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Antihyperglycemic, hypolipidemic and antioxidant activities of total saponins extracted from Aralia taibaiensis in experimental type 2 diabetic rats
Journal of Ethnopharmacology 152 (2014) 553–560
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Antihyperglycemic, hypolipidemic and antioxidant activities of total saponins extracted from Aralia taibaiensis in experimental type 2 diabetic rats Yan Weng a,1, Lu Yu b,1, Jia Cui a,1, Yan-Rong Zhu a,1, Chao Guo a,1, Guo Wei a, Jia-Lin Duan a, Ying Yin a, Yue Guan a, Yan-Hua Wang a, Zhi-Fu Yang a, Miao-Miao Xi a,n, Ai-Dong Wen a,nn a b
Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, No. 127 Changle West Road, Xi'an 710032, Shaanxi, PR China Department of Pathology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 22 August 2013 Received in revised form 1 January 2014 Accepted 3 February 2014 Available online 11 February 2014
Ethnopharmacological relevance: As a well-known traditional Chinese medicine the root bark of Aralia taibaiensis has multiple pharmacological activities, including relieving rheumatism, promoting blood circulation to arrest pain, inducing diuresis to reduce edema, and antidiabetic action. It has long been used as a folk medicine for the treatment of traumatic injury, rheumatic arthralgia, nephritis, edema, hepatitis and diabetes mellitus in China. Aim of study: To evaluate the antihyperglycemic, hypolipidemic and antioxidant activities of total saponins extracted from Aralia taibaiensis (SAT) in experimental type 2 diabetic mellitus (T2DM) rats. Materials and methods: Acute toxicity was studied in rats to determine the safe oral dose of SAT. Then, SAT was given orally to normal and streptozotocin–nicotinamide induced T2DM rats at 80, 160 and 320 mg/kg doses for a series of 28 days to determine the antihyperglycemic activity. Glibenclamide (600 μg/kg), a standard antidiabetic drug, was used as a positive control drug. At the end of treatment, biochemical parameters and antioxidant levels were measured to evaluate the hypolipidemic and antioxidant activities of SAT. Results: Oral administration of SAT did not exhibit toxicity and death at a dose not more than 2000 mg/ kg. SAT dose-dependently improved the symptoms of polydipsia, polyuria, polyphagia and weight loss in diabetic rats. Compared with diabetic control group, administration of 320 mg/kg SAT resulted in significant (Po 0.05) fall in the levels of fasting blood glucose, glycosylated hemoglobin, creatinine, urea, alanine transarninase, aspartate aminotransferase, total cholesterol, triglycerides, low density lipoprotein cholesterol and malondialdehyde, but significant (P o0.05) increase in the levels of serum insulin, superoxide dismutase and reduced glutathione. However, SAT did not have any effect on the normal rats. Conclusions: SAT had excellent antihyperglycemic, hypolipidemic and antioxidant activities in T2DM rats and might be a promising drug in the therapy of diabetes mellitus and its complications. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Saponins Aralia taibaiensis Type 2 diabetic mellitus Antihyperglycemic activity Hypolipidemic activity Antioxidant activity
1. Introduction Abbreviations: SAT, total saponins extracted from Aralia taibaiensis; T2DM, type 2 diabetic mellitus; STZ, streptozotocin; NA, nicotinamide; FBG, fasting blood glucose; FSI, fasting serum insulin; HbA1c, glycosylated hemoglobin; T2DM, type 2 diabetes mellitus; NIDDM, noninsulin-dependent diabetes mellitus; TCM, traditional Chinese medicine; CMC-Na, carboxymethyl cellulose-natrium; Gli, glibenclamide; IR, insulin resistance; HOMA-IR, hemostasis of model assessment-insulin resistance; TC, total cholesterol; TG, triglycerides; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; TP, total protein; ALT, alanine transarninase; AST, aspartate aminotransferase; VLDL-C, very low density lipoprotein cholesterol; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, reduced glutathione; HE, hematoxylin–eosin staining; CHD, coronary heart disease; ROS, reactive oxygen species; NO, nitric oxide n Corresponding author. Tel.: þ 86 29 84775475x8210; fax: þ 86 29 84773636. nn Corresponding author. Tel.: þ 86 29 84778101; fax: þ86 29 84773636. E-mail addresses: handsomfi[email protected] (M.-M. Xi), [email protected] (A.-D. Wen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jep.2014.02.001 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.
Diabetes mellitus (DM) is a chronic disease of endocrine metabolic disorder including alterations in carbohydrate, fat and protein metabolisms. It is characterized by hyperglycemia arising as a consequence of a relative or absolute deficiency of insulin secretion, resistance to insulin action or both (American Diabetes Association, 2010). DM can increase the risk of complications from vascular disease, so it is a major and growing public health problem throughout the world (Wild et al., 2004). Type 2 diabetes mellitus (T2DM), also named noninsulin-dependent diabetes mellitus (NIDDM) is the most common form of diabetes (Li et al., 2004). Its hallmark characteristic is insulin resistance. Nowadays, available therapies for diabetes include insulin and various oral antidiabetic agents such as sulfonylureas, biguanides and glinides.
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However, many of these usually produce serious side effects including hypoglycemia, drug-resistance, dropsy, weight gain (Tahrani et al., 2010). In addition, they are not suitable for use during pregnancy. It is reported that more than 800 plants have been used as traditional remedies for the treatment of diabetes (Alarcon-Aguilara et al., 1998). Furthermore, after the recommendations made by WHO on diabetes mellitus (World Health Organization, 1980), investigation on natural hypoglycemic agents from the medicinal plants has become more important. Aralia taibaiensis Z.Z. Wang et H.C. Zheng (Araliaceae) is a synonym of Aralia stipulata Franch. It is widely distributed in the Qinba Mountains of western China. The extract of root bark of Aralia taibaiensis has long been used as a folk medicine to treat diabetes mellitus in China (Xi et al., 2010) and its main active components are triterpenoid saponins (Tang et al., 1996, 1997). Many studies have demonstrated that triterpenoid saponins, such as christinin A, calendasaponins A, B, C, and D, can significantly decrease the levels of plasma glucose and triglyceride, beneficial to NIDDM (Abdel-Zaher et al., 2005; Yoshikawa et al., 2001). Lee et al. (2000) have also reported that kaikasaponin III possess antilipid peroxidation activity to protect the vascular endothelium and prevent diabetic complications. Our previous studies have proved that total saponins extracted from Aralia taibaiensis (SAT) outperformed other 11 antidiabetic and saponin-rich traditional Chinese medicine (TCM) extracts in the assays of antioxidant and antiglycation in vitro and ex vivo, and they also exhibited the best α-glucosidase and β-amylase inhibitory activities in vitro (Xi et al., 2008, 2010; Dou et al., 2013). In addition, we firstly found SAT could dramatically stimulate high-glucose-induced insulin secretion and its antidiabetic activity might be related to its high saponin content (Cui et al., 2013). However, there is little pharmacodynamic research to confirm the antidiabetic effect of SAT in vivo. Thus, the present study was undertaken to investigate the antihyperglycemic, hypolipidemic and antioxidant activities of SAT in the normal and T2DM rats.
2. Materials and methods 2.1. Chemicals and animals The following materials were purchased from the sources in brackets: STZ and NA (Sigma-Aldrichs, Hongkong, China), glibenclamide tablets (Pacific Pharmaceutical Ltd. Co., Tianjin, China), oleanolic acid (National institutes for Food and Drug Control, 110709-200505), citric acid (Fengchuan Chemical Reagent Science and Technology Ltd. Co., Tianjin, China), trisodium citrate (Fuchen Chemical Reagent Company, Tianjin, China), and carboxymethyl cellulose-natrium (CMC-Na) (Kemiou Chemical Reagent Development Center, Tianjin, China). All other chemicals and reagents used in study were analytical grade. Male wistar rats (ethical permission no.: SCSK(Jing)2009-0004) weighing 220–240 g were purchased from Beijing Huafukang BioTechnology Company (Beijing, China). All rats were housed in a standard laboratory conditions (temperature 25 72 1C and humidity 457 5% with 12 h light and 12 h dark cycle) during the experiments. They were fasted overnight before every experiment unless otherwise specified. The animal experiments were carried out after the approval of University Animal Ethics Committee in accordance with University Ethics Guidelines for the care and use of laboratory animals.
University). A voucher specimen (FMMUDP-Voucher no. SAP012) was deposited in the Herbarium of the Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, China. 2.3. Preparation of extract SAT was prepared according to the method described by Tang et al. (1996). Firstly, the dry and powdered root bark (100 g) was extracted three times with 10-fold (v/v) 80% ethanol under reflux for 1 h. Secondly, the alcoholic extracts were concentrated, suspended in distilled water and then partitioned successively with 3-fold (v/v) n-butanol saturated with water for three times. Lastly, the n-butanol extracts were combined and evaporated using a rotary evaporator at 60 1C. The yield was 15.91% (w/w). The extract was preserved in a refrigerator for further use. 2.4. Determination of total saponins The content of total saponins in SAT was determined approximately using the method described by Wang et al. (2007). To 10mL tubes, 0.05 mL oleanolic acid (1.5 mg/mL), 0.05 mL extract solution (4.0 mg/mL) and 0.05 mL methanol were added. Then, the solvents in each tube were evaporated at 60 1C in a water-bath. The residue was dissolved in 0.2 mL 5% vanillin–glacial acetic acid solution and 0.8 mL perchloric acid. After this, the tubes were transferred to a water-bath at 60 1C for 15 min and quickly cooled in ice water, and 5 mL acetic acid was added to each tube. The absorbance of each solution was measured by spectrophotometry at a maximum absorption wavelength of 554 nm. The content of total saponins in SAT was 90.34%. 2.5. Acute toxicity study Acute oral toxicity study was performed as per Organization for Economic Cooperation and Development guidelines 423 (acute toxic classic method) (OECD, 2001). 24 Male wistar rats starved overnight were divided into four groups (n¼ 6) and orally fed with SAT in increasing dose levels of 250, 500, 1000 and 2000 mg/kg. The rats were observed individually at least once during the first 30 min, periodically during the first 24 h, with special attention given during the first 4 h, and daily thereafter, they were observed for a total of 14 days for any physical signs of toxicity such as writhing, gasping, palpitation and decreased respiratory rate or mortality. After 14 days, the lethality or death was calculated. 2.6. Induction of NIDDM To the overnight fasted rats, NA was given intraperitoneally (110 mg/kg) beforehand. Then, 15 min after, STZ dissolved in 0.1 M cold citrate buffer (pH 4.5) was administered by intravenous route (65 mg/kg) immediately (Masiello et al., 1998). Control rats were similarly injected with vehicles only. After 7 days of administration, the level of fasting blood glucose (FBG) was measured from tail vein using an Accu-Chek Performa glucose meter (Roche Diagnostics Ltd. Co., Shanghai, China), and repeated three times at intervals of 7 days. The rats with stable FBG Z7.0 mmol/L accompanied by insulin resistance were considered T2DM (World Health Organization, 2006) and included in the following studies.
2.2. Plant materials 2.7. Experimental design The root bark of Aralia taibaiensis Z.Z. Wang et H.C. Zheng was collected in Taibai Mountain, Shaanxi Province of China, and botanically identified by Dr. Haifeng Tang (vice-professor of Department of Pharmacy, Xijing Hospital, Fourth Military Medical
In the antidiabetic experiment, totally 54 rats (24 normal and 30 diabetic rats) were divided into nine groups with six rats each and treated as follows: Group 1: normal rats treated with 0.5%
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carboxymethyl cellulose-natrium (CMC-Na) solution (CON); Group 2: diabetic rats treated with 0.5% CMC-Na solution (MOD); Group 3: diabetic rats treated with 80 mg/kg SAT suspension (MOD þL); Group 4: diabetic rats treated with 160 mg/kg SAT suspension (MOD þM); Group 5: diabetic rats treated with 320 mg/kg SAT suspension (MOD þ H); Group 6: diabetic rats treated with 600 μg/ kg glibenclamide (Gli) suspension (MOD þGli); Group 7: normal rats treated with 80 mg/kg SAT suspension (CONþL); Group 8: normal rats treated with 160 mg/kg SAT suspension (CONþM); Group 9: normal rats treated with 320 mg/kg SAT suspension (CONþH). SAT and Gli suspensions, prepared by dispersing certain drugs in 0.5% CMC-Na solution, were administered orally to their corresponding groups via a force feeding needle once a day continuously for 28 days. During the experimental period, body weight, food intake and FBG of all rats were monitored weekly. On day 28, the rats were sacrificed following blood sample collection from the abdominal aorta. The whole blood, anticoagulated with EDTA was used to determine the level of glycosylated hemoglobin (HbA1c) directly, and the serum, separated by centrifuging the blood samples at 4000 rpm for 15 min, was used for the analysis of insulin, lipid profile, biochemical parameters and antioxidant levels. At the same time, the tissues of pancreas were dissected out for histopathological examination. 2.8. Biochemical analysis 2.8.1. Determination of insulin level The fasting serum insulin (FSI) level was measured by a radioactive immune analysis kit obtained from Beijing North Institute of Biological Technology (Beijing, China). Insulin resistance (IR) was calculated according to the mathematical formula of Hemostasis of Model Assessment-Insulin Resistance (HOMA-IR) by dividing the multiplication of FSI (mIU/L) and FBG (mmol/L) by 22.5. Elevated HOMA-IR levels accounted for low insulin sensitivity (Selimoglu et al., 2009). 2.8.2. Measurement of biochemical parameters The HbA1c level was determined with a dedicated ion exchange HPLC system (D-10™ Analyzer, Bio-Rads, USA) (Bannon et al., 1984). The HbA1c result was calculated as a ratio to total hemoglobin. The levels of serum total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total protein (TP), creatinine, urea, alanine transarninase (ALT) and aspartate aminotransferase (AST) were analyzed using a 7180-automatic biochemical analyzer (Hitachi, Japan) (Luo and Song, 2010). The serum level of very low density lipoprotein cholesterol (VLDL-C) was calculated using the Friedewald formula: VLDL ¼TG/5 (Friedewald et al., 1972). The atherogenic index, a useful determinant of cardiovascular risk was assessed by TC/HDL-C ratio (Grover et al., 1999). 2.8.3. Estimation of antioxidant levels The serum malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH) levels were determined using commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2.8.4. Histopathological examination The excised pancreas tissues were immediately rinsed with normal saline and fixed in 10% neutral-buffered formalin solution. After embedding in paraffin, they were cut into 5 μm-thick sections. The sections were stained with hematoxylin–eosin staining (HE) for histopathological examination.
Fig. 1. Effect of SAT on body weight (A) and food intake (B) in normal and STZ–NA induced diabetic rats (n¼ 6). The values are expressed as mean 7 SD (n¼6). a P o 0.05 as compared with the initial body weight or food intake in CON group; # Po 0.05 as compared with the final body weight or food intake in CON group; n Po 0.05 as compared with the final body weight or food intake in MOD group.
2.9. Statistical analysis All data except food intake were expressed as mean 7 SD. The statistical difference versus the control group was determined by Student's independent sample t-test. Differences among groups were analyzed by one-way ANOVA using SPSS version 18.0. Values of P r0.05 were considered to be statistically significant.
3. Results 3.1. Acute toxicity study In rats, oral administration of SAT at four doses did not produce any drug-induced physical signs of toxicity and no death was registered up to 14 days, indicating that SAT was nontoxic in rats up to an oral dose of 2000 mg/kg. Therefore, investigation of antihyperglycemic activity of SAT at 80, 160 and 320 mg/kg dose levels was safe and feasible. 3.2. Effect of SAT on body weight and food intake Body weight and food intake were recorded in the afternoon postprandial state on the days of initial, 7th, 14th, 21th and 28th days of administration. Fig. 1 represents the changes of body weight and food intake before and after administration of SAT to the control and model rats, respectively. There was no significant intra-group variation in the basal body weight and food intake of
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all rats before the experiment. However, after induced to NIDDM, the model rats exhibited the symptoms of polydipsia, polyuria, polyphagia and rapid weight loss. Treatment with SAT improved the diabetic symptoms dose-dependently. 160 and 320 mg/kg SATs significantly (P o0.05) increased body weight and decreased food intake and urine volume of the diabetic rats. It was noteworthy that the maximum effect of SAT on improving diabetic symptoms was comparable to Gli. Interestingly, different doses of SAT had no effect on body weight and food intake of the normal rats. 3.3. Effect of SAT on FBG, HbA1c and FSI levels Induction of T2DM in the experimental rats was confirmed by the presence of high levels of FBG and HOMA-IR. As shown in Tables 1 and 2, there was a statistical (P o0.05) increase in the levels of FBG and HbA1c but significant (P o0.05) decrease in FSI level in MOD group. Hence, the value of HOMA-IR in MOD group
was significantly (P o0.05) large. Besides, SD value of FBG in MOD group was generally larger than that in CON group, so were those in SAT-treated model groups at 0th day. This was probably caused by the large individual difference of model rats whose body was in a state of disorder. However, daily administration of SAT to the diabetic rats for a series of 28 days led to a dose- and timedependent fall in levels of FBG, HbA1c and HOMA-IR but a rise in FSI. At the end of study, these four evaluation indices in MOD þH and MOD þGli groups all became nearly close to normal levels. Similarly SD value of FBG had a decreasing trend in SAT-treated model groups. It was worth noticing that though SD value of FBG in MOD þL group had always been large during the early treatment, the individual differences of the rats reduced greatly and SD value became much smaller after a treatment of 28 days. No modulation in these parameters was observed in normal rats treated with SAT at any dose. 3.4. Effect of SAT on serum lipid profiles
Table 1 Effect of SAT on FBG in control and model rats (mean7 SD, n¼ 6). Groups
CON MOD MOD þL MOD þM MOD þH MOD þGli CONþ L CONþ M CONþ H # n
Fasting blood glucose level (mmol/L) 0th Day
7th Day
14th Day
21th Day
28th Day
4.6 7 0.4 15.2 7 3.0# 16.17 8.1 16.6 7 6.3 15.5 7 6.7 15.8 7 5.1 4.6 7 0.3 4.3 7 0.9 4.17 0.9
4.4 7 0.2 16.6 7 2.2# 14.2 7 7.7 13.2 7 7.3 10.3 7 4.4n 8.7 7 3.3n 4.3 7 0.7 4.6 7 0.7 4.8 7 0.5
4.17 0.3 14.2 7 2.6# 12.3 7 8.6 10.5 7 6.3n 8.2 7 2.8n 7.5 7 2.0n 4.5 7 0.2 4.6 7 0.1 4.0 7 0.2
4.17 0.4 15.8 7 4.9# 10.17 9.0n 8.8 7 2.7n 6.4 7 2.5n 6.7 7 1.6n 4.4 7 0.6 5.0 7 0.1 4.17 0.2
4.17 0.3 16.17 1.3# 8.8 7 1.0n 6.3 7 0.8n 5.17 0.9n 4.9 7 0.6n 4.2 7 0.4 4.2 7 0.5 4.3 7 0.4
Po 0.05 compared with CON group. Po 0.05 compared with MOD group.
3.5. Effect of SAT on serum biochemical level
Table 2 Effect of SAT on HbA1c, FSI and HOMA-IR in control and model rats (mean 7 SD, n¼ 6). Groups
HbA1c (%)
Serum insulin (mIU/L)
HOMA-IR
CON MOD MOD þL MOD þM MOD þH MOD þGli CONþ L CONþ M CONþ H
10.82 7 0.29 20.50 7 0.67# 16.39 7 0.79n 14.007 1.04n 12.687 0.50n 11.52 7 0.85n 11.117 0.56 10.69 7 0.29 10.747 0.55
33.937 1.40 11.59 7 0.79# 17.56 7 1.02n 23.29 7 1.35n 28.167 1.73n 29.09 7 1.49n 32.95 7 1.35 33.257 1.27 32.22 7 1.18
6.187 0.03 8.29 7 0.05# 6.87 7 0.06n 6.52 7 0.05n 6.38 7 0.06n 6.34 7 0.07n 6.157 0.04 6.217 0.02 6.167 0.03
# n
Table 3 depicts the effect of SAT on serum lipid levels in the tested groups. The results showed that compared with the CON group, the levels of TC, TG, LDL-C and VLDL-C in MOD group increased significantly (Po0.05) while HDL-C level decreased slightly, which led to a 1.8-fold higher of atherogenic index in the diabetic rats. Treatment with different doses of SAT on the diabetic rats all resulted in significant (Po0.05) decrease in TC, TG, LDL-C and VLDL-C levels, but did not affect the normal rats. Consistent with this, a fall of 42%, 46% and 50% in atherogenic index was observed in graded doses of SAT-treated diabetic rats, and 47% in Gli-treated diabetic rats. Differently, there was only a small increment of 16% and 18% in HDL-C in the Gli- and high dose of SAT-treated diabetic rats.
Po 0.05 compared with CON group. Po 0.05 compared with MOD group.
The effect of SAT on the activities of hepatic and renal function markers is shown in Table 4. In comparison with the CON group, the levels of serum ALT, AST, urea and creatinine were increased significantly (Po 0.05) while serum TP level was slightly lower in MOD group. Besides, SD of ALT and AST with the values of 36.82 and 94.78 was large in the diabetic rats. However, SAT could significantly and dose-dependently change the biochemical parameters to different extents. Oral administration of SAT at 320 mg/ kg had a maximum effect to bring these values back to normal. Compared with MOD group, the enhanced levels of ALT, AST, urea and creatinine were brought down by 54%, 41%, 46%, and 22% in MODþ H group, comparable to those of 58%, 43%, 49% and 21% decrease in MODþ Gli group, respectively. Meanwhile, an increase of 12% and 14% TP was observed in MOD þH and MOD þGli groups, respectively. It was also found that SD value of ALT and
Table 3 Hypolipidemic activity of SAT in control and model rats (mean 7 SD, n¼ 6). Groups
TC (mmol/L)
TG (mmol/L)
HDL-C (mmol/L)
LDL-C (mmol/L)
VLDL-C (mmol/L)
Atherogenic index
CON MOD MOD þL MOD þM MOD þH MOD þGli CONþ L CONþ M CONþ H
1.09 70.06 1.80 70.12# 1.09 70.33n 1.0770.18n 1.06 70.39n 1.10 70.13n 1.09 70.11 1.08 70.29 1.0770.14
0.59 7 0.03 1.45 7 0.26# 0.687 0.03n 0.677 0.16n 0.647 0.12n 0.63 7 0.16n 0.617 0.09 0.52 7 0.03 0.55 7 0.04
0.55 7 0.03 0.50 7 0.02 0.517 0.04 0.54 7 0.03 0.59 7 0.03 0.58 7 0.02 0.577 0.05 0.56 7 0.09 0.55 7 0.09
0.23 7 0.02 0.497 0.05# 0.26 7 0.03n 0.25 7 0.02n 0.22 7 0.03n 0.217 0.02n 0.25 7 0.02 0.22 7 0.03 0.26 7 0.04
0.127 0.01 0.29 7 0.02# 0.147 0.02n 0.137 0.01n 0.137 0.02n 0.137 0.03n 0.127 0.03 0.107 0.01 0.117 0.02
1.98 7 0.19 3.60 7 0.27# 2.08 7 0.18n 1.93 7 0.21n 1.80 7 0.07n 1.90 7 0.10n 1.917 0.06 1.93 7 0.11 1.95 7 0.09
# n
Po 0.05 compared with CON group. Po 0.05 compared with MOD group.
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Table 4 Effect of SAT on serum biochemical parameters in control and model rats (mean 7 SD, n¼ 6). Groups
ALT (U/L)
AST (U/L)
TP (g/L)
Urea (mmol/L)
Creatinine (μmol/L)
CON MOD MOD þL MOD þM MOD þH MOD þGli CONþ L CONþ M CONþ H
24.48 7 2.61 77.337 36.82# 45.20 7 16.96n 42.65 7 19.42n 35.777 6.38n 32.38 7 12.68n 24.20 7 1.08 25.20 7 1.08 26.90 7 2.12
69.477 13.84 121.40 7 94.78# 78.737 15.65n 73.69 7 12.92n 71.43 7 24.07n 68.757 10.60n 72.50 7 16.34 70.907 19.67 67.107 13.14
58.53 7 1.39 50.157 3.99 51.60 7 2.72 54.46 7 2.43 56.36 7 3.84 57.007 4.68 59.90 7 0.79 58.707 1.51 57.50 7 0.90
5.63 7 0.52 12.69 7 1.66# 10.55 7 3.45 8.29 7 2.83n 6.86 7 5.87n 6.42 7 4.13n 5.62 7 0.55 5.69 7 0.01 5.677 0.56
53.83 7 2.48 68.677 16.01# 62.86 7 11.22 58.63 7 9.12n 53.25 7 12.69n 54.25 7 5.74n 52.88 7 4.36 52.23 7 7.07 51.90 7 2.00
# n
Po 0.05 compared with CON group. Po 0.05 compared with MOD group.
Fig. 2. Effect of SAT on serum MDA (A), SOD (B), and GSH levels (C) in STZ–NA induced diabetic and normal rats (n ¼6). The values are expressed as mean 7 SD (n ¼6). # P o 0.05 compared with CON group, and nPo 0.05 compared with MOD group.
AST became much smaller in SAT-treated model groups, which was attributable to the small individual difference of the rats after treatment with SAT. When it came to the normal rats, SAT did not produce any effect on the hepatic and renal function markers.
SAT almost did not change the antioxidant levels of the normal rats.
3.6. Antioxidant activity of SAT
3.7. Effects of SAT on histological changes
Fig. 2 shows the increased level of serum MDA and decreased levels of GSH and SOD in T2DM rats, but SAT exhibited an antioxidant effect in a dose-dependent manner in the diabetic rats. It was observed that 80 mg/kg SAT could improve the oxidative damage of the diabetic rats to a certain degree, but there was no statistical difference (P 40.05) compared with MOD group. However, administration of 160 and 320 mg/kg SAT to the diabetic rats significantly (P o0.05) increased SOD level by 65% and 109%, decreased MDA level by 27% and 37%, and increased GSH level by 49% and 104%, respectively. Gli-treated diabetic rats had a similar result of 42% decrease in MDA, 116% and 127% increase in GSH and SOD levels respectively. It was notable that
The results of histopathologic examination of pancreas in rats of all experimental groups are shown in Fig. 3, which supported all the findings above. Fig. 3A shows the normal appearance of islet cells in the pancreas, while in Fig. 3B, decreased number and size of pancreatic islets, ambiguity of their verges, karyopyknosis and degranulation of cells, vacuolation and invasion of connective tissues were detected in the diabetic rats. SAT dose-dependently enhanced the regeneration of islets of langerhans in the pancreas and restoration of normal cellular appearance and size of the islet with hyperplasia (Fig. 3C–E). The islet cells were restored to near normal upon treatment with high dose of SAT (Fig. 3E) and Gli (Fig. 3F). No such changes were found in normal rats (Fig. 3G–I).
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Fig. 3. Effect of SAT on pancreatic islet tissue of rats in model and control groups (HE 100). (A) Normal rats treated with vehicle alone; (B) T2DM rats treated with vehicle alone; (C) T2DM rats treated with SAT (80 mg/kg); (D) T2DM rats treated with SAT (160 mg/kg); (E) T2DM rats treated with SAT (320 mg/kg); (F) T2DM rats treated with glibenclamide (600 μg/kg); (G) normal rats treated with SAT (80 mg/kg); (H) normal rats treated with SAT (160 mg/kg); (I) normal rats treated with SAT (320 mg/kg).
4. Discussion The present study was a preliminary assessment of the antihyperglycemic effect of SAT using STZ–NA induced T2DM rats. On one hand, STZ damaged pancreatic β-cells by specifically inducing DNA strand breakage, inhibiting free radical scavenger-enzymes and enhancing the production of the superoxide radical (Maritim et al., 2003; Lenzen, 2007). On the other hand, NA could protect the islet cells from lysis and partially preserve their mitochondrial activity in the presence of reactive oxygen intermediates (Burkart et al., 1992). Hence, STZ–NA-induced diabetes only destructed partial β-cells of islets of langerhans, produced less pancreatic insulin stores and moderate and stable hyperglycemia (Masiello et al., 1998). In our study, the residual of low level insulin in the diabetic rats suggested partial destruction of β-cells. Furthermore, treatment with glibenclamide, a known insulin secretogogue, confirmed the presence of remnant β-cells in the diabetic rats. Therefore, the STZ–NA-induced diabetic model appeared closer to human T2DM, so was useful for pharmacological investigations of new antidiabetic agents. The extract of TCM is traditionally prepared by soaking the raw material in water or ethanol for a long time, so the crude extract commonly has low yield and purity. According to the records in “Chinese Materia Medica”, the root bark of Aralia taibaiensis with the weight from 15 g to 30 g is decocted in water to get the coarse aqueous extract, which has the saponin content of 2.0–5.0% and is used for oral treatment of traumatic injury, rheumatic arthralgia, nephritis edema, hepatitis and diabetes mellitus in folk medicine. In our study, SAT with high content of saponins above 90% is obtained by two-step extraction using ethanol and saturated nbutanol with water for three times, respectively. Furthermore, the
excellent antidiabetic effect of SAT at 80, 160 and 320 mg/kg in T2DM rats has been confirmed. In SAT 13 triterpenoid saponins have been successfully identified by extensive spectral analysis (IR, 1 H-NMR, 13C-NMR, DEPT, DQCOSY, TOCSY, NOESY, HMQC, HMBC, ESI–MS, HR-ESI–MS) and chemical evidence (GC/MS analysis of the acid hydrolysates) in our previous study (Dou et al., 2013). It is known that triterpenoid saponins are poorly absorbed from gastrointestinal tract and have low oral bioavailability because they are easily metabolized to aglycones by the acidic environment of gastric juice and the enzymes generated by the intestinal microbiota (Yan, 2001), so are SAT. Hence, oleanolic acids might be the real active ingredients which are absorbed into blood to educe the pharmacodynamic action after oral administration of SAT. In the diabetic rats, treatment with SAT showed dose- and time-dependent antihyperglycemic activity. At the end of study, there was a 45%, 61% and 68% reduction in FBG, 52%, 101% and 143% increase in FSI in MOD þL, MOD þM and MOD þH groups, respectively. Hence, in comparison with MOD group, HOMA-IR in SAT-treated rats was decreased by 17%, 21% and 23%. The maximum antihyperglycemic effect of SAT was comparable to the standard antidiabetic drug of Gli, which had a 151% increase in FSI and 70% and 24% reduction in FBG and HOMA-IR. Consistently, the islet cells were restored to near normal level upon treatment with high dose of SAT and Gli. The mechanism of the antihyperglycemic effect of SAT might be due to the protective effect on pancreatic islet cells and increase of insulin secretion from the remaining pancreatic β-cells. However, no effect was observed in normal rats, which could be explained by the very significantly (P o0.01) weak potentiation of insulin secretory response at low glucose concentration (Cui et al., 2013).
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The T2DM rats were associated with increased food intake but severe weight loss, which were due to poor glycemic control. Insulin was an important regulator of protein synthesis and proteolysis in skeletal muscle. During insulin resistance or deficiency, the excessive catabolism of protein provided amino-acids for gluconeogenesis and resulted in muscle wasting and weight loss in diabetic rats (Kasetti et al., 2010). Administration of SAT to the diabetic rats prevented the body weight loss and brought the food intake back to normal, which might owe to the protective effect of SAT in controlling muscle wasting i.e. reversal of gluconeogenesis and might also owe to the improvement in insulin secretion and glycemic control. HbA1c is a reliable index of glycemic control in diabetes. High level of HbA1c predicts high risks for the development and/or progression of retinopathy, nephropathy, neuropathy (Calisti and Tognetti, 2005). In our study, increased level of HbA1c in diabetic rats indicated the occurrence of glycosylation due to hyperglycemia. Administration of SAT and glibenclamide to the diabetic rats significantly reduced HbA1c levels. The explanation for this was assumed to be an improvement in insulin secretion. The increased level of insulin was directly proportional to FBG level, which was consistent with the results in SAT-treated diabetic rats. This suggested that SAT might have the potential to ameliorate the development of diabetes associated complications. In addition to the antihyperglycemic effect, SAT was also able to improve the lipid abnormalities in diabetic rats. High levels of TC and most importantly, VLDL-C are the predictors of atherosclerosis (Temme et al., 2002), but an increase in HDL level is associated with a decrease in coronary risk (Kasetti et al., 2010). In our study, the calculated atherogenic index in diabetic group was significantly (P o0.05) higher than that in normal group, but treatment with SAT effectively corrected the status of dyslipidemia via a dose-dependent manner. The reason for this might be directly attributable to the improvement in insulin, and besides, other lipid-lowing mechanism, such as repressing some key enzymes in cholesterol and/or fatty acids biosynthesis might also be involved in the hypolipidemic activity of SAT. Regulation of plasma or tissue lipid levels led to a decrease in the risk of micro- or macrovascular disease and related complications (Sakatani et al., 2005). Therefore, the hypolipidemic effect of SAT in diabetic rats supported its ability to prevent the CHD diseases associated with diabetes. ALT and AST are reliable markers of liver function (Kasetti et al., 2010). Elevated levels of ALT and AST in serum indicated that the liver was necrotized (Jasmine and Daisy, 2007). Kidney is playing a key role in removing the metabolic waste such as creatinine and urea from body, thereby helping to maintain body homeostasis of above substances (Ramachandran et al., 2012). Increased levels of urea and creatinine, as well as decreased level of TP in diabetic rats suggested impaired renal dysfunction. Administration of SAT and glibenclamide to diabetic rats significantly reduced levels of ALT, AST, creatinine and urea and slightly increased TP level, which represented the preventive action of SAT on liver and kidney damages in diabetic condition. Besides, large SD of ALT and AST also became smaller in SAT-treated model groups. Considering the large individual difference in model rats, increasing the sample size might be helpful to improve the accuracy of estimation and the power of test statistically. During T2DM, persistent hyperglycemia impairs prooxidant/ antioxidant balance, reduces antioxidant levels and increases production of reactive oxygen species (Aragno et al., 2004; Nishikawa et al., 2000). MDA, GSH (a major endogenous antioxidant) and SOD (a scavenger of free radicals), as the oxidative stress biomarkers, involve in the development of diabetic complications (Maritim et al., 2003). In our study, oral administration of SAT dose-dependently reduced serum MDA level and increased the levels of GSH and SOD in T2DM rats, but had no effect on
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normal rats. The results strongly supported a high antioxidant activity of SAT in diabetic condition and hence, SAT possessed a potential to reduce or prevent the diabetic micro and macrovascular complications.
5. Conclusions Our study confirmed that SAT had an excellent antidiabetic effect, which could be explained, at least in part, by its antihyperglycemic, hypolipidemic and antioxidant activities in T2DM rats. SAT also had the potential to ameliorate diabetic complications. It could prevent liver and kidney damages and decrease high levels of HOMA-IR and atherogenic index of the diabetic rats. However, SAT had no effect on normal rats, so it cannot lead to hypoglycemia in normal rats. All these findings provide scientific basis for the use of Aralia taibaiensis as a promising traditional Chinese medicine in the therapy of diabetes mellitus and its complications. Hence, further research is needed to develop SAT into a new drug for treating diabetes mellitus.
Acknowledgments The work is supported by grants from the National Natural Science Foundation of China (Nos. 81001673, 81303264 and 31201037) and the “13115” Technology Innovation Project of Shaanxi Province, PR China (No. 2010ZDKG-62) for the financial assistance. References Abdel-Zaher, A.O., Salim, S.Y., Assaf, M.H., Abdel-Hady, R.H., 2005. Antidiabetic activity and toxicity of Zizyphus spina-christi leaves. J. Ethnopharmacol. 101, 129–138. Alarcon-Aguilara, F.J., Roman-Ramos, R., Perez-Gutierrez, S., Aguilar-Contreras, A., Contreras-Weber, C.C., Flores-Saenz, J.L., 1998. Study of the anti-hyperglycemic effect of plants used as antidiabetics. J. Ethnopharmacol. 61, 101–110. American Diabetes Association, 2010. Diagnosis and classification of diabetes mellitus. Diabetes Care 34, S62–S69. Aragno, M., Mastrocola, R., Catalano, M.G., 2004. Oxidative stress impairs skeletal muscle repair in diabetic rats. Diabetes 53, 1082–1088. Bannon, P., Lessard, F., Lepage, R., Joly, J.G., Dufresne, L., 1984. Glycated hemoglobin in uremic patients as measured by affinity and ion exchange chromatography. Clin. Chem. 30, 485–486. Burkart, V., Koike, T., Brenner, H.H., 1992. Oxygen radicals generated by the enzyme xanthine oxidase lyse rat pancreatic islet cells in vitro. Diabetologia 35, 1028–1034. Calisti, L., Tognetti, S., 2005. Measure of glycosylated haemoglobin. Acta Biomed. 76, 59–62. Cui, J., Li, Y., Jia, N., Song, X., Duan, J., Weng, Y., Guan, Y., Zhou, D., Wang, L., Xi, M., Wen, A., 2013. Insulin-secretagogue activity of eleven plant extracts and twelve pure compounds isolated from Aralia taibaiensis. Life Sci. 92, 131–136. Dou, F., Xi, M., Wang, J., Tian, X., Hong, L., Tang, H., Wen, A., 2013. α-Glucosidase and β-amylase inhibitory activities of saponins from traditional Chinese medicines in the treatment of diabetes mellitus. Pharmazie 68, 1–5. Friedewald, W.T., Levy, R.I., Fredrickson, D.S., 1972. Estimation of the concentration of low-density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin. Chem. 18, 499–502. Grover, S.A., Levington, C., Paquet, S., 1999. Identifying adults at low risk for significant hyperlipidemia: a validated clinical index. J. Clin. Epidemiol. 52, 49–55. Jasmine, R., Daisy, P., 2007. Hypoglycemic and hepatoprotective activity of Eugenia jumbolana in streptozotocin-induced diabetic rats. Int. J. Biol. Chem. 1, 117–121. Kasetti, R.B., Rajasekhar, M.D., Kondeti, V.K., Fatima, S.S., Kumar, E.G., Swapna, S., Ramesh, B., Rao, C.A., 2010. Antihyperglycemic and antihyperlipidemic activities of methanol:water (4:1) fraction isolated from aqueous extract of Syzygium alternifolium seeds in streptozotocin induced diabetic rats. Food Chem. Toxicol. 48, 1078–1084. Lee, K.T., Sohn, I.C., Kim, D.H., Choi, J.W., Kwon, S.H., Park, H.J., 2000. Hypoglycemic and hypolipidemic effects of tectorigenin and kaikasaponin III in the streptozotocin-induced diabetic rat and their antioxidant activity in vitro. Arch. Pharm. Res. 23, 461–466. Lenzen, S., 2007. The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia 51, 216–226.
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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. J. Ethnopharmacol. 92, 1–21. Luo, F.Y., Song, Z.Q., 2010. Establishment of automatic biochemical analyzer calibration cycle of biochemical tests. J. Lab. Clin. Med. 7, 1471–1472. Maritim, A.C., Sanders, R.A., Watkins , J.B., 2003. Diabetes, oxidative stress, and antioxidants: a review. J. Biochem. Mol. Toxicol. 17, 24–38. Masiello, P., Broca, C., Gross, R., Roye, M., Manteghetti, M., Hillaire-Buys, D., Novelli, M., Ribes, G., 1998. Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes 47, 224–229. Nishikawa, T., Edelstein, D., Du, X.L., Yamagishi, S., Matsumura, T., Kaneda, Y., Yorek, M.A., Beebe, D., Oates, P.J., Hammes, H.P., Giardino, I., Brownlee, M., 2000. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404, 787–790. OECD, 2001. OECD Guideline for Testing of Chemicals 423: Acute Oral Toxicityacute Toxic Class Method. Ramachandran, S., Rajasekaran, A., Manisenthilkumar, K., 2012. Investigation of hypoglycemic, hypolipidemic and antioxidant activities of aqueous extract of Terminalia paniculata bark in diabetic rats, Asian Pac. J. Trop. Biomed. 2, 262–268. Sakatani, T., Shirayama, T., Suzaki, Y., 2005. The association between cholesterol and mortality in heart failure. Comparison between patients with and without coronary artery disease. Int. Heart J. 46, 619–629. Selimoglu, H., Duran, C., Kiyici, S., Guclu, M., Ersoy, C., Ozkaya, G., Erturk, E., Tuncel, E., Imamoglu, S., 2009. Comparison of composite whole body insulin sensitivity index derived from mixed meal test and oral glucose tolerance test in insulin resistant obese subjects. Endocrine 36, 299–304. Tahrani, A.A., Piya, M.K., Kennedy, A., Barnett, A.H., 2010. Glycaemic control in type 2 diabetes: targets and new therapies. Pharmacol. Ther. 125, 328–361. Tang, H., Yi, Y., Wang, Z., Hu, W., Li, Y., 1996. Studies on the triterpenoid saponins of the root bark of Aralia taibaiensis. Yao Xue Xue Bao 31, 517–523.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Antihyperglycemic, hypolipidemic and antioxidant activities of total saponins extracted from Aralia taibaiensis in experimental type 2 diabetic rats Yan Weng a,1, Lu Yu b,1, Jia Cui a,1, Yan-Rong Zhu a,1, Chao Guo a,1, Guo Wei a, Jia-Lin Duan a, Ying Yin a, Yue Guan a, Yan-Hua Wang a, Zhi-Fu Yang a, Miao-Miao Xi a,n, Ai-Dong Wen a,nn a b
Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, No. 127 Changle West Road, Xi'an 710032, Shaanxi, PR China Department of Pathology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 22 August 2013 Received in revised form 1 January 2014 Accepted 3 February 2014 Available online 11 February 2014
Ethnopharmacological relevance: As a well-known traditional Chinese medicine the root bark of Aralia taibaiensis has multiple pharmacological activities, including relieving rheumatism, promoting blood circulation to arrest pain, inducing diuresis to reduce edema, and antidiabetic action. It has long been used as a folk medicine for the treatment of traumatic injury, rheumatic arthralgia, nephritis, edema, hepatitis and diabetes mellitus in China. Aim of study: To evaluate the antihyperglycemic, hypolipidemic and antioxidant activities of total saponins extracted from Aralia taibaiensis (SAT) in experimental type 2 diabetic mellitus (T2DM) rats. Materials and methods: Acute toxicity was studied in rats to determine the safe oral dose of SAT. Then, SAT was given orally to normal and streptozotocin–nicotinamide induced T2DM rats at 80, 160 and 320 mg/kg doses for a series of 28 days to determine the antihyperglycemic activity. Glibenclamide (600 μg/kg), a standard antidiabetic drug, was used as a positive control drug. At the end of treatment, biochemical parameters and antioxidant levels were measured to evaluate the hypolipidemic and antioxidant activities of SAT. Results: Oral administration of SAT did not exhibit toxicity and death at a dose not more than 2000 mg/ kg. SAT dose-dependently improved the symptoms of polydipsia, polyuria, polyphagia and weight loss in diabetic rats. Compared with diabetic control group, administration of 320 mg/kg SAT resulted in significant (Po 0.05) fall in the levels of fasting blood glucose, glycosylated hemoglobin, creatinine, urea, alanine transarninase, aspartate aminotransferase, total cholesterol, triglycerides, low density lipoprotein cholesterol and malondialdehyde, but significant (P o0.05) increase in the levels of serum insulin, superoxide dismutase and reduced glutathione. However, SAT did not have any effect on the normal rats. Conclusions: SAT had excellent antihyperglycemic, hypolipidemic and antioxidant activities in T2DM rats and might be a promising drug in the therapy of diabetes mellitus and its complications. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Saponins Aralia taibaiensis Type 2 diabetic mellitus Antihyperglycemic activity Hypolipidemic activity Antioxidant activity
1. Introduction Abbreviations: SAT, total saponins extracted from Aralia taibaiensis; T2DM, type 2 diabetic mellitus; STZ, streptozotocin; NA, nicotinamide; FBG, fasting blood glucose; FSI, fasting serum insulin; HbA1c, glycosylated hemoglobin; T2DM, type 2 diabetes mellitus; NIDDM, noninsulin-dependent diabetes mellitus; TCM, traditional Chinese medicine; CMC-Na, carboxymethyl cellulose-natrium; Gli, glibenclamide; IR, insulin resistance; HOMA-IR, hemostasis of model assessment-insulin resistance; TC, total cholesterol; TG, triglycerides; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; TP, total protein; ALT, alanine transarninase; AST, aspartate aminotransferase; VLDL-C, very low density lipoprotein cholesterol; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, reduced glutathione; HE, hematoxylin–eosin staining; CHD, coronary heart disease; ROS, reactive oxygen species; NO, nitric oxide n Corresponding author. Tel.: þ 86 29 84775475x8210; fax: þ 86 29 84773636. nn Corresponding author. Tel.: þ 86 29 84778101; fax: þ86 29 84773636. E-mail addresses: handsomfi[email protected] (M.-M. Xi), [email protected] (A.-D. Wen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jep.2014.02.001 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.
Diabetes mellitus (DM) is a chronic disease of endocrine metabolic disorder including alterations in carbohydrate, fat and protein metabolisms. It is characterized by hyperglycemia arising as a consequence of a relative or absolute deficiency of insulin secretion, resistance to insulin action or both (American Diabetes Association, 2010). DM can increase the risk of complications from vascular disease, so it is a major and growing public health problem throughout the world (Wild et al., 2004). Type 2 diabetes mellitus (T2DM), also named noninsulin-dependent diabetes mellitus (NIDDM) is the most common form of diabetes (Li et al., 2004). Its hallmark characteristic is insulin resistance. Nowadays, available therapies for diabetes include insulin and various oral antidiabetic agents such as sulfonylureas, biguanides and glinides.
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However, many of these usually produce serious side effects including hypoglycemia, drug-resistance, dropsy, weight gain (Tahrani et al., 2010). In addition, they are not suitable for use during pregnancy. It is reported that more than 800 plants have been used as traditional remedies for the treatment of diabetes (Alarcon-Aguilara et al., 1998). Furthermore, after the recommendations made by WHO on diabetes mellitus (World Health Organization, 1980), investigation on natural hypoglycemic agents from the medicinal plants has become more important. Aralia taibaiensis Z.Z. Wang et H.C. Zheng (Araliaceae) is a synonym of Aralia stipulata Franch. It is widely distributed in the Qinba Mountains of western China. The extract of root bark of Aralia taibaiensis has long been used as a folk medicine to treat diabetes mellitus in China (Xi et al., 2010) and its main active components are triterpenoid saponins (Tang et al., 1996, 1997). Many studies have demonstrated that triterpenoid saponins, such as christinin A, calendasaponins A, B, C, and D, can significantly decrease the levels of plasma glucose and triglyceride, beneficial to NIDDM (Abdel-Zaher et al., 2005; Yoshikawa et al., 2001). Lee et al. (2000) have also reported that kaikasaponin III possess antilipid peroxidation activity to protect the vascular endothelium and prevent diabetic complications. Our previous studies have proved that total saponins extracted from Aralia taibaiensis (SAT) outperformed other 11 antidiabetic and saponin-rich traditional Chinese medicine (TCM) extracts in the assays of antioxidant and antiglycation in vitro and ex vivo, and they also exhibited the best α-glucosidase and β-amylase inhibitory activities in vitro (Xi et al., 2008, 2010; Dou et al., 2013). In addition, we firstly found SAT could dramatically stimulate high-glucose-induced insulin secretion and its antidiabetic activity might be related to its high saponin content (Cui et al., 2013). However, there is little pharmacodynamic research to confirm the antidiabetic effect of SAT in vivo. Thus, the present study was undertaken to investigate the antihyperglycemic, hypolipidemic and antioxidant activities of SAT in the normal and T2DM rats.
2. Materials and methods 2.1. Chemicals and animals The following materials were purchased from the sources in brackets: STZ and NA (Sigma-Aldrichs, Hongkong, China), glibenclamide tablets (Pacific Pharmaceutical Ltd. Co., Tianjin, China), oleanolic acid (National institutes for Food and Drug Control, 110709-200505), citric acid (Fengchuan Chemical Reagent Science and Technology Ltd. Co., Tianjin, China), trisodium citrate (Fuchen Chemical Reagent Company, Tianjin, China), and carboxymethyl cellulose-natrium (CMC-Na) (Kemiou Chemical Reagent Development Center, Tianjin, China). All other chemicals and reagents used in study were analytical grade. Male wistar rats (ethical permission no.: SCSK(Jing)2009-0004) weighing 220–240 g were purchased from Beijing Huafukang BioTechnology Company (Beijing, China). All rats were housed in a standard laboratory conditions (temperature 25 72 1C and humidity 457 5% with 12 h light and 12 h dark cycle) during the experiments. They were fasted overnight before every experiment unless otherwise specified. The animal experiments were carried out after the approval of University Animal Ethics Committee in accordance with University Ethics Guidelines for the care and use of laboratory animals.
University). A voucher specimen (FMMUDP-Voucher no. SAP012) was deposited in the Herbarium of the Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, China. 2.3. Preparation of extract SAT was prepared according to the method described by Tang et al. (1996). Firstly, the dry and powdered root bark (100 g) was extracted three times with 10-fold (v/v) 80% ethanol under reflux for 1 h. Secondly, the alcoholic extracts were concentrated, suspended in distilled water and then partitioned successively with 3-fold (v/v) n-butanol saturated with water for three times. Lastly, the n-butanol extracts were combined and evaporated using a rotary evaporator at 60 1C. The yield was 15.91% (w/w). The extract was preserved in a refrigerator for further use. 2.4. Determination of total saponins The content of total saponins in SAT was determined approximately using the method described by Wang et al. (2007). To 10mL tubes, 0.05 mL oleanolic acid (1.5 mg/mL), 0.05 mL extract solution (4.0 mg/mL) and 0.05 mL methanol were added. Then, the solvents in each tube were evaporated at 60 1C in a water-bath. The residue was dissolved in 0.2 mL 5% vanillin–glacial acetic acid solution and 0.8 mL perchloric acid. After this, the tubes were transferred to a water-bath at 60 1C for 15 min and quickly cooled in ice water, and 5 mL acetic acid was added to each tube. The absorbance of each solution was measured by spectrophotometry at a maximum absorption wavelength of 554 nm. The content of total saponins in SAT was 90.34%. 2.5. Acute toxicity study Acute oral toxicity study was performed as per Organization for Economic Cooperation and Development guidelines 423 (acute toxic classic method) (OECD, 2001). 24 Male wistar rats starved overnight were divided into four groups (n¼ 6) and orally fed with SAT in increasing dose levels of 250, 500, 1000 and 2000 mg/kg. The rats were observed individually at least once during the first 30 min, periodically during the first 24 h, with special attention given during the first 4 h, and daily thereafter, they were observed for a total of 14 days for any physical signs of toxicity such as writhing, gasping, palpitation and decreased respiratory rate or mortality. After 14 days, the lethality or death was calculated. 2.6. Induction of NIDDM To the overnight fasted rats, NA was given intraperitoneally (110 mg/kg) beforehand. Then, 15 min after, STZ dissolved in 0.1 M cold citrate buffer (pH 4.5) was administered by intravenous route (65 mg/kg) immediately (Masiello et al., 1998). Control rats were similarly injected with vehicles only. After 7 days of administration, the level of fasting blood glucose (FBG) was measured from tail vein using an Accu-Chek Performa glucose meter (Roche Diagnostics Ltd. Co., Shanghai, China), and repeated three times at intervals of 7 days. The rats with stable FBG Z7.0 mmol/L accompanied by insulin resistance were considered T2DM (World Health Organization, 2006) and included in the following studies.
2.2. Plant materials 2.7. Experimental design The root bark of Aralia taibaiensis Z.Z. Wang et H.C. Zheng was collected in Taibai Mountain, Shaanxi Province of China, and botanically identified by Dr. Haifeng Tang (vice-professor of Department of Pharmacy, Xijing Hospital, Fourth Military Medical
In the antidiabetic experiment, totally 54 rats (24 normal and 30 diabetic rats) were divided into nine groups with six rats each and treated as follows: Group 1: normal rats treated with 0.5%
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carboxymethyl cellulose-natrium (CMC-Na) solution (CON); Group 2: diabetic rats treated with 0.5% CMC-Na solution (MOD); Group 3: diabetic rats treated with 80 mg/kg SAT suspension (MOD þL); Group 4: diabetic rats treated with 160 mg/kg SAT suspension (MOD þM); Group 5: diabetic rats treated with 320 mg/kg SAT suspension (MOD þ H); Group 6: diabetic rats treated with 600 μg/ kg glibenclamide (Gli) suspension (MOD þGli); Group 7: normal rats treated with 80 mg/kg SAT suspension (CONþL); Group 8: normal rats treated with 160 mg/kg SAT suspension (CONþM); Group 9: normal rats treated with 320 mg/kg SAT suspension (CONþH). SAT and Gli suspensions, prepared by dispersing certain drugs in 0.5% CMC-Na solution, were administered orally to their corresponding groups via a force feeding needle once a day continuously for 28 days. During the experimental period, body weight, food intake and FBG of all rats were monitored weekly. On day 28, the rats were sacrificed following blood sample collection from the abdominal aorta. The whole blood, anticoagulated with EDTA was used to determine the level of glycosylated hemoglobin (HbA1c) directly, and the serum, separated by centrifuging the blood samples at 4000 rpm for 15 min, was used for the analysis of insulin, lipid profile, biochemical parameters and antioxidant levels. At the same time, the tissues of pancreas were dissected out for histopathological examination. 2.8. Biochemical analysis 2.8.1. Determination of insulin level The fasting serum insulin (FSI) level was measured by a radioactive immune analysis kit obtained from Beijing North Institute of Biological Technology (Beijing, China). Insulin resistance (IR) was calculated according to the mathematical formula of Hemostasis of Model Assessment-Insulin Resistance (HOMA-IR) by dividing the multiplication of FSI (mIU/L) and FBG (mmol/L) by 22.5. Elevated HOMA-IR levels accounted for low insulin sensitivity (Selimoglu et al., 2009). 2.8.2. Measurement of biochemical parameters The HbA1c level was determined with a dedicated ion exchange HPLC system (D-10™ Analyzer, Bio-Rads, USA) (Bannon et al., 1984). The HbA1c result was calculated as a ratio to total hemoglobin. The levels of serum total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total protein (TP), creatinine, urea, alanine transarninase (ALT) and aspartate aminotransferase (AST) were analyzed using a 7180-automatic biochemical analyzer (Hitachi, Japan) (Luo and Song, 2010). The serum level of very low density lipoprotein cholesterol (VLDL-C) was calculated using the Friedewald formula: VLDL ¼TG/5 (Friedewald et al., 1972). The atherogenic index, a useful determinant of cardiovascular risk was assessed by TC/HDL-C ratio (Grover et al., 1999). 2.8.3. Estimation of antioxidant levels The serum malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH) levels were determined using commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2.8.4. Histopathological examination The excised pancreas tissues were immediately rinsed with normal saline and fixed in 10% neutral-buffered formalin solution. After embedding in paraffin, they were cut into 5 μm-thick sections. The sections were stained with hematoxylin–eosin staining (HE) for histopathological examination.
Fig. 1. Effect of SAT on body weight (A) and food intake (B) in normal and STZ–NA induced diabetic rats (n¼ 6). The values are expressed as mean 7 SD (n¼6). a P o 0.05 as compared with the initial body weight or food intake in CON group; # Po 0.05 as compared with the final body weight or food intake in CON group; n Po 0.05 as compared with the final body weight or food intake in MOD group.
2.9. Statistical analysis All data except food intake were expressed as mean 7 SD. The statistical difference versus the control group was determined by Student's independent sample t-test. Differences among groups were analyzed by one-way ANOVA using SPSS version 18.0. Values of P r0.05 were considered to be statistically significant.
3. Results 3.1. Acute toxicity study In rats, oral administration of SAT at four doses did not produce any drug-induced physical signs of toxicity and no death was registered up to 14 days, indicating that SAT was nontoxic in rats up to an oral dose of 2000 mg/kg. Therefore, investigation of antihyperglycemic activity of SAT at 80, 160 and 320 mg/kg dose levels was safe and feasible. 3.2. Effect of SAT on body weight and food intake Body weight and food intake were recorded in the afternoon postprandial state on the days of initial, 7th, 14th, 21th and 28th days of administration. Fig. 1 represents the changes of body weight and food intake before and after administration of SAT to the control and model rats, respectively. There was no significant intra-group variation in the basal body weight and food intake of
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all rats before the experiment. However, after induced to NIDDM, the model rats exhibited the symptoms of polydipsia, polyuria, polyphagia and rapid weight loss. Treatment with SAT improved the diabetic symptoms dose-dependently. 160 and 320 mg/kg SATs significantly (P o0.05) increased body weight and decreased food intake and urine volume of the diabetic rats. It was noteworthy that the maximum effect of SAT on improving diabetic symptoms was comparable to Gli. Interestingly, different doses of SAT had no effect on body weight and food intake of the normal rats. 3.3. Effect of SAT on FBG, HbA1c and FSI levels Induction of T2DM in the experimental rats was confirmed by the presence of high levels of FBG and HOMA-IR. As shown in Tables 1 and 2, there was a statistical (P o0.05) increase in the levels of FBG and HbA1c but significant (P o0.05) decrease in FSI level in MOD group. Hence, the value of HOMA-IR in MOD group
was significantly (P o0.05) large. Besides, SD value of FBG in MOD group was generally larger than that in CON group, so were those in SAT-treated model groups at 0th day. This was probably caused by the large individual difference of model rats whose body was in a state of disorder. However, daily administration of SAT to the diabetic rats for a series of 28 days led to a dose- and timedependent fall in levels of FBG, HbA1c and HOMA-IR but a rise in FSI. At the end of study, these four evaluation indices in MOD þH and MOD þGli groups all became nearly close to normal levels. Similarly SD value of FBG had a decreasing trend in SAT-treated model groups. It was worth noticing that though SD value of FBG in MOD þL group had always been large during the early treatment, the individual differences of the rats reduced greatly and SD value became much smaller after a treatment of 28 days. No modulation in these parameters was observed in normal rats treated with SAT at any dose. 3.4. Effect of SAT on serum lipid profiles
Table 1 Effect of SAT on FBG in control and model rats (mean7 SD, n¼ 6). Groups
CON MOD MOD þL MOD þM MOD þH MOD þGli CONþ L CONþ M CONþ H # n
Fasting blood glucose level (mmol/L) 0th Day
7th Day
14th Day
21th Day
28th Day
4.6 7 0.4 15.2 7 3.0# 16.17 8.1 16.6 7 6.3 15.5 7 6.7 15.8 7 5.1 4.6 7 0.3 4.3 7 0.9 4.17 0.9
4.4 7 0.2 16.6 7 2.2# 14.2 7 7.7 13.2 7 7.3 10.3 7 4.4n 8.7 7 3.3n 4.3 7 0.7 4.6 7 0.7 4.8 7 0.5
4.17 0.3 14.2 7 2.6# 12.3 7 8.6 10.5 7 6.3n 8.2 7 2.8n 7.5 7 2.0n 4.5 7 0.2 4.6 7 0.1 4.0 7 0.2
4.17 0.4 15.8 7 4.9# 10.17 9.0n 8.8 7 2.7n 6.4 7 2.5n 6.7 7 1.6n 4.4 7 0.6 5.0 7 0.1 4.17 0.2
4.17 0.3 16.17 1.3# 8.8 7 1.0n 6.3 7 0.8n 5.17 0.9n 4.9 7 0.6n 4.2 7 0.4 4.2 7 0.5 4.3 7 0.4
Po 0.05 compared with CON group. Po 0.05 compared with MOD group.
3.5. Effect of SAT on serum biochemical level
Table 2 Effect of SAT on HbA1c, FSI and HOMA-IR in control and model rats (mean 7 SD, n¼ 6). Groups
HbA1c (%)
Serum insulin (mIU/L)
HOMA-IR
CON MOD MOD þL MOD þM MOD þH MOD þGli CONþ L CONþ M CONþ H
10.82 7 0.29 20.50 7 0.67# 16.39 7 0.79n 14.007 1.04n 12.687 0.50n 11.52 7 0.85n 11.117 0.56 10.69 7 0.29 10.747 0.55
33.937 1.40 11.59 7 0.79# 17.56 7 1.02n 23.29 7 1.35n 28.167 1.73n 29.09 7 1.49n 32.95 7 1.35 33.257 1.27 32.22 7 1.18
6.187 0.03 8.29 7 0.05# 6.87 7 0.06n 6.52 7 0.05n 6.38 7 0.06n 6.34 7 0.07n 6.157 0.04 6.217 0.02 6.167 0.03
# n
Table 3 depicts the effect of SAT on serum lipid levels in the tested groups. The results showed that compared with the CON group, the levels of TC, TG, LDL-C and VLDL-C in MOD group increased significantly (Po0.05) while HDL-C level decreased slightly, which led to a 1.8-fold higher of atherogenic index in the diabetic rats. Treatment with different doses of SAT on the diabetic rats all resulted in significant (Po0.05) decrease in TC, TG, LDL-C and VLDL-C levels, but did not affect the normal rats. Consistent with this, a fall of 42%, 46% and 50% in atherogenic index was observed in graded doses of SAT-treated diabetic rats, and 47% in Gli-treated diabetic rats. Differently, there was only a small increment of 16% and 18% in HDL-C in the Gli- and high dose of SAT-treated diabetic rats.
Po 0.05 compared with CON group. Po 0.05 compared with MOD group.
The effect of SAT on the activities of hepatic and renal function markers is shown in Table 4. In comparison with the CON group, the levels of serum ALT, AST, urea and creatinine were increased significantly (Po 0.05) while serum TP level was slightly lower in MOD group. Besides, SD of ALT and AST with the values of 36.82 and 94.78 was large in the diabetic rats. However, SAT could significantly and dose-dependently change the biochemical parameters to different extents. Oral administration of SAT at 320 mg/ kg had a maximum effect to bring these values back to normal. Compared with MOD group, the enhanced levels of ALT, AST, urea and creatinine were brought down by 54%, 41%, 46%, and 22% in MODþ H group, comparable to those of 58%, 43%, 49% and 21% decrease in MODþ Gli group, respectively. Meanwhile, an increase of 12% and 14% TP was observed in MOD þH and MOD þGli groups, respectively. It was also found that SD value of ALT and
Table 3 Hypolipidemic activity of SAT in control and model rats (mean 7 SD, n¼ 6). Groups
TC (mmol/L)
TG (mmol/L)
HDL-C (mmol/L)
LDL-C (mmol/L)
VLDL-C (mmol/L)
Atherogenic index
CON MOD MOD þL MOD þM MOD þH MOD þGli CONþ L CONþ M CONþ H
1.09 70.06 1.80 70.12# 1.09 70.33n 1.0770.18n 1.06 70.39n 1.10 70.13n 1.09 70.11 1.08 70.29 1.0770.14
0.59 7 0.03 1.45 7 0.26# 0.687 0.03n 0.677 0.16n 0.647 0.12n 0.63 7 0.16n 0.617 0.09 0.52 7 0.03 0.55 7 0.04
0.55 7 0.03 0.50 7 0.02 0.517 0.04 0.54 7 0.03 0.59 7 0.03 0.58 7 0.02 0.577 0.05 0.56 7 0.09 0.55 7 0.09
0.23 7 0.02 0.497 0.05# 0.26 7 0.03n 0.25 7 0.02n 0.22 7 0.03n 0.217 0.02n 0.25 7 0.02 0.22 7 0.03 0.26 7 0.04
0.127 0.01 0.29 7 0.02# 0.147 0.02n 0.137 0.01n 0.137 0.02n 0.137 0.03n 0.127 0.03 0.107 0.01 0.117 0.02
1.98 7 0.19 3.60 7 0.27# 2.08 7 0.18n 1.93 7 0.21n 1.80 7 0.07n 1.90 7 0.10n 1.917 0.06 1.93 7 0.11 1.95 7 0.09
# n
Po 0.05 compared with CON group. Po 0.05 compared with MOD group.
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Table 4 Effect of SAT on serum biochemical parameters in control and model rats (mean 7 SD, n¼ 6). Groups
ALT (U/L)
AST (U/L)
TP (g/L)
Urea (mmol/L)
Creatinine (μmol/L)
CON MOD MOD þL MOD þM MOD þH MOD þGli CONþ L CONþ M CONþ H
24.48 7 2.61 77.337 36.82# 45.20 7 16.96n 42.65 7 19.42n 35.777 6.38n 32.38 7 12.68n 24.20 7 1.08 25.20 7 1.08 26.90 7 2.12
69.477 13.84 121.40 7 94.78# 78.737 15.65n 73.69 7 12.92n 71.43 7 24.07n 68.757 10.60n 72.50 7 16.34 70.907 19.67 67.107 13.14
58.53 7 1.39 50.157 3.99 51.60 7 2.72 54.46 7 2.43 56.36 7 3.84 57.007 4.68 59.90 7 0.79 58.707 1.51 57.50 7 0.90
5.63 7 0.52 12.69 7 1.66# 10.55 7 3.45 8.29 7 2.83n 6.86 7 5.87n 6.42 7 4.13n 5.62 7 0.55 5.69 7 0.01 5.677 0.56
53.83 7 2.48 68.677 16.01# 62.86 7 11.22 58.63 7 9.12n 53.25 7 12.69n 54.25 7 5.74n 52.88 7 4.36 52.23 7 7.07 51.90 7 2.00
# n
Po 0.05 compared with CON group. Po 0.05 compared with MOD group.
Fig. 2. Effect of SAT on serum MDA (A), SOD (B), and GSH levels (C) in STZ–NA induced diabetic and normal rats (n ¼6). The values are expressed as mean 7 SD (n ¼6). # P o 0.05 compared with CON group, and nPo 0.05 compared with MOD group.
AST became much smaller in SAT-treated model groups, which was attributable to the small individual difference of the rats after treatment with SAT. When it came to the normal rats, SAT did not produce any effect on the hepatic and renal function markers.
SAT almost did not change the antioxidant levels of the normal rats.
3.6. Antioxidant activity of SAT
3.7. Effects of SAT on histological changes
Fig. 2 shows the increased level of serum MDA and decreased levels of GSH and SOD in T2DM rats, but SAT exhibited an antioxidant effect in a dose-dependent manner in the diabetic rats. It was observed that 80 mg/kg SAT could improve the oxidative damage of the diabetic rats to a certain degree, but there was no statistical difference (P 40.05) compared with MOD group. However, administration of 160 and 320 mg/kg SAT to the diabetic rats significantly (P o0.05) increased SOD level by 65% and 109%, decreased MDA level by 27% and 37%, and increased GSH level by 49% and 104%, respectively. Gli-treated diabetic rats had a similar result of 42% decrease in MDA, 116% and 127% increase in GSH and SOD levels respectively. It was notable that
The results of histopathologic examination of pancreas in rats of all experimental groups are shown in Fig. 3, which supported all the findings above. Fig. 3A shows the normal appearance of islet cells in the pancreas, while in Fig. 3B, decreased number and size of pancreatic islets, ambiguity of their verges, karyopyknosis and degranulation of cells, vacuolation and invasion of connective tissues were detected in the diabetic rats. SAT dose-dependently enhanced the regeneration of islets of langerhans in the pancreas and restoration of normal cellular appearance and size of the islet with hyperplasia (Fig. 3C–E). The islet cells were restored to near normal upon treatment with high dose of SAT (Fig. 3E) and Gli (Fig. 3F). No such changes were found in normal rats (Fig. 3G–I).
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Fig. 3. Effect of SAT on pancreatic islet tissue of rats in model and control groups (HE 100). (A) Normal rats treated with vehicle alone; (B) T2DM rats treated with vehicle alone; (C) T2DM rats treated with SAT (80 mg/kg); (D) T2DM rats treated with SAT (160 mg/kg); (E) T2DM rats treated with SAT (320 mg/kg); (F) T2DM rats treated with glibenclamide (600 μg/kg); (G) normal rats treated with SAT (80 mg/kg); (H) normal rats treated with SAT (160 mg/kg); (I) normal rats treated with SAT (320 mg/kg).
4. Discussion The present study was a preliminary assessment of the antihyperglycemic effect of SAT using STZ–NA induced T2DM rats. On one hand, STZ damaged pancreatic β-cells by specifically inducing DNA strand breakage, inhibiting free radical scavenger-enzymes and enhancing the production of the superoxide radical (Maritim et al., 2003; Lenzen, 2007). On the other hand, NA could protect the islet cells from lysis and partially preserve their mitochondrial activity in the presence of reactive oxygen intermediates (Burkart et al., 1992). Hence, STZ–NA-induced diabetes only destructed partial β-cells of islets of langerhans, produced less pancreatic insulin stores and moderate and stable hyperglycemia (Masiello et al., 1998). In our study, the residual of low level insulin in the diabetic rats suggested partial destruction of β-cells. Furthermore, treatment with glibenclamide, a known insulin secretogogue, confirmed the presence of remnant β-cells in the diabetic rats. Therefore, the STZ–NA-induced diabetic model appeared closer to human T2DM, so was useful for pharmacological investigations of new antidiabetic agents. The extract of TCM is traditionally prepared by soaking the raw material in water or ethanol for a long time, so the crude extract commonly has low yield and purity. According to the records in “Chinese Materia Medica”, the root bark of Aralia taibaiensis with the weight from 15 g to 30 g is decocted in water to get the coarse aqueous extract, which has the saponin content of 2.0–5.0% and is used for oral treatment of traumatic injury, rheumatic arthralgia, nephritis edema, hepatitis and diabetes mellitus in folk medicine. In our study, SAT with high content of saponins above 90% is obtained by two-step extraction using ethanol and saturated nbutanol with water for three times, respectively. Furthermore, the
excellent antidiabetic effect of SAT at 80, 160 and 320 mg/kg in T2DM rats has been confirmed. In SAT 13 triterpenoid saponins have been successfully identified by extensive spectral analysis (IR, 1 H-NMR, 13C-NMR, DEPT, DQCOSY, TOCSY, NOESY, HMQC, HMBC, ESI–MS, HR-ESI–MS) and chemical evidence (GC/MS analysis of the acid hydrolysates) in our previous study (Dou et al., 2013). It is known that triterpenoid saponins are poorly absorbed from gastrointestinal tract and have low oral bioavailability because they are easily metabolized to aglycones by the acidic environment of gastric juice and the enzymes generated by the intestinal microbiota (Yan, 2001), so are SAT. Hence, oleanolic acids might be the real active ingredients which are absorbed into blood to educe the pharmacodynamic action after oral administration of SAT. In the diabetic rats, treatment with SAT showed dose- and time-dependent antihyperglycemic activity. At the end of study, there was a 45%, 61% and 68% reduction in FBG, 52%, 101% and 143% increase in FSI in MOD þL, MOD þM and MOD þH groups, respectively. Hence, in comparison with MOD group, HOMA-IR in SAT-treated rats was decreased by 17%, 21% and 23%. The maximum antihyperglycemic effect of SAT was comparable to the standard antidiabetic drug of Gli, which had a 151% increase in FSI and 70% and 24% reduction in FBG and HOMA-IR. Consistently, the islet cells were restored to near normal level upon treatment with high dose of SAT and Gli. The mechanism of the antihyperglycemic effect of SAT might be due to the protective effect on pancreatic islet cells and increase of insulin secretion from the remaining pancreatic β-cells. However, no effect was observed in normal rats, which could be explained by the very significantly (P o0.01) weak potentiation of insulin secretory response at low glucose concentration (Cui et al., 2013).
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The T2DM rats were associated with increased food intake but severe weight loss, which were due to poor glycemic control. Insulin was an important regulator of protein synthesis and proteolysis in skeletal muscle. During insulin resistance or deficiency, the excessive catabolism of protein provided amino-acids for gluconeogenesis and resulted in muscle wasting and weight loss in diabetic rats (Kasetti et al., 2010). Administration of SAT to the diabetic rats prevented the body weight loss and brought the food intake back to normal, which might owe to the protective effect of SAT in controlling muscle wasting i.e. reversal of gluconeogenesis and might also owe to the improvement in insulin secretion and glycemic control. HbA1c is a reliable index of glycemic control in diabetes. High level of HbA1c predicts high risks for the development and/or progression of retinopathy, nephropathy, neuropathy (Calisti and Tognetti, 2005). In our study, increased level of HbA1c in diabetic rats indicated the occurrence of glycosylation due to hyperglycemia. Administration of SAT and glibenclamide to the diabetic rats significantly reduced HbA1c levels. The explanation for this was assumed to be an improvement in insulin secretion. The increased level of insulin was directly proportional to FBG level, which was consistent with the results in SAT-treated diabetic rats. This suggested that SAT might have the potential to ameliorate the development of diabetes associated complications. In addition to the antihyperglycemic effect, SAT was also able to improve the lipid abnormalities in diabetic rats. High levels of TC and most importantly, VLDL-C are the predictors of atherosclerosis (Temme et al., 2002), but an increase in HDL level is associated with a decrease in coronary risk (Kasetti et al., 2010). In our study, the calculated atherogenic index in diabetic group was significantly (P o0.05) higher than that in normal group, but treatment with SAT effectively corrected the status of dyslipidemia via a dose-dependent manner. The reason for this might be directly attributable to the improvement in insulin, and besides, other lipid-lowing mechanism, such as repressing some key enzymes in cholesterol and/or fatty acids biosynthesis might also be involved in the hypolipidemic activity of SAT. Regulation of plasma or tissue lipid levels led to a decrease in the risk of micro- or macrovascular disease and related complications (Sakatani et al., 2005). Therefore, the hypolipidemic effect of SAT in diabetic rats supported its ability to prevent the CHD diseases associated with diabetes. ALT and AST are reliable markers of liver function (Kasetti et al., 2010). Elevated levels of ALT and AST in serum indicated that the liver was necrotized (Jasmine and Daisy, 2007). Kidney is playing a key role in removing the metabolic waste such as creatinine and urea from body, thereby helping to maintain body homeostasis of above substances (Ramachandran et al., 2012). Increased levels of urea and creatinine, as well as decreased level of TP in diabetic rats suggested impaired renal dysfunction. Administration of SAT and glibenclamide to diabetic rats significantly reduced levels of ALT, AST, creatinine and urea and slightly increased TP level, which represented the preventive action of SAT on liver and kidney damages in diabetic condition. Besides, large SD of ALT and AST also became smaller in SAT-treated model groups. Considering the large individual difference in model rats, increasing the sample size might be helpful to improve the accuracy of estimation and the power of test statistically. During T2DM, persistent hyperglycemia impairs prooxidant/ antioxidant balance, reduces antioxidant levels and increases production of reactive oxygen species (Aragno et al., 2004; Nishikawa et al., 2000). MDA, GSH (a major endogenous antioxidant) and SOD (a scavenger of free radicals), as the oxidative stress biomarkers, involve in the development of diabetic complications (Maritim et al., 2003). In our study, oral administration of SAT dose-dependently reduced serum MDA level and increased the levels of GSH and SOD in T2DM rats, but had no effect on
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normal rats. The results strongly supported a high antioxidant activity of SAT in diabetic condition and hence, SAT possessed a potential to reduce or prevent the diabetic micro and macrovascular complications.
5. Conclusions Our study confirmed that SAT had an excellent antidiabetic effect, which could be explained, at least in part, by its antihyperglycemic, hypolipidemic and antioxidant activities in T2DM rats. SAT also had the potential to ameliorate diabetic complications. It could prevent liver and kidney damages and decrease high levels of HOMA-IR and atherogenic index of the diabetic rats. However, SAT had no effect on normal rats, so it cannot lead to hypoglycemia in normal rats. All these findings provide scientific basis for the use of Aralia taibaiensis as a promising traditional Chinese medicine in the therapy of diabetes mellitus and its complications. Hence, further research is needed to develop SAT into a new drug for treating diabetes mellitus.
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