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2.11. Measurement of MDA levels and SOD activity in liver The livers of mice were taken immediately after being sacrificed and washed with 0.9% NaCl, and then homogenized in 0.25 M sucrose of 4.5 mL using a Teflon homogenizer. The cytosolic fraction was obtained by a two-step centrifugation first at 1000 rpm for 10 min and then at 2000 rpm for 30 min at 4 1C. The MDA level and the SOD activity were determined at 450 nm and 550 nm by commercially available kits according to the manufacturer’s instructions.

2.12. UPLC/Q-TOF–MS analysis The components of the extracts were analyzed by Ultraperformance Liquid Chromatography/Quadrupole-Time-of-Flight Mass Spectrometry (UPLC/Q-TOF–MS). Each 30 mg of the POL extracts powder was dissolved in 5 mL distilled water by sonication for 5 min. The solutions were then mixed by vortexing for 5 min before centrifugation at 11 000 rpm for 10 min. Subsequently, 2 μL aliquots of the supernatant were injected into the chromatographic system for analysis. Chromatography was performed by ACQUITY UPLC system (Waters Corp., Milford, MA, USA) equipped with a samples thermostatic chamber at 4 1C. The separation was performed on an Acquity UPLC BEH C18 column (i.d., 2.1 mm 50 mm; 1.7 μm; Waters, Milford, MA, USA). The column temperature was maintained at 30 1C, and the flow rate was 0.3 mL/min. Chromatographic conditions were optimized to obtain high sensitivity and short run time for simultaneous analysis. The mobile phase consisted of acetonitrile (solvent A)-0.1% formic acid–water (solvent B) with a linear gradient elution program: 0–2 min, 2–2%A; 2–10 min, 2–15% A; 10–18 min, 15–25% A; 18–22 min, 25–80% A; 22–26 min, 80–98% A. The high-mass resolution experiments analysis was performed on a Q-TOF Synapt system (Waters, Milford, MA, USA), equipped with electrospray ionization (ESI) source, and conducted in negative ion detection mode. The desolvation gas, nitrogen (N2), was at a flow rate of 700 L/h with the temperature at 350 1C, while the source temperature was set at 120 1C. The capillary and cone voltages were 2.5 kV and 40 V. The mass range was recorded from 50 to1200 Da within 0.2 s. The transfer collision energy (EC) was 4 V. The raw data was acquired and screened by MassLynx to analyze the final compounds (version 4.1; Waters)

2.13. Statistical analyses Data were presented as means 7standard deviation (S.D), and the statistical analysis was performed using one-way ANOVA followed by Tukey’s test as a post hoc test with the SPSS 16.0 software. P o0.05 was considered statistically significant.

2.9. Glucose tolerance test The day before sacrificed, oral glucose tolerance test of mice was performed after an overnight fast. After being administered orally with glucose (2.0 g/kg), blood of mice was drawn from the tail vein at 0, 30, 60, 120 min, and then plasma glucose concentrations were measured using a Glucometer-elite commercial test. 2.10. Measurement of insulin levels in serum Blood samples of mice were collected from the orbital vein before being sacrificed on 21th day and then centrifuged at 3500 rpm for 20 min. The insulin level in serum was determined using ELISA kit at 450 nm by microplate reader.

3. Results 3.1. Cytotoxicity of the fresh and dried POL at different concentrations on HepG2 cells As shown in Fig. 1, when the concentration of POL was higher than 1 mg/mL, cell viability was lower than 95%, which means that drugs at these concentrations show toxic side effects on cells. At the concentration of 1 mg/mL, cell viability of POL was nearly 95% (the fresh 95.21 73.01%, the dried POL 94.88 72.05%). So the concentration of 1 mg/mL was not toxic to cells. Finally, 1 mg/mL was set as the high-dose of both fresh and dried POL, 0.5 mg/mL the middle-dose, 0.25 mg/mL the low one.


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3.2. Effects of the fresh and dried POL on glucose consumption in HepG2 cells The insulin-resistant was highly correlated with the consumption of extracellular glucose due to the decrease of the sensitivity of insulin receptors (Zakula et al., 2011). Exposure of HepG2 cells to 10 9 mol/L insulin, the consumption of extracellular glucose was significantly decreased, compared with normal control group (P o0.05). The positive control metformin (0.086 mg/mL) reversed remarkably the glucose consumption to normal level. As expected, the treatment with fresh (1.0 mg/mL and 0.5 mg/mL) and dried (1.0 mg/mL) POL increased significantly the GC in insulin-resistant HepG2 cells (Fig. 2). Interestingly, the fresh extract increased significantly extracellular glucose consumption, compared with the dried extract groups (P o0.05, FrH vs DrH, FrM vs DrM) (Fig. 2).

These data suggested that both fresh and dried POL extracts were able to improve extracellular glucose consumption in insulinresistant HepG2 cells, more importantly, the fresh extract exhibited stronger effect than the dried one. 3.3. Effects of the fresh and dried POL on fasting blood glucose levels in STZ-induced diabetic mice In STZ-induced diabetic mice, the fasting blood glucose levels were increased significantly by STZ with the dosage of 35 mg/kg. After being treated with fresh and dried POL, the fasting blood glucose levels were reduced significantly at the 3rd week, compared with diabetic control group (Po 0.05). As shown in Fig. 3, the treatment of the fresh extract (400 mg/kg and 200 mg/kg) reduced significantly the fasting blood glucose levels by 31.46% and 18.13% at the 3rd week (P o0.05, P o0.01). However, dried extract exhibited a significant hypoglycemic activity only at the dose of 400 mg/kg, with merely 14.00% decrease in blood glucose levels (P o0.05). Positive control metformin (250 mg/kg) reduced the blood glucose levels remarkably by 38.89%. Importantly, the fasting blood glucose levels decreased significantly in fresh POL (400 mg/kg and 200 mg/kg) compared to dried one (P o0.05). These results suggested that both fresh and dried POL showed hypoglycemic activity in STZ-induced diabetic mice and the fresh POL had better effect. 3.4. Effects of the fresh and dried POL on glucose tolerance in diabetic mice

Fig. 1. Cytotoxicity of the fresh and dried POL at different concentrations on HepG2 cells.

The glucose tolerance test was conducted to evaluate the regulatory effect of fresh and dried POL on blood glucose in diabetic mice. As described in Fig. 4, the blood glucose was

Fig. 2. Effects of the fresh and dried POL on glucose consumption in HepG2 cells. (A) The OD value of the fresh and dried POL of HepG2 cells by MTT; (B) Glucose consumptions of the fresh and dried POL of HepG2 cells; (C) The ratio of the GC and MTT (GC/MTT) to reflect effects of the fresh and dried POL on glucose consumption in HepG2 cells. NC¼ normal control group, IRC ¼insulin-resistant control group, MH¼IRC treated with metformin (0.086 mg/mL), FrH (or DrH) ¼IRC treated with high-dose fresh (or dried) extract (1.0 mg/mL); FrM (or DrM)¼ IRC treated with middle-dose fresh (or dried) extract (0.5 mg/mL); FrL (or DrL)¼ IRC treated with low-dose fresh (or dried) extract (0.25 mg/mL). #Po 0.05, ##Po 0.01, IRC vs NC;nPo 0.05, nnP o0.01 IRC vs POL administration; $Po 0.05 FrH vs DrH, FrM vs DrM. Data were expressed as mean 7SD (n¼ 6).


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Time (min) Fig. 4. Effects of the fresh and dried extracts on glucose tolerance in diabetic mice. NC¼normal control, DC ¼ diabetic control; MH¼metformin, FrH (or DrH) ¼ fresh (or dried) extract (400 mg/kg, ig); FrL (or DrL)¼ fresh (or dried) extract (200 mg/kg, ig). Data were expressed as means 7 SD (n¼ 6).

increased remarkably within 30 min (near to the highest level) and then reached the highest level within 60 min in diabetic control mice (P o 0.05). After treatment with drugs for 60 min, the blood glucose level decreased gradually with time. Metformin (250 mg/kg) prevented significantly the increase of the blood glucose levels after glucose administration at 30, 60 and 120 min, compared with the diabetic control group (P o 0.05). Interestingly, the fresh and dried POL significantly prevented an increase in blood glucose levels after glucose ingestion, compared with the diabetic controls (P o 0.05). Moreover, the fresh POL significantly prevented this increase within 30 min while the dried one only after 30 min. The data indicated that the fresh POL had a better regulatory function on blood glucose than the dried one.

3.5. Effects of the fresh and dried POL on serum insulin level in diabetic mice The insulin level may be related to the high blood glucose level in STZ-induced diabetic mice (Kim Kim, 2006). Accordingly, STZinduced diabetic mice were developed insulin deficiency as evidenced by insulin levels (Fig. 5). With the 21 days administration of fresh POL at the doses of 400 and 200 mg/kg significantly increased insulin levels after treatment while the dried POL only increased significantly the insulin levels at the doses of 400 mg/kg. The results indicated that POL might stimulate insulin secretion to improve the insulin levels in diabetes. Importantly, the fresh POL had a better regulatory function on insulin secretion than the dried one.

Serum insulin (uIU/mL) .

Fig. 3. Effects of fresh and dried extracts on fasting blood glucose levels in diabetic mice. NC¼normal control, DC ¼diabetic control; MH ¼metformin, FrH (or DrH) ¼fresh (or dried) POL (400 mg/kg, ig); FrL (or DrL) ¼fresh (or dried) POL (200 mg/kg, ig). #P o 0.01, DC vs NC;nP o0.05, fresh (or dried) POL vs DC; nnP o0.01, fresh (or dried) POL vs DC; $ Po 0.05, fresh extract vs dried extract. Data were expressed as Mean 7 SD (n¼ 6).

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Fig. 5. Effects of fresh and dried extracts on serum insulin level in diabetic mice. NC¼ normal control, DC ¼diabetic control; MH ¼metformin, FrH (or DrH) ¼fresh (or dried) extract (400 mg/kg, ig); FrL (or DrL) ¼ fresh (or dried) extract (200 mg/kg, ig). #Po 0.01, DC vs NC; nPo 0.05, fresh (or dried) POL vs DC; nnPo 0.01, fresh (or dried) POL vs DC; $Po 0.05, fresh POL vs dried POL. Data were expressed as means 7SD (n¼ 6).

3.6. Effects of the fresh and dried POL on MDA level and SOD activity in liver Oxidative stress has been implicated in the pathogenesis of diabetes mellitus and diabetic chronic complications (Shivananjappa and Muralidhara, 2012; Savu et al., 2012). The imbalance of oxidative stress in liver has been found to contribute to the glucose metabolism and to be associated with the onset and development of diabetes mellitus (Kim et al., 2013). The increase of hepatic MDA levels and the decrease of SOD activity were observed in STZ-induced diabetic mice based on the imbalance of oxidative defense system (He et al., 2011). As shown in Fig. 6A and B, the hepatic MDA level was increased significantly whereas the SOD activity was decreased significantly in STZ-induced diabetic mice, compared with normal mice (P o0.05). However, the treatment of fresh POL (400 and 200 mg/kg), metformin (250 mg/kg) and dried POL (400 mg/kg) decreased significantly the hepatic MDA level and increased significantly SOD activity in liver compared with diabetic mice (P o0.05). Furthermore, dried POL of 200 mg/kg also increased significantly the SOD activity, but did not affect markedly on the MDA levels (Fig. 6). These finding suggested that the fresh and dried POL had antioxidative activity on STZinduced diabetic mice and the fresh POL showed stronger activity. 3.7. Comparison on main active components of fresh and dried POL extracts In order to reveal the antidiabetic activity difference, the main components in fresh and dried POL extracts were analyzed by UPLC/Q-TOF–MS method. As depicted in Fig. 6, fresh POL contains more ingredients than dry herbs. The UPLC/Q-TOF–MS results revealed that the varieties, as well as relative levels of phenolic


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Fig. 6. Effects of the fresh and dried extracts on SOD activity and MDA level in liver. (A) Measurement of SOD activity; (B) measurement of MDA level. NC¼normal control, DC ¼diabetic control; MH¼ metformin, FrH (or DrH) ¼fresh (or dried) extract (400 mg/kg, ig); FrL (or DrL) ¼fresh (or dried) extract (200 mg/kg). #Po 0.01 DC vs NC; nP o0.05 treatment groups vs DC; nnPo 0.01 treatment groups vs DC; $ Po 0.05 Fresh extract groups vs Dried extract groups. Data were expressed as Means 7 SD (n¼6).

acids and flavonoids components in the fresh POL were more abundant than the dried one. In addition, as for catecholamines components, the contents of noradrenaline (NA) and levodopa (L-dopa) were existed in the fresh POL rather than the dried one (Figs. 7 and 8). In polyphenols components, ferulic acid and caffeic acid were observed in the fresh POL at the retention time of 6.883 min and 11.107 min, but not in the dried one. Flavonoids such as luteolin, myricetin, quercetin, apigenin at 12.330, 13.452, 14.032, 18.047 min retention time in the fresh POL were more abundant than the dried one (Fig. 7 and Table 1). However, we only identified nine ingredients owing to the limit of our test method and knowledge. In order to provide more convincing evidence for further application of the fresh POL, we will try to figure out the other compounds. Combined with the relevant literature, we speculated that the major bioactive components of POL might be the abundant catecholamines, phenolic acids and flavonoids, such as L-dopa, DA, ferulic acid, caffeic acid and luteolin, myricetin, quercetin, apigenin (del Campo et al., 2011; Okezie et al., 2007). The contents of Norepinephrine; Ferulic acid; Levodopa; Caffeic acid; Luteolin; Myricetin; Quercetin; Apigenin; Friedelin in fresh extract and were 16.73, 17.68, 7.44, 9.67, 23.55, 11.54, 26.72, 3.37, 4.57 μg/g crude drug, and were 0.31, 0.16, 0.09, 0.13, 1.42, 1.19, 2.25, 0.09, 0.07 μg/g crude drug in dried extract.

4. Discussion Traditionally, herbs are used as fresh or dried. Many reports showed differences on pharmacological activity of fresh and dried medicinal materials. Heat-labile, vulnerable oxidative and unstable ingredients are easily destroyed during drying process which could result in changes of phytochemical composition and impaired activities. Our survey from Chinese folk indicated that many people used fresh POL to relieve the symptoms of diabetes and it really exhibited significant effects. This survey was confirmed in current study. The liver is the center of glucose metabolism. HepG2, a human hepatocellular carcinoma line with liver cell morphology and

Fig. 7. The chromatogram of the extracts of fresh and dried POL. (A) Fresh POL extracts; (B) dried POL extracts.

function (Frittitta et al., 2000), was used to study and compare the hypoglycemic effects of the fresh and dry POL extracts in hepatocytes. The tetrazolium salt (MTT) method is comprehensive measurements of cell growth/cell kill and is widely used to monitor cell proliferation (Vande-Loosdrecht et al., 1991). Glucose consumption due to cell proliferation can be deducted by calculating the ratio of the GC and MTT. Our results showed that both the fresh and dried POL extracts were able to increase the glucose consumption in insulin-resistant HepG2 cells within certain concentrations. However, the fresh POL extract is more effective on glucose uptake in HepG2 cells after correction by MTT, compared with the dried one. These studies indicated that the fresh POL extract exhibited stronger hypoglycemic effect than the dried one in vitro. STZ-induced diabetes is characterized by hypoinsulinemia, polydipsia, polyuria and decreased body weight (Lenzen, 2008; Wu and Huan, 2008). Metformin was selected as positive control for evaluating hypoglycemic activity because it was documented as antidiabetic (Islam and Loots, 2009). In our experiment, we found that the treatment with metformin or the POL extracts greatly improved diabetic mice polyuria and polydipsia, indicating improvement in the diabetic conditions. In diabetic mice, the fresh extract produced a significant antihyperglycemic effect from the 1st week to the end of the 3rd week, while the dried extract from the 2nd week to the end of the 3nd week. However, at the end of the 3-week treatment, 31.46% and 14.00% reduction were found in the fresh and dried extracts. The result indicates that the fresh extract is more potent than the dried one on hyperglycemic amelioration in diabetic mice. The result is consistent with cell test results in vitro.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Insulin is the leading hormone that regulates blood glucose homeostasis by stimulating the utilization of glucose by liver, muscle and adipose tissue, coupled with stimulation of anabolic processes such as glucose, protein and lipid synthesis (Zheng, et al., 2011a, 2011b). STZ-induced diabetes is characterized by apoptosis of pancreatic β-cells and by attenuating insulin gene expression and reducing insulin synthesis (Mythili et al., 2004; Sharma et al., 2006). Under normal physiological conditions, pancreatic β-cells normally maintain blood glucose concentrations within a narrow range by modulating their insulin secretion rate in response to the glucose concentration in blood (Patel et al., 2006; Yin et al., 2006; Priel et al., 2007; Jiang et al., 2007; Zhang et al., 2004). Apoptosis of pancreatic β-cells is traditionally considered as the primary factor that ultimately causes hyperglycemia. We assumed that hypoglycemic effect of POL might be through the preservation of pancreatic β-cells and stimulating insulin secretion in diabetic mice. It was confirmed by controlling the serum insulin levels which were decreased in diabetic mice of all test groups compared to the normal mice. Serum insulin was increased by the fresh POL extract at the dosage of 400 and 200 mg/kg and dry POL extract at the dosage of 400 mg/kg. This effect is probably caused by the regeneration of pancreatic β-cells which were destroyed by streptotozin (Eidi et al., 2006). However,

7

this proposed mechanism need to be proved in further study. Components of POL extracts such as flavonoids with their antioxidant activity may be responsible for this effect (Giang et al., 2005; Khang et al., 2011; Fang et al., 2008). Oxidative stress that leads to an increase in the production of reactive oxygen species (ROS) and cellular lipid peroxidation, has been found to play an important role in the development of diabetes mellitus (Johansen et al., 2005; Fang et al., 2002; Pastore et al., 2003). Evidence suggests that complex alterations in the activities of antioxidant enzymes as well as the oxidative stress marker MDA are observed in diabetic mice (Scott and King, 2004). In the anti-oxidative defense system, SOD is the first enzyme of the scavenger enzyme series to protect tissues against oxygen free radicals by catalyzing the removal of superoxide radicals, which damages the membrane and biological structures (Arivazhagan et al., 2000). MDA reflected the degree of lipid peroxidation and played an important role in the progression of diabetic pancreas damage (Ilhan et al., 2001). Therefore, we determined the effects of extracts on the SOD activity and MDA level in diabetic mice (Evans, 2007). Besides this, mice treated with STZ have increased hepatic MDA levels and decreased the activity of hepatic SOD concentration. In the present study, the decrease of hepatic SOD and increase of hepatic MDA level were

Fig. 8. The mass spectra of metabolites of fresh and dried POL. (1) Norepinephrine; (2) ferulic acid; (3) levodopa; (4) caffeic acid; (5) luteolin; (6) myricetin; (7) quercetin; (8) apigenin; (9) friedelin.


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8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66


Fig. 8. (continued)

observed in diabetic mice compared with normal mice. Oral administration of fresh extract-treated group for 21 days caused a significant increase of SOD activity and a decrease of MDA level in liver. However, the dried extract only significantly decreased MDA level in liver, indicating that the fresh POL extract exhibited

better anti-oxidative effect than the dried one. Therefore, antioxidative stress might be one of the underlying mechanisms for POL to alleviate the high blood glucose status in diabetic mice. Furthermore, the results of researchers showed that the hypoglycemic effect of alkaloids of POL could be achieved by inhibiting


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Q6 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Q2 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Table 1 Identification of different compounds in fresh POL extracts and dry one. No.

tR (min)

[M–H]-

( )ESI-MS m/z (% base peak)

Compounds

1 2 3 4 5 6 7 8 9

6.548 6.883 10.518 11.107 12.330 13.452 14.032 18.047 22.194

168 193 198 179 285 317 301 269 425

168, 153, 137,121,105 193, 179, 163, 147, 103 198, 181, 165, 149, 105 179, 163,147, 103 285, 253,187, 171, 153 317, 253, 177, 161, 145 301, 269, 193, 177, 161, 145 269, 177, 161, 145 425, 409, 395, 353, 325, 269, 255

Norepinephrine Ferulic acid Levodopa Caffeic acid Luteolin Myricetin Quercetin Apigenin Friedelin

α-glucosidase activity. In addtion, this hypoglycemic activity might also be related to its hypolipidaemic and insulin resistance reducer effects (El-Sayed, 2011a,b). Different hypoglycemic activities of fresh and dried extracts may be associated with their different components. In this study, we compared the chemical compositions of fresh and dried POL. Our results showed that the fresh POL extract contained some components of catecholamines and phenolic acids which were not existed in the dried one. Furthermore, the contents of some flavonoids in the fresh POL extract were higher than the dried one. These results suggested that the difference between the fresh and dried POL on hypoglycemic effects might be related to these components. However, this result need to be further confirmed.

5. Conclusions In conclusion, our study confirms the difference between the fresh and dried POL on hypoglycemic effects. Interestingly, the fresh POL exhibited stronger hyperglycemic amelioration than the dried one. This difference in efficacy might be related to the components of catecholamines, phenolic acids and flavonoids in the fresh POL. Our findings provided evidence for more effective application of the fresh POL in the treatment of diabetes mellitus. Further studies were undertaken to confirm and clarify the mechanism of this activity.

Uncited references Chen et al. (2003), Mustafa et al. (2010), NoorShahida et al. (2009).

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