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Saponin rich fractions from Polygonatum odoratum (Mill.) Druce with more potential hypoglycemic effects
Journal of Ethnopharmacology 141 (2012) 228–233
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Saponin rich fractions from Polygonatum odoratum (Mill.) Druce with more potential hypoglycemic effects Yafei Deng a , Kai He b , Xiaoli Ye b , Xin Chen a , Jing Huang a , Xuegang Li a,1 , Lujiang Yuan a,∗ , Yalan Jin a , Qing Jin a , Panpan Li a a b
Chemistry Institute of Pharmaceutical Resources, College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, PR China School of Life Science, Southwest University, Chongqing 400715, PR China
a r t i c l e
i n f o
Article history: Received 25 September 2011 Received in revised form 1 February 2012 Accepted 2 February 2012 Available online 17 February 2012 Keywords: Saponin Flavonoids ␣-Glycosidase Hypoglycemic Polygonatum odoratum (Mill.) Druce
a b s t r a c t Aims: The root of Polygonatum odoratum (YuZhu), also a medicinal food has long been used for the treatment of diabetes. The objective of the study was to characterize the anti-diabetic active fractions or compounds in this herb. Materials and methods: Fractions with a different polarity were prepared by solvent extraction and macroporous absorptive resin (D101) column and their anti-diabetic potentials were evaluated by glucose uptake in HepG2 cells and STZ-induced diabetic rats. In addition, ␣-glycosidase inhibitory activities of active fractions were measured in vitro and chemical compositions including saponin, total flavonoids and total sugar in the fractions were determined. Results: The n-buthanol fraction, a saponin-rich fraction obtained by partitioning the ethanol extract with n-buthanol after petroleum ether and acetic ether showed the highest anti-diabetic potential in glucose uptake in HepG2 cells followed by acetic ether fraction which was rich in flavonoids. Further fractionation the saponin-rich fraction using macroporous resin column (D101), polysaccharide, flavonoid and saponin rich fractions were obtained by elution with water, 40% and 60% ethanol, respectively and their antidiabetic potentials proved by glucose uptake test in HepG2 cells and STZ-induced diabetic rats were in the order of saponin rich fraction > flavonoid rich fraction > polysaccharide rich fraction. Long-term therapy test (60 d) in severe diabetic rats indicated that saponin-rich fraction significantly ameliorated clinical symptoms of diabetes including the elevated blood glucose, body weight loss as well as the increased food and water intake while flavonoid-rich fraction was more potential than saponin-rich fraction to increase superoxide dismutase (SOD) activity and decrease malondialdehyde (MDA) level in rat plasma. Additionally, saponin-rich fraction and flavonoid-rich fraction showed ␣-glycosidase inhibitory activity with IC50 value of 2.05 ± 0.32 and 3.92 ± 0.65 mg/ml, respectively. Conclusion: The results suggested that saponin in this herb was more important than flavonoid in exhibiting anti-diabetic activity and flavonoid contributed more to anti-oxidant activity in vivo. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a group of chronic metabolic disorders characterized by hyperglycemia resulting from defects in insulin
Abbreviations: AUC, areas under the curve; CAT, catalase; DM, diabetes mellitus; DMSO, dimethyl sulfoxide; FBG, fasting blood glucose; FBS, fetal bovine serum; GFR, glomerular filtration rate; MDA, malondialdehyde; MTT, methylthiontrazole; OGTT, oral glucose tolerance test; PBG, postprandial blood glucose; RPO, rhizomes of Polygonatum odoratum; ROS, reactive oxygen species; SOD, superoxide dismutase; STZ, streptozotocin; pNPG, p-nitrophenyl-␣-d-glucopyranoside. ∗ Corresponding author. Tel.: +86 23 68251503; fax: +86 23 68251225. E-mail address: [email protected] (L. Yuan). 1 This author contributed equally to the work. 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.02.023
secretion, action or both and the chronic hyperglycemia causes serious body damage such as blood vessels and nerves damages (O’Connell et al., 2008). Current knowledge strongly supports that control of hyperglycemia is critical in the treatment of not only diabetic patients but also individuals with impaired glucose tolerance (Laakso, 1999). So far a number of hypoglycemic agents including insulin injection and oral drugs such as glycosidase inhibitors, biguanides and sulphonylureas have been clinically used to maintain blood glucose level (Scheen and Lefebvre, 1998). Unexpectedly, some adverse effects had been seen in the use of those agents. Pioglitazone, for instance, may induce hepatocellular-cholestatic liver injury (May et al., 2002) and metformin should be stopped for the therapy of diabetic nephropathy when glomerular filtration rate (GFR) is lowered than 60 ml/min (Phillips and Braddon, 2002).
Y. Deng et al. / Journal of Ethnopharmacology 141 (2012) 228–233
Therefore, it is believed that herbal medicine is a valuable reservoir for novel drugs to deal with this disease due to its few side effects (Li et al., 2004; Jung et al., 2007). The rhizome of Polygonatum odoratum (Mill.) Druc (RPO), an edible medicinal herb has long been used to treat various diseases including DM (China Pharmacopoeia Committee, 2010). Studies had shown that RPO had the beneficial effects on hyperglycemia. Chen et al. (2001) reported that the water extract of RPO decreased the blood glucose level in starch loaded mice. Meanwhile, the n-butanol fraction from the methanol extract of this herb exhibited dramatic hypoglycemic effects in STZ-induced diabetic mice (Kato and Miura, 1994). Although a few studies had revealed that flavonoid, saponin and polysaccharide were crucial compounds (Choi and Park, 2002; Shu et al., 2009), there was no definite conclusion about the active constituents. The objective of this study is to characterize the anti-diabetic constituents or fractions by evaluation anti-diabetic potential of different fractions prepared systematically.
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2.3. Preparation RPO fractions The dried RPO (5 kg) were extracted with 80% ethanol (40 L × 3) at 80 ◦ C for 2 h, and then ethanol was evaporated under vacuum condition to give a brown residue (25.00% yield, labeled as Fr.0). The residue was suspended in water and successively partitioned with petroleum ether (0.47% yield, labeled as Fr.1), acetic ether (0.31% yield, labeled as Fr.2), n-buthanol (2.08% yield, labeled as Fr.3). The remaining aqueous fraction was labeled as Fr.4 (21.09% yield). The fractions, then, were subjected to glucose uptake test in HepG2 cells. Fr.3 with the highest ability to enhance glucose uptake in HepG2 cells was further fractionated with D101 macroporous resin column in which water and ethanol at various concentrations (20%, 40%, 60% and 80%) were used as eluent. Sequentially, Fr.3-1 (1.58%), Fr.3-2 (0.12%), Fr.3-3 (0.08%), Fr.3-4 (0.09%) and Fr.3-5 (0.03%) were obtained. The detailed process was shown in Fig. 1.
2.4. Test of glucose uptake in HepG2 cells
2. Materials and methods 2.1. Materials and chemicals The rhizomes of Polygonatum odoratum were purchased from Tongjunge Drugstore of Chongqing (Chongqing, China). The taxonomic identification of the plant material was confirmed by a group of professors in pharmacognosy laboratory of the college of pharmaceutical sciences, Southwest University and a voucher of specimen (No. 20090031) was deposited there. Streptozotocin (STZ) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). RPMI-1640 culture medium and fetal bovine serum (FBS) were obtained from Gibco Life Technologies (Burlington, Ontario, Canada). Superoxide dismutase (SOD), malondialdehyde (MDA) and glucose assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Metformin was purchased from Beijing Coway Pharmaceutical Factory (Beijing, China). ␣-Glucosidase and pnitrophenyl-␣-d-glucopyranoside (pNPG) were purchased from Sigma Chemical Co. All other reagents were of the highest available purity and were used as purchased.
2.2. Experimental animals Male Sprague-Dawley rats (200 ± 20 g) and diets were purchased from Animal Breeding Center of the Third Military Medical University (Chongqing, China) and the use and care of animals were in accordance with National Research Council’s guidelines for the use of experimental animals and permitted by Chongqing Animal Care Committee (SYXK-(YU), 2009-0002). Animals were housed under standard environmental conditions (24 ± 2 ◦ C, 55 ± 10% relative humidity, 15 times air changes in 1 h, 12 h light-dark cycle) and adapted to diet for one week before the experiment. Diabetes was induced by single injection of STZ (40 mg/kg for mild diabetes and 55 mg/kg for severe diabetes) in citrate buffer (0.1 M sodium citrate and 0.1 M citric acid, pH 4.5) in overnight fasted rats (Brosky and Logothetopoulos, 1969; Dias et al., 2010). Fasting blood glucose (FBG) level was evaluated 3 d after STZ administration using glucose assay kits and rats with FBG value of 120–250 mg/dl were included in mild diabetic group and with FBG value of 300 mg/dl or above were included in severe diabetic groups (Gupta et al., 2005; Kesari et al., 2006).
HepG2 cells were seeded into 48-well plates in 1640culture medium supplemented with 10% heat-inactivated FBS and penicillin-streptomycin (100 U/ml, each) and cultured in an incubator (5% CO2 ) at 37 ◦ C for 24 h. Then the medium was replaced by 1640-culture medium without FBS and extracts with different concentrations as well as metformin (0.01 mg/ml) were added to corresponding wells. After 24 h incubation, the glucose concentrations in the medium were determined using commercially available kits. Glucose uptake was calculated by subtracting the glucose concentration of control groups from treated groups. MTT assay (van de Loosdrecht et al., 1991; Zheng et al., 2011) was subsequently carried out after the glucose uptake test to estimate the influence of extracts on cell survival. Briefly, 15 l MTT (5 mg/ml in DMEM) was added to each well and incubated at 37 ◦ C for 4 h. The corresponding supernatant, then were discarded and cells were washed. DMSO (200 l) was added to each well to extract the dye and the microplate was placed on a shaker for well dissolution. After 10 min, the optical density (OD) of each well was measured at 490 nm on a microplate reader.
2.5. Chemical analysis Total saponin content was determined using a spectrophotometric method and oleanic acid was used to prepare the calibration curve (Hiai et al., 1976). Total sugar was determined by phenolsulfuric acid method (Rao and Pattabiraman, 1989). Meanwhile, total flavonoids content was determined by the aluminum chloride colorimetric method and rutin was used to prepare the calibration curve (Chang et al., 2002).
2.6. Oral glucose tolerance test The mild diabetic rats were divided into seven groups (n = 6) and oral glucose tolerance test (OGTT) was carried out after overnight fasting. Control group was given vehicle (distilled water) only and positive group received metformin (300 mg/kg). Meanwhile, other groups received different fractions (Fr.3-1, Fr.3-2, Fr.3-3, Fr.3-4 and Fr.3-5) suspended in distilled water at a dose of 500 mg/kg. After 30 min of administration, each rat received orally a glucose solution (2 g/kg). The blood glucose levels were measured before and 30, 60 and 120 min after glucose loading using commercially available kits. Total glycemic responses to OGTT were calculated from respective areas under the curve (AUC) of glycemia during the 120 min observation period.
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Y. Deng et al. / Journal of Ethnopharmacology 141 (2012) 228–233
Polygonatum odoratum (Mill.) Druce (dried roots) extract with ethanol
Ethanol extracts ( Fr.0, 25.00%)
Petroleum ether
Acetic ether
(Fr.1, 0.47%)
H2O
n-Buthanol
(Fr.4, 21.09%)
(Fr.3, 2.08%)
(Fr.2, 0.31%)
D101 macroporous resin column
H2O (Fr.3-1, 1.58%)
20%Ethanol
40%Ethanol
60%Ethanol
80%Ethanol
(Fr.3-2, 0.12%)
(Fr.3-3, 0.08%)
(Fr.3-4, 0.09%)
(Fr.3-5, 0.03%)
Fig. 1. Preparation scheme of the fractions from Polygonatum odoratum.
2.7. Hypoglycemic effects of selected sub-fractions on severe diabetic rats The severe diabetic rats were randomly divided into five groups of 10 rats each. The control group was given vehicle solution (distilled water) and the treated groups received the sub-fractions of Fr.3-2, Fr.3-3 and Fr.3-4 at a dose of 500 mg/kg as well as metformin (300 mg/kg) orally for 60 d. In the treatment process, FBG levels were monitored every 10 d. Meanwhile, body weight, food and water intake were recorded every 15 d. At the end of the experiment, SOD activity and MDA level in plasma were estimated using commercially available kits. 2.8. Assay for ˛-glycosidase inhibitory activity The ␣-glycosidase inhibitory assay was performed according to the procedures reported by Matsu et al. (1996) and Wu et al. (2011). Briefly, Sample solution (20 l), ␣-glycosidase (200 l, 0.5 U/ml) in 0.1 M phosphate buffer (pH 6.9), and 1740 l sodium phosphate buffer (pH 6.9) were mixed and maintained at 37 ◦ C for 10 min. Then, pNPG (40 l) was added into the reaction mixture and incubated at 37 ◦ C for another 60 min. The absorbance at 400 nm was measured. Acarbose was used as positive control. The following equation was used to calculate the inhibitory activity of the extracts.
Inhibition activity (%) =
S400
nm, control
S400
− S400
nm, sample
nm, control
× 100%
highest content of saponin in Fr.3, flavonoid in Fr.2 and sugar in Fr.4 were observed. The highest content of sugar in Fr.3-1, the highest flavonoid in Fr.3-3 (40% ethanol eluent) and the highest saponin in Fr.3-4 (60% ethanol eluent) were observed. Saponin in Fr.3-1 (20% ethanol eluent) and sugar in Fr.3-5 (80% ethanol eluent) were not found.
3.2. Effects of the RPO fractions on glucose uptake in HepG2 cells All the fractions at the concentration lower than 0.1 mg/ml showed no influence on cell survival and proliferation (data not show). Hence, the maximum concentration of fractions for the glucose uptake test was set to 0.1 mg/ml and the results were shown in Fig. 2. As shown in Fig. 2a, the glucose uptakes in HepG2 cells mediated by Fr.2 and Fr.3 were significantly higher, and mediated by Fr.1 and Fr.4 were lower than that by Fr.0. The mediation was in dose-dependent manner. The highest glucose uptake was mediated by Fr.3 which was rich in saponin followed by Fr.2, a flavonoid rich fraction. Based on these results, the saponin rich fraction (Fr.3) was further fractionated into five sub-fractions using D101 macroporous resin column and their results of glucose uptake test were shown in Fig. 2b. All the sub-fractions mediated a higher glucose uptake in HepG2 cells than the starting Fr.3 and the mediations were in a dose-dependent manner. At the dose of 0.025 mg/ml, the values of glucose uptake promoted by Fr.3-1, Fr.3-2, Fr.3-3, Fr.34 and Fr.3-5 were 1.60 ± 0.13, 2.20 ± 0.18, 2.60 ± 0.22, 3.02 ± 0.25 and 1.90 ± 0.16 mmol/L, respectively. The data indicated that Fr.34, again a saponin rich fraction had the highest ability to enhance glucose uptake in HepG2 cells. Accordingly, all of fractions were subjected to oral glucose tolerance test.
2.9. Data analysis All results were expressed as mean ± SD and were analyzed by SPSS for Windows, version 13.0 (SPSS Inc., Chicago, IL). Statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test and a value of p < 0.05 was considered statistically significant. 3. Results 3.1. Chemical compositions The results of chemical analysis were listed in Table 1. Saponin, flavonoid and sugar were found in ethanol (Fr.0) and n-buthanol fractions (Fr.3), while none of them existed in petroleum ether fraction (Fr.1). Acetic ether fraction (Fr.2) contained only flavonoid, and both saponin and sugar existed in water remaining phase (Fr.4). The
Table 1 Chemical compositions of the extracts from Polygonatum odoratum (mg/g). Extracts
Saponin
Fr.0 Fr.1 Fr.2 Fr.3 Fr.4 Fr.3-1 Fr.3-2 Fr.3-3 Fr.3-4 Fr.3-5
4.2 ± – – 34.4 ± 1.2 ± – 95.6 ± 183.3 ± 451.6 ± 107.4 ±
0.5e
3.0d 0.1f 12.2c 22.1b 37.3a 14.9c
Total flavonoids
Total sugar
3.5 ± – 95.5 ± 32.8 ± + 5.1 ± 166.0 ± 305.7 ± 143.1 ± 34.0 ±
392.4 ± – + 469.3 ± 548.0 ± 389.4 ± 175.3 ± 61.8 ± 31.2 ± –
0.7e 8.5c 1.9d 1.2e 19.7b 36.2a 15.4b 2.4d
24.1c
33.2b 44.8a 38.0c 11.2d 7.2e 3.7f
“+” Positive reaction, “−” negative reaction; Data in the same column marked with different letters were significantly different (p < 0.05)
Y. Deng et al. / Journal of Ethnopharmacology 141 (2012) 228–233
4
a 240
0.01 mg/ml 0.025 mg/ml 0.05 mg/ml 0.10 mg/ml
3
Blood glucose level (mg/dl)
Glucose uptake (mmol/L)
a
2 1
4
Fr.0
Fr.1
Fr.2
Fr.3
Fr.4 Metformin
0.010 mg/ml 0.006 mg/ml 0.012 mg/ml 0.025 mg/ml
*
200
* ** **
180
** **
160 140
3
b 25000
0
20
40 *
20000
AUC (mg min/dl)
Glucose uptake (mmol/L)
Control Fr.3-1 Fr.3-2 Fr.3-3 Fr.3-4 Fr.3-5 Metformin
220
120
0
b
231
2
60 80 Time (min)
100
120
* **
**
15000
10000
5000
1 0
Control
0 Fr.3-1
Fr.3-2
Fr.3-3
Fr.3-4 Fr.3-5 Metformin
Fig. 2. Effects of solvent extracts (a) of Polygonatum odoratum and five sub-fractions (b) obtained by D101 macroporous resin separation of the n-butanol fraction on glucose uptake in HepG2 cells.
Fr.3-1
Fr.3-2
Fr.3-3
Fr.3-4
Fr.3-5 Metformin
Fig. 3. Oral glucose tolerance tests (OGTT) in STZ-induced mild diabetic rats (a). Area under the glycaemic curve (AUC) obtained using the trapezoid method through OGTT (b). Results are mean ± SD (n = 6). Significant difference from control: *p < 0.05; **p < 0.01.
3.4. Hypoglycemic effects of Fr.3-2, Fr.3-3 and Fr.3-4 on severe diabetic rats 3.3. Effects of the sub-fractions from Fr.3 on mild diabetic rats The effects of sub-fractions from Fr.3 on the postprandial blood glucose (PBG) levels in the mild diabetic rats were shown in Fig. 3a. The PBG values in untreated diabetic groups increased from 134 to 218 mg/dl in 30 min after glucose loading and decreased thereafter. However, the increase in PBG level was significantly suppressed when Fr.3-1, 3-2, 3-3, 3-4, 3-5 and metformin were administrated. The calculated AUC values for glucose response in the order of control > Fr.3-1 > Fr.3-5 > Fr.3-2 > Fr.33 > Fr.3-4 > metformin (Fig. 3b) suggested the ability to suppress the rise of glucose concentration was in the order of metformin > Fr.34 > Fr.3-3 > Fr.3-2 > Fr.3-5 > Fr.3-1 which was in agreement with the results of glucose uptake test. Fr.3-4 (p < 0.01) and Fr.3-2, Fr.3-3 (p < 0.05) improved glucose tolerance significantly and no remarkable effect for Fr.3-1 and Fr.3-5 was observed. Therefore, Fr.3-2, Fr.3-3 and Fr.3-4 were further evaluated in severe diabetic rats.
Hypoglycemic effects of Fr.3-2, Fr.3-3 and Fr.3-4 on severe diabetic rats were listed in Table 2. The body weight, food and water intake of each rat during the 60 d treatment were also recorded (Table 3). The FBG levels of diabetic rats were significantly higher than normal rats (p < 0.01) during the experimental period. Significant decrease in FBG levels (p < 0.05) for the treated diabetic rats was observed 10 d after administration of metformin, Fr.3-3 and Fr.3-4. At the end of the treatment (60 d), FBG values for metformin treated rats decreased by 63.4%, for Fr.3-4 by 46.3%, for Fr.3-3 by 37.3%, for Fr.3-2 by 26.1%, respectively. A continuous decrease in body weight for diabetic rats and continuous increase in body weight for normal rats were observed (p < 0.01). After 60 d, the average body weight for untreated diabetic rats, diabetic rats treated with metformin and Fr.3-4 was 37.2%, 48.8% and 46.4% of normal rats, respectively, which indicated that treatments with metformin and Fr.3-4 prevented the body weight loss. Fr.3-2 and Fr.3-3 did not show appreciable effects
Table 2 Effects of Fr.3-2, Fr.3-3 and Fr.3-4 on FBG levels in severe diabetic rats. Groups
FBG levels after treatment (mg/dl) 0d
Normal comtrol Diabetic control Metformin Fr.3-2 Fr.3-3 Fr.3-4
84.9 416.7 422.2 410.5 421.7 408.0
10 d ± ± ± ± ± ±
8.2 26.2# 21.3 30.4 26.4 28.5
85.4 420.2 301.9 391.6 380.3 354.7
20 d ± ± ± ± ± ±
7.2 38.6# 32.7** 30.4 29.7* 34.2**
82.6 425.7 254.3 374.0 331.4 301.8
30 d ± ± ± ± ± ±
12.4 31.7# 27.6** 38.1* 32.6** 35.6**
The data are expressed in mean ± SD; n = 6–10 in each group. # p < 0.01 compared with the corresponding value for normal control group. * p < 0.05 compared with the corresponding value for diabetic control group. ** p < 0.01 compared with the corresponding value for diabetic control group.
83.8 428.2 211.7 362.0 304.8 262.6
40 d ± ± ± ± ± ±
14.3 37.1# 33.9** 36.3** 29.7** 37.4**
86.9 427.5 191.9 349.5 282.2 253.3
50 d ± ± ± ± ± ±
12.0 42.0# 34.4** 35.3** 33.6** 28.9**
81.8 409.1 172.2 322.7 272.4 230.9
60 d ± ± ± ± ± ±
9.3 31.6# 28.2** 40.4** 34.9** 27.8**
80.2 405.1 154.4 303.5 264.5 218.9
± ± ± ± ± ±
15.3 39.8# 30.0** 38.7** 29.7** 34.9**
232
Y. Deng et al. / Journal of Ethnopharmacology 141 (2012) 228–233
Table 3 Effects of Fr.3-2, Fr.3-3 and Fr.3-4 on body weight, food and water intake in each group. Group
0d
Body weight (g)
Normal comtrol Diabetic control Metformin Fr.3-2 Fr.3-3 Fr.3-4
225.4 217.5 210.1 207.6 214.2 217.4
± ± ± ± ± ±
17.3 19.1 17.5 20.4 18.7 15.8
15 d 260.1 200.1 225.5 198.7 194.2 211.5
± ± ± ± ± ±
21.4 24.6# 18.3* 19.2 17.4 16.9
30 d
Food intake (g)
Normal comtrol Diabetic control Metformin Fr.3-2 Fr.3-3 Fr.3-4
25.7 32.3 31.8 33.1 33.0 32.7
± ± ± ± ± ±
3.0 2.8# 2.5 5.5 5.5 4.2
26.2 38.6 34.2 37.7 37.3 35.8
± ± ± ± ± ±
Water intake (g)
Normal comtrol Diabetic control Metformin Fr.3-2 Fr.3-3 Fr.3-4
45.5 75.2 73.3 76.8 76.3 73.9
± ± ± ± ± ±
5.7 4.7# 6.6 8.3 7.2 7.1
48.7 88.9 81.1 88.7 84.8 81.7
± ± ± ± ± ±
45 d
306.1 186.0 216.2 190.2 184.3 206.1
± ± ± ± ± ±
23.4 25.5# 19.9** 16.8 18.6 15.4*
2.6 3.3# 3.9* 4.1 2.3 3.1*
26.8 42.2 38.2 41.0 40.1 38.3
± ± ± ± ± ±
4.9 4.2# 5.4** 8.6 6.7 5.1*
48.9 112.3 87.3 101.9 92.3 88.7
± ± ± ± ± ±
60 d
366.1 166.5 207.6 177.0 178.0 198.5
± ± ± ± ± ±
20.2 26.4# 18.2** 17.2 19.3 17.5**
3.3 4.0# 3.1* 2.9 2.6 3.7*
27.9 49.0 41.1 47.1 46.5 43.1
± ± ± ± ± ±
3.4 3.9# 2.3** 3.0 2.9* 2.2**
5.2 8.1# 4.0** 6.6* 5.6** 6.0**
48.7 128.6 94.0 108.0 101.2 94.9
± ± ± ± ± ±
8.3 10.8# 4.5** 5.6** 4.0** 4.9**
414.9 154.3 202.3 160.5 167.7 192.5
± ± ± ± ± ±
25.4 22.0# 16.8** 23.8 18.6 16.0**
28.3 56.1 45.1 52.5 51.7 46.2
± ± ± ± ± ±
3.5 6.7# 2.5** 3.2* 3.2* 3.6**
51.8 140.9 98.8 112.9 108.0 99.7
± ± ± ± ± ±
6.6 7.5# 3.2** 6.7** 4.2** 5.2**
The data are expressed in mean ± SD; n = 6–10 in each group. # p < 0.01 compared with the corresponding value for normal control group. * p < 0.05 compared with the corresponding value for diabetic control group. ** p < 0.01 compared with the corresponding value for diabetic control group.
on preventing weight loss (Table 3). Still in Table 3, the diabetic rats consumed much more food and water than normal rats (p < 0.01). Metformin and Fr.3-4 decreased the food and water consumption significantly after 15 d treatment (p < 0.05). Fr.3-2 and Fr.3-3 treatment also ameliorated the increase in food and water intake at the end of the treatment.
3.5. Effects of Fr.3-2, Fr.3-3 and Fr.3-4 on SOD activity and MDA level in plasma To assess the antioxidant ability of the fractions, the SOD activity and MDA level in plasma of different groups were measured at the end of the treatment and the results were summarized in Table 4. An obvious decrease in SOD activity and increase in MDA level in diabetic rats were observed (p < 0.01) compared to normal rats. Fr.3-3 and Fr.3-4 increased SOD activity and decrease MDA level significantly in plasma compared to diabetic rats (p < 0.01). However, the potency for the latter is slightly weak. 3.6. Effects of Fr.3-2, Fr.3-3 and Fr.3-4 on ˛-glycosidase inhibitory activity in vitro The results of ␣-glycosidase inhibitory activity are shown in Table 5. As can be seen, Fr.3-4 with IC50 value of 2.05 ± 0.32 mg/ml was more potential than Fr.3-3 (IC50 , 3.92 ± 0.65) on the inhibition of ␣-glycosidase activity.
Table 4 Effects of Fr.3-2, Fr.3-3 and Fr.3-4 on SOD activity and MDA level in plasma. Groups
SOD activity (U/ml)
Normal comtrol Diabetic control Metformin Fr.3-2 Fr.3-3 Fr.3-4
139.5 86.1 129.3 90.6 124.2 110.0
± ± ± ± ± ±
8.8 12.7# 10.0** 14.3 10.5** 9.7*
MDA level (nmol/ml) 2.8 7.4 4.0 6.9 4.3 5.3
± ± ± ± ± ±
0.7 0.8# 0.9** 1.0 0.5** 0.7*
The data are expressed in mean ± SD; n = 6–10 in each group. # p < 0.01 compared with the corresponding value for normal control group. * p < 0.05 compared with the corresponding value for diabetic control group. ** p < 0.01 compared with the corresponding value for diabetic control group.
4. Discussion The term for diabetes in ancient China was Xiaoke Zheng or Xiaodan Zheng resulted from lung heat, excessive fire in the stomach, deficiency of kidney Yin or both of Yin and Yang (Ko et al., 2004). According to those, treatment strategies for diabetes in Chinese history were eliminating heat by nourishing yin, moistening dryness and promoting fluid production (Li et al., 2004). Yuzhu, rhizomes of Polygonatum odoratum (Mill.) Druce distributed widely over southern area of China is an important nourishing yin herb and has long been used, since then, to improve health status of patients with Xiaokezheng. Our results that the extracts of this herb reduced the FBG levels, prevented body weight loss, food and water intake of diabetic rats confirmed the traditional use of this herb. Traditionally, this herb was used as decoction which may result in loss of activity and excessive volume of preparation for patients to consume. Hence, it is essential to characterize the pharmacological constitutes or active fraction in order to reduce the dosage. Our findings that Fr.3-4 and Fr.3-3 had strong anti-diabetic potential and Fr.3-4 had the highest content of saponin, and Fr.3-3 had the highest content of flavonoid suggested that both saponin and flavonoid in this herb were the main active compounds contributing to hypoglycemic activity, and saponin contributed even more. Further research on technologies that can efficiently extract saponin and flavonoid from this herb is urgently needed. Hyperglycemia is an important factor responsible for the generation of reactive oxygen species (ROS) (Ihara et al., 1999; Dave and Kalia, 2007). The excessive ROS leads to sharp reduction in antioxidant defense, such as decreased activity of SOD, catalase (CAT) (Mates et al., 1999) and so on. Accordingly, the increased level of plasma MDA in the post meal state and the reduced activities of SOD as indicators of the tissue damage in diabetic patients had
Table 5 The inhibition potency to ␣-glucosidase for the active extracts. Acarbose IC50 (mg/ml)
Fr.3-2
0.15 ± 0.05
d
Fr.3-3
5.17 ± 0.94
c
Fr.3-4
3.92 ± 0.65
b
2.05 ± 0.32a
IC50 is the concentration required to produce 50% inhibition of the enzyme activity; data in the same row marked with different letters were significantly different (p < 0.05).
Y. Deng et al. / Journal of Ethnopharmacology 141 (2012) 228–233
been reported (Ceriello et al., 1998). Moreover, flavonoids are well known antioxidants either through their own reducing capacities or through their possible influences on intracellular redox status (Heim et al., 2002; Williams et al., 2004). Our results that Fr.3-3 had the highest content of flavonoid explained the increase SOD activity and decrease MDA level in plasma of rats administered Fr.3-3. Meanwhile, alleviation the high blood glucose level by Fr.3-3 and Fr.3-4 can also contribute to improved antioxidant status. These results suggest that RPO saponin and flavonoid deserve further investigation on prevention of diabetic patient from body damage or the development of diabetic complications. In conclusion, the results obtained in the present work suggest that the RPO has hypoglycemic effects. Saponin is the most active constituent followed by flavonoid. Therefore, research on isolation and characterization of the active molecule is urgently needed to better understand the mechanism of action of the herb as an agent against hyperglycemia. Acknowledgments This work was founded by National Science and Technology Pillar program of China (2011BAI13B02-1) and National Key Technologies R & D Program of China during the 11th Five-Year Plan Period (2010ZX09401-306-3-10). References Brosky, G., Logothetopoulos, J., 1969. Streptozotocin diabetes in the mouse and guinea pig. Diabetes 18, 606–611. Ceriello, A., Bortolotti, N., Motz, E., Crescentini, A., Lizzio, S., Russo, A., Tonutti, L., Taboga, C., 1998. Meal-generated oxidative stress in type 2 diabetic patients. Diabetes Care 21, 1529–1533. Chang, C.C., Yang, M.H., Wen, H.M., Chern, J.C., 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis 10, 178–182. China Pharmacopoeia Committee, 2010. Pharmacopoeia of the People’s Republic of China, the first division of 2010 ed. China Chemical Industry Press, Beijing, pp. 78–79. Chen, H., Feng, R., Guo, Y., Sun, L., Jiang, J., 2001. Hypoglycemic effects of aqueous extract of Rhizoma Polygonati Odorati in mice and rats. Journal of Ethnopharmacology 74, 225–229. Choi, S.B., Park, S., 2002. A steroidal glycoside from Polygonatum odoratum (Mill.) Druce. improves insulin resistance but does not alter insulin secretion in 90% pancreatectomized rats. Bioscience Biotechnology and Biochemistry 66, 2036–2043. Dave, G.S., Kalia, K., 2007. Hyperglycemia induced oxidative stress in type-1 and type-2 diabetic patients with and without nephropathy. Cellular and Molecular Biology 53, 68–78. Dias, T., Bronze, M.R., Houghton, P.J., Mota-Filipe, H., Paulo, A., 2010. The flavonoidrich fraction of Coreopsis tinctoria promotes glucose tolerance regain through pancreatic function recovery in streptozotocin-induced glucose-intolerant rats. Journal of Ethnopharmacology 132, 483–490.
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Gupta, R.K., Kesari, A.N., Murthy, P.S., Chandra, R., Tandon, V., Watal, G., 2005. Hypoglycemic and antidiabetic effect of ethanolic extract of leaves of Annona squamosa L. in experimental animals. Journal of Ethnopharmacology 99, 75–81. Heim, K.E., Tagliaferro, A.R., Bobilya, D.J., 2002. Flavonoid antioxidants: chemistry, metabolism and structure–activity relationships. Journal of Nutritional Biochemistry 13, 572–584. Hiai, S., Oura, H., Nakajima, T., 1976. Color reaction of some sapogenins and saponins with vanillin and sulfuric acid. Planta Medica 29, 116–122. Ihara, Y., Toyokuni, S., Uchida, K., Odaka, H., Tanaka, T., Ikeda, H., Hiai, H., Seino, Y., Yamada, Y., 1999. Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes. Diabetes 48, 927–932. Jung, U.J., Baek, N.I., Chung, H.G., Bang, M.H., Yoo, J.S., Jeong, T.S., Lee, K.T., Kang, Y.J., Lee, M.K., Kim, H.J., Yeo, J.Y., Choi, M.S., 2007. The anti-diabetic effects of ethanol extract from two variants of Artemisia princeps Pampanini in C57BL/KsJ-db/db mice. Food and Chemical Toxicology 45, 2022–2029. Kato, A., Miura, T., 1994. Hypoglycemic action of the rhizomes of Polygonatum officinale in normal and diabetic mice. Planta Medica 60, 201–203. Kesari, A.N., Gupta, R.K., Singh, S.K., Diwakar, S., Watal, G., 2006. Hypoglycemic and antihyperglycemic activity of Aegle marmelos seed extract in normal and diabetic rats. Journal of Ethnopharmacology 107, 374–379. Ko, K.M., Mak, D.H., Chiu, P.Y., Poon, M.K., 2004. Pharmacological basis of ‘Yanginvigoration’ in Chinese medicine. Trends in Pharmacological Sciences 25, 3–6. Laakso, M., 1999. Hyperglycemia and cardiovascular disease in type 2 diabetes. Diabetes 48, 937–942. Li, W.L., Zheng, H.C., Bukuru, J., De Kimpe, N., 2004. Natural medicines used in the traditional Chinese medical system for therapy of diabetes mellitus. Journal of Ethnopharmacology 92, 1–21. Mates, J.M., Perez-Gomez, C., Nunez de Castro, I., 1999. Antioxidant enzymes and human diseases. Clinical Biochemistry 32, 595–603. Matsu, T.I., Yashimoto, C., Osajimo, K., Ok, T.I., Psajima, Y., 1996. In vitro survey of ␣-glucosidase inhibitory food components. Bioscience Biotechnology and Biochemistry 60, 2019–2022. May, L.D., Lefkowitch, J.H., Kram, M.T., Rubin, D.E., 2002. Mixed hepatocellularcholestatic liver injury after pioglitazone therapy. Annals of Internal Medicine 136, 449–452. O’Connell, P.A., Taba, M., Nomizo, A., Foss Freitas, M.C., Suaid, F.A., Uyemura, S.A., Trevisan, G.L., Novaes, A.B., Souza, S.L., Palioto, D.B., Grisi, M.F., 2008. Effects of periodontal therapy on glycemic control and inflammatory markers. Journal of Periodontology 79, 774–783. Phillips, P., Braddon, J., 2002. Oral hypoglycaemics. When not to use what. Australian Family Physician 31, 637–643. Rao, P., Pattabiraman, T.N., 1989. Reevaluation of the phenol-sulfuric acid reaction for the estimation of hexoses and pentoses. Analytical Biochemistry 181, 18–22. Scheen, A.J., Lefebvre, P.J., 1998. Oral antidiabetic agents. A guide to selection. Drugs 55, 225–236. Shu, X.S., Lv, J.H., Tao, J., Li, G.M., Li, H.D., Ma, N., 2009. Antihyperglycemic effects of total flavonoids from Polygonatum odoratum in STZ and alloxan-induced diabetic rats. Journal of Ethnopharmacology 124, 539–543. van de Loosdrecht, A.A., Nennie, E., Ossenkoppele, G.J., Beelen, R.H., Langenhuijsen, M.M., 1991. Cell mediated cytotoxicity against U 937 cells by human monocytes and macrophages in a modified colorimetric MTT assay. A methodological study. Journal of Immunological Methods 141, 15–22. Williams, R.J., Spencer, J.P., Rice-Evans, C., 2004. Flavonoids: antioxidants or signalling molecules? Free Radical Biology and Medicine 36, 838–849. Wu, T.T., Zhou, X.T., Deng, Y.F., Jing, Q., Li, M., Yuan, L.J., 2011. In vitro studies of Gynura divaricata (L.) DC extracts as inhibitors of key enzymes relevant for type 2 diabetes and hypertension. Journal of Ethnopharmacology 136, 305–308. Zheng, X.K., Li, Y.J., Zhang, L., Feng, W.S., Zhang, X., 2011. Antihyperglycemic activity of Selaginella tamariscina (Beauv.) Spring. Journal of Ethnopharmacology 133, 531–537.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Saponin rich fractions from Polygonatum odoratum (Mill.) Druce with more potential hypoglycemic effects Yafei Deng a , Kai He b , Xiaoli Ye b , Xin Chen a , Jing Huang a , Xuegang Li a,1 , Lujiang Yuan a,∗ , Yalan Jin a , Qing Jin a , Panpan Li a a b
Chemistry Institute of Pharmaceutical Resources, College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, PR China School of Life Science, Southwest University, Chongqing 400715, PR China
a r t i c l e
i n f o
Article history: Received 25 September 2011 Received in revised form 1 February 2012 Accepted 2 February 2012 Available online 17 February 2012 Keywords: Saponin Flavonoids ␣-Glycosidase Hypoglycemic Polygonatum odoratum (Mill.) Druce
a b s t r a c t Aims: The root of Polygonatum odoratum (YuZhu), also a medicinal food has long been used for the treatment of diabetes. The objective of the study was to characterize the anti-diabetic active fractions or compounds in this herb. Materials and methods: Fractions with a different polarity were prepared by solvent extraction and macroporous absorptive resin (D101) column and their anti-diabetic potentials were evaluated by glucose uptake in HepG2 cells and STZ-induced diabetic rats. In addition, ␣-glycosidase inhibitory activities of active fractions were measured in vitro and chemical compositions including saponin, total flavonoids and total sugar in the fractions were determined. Results: The n-buthanol fraction, a saponin-rich fraction obtained by partitioning the ethanol extract with n-buthanol after petroleum ether and acetic ether showed the highest anti-diabetic potential in glucose uptake in HepG2 cells followed by acetic ether fraction which was rich in flavonoids. Further fractionation the saponin-rich fraction using macroporous resin column (D101), polysaccharide, flavonoid and saponin rich fractions were obtained by elution with water, 40% and 60% ethanol, respectively and their antidiabetic potentials proved by glucose uptake test in HepG2 cells and STZ-induced diabetic rats were in the order of saponin rich fraction > flavonoid rich fraction > polysaccharide rich fraction. Long-term therapy test (60 d) in severe diabetic rats indicated that saponin-rich fraction significantly ameliorated clinical symptoms of diabetes including the elevated blood glucose, body weight loss as well as the increased food and water intake while flavonoid-rich fraction was more potential than saponin-rich fraction to increase superoxide dismutase (SOD) activity and decrease malondialdehyde (MDA) level in rat plasma. Additionally, saponin-rich fraction and flavonoid-rich fraction showed ␣-glycosidase inhibitory activity with IC50 value of 2.05 ± 0.32 and 3.92 ± 0.65 mg/ml, respectively. Conclusion: The results suggested that saponin in this herb was more important than flavonoid in exhibiting anti-diabetic activity and flavonoid contributed more to anti-oxidant activity in vivo. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a group of chronic metabolic disorders characterized by hyperglycemia resulting from defects in insulin
Abbreviations: AUC, areas under the curve; CAT, catalase; DM, diabetes mellitus; DMSO, dimethyl sulfoxide; FBG, fasting blood glucose; FBS, fetal bovine serum; GFR, glomerular filtration rate; MDA, malondialdehyde; MTT, methylthiontrazole; OGTT, oral glucose tolerance test; PBG, postprandial blood glucose; RPO, rhizomes of Polygonatum odoratum; ROS, reactive oxygen species; SOD, superoxide dismutase; STZ, streptozotocin; pNPG, p-nitrophenyl-␣-d-glucopyranoside. ∗ Corresponding author. Tel.: +86 23 68251503; fax: +86 23 68251225. E-mail address: [email protected] (L. Yuan). 1 This author contributed equally to the work. 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.02.023
secretion, action or both and the chronic hyperglycemia causes serious body damage such as blood vessels and nerves damages (O’Connell et al., 2008). Current knowledge strongly supports that control of hyperglycemia is critical in the treatment of not only diabetic patients but also individuals with impaired glucose tolerance (Laakso, 1999). So far a number of hypoglycemic agents including insulin injection and oral drugs such as glycosidase inhibitors, biguanides and sulphonylureas have been clinically used to maintain blood glucose level (Scheen and Lefebvre, 1998). Unexpectedly, some adverse effects had been seen in the use of those agents. Pioglitazone, for instance, may induce hepatocellular-cholestatic liver injury (May et al., 2002) and metformin should be stopped for the therapy of diabetic nephropathy when glomerular filtration rate (GFR) is lowered than 60 ml/min (Phillips and Braddon, 2002).
Y. Deng et al. / Journal of Ethnopharmacology 141 (2012) 228–233
Therefore, it is believed that herbal medicine is a valuable reservoir for novel drugs to deal with this disease due to its few side effects (Li et al., 2004; Jung et al., 2007). The rhizome of Polygonatum odoratum (Mill.) Druc (RPO), an edible medicinal herb has long been used to treat various diseases including DM (China Pharmacopoeia Committee, 2010). Studies had shown that RPO had the beneficial effects on hyperglycemia. Chen et al. (2001) reported that the water extract of RPO decreased the blood glucose level in starch loaded mice. Meanwhile, the n-butanol fraction from the methanol extract of this herb exhibited dramatic hypoglycemic effects in STZ-induced diabetic mice (Kato and Miura, 1994). Although a few studies had revealed that flavonoid, saponin and polysaccharide were crucial compounds (Choi and Park, 2002; Shu et al., 2009), there was no definite conclusion about the active constituents. The objective of this study is to characterize the anti-diabetic constituents or fractions by evaluation anti-diabetic potential of different fractions prepared systematically.
229
2.3. Preparation RPO fractions The dried RPO (5 kg) were extracted with 80% ethanol (40 L × 3) at 80 ◦ C for 2 h, and then ethanol was evaporated under vacuum condition to give a brown residue (25.00% yield, labeled as Fr.0). The residue was suspended in water and successively partitioned with petroleum ether (0.47% yield, labeled as Fr.1), acetic ether (0.31% yield, labeled as Fr.2), n-buthanol (2.08% yield, labeled as Fr.3). The remaining aqueous fraction was labeled as Fr.4 (21.09% yield). The fractions, then, were subjected to glucose uptake test in HepG2 cells. Fr.3 with the highest ability to enhance glucose uptake in HepG2 cells was further fractionated with D101 macroporous resin column in which water and ethanol at various concentrations (20%, 40%, 60% and 80%) were used as eluent. Sequentially, Fr.3-1 (1.58%), Fr.3-2 (0.12%), Fr.3-3 (0.08%), Fr.3-4 (0.09%) and Fr.3-5 (0.03%) were obtained. The detailed process was shown in Fig. 1.
2.4. Test of glucose uptake in HepG2 cells
2. Materials and methods 2.1. Materials and chemicals The rhizomes of Polygonatum odoratum were purchased from Tongjunge Drugstore of Chongqing (Chongqing, China). The taxonomic identification of the plant material was confirmed by a group of professors in pharmacognosy laboratory of the college of pharmaceutical sciences, Southwest University and a voucher of specimen (No. 20090031) was deposited there. Streptozotocin (STZ) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). RPMI-1640 culture medium and fetal bovine serum (FBS) were obtained from Gibco Life Technologies (Burlington, Ontario, Canada). Superoxide dismutase (SOD), malondialdehyde (MDA) and glucose assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Metformin was purchased from Beijing Coway Pharmaceutical Factory (Beijing, China). ␣-Glucosidase and pnitrophenyl-␣-d-glucopyranoside (pNPG) were purchased from Sigma Chemical Co. All other reagents were of the highest available purity and were used as purchased.
2.2. Experimental animals Male Sprague-Dawley rats (200 ± 20 g) and diets were purchased from Animal Breeding Center of the Third Military Medical University (Chongqing, China) and the use and care of animals were in accordance with National Research Council’s guidelines for the use of experimental animals and permitted by Chongqing Animal Care Committee (SYXK-(YU), 2009-0002). Animals were housed under standard environmental conditions (24 ± 2 ◦ C, 55 ± 10% relative humidity, 15 times air changes in 1 h, 12 h light-dark cycle) and adapted to diet for one week before the experiment. Diabetes was induced by single injection of STZ (40 mg/kg for mild diabetes and 55 mg/kg for severe diabetes) in citrate buffer (0.1 M sodium citrate and 0.1 M citric acid, pH 4.5) in overnight fasted rats (Brosky and Logothetopoulos, 1969; Dias et al., 2010). Fasting blood glucose (FBG) level was evaluated 3 d after STZ administration using glucose assay kits and rats with FBG value of 120–250 mg/dl were included in mild diabetic group and with FBG value of 300 mg/dl or above were included in severe diabetic groups (Gupta et al., 2005; Kesari et al., 2006).
HepG2 cells were seeded into 48-well plates in 1640culture medium supplemented with 10% heat-inactivated FBS and penicillin-streptomycin (100 U/ml, each) and cultured in an incubator (5% CO2 ) at 37 ◦ C for 24 h. Then the medium was replaced by 1640-culture medium without FBS and extracts with different concentrations as well as metformin (0.01 mg/ml) were added to corresponding wells. After 24 h incubation, the glucose concentrations in the medium were determined using commercially available kits. Glucose uptake was calculated by subtracting the glucose concentration of control groups from treated groups. MTT assay (van de Loosdrecht et al., 1991; Zheng et al., 2011) was subsequently carried out after the glucose uptake test to estimate the influence of extracts on cell survival. Briefly, 15 l MTT (5 mg/ml in DMEM) was added to each well and incubated at 37 ◦ C for 4 h. The corresponding supernatant, then were discarded and cells were washed. DMSO (200 l) was added to each well to extract the dye and the microplate was placed on a shaker for well dissolution. After 10 min, the optical density (OD) of each well was measured at 490 nm on a microplate reader.
2.5. Chemical analysis Total saponin content was determined using a spectrophotometric method and oleanic acid was used to prepare the calibration curve (Hiai et al., 1976). Total sugar was determined by phenolsulfuric acid method (Rao and Pattabiraman, 1989). Meanwhile, total flavonoids content was determined by the aluminum chloride colorimetric method and rutin was used to prepare the calibration curve (Chang et al., 2002).
2.6. Oral glucose tolerance test The mild diabetic rats were divided into seven groups (n = 6) and oral glucose tolerance test (OGTT) was carried out after overnight fasting. Control group was given vehicle (distilled water) only and positive group received metformin (300 mg/kg). Meanwhile, other groups received different fractions (Fr.3-1, Fr.3-2, Fr.3-3, Fr.3-4 and Fr.3-5) suspended in distilled water at a dose of 500 mg/kg. After 30 min of administration, each rat received orally a glucose solution (2 g/kg). The blood glucose levels were measured before and 30, 60 and 120 min after glucose loading using commercially available kits. Total glycemic responses to OGTT were calculated from respective areas under the curve (AUC) of glycemia during the 120 min observation period.
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Y. Deng et al. / Journal of Ethnopharmacology 141 (2012) 228–233
Polygonatum odoratum (Mill.) Druce (dried roots) extract with ethanol
Ethanol extracts ( Fr.0, 25.00%)
Petroleum ether
Acetic ether
(Fr.1, 0.47%)
H2O
n-Buthanol
(Fr.4, 21.09%)
(Fr.3, 2.08%)
(Fr.2, 0.31%)
D101 macroporous resin column
H2O (Fr.3-1, 1.58%)
20%Ethanol
40%Ethanol
60%Ethanol
80%Ethanol
(Fr.3-2, 0.12%)
(Fr.3-3, 0.08%)
(Fr.3-4, 0.09%)
(Fr.3-5, 0.03%)
Fig. 1. Preparation scheme of the fractions from Polygonatum odoratum.
2.7. Hypoglycemic effects of selected sub-fractions on severe diabetic rats The severe diabetic rats were randomly divided into five groups of 10 rats each. The control group was given vehicle solution (distilled water) and the treated groups received the sub-fractions of Fr.3-2, Fr.3-3 and Fr.3-4 at a dose of 500 mg/kg as well as metformin (300 mg/kg) orally for 60 d. In the treatment process, FBG levels were monitored every 10 d. Meanwhile, body weight, food and water intake were recorded every 15 d. At the end of the experiment, SOD activity and MDA level in plasma were estimated using commercially available kits. 2.8. Assay for ˛-glycosidase inhibitory activity The ␣-glycosidase inhibitory assay was performed according to the procedures reported by Matsu et al. (1996) and Wu et al. (2011). Briefly, Sample solution (20 l), ␣-glycosidase (200 l, 0.5 U/ml) in 0.1 M phosphate buffer (pH 6.9), and 1740 l sodium phosphate buffer (pH 6.9) were mixed and maintained at 37 ◦ C for 10 min. Then, pNPG (40 l) was added into the reaction mixture and incubated at 37 ◦ C for another 60 min. The absorbance at 400 nm was measured. Acarbose was used as positive control. The following equation was used to calculate the inhibitory activity of the extracts.
Inhibition activity (%) =
S400
nm, control
S400
− S400
nm, sample
nm, control
× 100%
highest content of saponin in Fr.3, flavonoid in Fr.2 and sugar in Fr.4 were observed. The highest content of sugar in Fr.3-1, the highest flavonoid in Fr.3-3 (40% ethanol eluent) and the highest saponin in Fr.3-4 (60% ethanol eluent) were observed. Saponin in Fr.3-1 (20% ethanol eluent) and sugar in Fr.3-5 (80% ethanol eluent) were not found.
3.2. Effects of the RPO fractions on glucose uptake in HepG2 cells All the fractions at the concentration lower than 0.1 mg/ml showed no influence on cell survival and proliferation (data not show). Hence, the maximum concentration of fractions for the glucose uptake test was set to 0.1 mg/ml and the results were shown in Fig. 2. As shown in Fig. 2a, the glucose uptakes in HepG2 cells mediated by Fr.2 and Fr.3 were significantly higher, and mediated by Fr.1 and Fr.4 were lower than that by Fr.0. The mediation was in dose-dependent manner. The highest glucose uptake was mediated by Fr.3 which was rich in saponin followed by Fr.2, a flavonoid rich fraction. Based on these results, the saponin rich fraction (Fr.3) was further fractionated into five sub-fractions using D101 macroporous resin column and their results of glucose uptake test were shown in Fig. 2b. All the sub-fractions mediated a higher glucose uptake in HepG2 cells than the starting Fr.3 and the mediations were in a dose-dependent manner. At the dose of 0.025 mg/ml, the values of glucose uptake promoted by Fr.3-1, Fr.3-2, Fr.3-3, Fr.34 and Fr.3-5 were 1.60 ± 0.13, 2.20 ± 0.18, 2.60 ± 0.22, 3.02 ± 0.25 and 1.90 ± 0.16 mmol/L, respectively. The data indicated that Fr.34, again a saponin rich fraction had the highest ability to enhance glucose uptake in HepG2 cells. Accordingly, all of fractions were subjected to oral glucose tolerance test.
2.9. Data analysis All results were expressed as mean ± SD and were analyzed by SPSS for Windows, version 13.0 (SPSS Inc., Chicago, IL). Statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test and a value of p < 0.05 was considered statistically significant. 3. Results 3.1. Chemical compositions The results of chemical analysis were listed in Table 1. Saponin, flavonoid and sugar were found in ethanol (Fr.0) and n-buthanol fractions (Fr.3), while none of them existed in petroleum ether fraction (Fr.1). Acetic ether fraction (Fr.2) contained only flavonoid, and both saponin and sugar existed in water remaining phase (Fr.4). The
Table 1 Chemical compositions of the extracts from Polygonatum odoratum (mg/g). Extracts
Saponin
Fr.0 Fr.1 Fr.2 Fr.3 Fr.4 Fr.3-1 Fr.3-2 Fr.3-3 Fr.3-4 Fr.3-5
4.2 ± – – 34.4 ± 1.2 ± – 95.6 ± 183.3 ± 451.6 ± 107.4 ±
0.5e
3.0d 0.1f 12.2c 22.1b 37.3a 14.9c
Total flavonoids
Total sugar
3.5 ± – 95.5 ± 32.8 ± + 5.1 ± 166.0 ± 305.7 ± 143.1 ± 34.0 ±
392.4 ± – + 469.3 ± 548.0 ± 389.4 ± 175.3 ± 61.8 ± 31.2 ± –
0.7e 8.5c 1.9d 1.2e 19.7b 36.2a 15.4b 2.4d
24.1c
33.2b 44.8a 38.0c 11.2d 7.2e 3.7f
“+” Positive reaction, “−” negative reaction; Data in the same column marked with different letters were significantly different (p < 0.05)
Y. Deng et al. / Journal of Ethnopharmacology 141 (2012) 228–233
4
a 240
0.01 mg/ml 0.025 mg/ml 0.05 mg/ml 0.10 mg/ml
3
Blood glucose level (mg/dl)
Glucose uptake (mmol/L)
a
2 1
4
Fr.0
Fr.1
Fr.2
Fr.3
Fr.4 Metformin
0.010 mg/ml 0.006 mg/ml 0.012 mg/ml 0.025 mg/ml
*
200
* ** **
180
** **
160 140
3
b 25000
0
20
40 *
20000
AUC (mg min/dl)
Glucose uptake (mmol/L)
Control Fr.3-1 Fr.3-2 Fr.3-3 Fr.3-4 Fr.3-5 Metformin
220
120
0
b
231
2
60 80 Time (min)
100
120
* **
**
15000
10000
5000
1 0
Control
0 Fr.3-1
Fr.3-2
Fr.3-3
Fr.3-4 Fr.3-5 Metformin
Fig. 2. Effects of solvent extracts (a) of Polygonatum odoratum and five sub-fractions (b) obtained by D101 macroporous resin separation of the n-butanol fraction on glucose uptake in HepG2 cells.
Fr.3-1
Fr.3-2
Fr.3-3
Fr.3-4
Fr.3-5 Metformin
Fig. 3. Oral glucose tolerance tests (OGTT) in STZ-induced mild diabetic rats (a). Area under the glycaemic curve (AUC) obtained using the trapezoid method through OGTT (b). Results are mean ± SD (n = 6). Significant difference from control: *p < 0.05; **p < 0.01.
3.4. Hypoglycemic effects of Fr.3-2, Fr.3-3 and Fr.3-4 on severe diabetic rats 3.3. Effects of the sub-fractions from Fr.3 on mild diabetic rats The effects of sub-fractions from Fr.3 on the postprandial blood glucose (PBG) levels in the mild diabetic rats were shown in Fig. 3a. The PBG values in untreated diabetic groups increased from 134 to 218 mg/dl in 30 min after glucose loading and decreased thereafter. However, the increase in PBG level was significantly suppressed when Fr.3-1, 3-2, 3-3, 3-4, 3-5 and metformin were administrated. The calculated AUC values for glucose response in the order of control > Fr.3-1 > Fr.3-5 > Fr.3-2 > Fr.33 > Fr.3-4 > metformin (Fig. 3b) suggested the ability to suppress the rise of glucose concentration was in the order of metformin > Fr.34 > Fr.3-3 > Fr.3-2 > Fr.3-5 > Fr.3-1 which was in agreement with the results of glucose uptake test. Fr.3-4 (p < 0.01) and Fr.3-2, Fr.3-3 (p < 0.05) improved glucose tolerance significantly and no remarkable effect for Fr.3-1 and Fr.3-5 was observed. Therefore, Fr.3-2, Fr.3-3 and Fr.3-4 were further evaluated in severe diabetic rats.
Hypoglycemic effects of Fr.3-2, Fr.3-3 and Fr.3-4 on severe diabetic rats were listed in Table 2. The body weight, food and water intake of each rat during the 60 d treatment were also recorded (Table 3). The FBG levels of diabetic rats were significantly higher than normal rats (p < 0.01) during the experimental period. Significant decrease in FBG levels (p < 0.05) for the treated diabetic rats was observed 10 d after administration of metformin, Fr.3-3 and Fr.3-4. At the end of the treatment (60 d), FBG values for metformin treated rats decreased by 63.4%, for Fr.3-4 by 46.3%, for Fr.3-3 by 37.3%, for Fr.3-2 by 26.1%, respectively. A continuous decrease in body weight for diabetic rats and continuous increase in body weight for normal rats were observed (p < 0.01). After 60 d, the average body weight for untreated diabetic rats, diabetic rats treated with metformin and Fr.3-4 was 37.2%, 48.8% and 46.4% of normal rats, respectively, which indicated that treatments with metformin and Fr.3-4 prevented the body weight loss. Fr.3-2 and Fr.3-3 did not show appreciable effects
Table 2 Effects of Fr.3-2, Fr.3-3 and Fr.3-4 on FBG levels in severe diabetic rats. Groups
FBG levels after treatment (mg/dl) 0d
Normal comtrol Diabetic control Metformin Fr.3-2 Fr.3-3 Fr.3-4
84.9 416.7 422.2 410.5 421.7 408.0
10 d ± ± ± ± ± ±
8.2 26.2# 21.3 30.4 26.4 28.5
85.4 420.2 301.9 391.6 380.3 354.7
20 d ± ± ± ± ± ±
7.2 38.6# 32.7** 30.4 29.7* 34.2**
82.6 425.7 254.3 374.0 331.4 301.8
30 d ± ± ± ± ± ±
12.4 31.7# 27.6** 38.1* 32.6** 35.6**
The data are expressed in mean ± SD; n = 6–10 in each group. # p < 0.01 compared with the corresponding value for normal control group. * p < 0.05 compared with the corresponding value for diabetic control group. ** p < 0.01 compared with the corresponding value for diabetic control group.
83.8 428.2 211.7 362.0 304.8 262.6
40 d ± ± ± ± ± ±
14.3 37.1# 33.9** 36.3** 29.7** 37.4**
86.9 427.5 191.9 349.5 282.2 253.3
50 d ± ± ± ± ± ±
12.0 42.0# 34.4** 35.3** 33.6** 28.9**
81.8 409.1 172.2 322.7 272.4 230.9
60 d ± ± ± ± ± ±
9.3 31.6# 28.2** 40.4** 34.9** 27.8**
80.2 405.1 154.4 303.5 264.5 218.9
± ± ± ± ± ±
15.3 39.8# 30.0** 38.7** 29.7** 34.9**
232
Y. Deng et al. / Journal of Ethnopharmacology 141 (2012) 228–233
Table 3 Effects of Fr.3-2, Fr.3-3 and Fr.3-4 on body weight, food and water intake in each group. Group
0d
Body weight (g)
Normal comtrol Diabetic control Metformin Fr.3-2 Fr.3-3 Fr.3-4
225.4 217.5 210.1 207.6 214.2 217.4
± ± ± ± ± ±
17.3 19.1 17.5 20.4 18.7 15.8
15 d 260.1 200.1 225.5 198.7 194.2 211.5
± ± ± ± ± ±
21.4 24.6# 18.3* 19.2 17.4 16.9
30 d
Food intake (g)
Normal comtrol Diabetic control Metformin Fr.3-2 Fr.3-3 Fr.3-4
25.7 32.3 31.8 33.1 33.0 32.7
± ± ± ± ± ±
3.0 2.8# 2.5 5.5 5.5 4.2
26.2 38.6 34.2 37.7 37.3 35.8
± ± ± ± ± ±
Water intake (g)
Normal comtrol Diabetic control Metformin Fr.3-2 Fr.3-3 Fr.3-4
45.5 75.2 73.3 76.8 76.3 73.9
± ± ± ± ± ±
5.7 4.7# 6.6 8.3 7.2 7.1
48.7 88.9 81.1 88.7 84.8 81.7
± ± ± ± ± ±
45 d
306.1 186.0 216.2 190.2 184.3 206.1
± ± ± ± ± ±
23.4 25.5# 19.9** 16.8 18.6 15.4*
2.6 3.3# 3.9* 4.1 2.3 3.1*
26.8 42.2 38.2 41.0 40.1 38.3
± ± ± ± ± ±
4.9 4.2# 5.4** 8.6 6.7 5.1*
48.9 112.3 87.3 101.9 92.3 88.7
± ± ± ± ± ±
60 d
366.1 166.5 207.6 177.0 178.0 198.5
± ± ± ± ± ±
20.2 26.4# 18.2** 17.2 19.3 17.5**
3.3 4.0# 3.1* 2.9 2.6 3.7*
27.9 49.0 41.1 47.1 46.5 43.1
± ± ± ± ± ±
3.4 3.9# 2.3** 3.0 2.9* 2.2**
5.2 8.1# 4.0** 6.6* 5.6** 6.0**
48.7 128.6 94.0 108.0 101.2 94.9
± ± ± ± ± ±
8.3 10.8# 4.5** 5.6** 4.0** 4.9**
414.9 154.3 202.3 160.5 167.7 192.5
± ± ± ± ± ±
25.4 22.0# 16.8** 23.8 18.6 16.0**
28.3 56.1 45.1 52.5 51.7 46.2
± ± ± ± ± ±
3.5 6.7# 2.5** 3.2* 3.2* 3.6**
51.8 140.9 98.8 112.9 108.0 99.7
± ± ± ± ± ±
6.6 7.5# 3.2** 6.7** 4.2** 5.2**
The data are expressed in mean ± SD; n = 6–10 in each group. # p < 0.01 compared with the corresponding value for normal control group. * p < 0.05 compared with the corresponding value for diabetic control group. ** p < 0.01 compared with the corresponding value for diabetic control group.
on preventing weight loss (Table 3). Still in Table 3, the diabetic rats consumed much more food and water than normal rats (p < 0.01). Metformin and Fr.3-4 decreased the food and water consumption significantly after 15 d treatment (p < 0.05). Fr.3-2 and Fr.3-3 treatment also ameliorated the increase in food and water intake at the end of the treatment.
3.5. Effects of Fr.3-2, Fr.3-3 and Fr.3-4 on SOD activity and MDA level in plasma To assess the antioxidant ability of the fractions, the SOD activity and MDA level in plasma of different groups were measured at the end of the treatment and the results were summarized in Table 4. An obvious decrease in SOD activity and increase in MDA level in diabetic rats were observed (p < 0.01) compared to normal rats. Fr.3-3 and Fr.3-4 increased SOD activity and decrease MDA level significantly in plasma compared to diabetic rats (p < 0.01). However, the potency for the latter is slightly weak. 3.6. Effects of Fr.3-2, Fr.3-3 and Fr.3-4 on ˛-glycosidase inhibitory activity in vitro The results of ␣-glycosidase inhibitory activity are shown in Table 5. As can be seen, Fr.3-4 with IC50 value of 2.05 ± 0.32 mg/ml was more potential than Fr.3-3 (IC50 , 3.92 ± 0.65) on the inhibition of ␣-glycosidase activity.
Table 4 Effects of Fr.3-2, Fr.3-3 and Fr.3-4 on SOD activity and MDA level in plasma. Groups
SOD activity (U/ml)
Normal comtrol Diabetic control Metformin Fr.3-2 Fr.3-3 Fr.3-4
139.5 86.1 129.3 90.6 124.2 110.0
± ± ± ± ± ±
8.8 12.7# 10.0** 14.3 10.5** 9.7*
MDA level (nmol/ml) 2.8 7.4 4.0 6.9 4.3 5.3
± ± ± ± ± ±
0.7 0.8# 0.9** 1.0 0.5** 0.7*
The data are expressed in mean ± SD; n = 6–10 in each group. # p < 0.01 compared with the corresponding value for normal control group. * p < 0.05 compared with the corresponding value for diabetic control group. ** p < 0.01 compared with the corresponding value for diabetic control group.
4. Discussion The term for diabetes in ancient China was Xiaoke Zheng or Xiaodan Zheng resulted from lung heat, excessive fire in the stomach, deficiency of kidney Yin or both of Yin and Yang (Ko et al., 2004). According to those, treatment strategies for diabetes in Chinese history were eliminating heat by nourishing yin, moistening dryness and promoting fluid production (Li et al., 2004). Yuzhu, rhizomes of Polygonatum odoratum (Mill.) Druce distributed widely over southern area of China is an important nourishing yin herb and has long been used, since then, to improve health status of patients with Xiaokezheng. Our results that the extracts of this herb reduced the FBG levels, prevented body weight loss, food and water intake of diabetic rats confirmed the traditional use of this herb. Traditionally, this herb was used as decoction which may result in loss of activity and excessive volume of preparation for patients to consume. Hence, it is essential to characterize the pharmacological constitutes or active fraction in order to reduce the dosage. Our findings that Fr.3-4 and Fr.3-3 had strong anti-diabetic potential and Fr.3-4 had the highest content of saponin, and Fr.3-3 had the highest content of flavonoid suggested that both saponin and flavonoid in this herb were the main active compounds contributing to hypoglycemic activity, and saponin contributed even more. Further research on technologies that can efficiently extract saponin and flavonoid from this herb is urgently needed. Hyperglycemia is an important factor responsible for the generation of reactive oxygen species (ROS) (Ihara et al., 1999; Dave and Kalia, 2007). The excessive ROS leads to sharp reduction in antioxidant defense, such as decreased activity of SOD, catalase (CAT) (Mates et al., 1999) and so on. Accordingly, the increased level of plasma MDA in the post meal state and the reduced activities of SOD as indicators of the tissue damage in diabetic patients had
Table 5 The inhibition potency to ␣-glucosidase for the active extracts. Acarbose IC50 (mg/ml)
Fr.3-2
0.15 ± 0.05
d
Fr.3-3
5.17 ± 0.94
c
Fr.3-4
3.92 ± 0.65
b
2.05 ± 0.32a
IC50 is the concentration required to produce 50% inhibition of the enzyme activity; data in the same row marked with different letters were significantly different (p < 0.05).
Y. Deng et al. / Journal of Ethnopharmacology 141 (2012) 228–233
been reported (Ceriello et al., 1998). Moreover, flavonoids are well known antioxidants either through their own reducing capacities or through their possible influences on intracellular redox status (Heim et al., 2002; Williams et al., 2004). Our results that Fr.3-3 had the highest content of flavonoid explained the increase SOD activity and decrease MDA level in plasma of rats administered Fr.3-3. Meanwhile, alleviation the high blood glucose level by Fr.3-3 and Fr.3-4 can also contribute to improved antioxidant status. These results suggest that RPO saponin and flavonoid deserve further investigation on prevention of diabetic patient from body damage or the development of diabetic complications. In conclusion, the results obtained in the present work suggest that the RPO has hypoglycemic effects. Saponin is the most active constituent followed by flavonoid. Therefore, research on isolation and characterization of the active molecule is urgently needed to better understand the mechanism of action of the herb as an agent against hyperglycemia. Acknowledgments This work was founded by National Science and Technology Pillar program of China (2011BAI13B02-1) and National Key Technologies R & D Program of China during the 11th Five-Year Plan Period (2010ZX09401-306-3-10). References Brosky, G., Logothetopoulos, J., 1969. Streptozotocin diabetes in the mouse and guinea pig. Diabetes 18, 606–611. Ceriello, A., Bortolotti, N., Motz, E., Crescentini, A., Lizzio, S., Russo, A., Tonutti, L., Taboga, C., 1998. Meal-generated oxidative stress in type 2 diabetic patients. Diabetes Care 21, 1529–1533. Chang, C.C., Yang, M.H., Wen, H.M., Chern, J.C., 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis 10, 178–182. China Pharmacopoeia Committee, 2010. Pharmacopoeia of the People’s Republic of China, the first division of 2010 ed. China Chemical Industry Press, Beijing, pp. 78–79. Chen, H., Feng, R., Guo, Y., Sun, L., Jiang, J., 2001. Hypoglycemic effects of aqueous extract of Rhizoma Polygonati Odorati in mice and rats. Journal of Ethnopharmacology 74, 225–229. Choi, S.B., Park, S., 2002. A steroidal glycoside from Polygonatum odoratum (Mill.) Druce. improves insulin resistance but does not alter insulin secretion in 90% pancreatectomized rats. Bioscience Biotechnology and Biochemistry 66, 2036–2043. Dave, G.S., Kalia, K., 2007. Hyperglycemia induced oxidative stress in type-1 and type-2 diabetic patients with and without nephropathy. Cellular and Molecular Biology 53, 68–78. Dias, T., Bronze, M.R., Houghton, P.J., Mota-Filipe, H., Paulo, A., 2010. The flavonoidrich fraction of Coreopsis tinctoria promotes glucose tolerance regain through pancreatic function recovery in streptozotocin-induced glucose-intolerant rats. Journal of Ethnopharmacology 132, 483–490.
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