Anti-hyperglycemic And Hypolipidemic Effects Of Cistanche Tubulosa

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Anti-hyperglycemic and hypolipidemic effects of Cistanche tubulosa in type 2 diabetic db/db mice

Wen-Ting Xiong, et al

Abstract

Ethnopharmacological relevance: The dried succulent stem of Cistanche tubulosa (Schenk) R. Wight is one component of traditional Chinese medicine prescriptions for diabetes. However, there have been no modern scientific reports to confirm this traditional claim for the Cistanche species until now. Thus, we investigated the effects of Cistanche tubulosa on glucose homeostasis and serum lipids in male BKS. Cg-Dock7m þ/þ Leprdb/J (db/db) mice, a model of type 2 diabetes.

Materials and methods: The verbascoside and echinacoside contents of Cistanche tubulosa powder were evaluated using HPLC. The total phenolic content, polysaccharide content, and antioxidant activity ofCistanche tubulosa powder were also evaluated. Then, different doses of Cistanche tubulosa (equivalent to 120.9, 72.6, or 24.2 mg verbascoside/kg) were administered orally once daily for 45 days to male db/db mice. Age-matched db/þ mice were used as normal controls. Bodyweight, fasting blood glucose, postprandial blood glucose, and insulin tolerance tests were measured during the experiment. At the time of sacrifice, blood was collected for measurement of insulin level, the homeostatic model assessment of insulin resistance (HOMA-IR), and total cholesterol, triglyceride, HDL-c, LDL-c, and VLDL-c levels; liver and muscle were harvested for measurement of glycogen levels.

Results: Cistanche tubulosa significantly suppressed the elevated fasting blood glucose and postprandial blood glucose levels, improved insulin resistance and dyslipidemia, and suppressed body weight loss in db/db mice. However, Cistanche tubulosa did not significantly affect serum insulin levels or hepatic and muscle glycogen levels.

Conclusion: This study provides scientific evidence for the traditional use of Cistanche tubulosa to treat diabetes, suggesting that Cistanche tubulosa has the potential for development into a functional food ingredient or drug to prevent hyperglycemia and treat hyperlipidemia.

Keywords: Cistanche tubulosa, Type 2 diabetes, Anti-hyperglycemic effect, Hypolipidemic effect

1. Introduction

Diabetes mellitus (DM) is a progressive and complex metabolic disorder that is mainly characterized by abnormally high blood glucose levels (hyperglycemia) (Tiwari and Rao, 2002; Stumvoll et al., 2005; Nathan et al., 2009). Approximately 90% of the 347 million patients with diabetes worldwide have type 2 (non-insulin-dependent) DM (Danaei et al., 2011). Three major metabolic defects consistently contribute to hyperglycemia in patients with type 2 DM: deficient insulin secretion, a high hepatic glucose output, and insulin resistance (Bavenholm et al., 2001). Type 2 DM can shorten life expectancy and represents a significant medical and economic burden (Gakidou et al., 2011)

The pathogenesis of type 2 DM is multi-factorial and has been linked to both genetic and lifestyle factors (Stumvoll et al., 2005). Lifestyle interventions combined with oral anti-hyperglycemic drugs are the typical method of managing type 2 DM. Oral drugs exert an anti-hyperglycemic effect by either ameliorating the three major metabolic defects that cause hyperglycemia or by reducing the postprandial increase in glycemic levels through impeding carbohydrate digestion and absorption in the gut.

Although multiple classes of effective drugs have become recognized choices for medical therapy in most type 2 DM patients, several of these synthetic drugs have serious side effects. For example, rosiglitazone and pioglitazone increase the incidence of myocardial infarction, congestive heart failure, and peripheral edema, and are associated with an increased risk of carcinogenesis and even death after long-term use (Nissen and Wolski, 2007; Zhang et al., 2007; Home et al., 2009). Therefore, a critical need exists for the discovery of alternative agents that would exert antihyperglycemic activity but exert fewer side effects. Compared to synthetic chemicals, naturally derived anti-hyperglycemic agents, such as herbs and traditional medicines, have a long history of usage and may represent generally safe potential sources of novel anti-hyperglycemic drugs (Yeh et al., 2003).

Cistanche, a genus of parasitic plants that belong to the Orobanchaceae family, is commonly used as a traditional medicine to cure renal disorders and infertility, according to the Chinese Pharmacopeia (Chinese Pharmacopoeia Commission, 2010). As recorded in Chinese medicine books such as BenCao TuJing and Nature of Drug, the Cistanche genus has also been boiled with mutton, potato, or rice to produce a tonic food to treat overstrain-induced impairment for hundreds of years, suggesting that the Cistanche genus is safe to take orally.

In traditional Chinese medicine, DM is referred to as the symptom “Xiao Ke”, and has been treated for centuries using Chinese herbs. The dried succulent stem of the Cistanche species is traditionally used as a component of nine anti-diabetes prescriptions (“Xiao Ke Fang”) in the traditional Chinese medicine book Sengji Zonglu. However, until now, there have been no modern scientific reports to confirm this traditional claim for the Cistanche species.

Cistanche tubulosa (Schenk) R. Wight, a Cistanche species that parasitizes the roots of the Tamarix plant – which is mainly cultivated around the Taklamakan desert – has recently been used to produce an instant tea for use as a functional food (DaZhongWeiShengBao, 2010; Baidu Pedia, 2011).

Cistanche tubulosa contains appreciable amounts of phenylethanoid glycosides (PhGs), iridoids, lignans, alditols, oligosaccharides, polysaccharides, and volatile compounds like palmitic acid and linoleic acid (Song et al., 2000; Jiang and Tu, 2009). The major active components of the Cistanche species are thought to be PhGs (Xie et al., 2006; Jiang and Tu, 2009). Cistanche tubulosa and its components, such as PhGs, polysaccharides, and polyphenols, have been reported to display good anti-oxidative (Deng et al., 2004; Pellati et al., 2004; Frum et al., 2007; Sui et al., 2011; Wang et al., 2011), anti-hypertension (Ahmad et al., 1995) and hypo cholesterol (Shimoda et al., 2009) effects. Numerous reports have indicated that PhGs, polysaccharides, and polyphenols exert anti-hyperglycemic effects or have a beneficial influence in type 2 DM through free radical scavenging and the modulation of glucose-induced oxidative stress (Pinto et al., 2010; Saltiel, 2001; Gulati et al., 2012). These components and bioactivities ofCistanche tubulosa suggest that it may potentially be used for the treatment of type 2 DM. Due to the absence of reports describing the effects of Cistanche tubulosa in the treatment of DM, we thought it worthwhile to investigate the anti-diabetic-related activity of Cistanche tubulosa. Thus, we examined the effects ofCistanche tubulosa on hyperglycemia and dyslipidemia in BKS db/db mice (BKS.Cg-Dock7m þ/þ Leprdb/J, Jackson Laboratory stock number 00642), a model of type 2 DM that displays many characteristics of human DM including progressive hyperglycemia, insulin resistance and obesity (Hummel et al., 1966; Nishina et al., 1994; Uchida et al., 2005; Srinivasan and Ramarao, 2007).

2. Materials and methods

2.1. Chemicals

Verbascoside (purity 493%) and echinacoside (purity 493%) were purchased from the National Institutes for Food and Drug Control (Beijing, China). HPLC grade methanol was purchased from Sigma-Aldrich (St. Louis, MO, USA). Acarbose was purchased from Bayer (West Haven, CT, USA). Purified water was produced using a Millipore Milli-Q Integral 3 System and Q-POD Milli-Q System (Bedford, MA, USA).

2.2. Cistanche tubulosa powder preparation

The dried succulent stems of Cistanche tubulosa (Sample Code 109092, cultivated in Hetian, Xinjiang, China) were purchased from Xinjiang Guru Biology Co. Ltd. (Hetian, Xinjiang, China). The dried roots were crushed into a powder, sieved through a 200-mesh sieve, mixed thoroughly, sealed in small packages to isolate the powder from light and humidity, and stored at 4 1C.

2.3. Identification of Cistanche tubulosa

The dried succulent stem of sample 109092 was identified from their morphological characteristic by Prof. DeXiang Gu and Prof. Xin Liu, who are experts on Cistanche species. The samples were further identified by DNA sequencing. Total genomic DNA was isolated from sample 109,092 using the AxyPrep™ Multisource Genomic DNA Kit (Axygen, CA, USA). The ITS region containing the 5.8S rDNA fragment was amplified using primer sequences of ITS1/ ITS2 5′ CGT AAC AAG GTT TCC GTA GAA 3′ and 5′ TTA TTG ATA TGC TTA AAC TCA GCG GG 3′ (BGI Genomics, Shenzhen, China), with a method described by Schneeweiss et al. (Schneeweiss et al., 2004). The amplified DNA was sequenced by BGI (BGI Genomics, Shenzhen, China), and the result was compared with the Cistanchetubulosa 5.8s rDNA sequence on GenBank.

2.4. HPLC analysis

Two PhGs, echinacoside and verbascoside, were used as quality standards for Cistanche tubulosa, according to the Chinese Pharmacopeia (Chinese Pharmacopoeia Commission, 2010). HPLC is the most frequently used one for the qualitative and quantitation of PhGs. Thus, the verbascoside and echinacoside contents ofCistanche tubulosa were determined following our previously published HPLC method (Zhao et al., 2011). Briefly, 0.1 g Cistanchetubulosa powder was added to 10 ml purified water and intensively vortex-stirred on an orbital shaker (Vortex-Genie 2, Scientific Industries, Bohemia, New York, USA) for 10 min at room temperature. The solid was re-extracted twice under the same conditions. The extracts were combined, adjusted to 50 ml, and centrifuged at 12,000g for 5 min. The supernatant was filtered through a 0.45 μm-pore polytetrafluoroethylene (PTFE) syringe filter (Pall, Ann Arbor, MI, USA). Then, 20 μl of the supernatant was injected into the HPLC System (Binary HPLC Pump 1525, Refractive Index Detector 2414, Photodiode Array Detector 2996; Waters, Milford, MA, USA) which included a Symmetry C18 column (4.6 mm  250 mm, 5 μm; Waters) and a Guard column (3.9 mm 20 mm, 5 mm; Waters); the system was controlled using Empower Pros software (Waters, Milford, MA, USA) with a flow rate of 1 ml/min at 30 1C. The mobile phase was made by mixing solvent A (0.5% (V/V) acetic acid aqueous solution) and solvent B (methanol) to form a linear gradient of 25–40% B in 0–10 min. The retention times for echinacoside and verbascoside were 10.4 min and 13.6 min, at a maximum UV absorbance of 330 nm.

2.5. Analysis of the total phenolic content, polysaccharide content, and antioxidant activity of Cistanche tubulosa

2.5.1. Sample preparation

Cistanche tubulosa extract was prepared with purified water using our previously published method (Zhao et al., 2011) with minor modifications, for the determination of total phenolic content, polysaccharide content, and antioxidant activity (Fig. 1). Briefly, 1 g Cistanche tubulosa powder was added into 15 ml purified water at room temperature, intensively vortex-stirred using an orbital shaker for 5 min, and the supernatant was recovered by centrifugation at 3000g for 15 min. The precipitation was re-extracted twice under the same condition. The combined supernatant was adjusted to 50 ml with purified water and passed through a 0.45 μm-pore PTFE syringe filter (Pall) before analysis.

2.5.2. Analysis of total phenolic content

Total phenolic content was determined using Folin–Ciocalteu reagent and gallic acid as a reference compound, as described by Singleton and Rossi (Dubois et al., 1956; Singleton and Rossi, 1965) with minor modifications. Briefly, 0.1 mL of Cistanche tubulosa extract was mixed with 0.9 mL purified water and 5 mL Folin– Ciocalteu reagent (previously diluted 10-fold with distilled water), allowed to stand at 25 1C for 3.5 min, then 4 mL of sodium bicarbonate (7.5% w/v) solution was added to the mixture and incubated at 25 1C for 60 min. Absorbance was measured at 765 nm using a spectrophotometer (Perkin Elmer λ 25 UV–visible, Waltham, MA, USA). The total phenolic content was determined using a standard curve created with gallic acid (0–0.6 mg/mL) as a standard. Results were calculated as milligram gallic acid equivalents (GAE)/1 g Cistanche tubulosa crude powder. All tests were replicated four times.

2.5.3. Analysis of polysaccharide content

The polysaccharide content of Cistanche tubulosa was measured using the phenol–sulfuric acid method (Dubois et al., 1951; Dubois et al., 1956). Briefly, 1 ml of appropriately diluted Cistanche tubulosa extract and 0.5 mL phenol solution were placed into screw cap tubes, which were capped and vortex-stirred. Then, 2.5 ml of concentrated sulfuric acid was added directly to the liquid surface within 5 s. The tubes were then closed, vortexstirred for 5 s, incubated for 2 min at 100 1C, and allowed to cool down to room temperature before analysis. Absorbance was measured at 490 nm using a spectrophotometer (Perkin Elmer λ 25 UV–visible) against a 70% methanol blank. The polysaccharide content was determined using a standard curve created with glucose (0–100 mg/mL) as a standard. Results were calculated as % polysaccharide/Cistanche tubulosa crude powder. All tests were replicated three times.

2.5.4. Analysis of total antioxidant activity

The antioxidant activity of Cistanche tubulosa extract, acarbose, and rosiglitazone was determined by the 2,2′-amino-di(2-ethylbenzothiazoline sulfonic acid-6)ammonium salt (ABTS) assay and ferric ion (Fe3þ) reducing antioxidant power assay (FRAP) using commercial kits (Beyotime, Haimen, China). With the appropriate oxidants, ABTS can be oxidized into green ABTS  þ; antioxidants can inhibit this process. The total antioxidant activity of antioxidants can be reflected by the absorbance of ABTS  þ at 734 nm. Absorbance was measured at 734 nm (ABTS) using a microplate reader (Epoch, Bio-Tek, Richmond, VT, USA), with Trolox (ABTS) as an antioxidant standard. The results of the ABTS assay were calculated as mM Trolox Equivalent Antioxidant Capacity (TEAC) /1 g powder.

Under acidic conditions, antioxidants can restore ferrictripyridyltriazine (Fe3þ-TPTZ) into blue Fe2þ-TPTZ. The total antioxidant activity of antioxidants can be reflected by the absorbance of Fe2þ-TPTZ at 593 nm. Absorbance was measured at 593 nm (FRAP) using a microplate reader (Epoch), with FeSO4 (FRAP) as the antioxidant standard. The results of the FRAP assay were calculated as mM FeSO4 Equivalent Antioxidant Capacity (FEAC) /1 g powder. All tests were replicated four times.

2.6. Effects of Cistanche tubulosa on hyperglycemia in db/db mice

2.6.1. Animals and treatment groups

Male BKS.Cg-Dock7m þ/þ Leprdb/J (db/db) mice and their age-matched non-diabetic heterozygote littermates (db/þ) were kindly provided at 5–6 weeks of age by the National Resource Center for Mutant Mice Model Animal Research Center of Nanjing University (Nanjing, China). The animal care and experimental procedures were approved by the Laboratory Animal Ethics Committee of the School of Life Sciences, Sun Yat-sen University (approval code 0046500), and were performed according to the Regulations for Animal Experiments of China. The animals were housed in a 2272 1C specific pathogen-free (SPF)-grade room at 6075% humidity under a 12 h light-dark cycle, and fed a purified AIN-93G diet (Trophic Animal Feed High-tech Co., Ltd., Nantong, China) and provided purified water ad libitum.

Seven days after arrival, the non-diabetic db/þ mice were given purified water (vehicle) as a normal control group for the diabetic groups (n¼7), while the diabetic db/db mice were weighed and randomly divided into 6 groups (n¼8) to investigate the effects ofCistanche tubulosa in type 2 diabetic db/db mice (Table 1). The db/þ mice were orally administered the vehicle, and db/db mice were orally administered the vehicle, rosiglitazone (0.52 mg/ kg/day), acarbose (19.5 mg/kg/day), or three doses of Cistanchetubulosa (equivalent to 120.9, 72.6 or 24.2 mg verbascoside/kg/day and referred to as CT120.9, CT72.6 or CT24.2, respectively) for a duration of 45 days. The doses of Cistanche tubulosa were set according to the crude powder dose recommended by the Pharmacopeia of the People's Republic of China (PPRC); Cistanchetubulosa doses of 120.9, 72.6, and 24.2 mg verbascoside/kg/day corresponding to 5-fold, 3-fold, and 1-fold dilutions of the crude powder dose recommended by the PPRC. All test samples for oral administration were dissolved in purified water at suitable concentrations in order to administer similar volumes of test samples to mice with the same body weight. The total volumes administered were o1 mL, and the test samples were orally administered to each group of mice every day between 16:30 and 17:30 for 45 days. Bodyweight and blood glucose levels were monitored throughout the experimental period. At the end of the experiment (day 50), the mice fasted for 14 h, and blood samples were collected from the ophthalmic vein, incubated at room temperature for 30 min, centrifuged and the serum samples were stored at 70 1C for further assay. Subsequently, the mice were euthanized. The liver and skeletal muscle were harvested, washed in ice-cold saline to remove the blood, snap-frozen in liquid nitrogen, and stored at 70 1C for further assay.

2.6.2. Assay of fasting blood glucose level (FBG), inter-peritoneal insulin tolerance test (ITT), and fasting serum insulin level (FINS)

Fasting blood glucose levels were measured after a 14 h fast before the start of the animal experiment (0 days), on day 10 and every 5 days during days 20–45 of the experiment using a blood glucose monitoring system (ACCU-CHEK Active, Roche Diagnostics GmbH, Mannheim, Germany). The percentage increases in the FBG values (FBG percentage increase values: FBG-increase %) on day x (day 20–45) were calculated using (BGday xBGday 0)/BG0 day 100, to represent the increases in the FBG values for each group compared to their initial FBG values. The postprandial blood glucose levels were determined 120 min after oral administration of glucose (2 g/kg BW) on day 40.

The ITT was carried out on day 45 of the experiment, following an overnight fast (14 h). The blood glucose levels of tail vein blood were measured before insulin injection (0 min) and at 30 min, 60 min after inter-peritoneal injection of insulin (0.1 IU/kg BW). Glucose responses during the ITT were expressed as integrated areas under curves for insulin (AUCinsulin), calculated using the trapezoid rule (Vishwakarma et al., 2003). At the end of the experiment, the fasting serum insulin levels were analyzed using the rat/mouse insulin ELISA kit from Millipore (Bedford, MA, USA); the serum samples were prepared following the kit's instructions. The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index was calculated using the following formula: HOMA-IR¼ fasting insulin (ng/ml)  fasting blood glucose (mmol/L)/22.5 (Matthews et al., 1985).

2.6.3. Assay of serum lipid levels

The serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), and triglyceride (TG) concentrations were determined using commercial enzymatic kits (Biosino Biotechnology & Science Inc., Beijing, China). Low-density lipoprotein cholesterol (LDL-c) levels were calculated as TC-TG/5 – HDL-c, whereas very-low-density lipoprotein cholesterol (VLDL-c) levels were calculated as TG/5, according to Friedewald's equation (Friedewald et al., 1972). The atherogenic index was calculated as (TCHDL-c)/HDL-c.

2.6.4. Assay of liver and skeletal muscle glycogen levels

Frozen liver and skeletal muscles were processed for glycogen analysis immediately after removal from the 70 1C freezers. The glycogen contents of the liver and skeletal muscles were measured using a commercial glycogen kit (Jiancheng, Nanjing, China) based on Keppler and Decker's method (Keppler and Decker, 1974). The absorbance of the samples at 505 nm was measured after cooling the tubes to room temperature. The amount of glycogen in each tissue sample was expressed as mg glucose per g of tissue.

2.7. Statistical analysis

All results are expressed as the mean7SEM and were analyzed with ANOVA of statistical packages Social Science Software (SPSS 16.0, Chicago, IL, USA) or GraphPad Prism 5 (La Jolla, CA, USA). The HOMA-IR indexes were also assessed using the two-sample independent t-test after logarithmic transformation. The AUC values were calculated using GraphPad Prism 5. P values of less than 0.05 were considered to indicate statistically significant differences.

3. Results

3.1. Identification of Cistanche sample

The sequence alignment results indicate that the amplified sequence is the same with Cistanche tubulosa genes for 5.8S rDNA partial and complete sequence (614 nucleotides; Gen-Bank accession number AB217871). On the basis of its morphological characteristics and 5.8S rDNA gene sequence, sample 109,092 is identified as Cistanchetubulosa.

3.2. Quantitative determination of the verbascoside content and echinacoside content of Cistanche tubulosa

The contents of verbascoside and echinacoside in Cistanchetubulosa were quantified by using the appropriate standards. The verbascoside and echinacoside contents of Cistanche tubulosa were 2.66% and 11.59%, respectively. The HPLC chromatogram ofCistanche tubulosa extract is shown in Fig. 2.

3.3. Total phenolic content, polysaccharide content, and antioxidant activity of Cistanche tubulosa

The total phenolic content of Cistanche tubulosa was 66.297 0.44 mg GA/g. The polysaccharide content of Cistanche tubules was 10.1570.26%. The ABTS and FRAP assays indicated that the antioxidant activities were 1.3970.06 mM TEAC/g and 1.617 0.04 mM FEAC/g for Cistanche tubulosa, 0.1570.01 mM TEAC/g and 0.1270.01 mM FEAC/g for acarbose, and 0.1670.01 mM TEAC/g and 0.1370.01 mM FEAC/g for rosiglitazone, respectively.

3.4. Anti-hyperglycemic effects of Cistanche tubulosa in db/db mice

Our results showed that Cistanche tubulosa could suppress the elevated fasting blood glucose levels and elevated postprandial blood glucose levels, increase insulin sensitivity, and attenuate the loss in body weight (BW) of diabetic db/db mice. Generally, Cistanche tubulosa was more effective at decreasing hyperglycemia than acarbose during the experiment, and furthermore, Cistanche tubulosa was superior to acarbose for improving insulin resistance (the p values are indicated below).

3.4.1. Effects of Cistanche tubulosa on body and liver weight

In the diabetic control group, BW constantly increased within the first 25 days. However, with the progression of diabetes, the BW of the diabetic control mice decreased slightly by day 30 (Supplementary Table 1). In the Cistanche tubulosa groups, all three doses of Cistanche tubulosa reduced the rate of weight loss compared to diabetic control mice (without significance, Supplementary Table 1). In the positive control group, rosiglitazone led to a significant increase in body weight from day 20 compared to the diabetic control group (po0.05). In contrast, acarbose slowed the rate of weight loss compared to the diabetic control group. In the normal control group, bodyweight constantly increased during the experiment and was significantly lower than the diabetic control group (po0.001).

At the end of the experiment, liver weight and the liver/body weight ratio were only significantly increased in the rosiglitazone group (po0.001) compared to the diabetic control group.

3.4.2. Effect of Cistanche tubulosa on blood glucose levels

Cistanche tubulosa displayed anti-hyperglycemic effects in db/db mice after 20 days' oral administration and could control FBG level and FBG-increase % value for a long period of time on db/db mice in a dose-dependent manner, as indicated by significantly decreased fasting and postprandial blood glucose levels (Fig. 3, Table 2). In the diabetic control group, the FBG level increased constantly during the experiment and was about 6 times higher than the normal control group on day 45 (28.3071.027 mmol/L vs. 4.6670.350 mmol/L; po0.001). All three doses of Cistanche tubulosa significantly inhibited the tendency of FBG to increase in db/db mice during the entire experiment, with CT120.9 showing the strongest hypoglycemic effect (day 20, 35, and 45: po0.001; day 40: po0.05 vs. diabetic control). In the positive control groups, rosiglitazone (0.52 mg/kg) and acarbose (19.5 mg/kg) also significantly inhibited the tendency of FBG to increase in db/db mice during the entire experiment (day 20 to 45: po0.001, day 10: po0.05, rosiglitazone vs. diabetic control; day 35: po0.001, day 40: po0.01, day 20 and 45: po0.05, acarbose vs. diabetic control). However, acarbose (19.5 mg/kg) was less effective than rosiglitazone (day 20: po0.05; day 30: po0.01; day 35–45: po0.001) and CT120.9 (day 45: po0.001). In the normal control group, the FBG level remained constant during the entire experiment and was significantly lower than that of the diabetic control group (po0.001).

After administration for 20 days, Cistanche tubulosa significantly inhibited the increase in blood glucose levels in db/db mice (Fig. 3). The FBG-increase % values of the diabetic control group and acarbose (19.5 mg/kg BW) group increased during the experiment. The FBG-increase % values of the three Cistanche tubulosa groups only increased slightly at day 30. The FBG-increase % values of the CT120.9 group remained constant between day 30 and day 45, while CT72.6 and CT24.2 increased insignificantly (p40.05) by day 45. By the end of the experiment, the three doses of Cistanchetubulosa (CT120.9, CT72.6, and CT24.2) significantly suppressed the increase in the FBG-increase % values by 181.5%, 151.72%, and 123.58% in comparison with the diabetic control group (PO 0.001), respectively. From day 20 to day 40, the FBG-increase % values of the three Cistanche tubulosa groups were lower or similar to the acarbose group (p40.05). At day 45, the FBG-increase % of the CT120.9 group was significantly lower than the acarbose group (po0.01).

Cistanche tubulosa displayed dose-dependent hypoglycemic effects (Table 2). At the end of the experiment, the FBG levels of the CT120.9 group were significantly lower than the diabetic control, acarbose, and two other Cistanche tubulosa groups (CT120.9 vs. acarbose, CT72.6, and CT24.2, po0.01; CT120.9 vs. diabetic control, po0.001; Table 2). By the end of the experiment, the FBG levels of the three Cistanche tubulosa groups (CT120.9, CT72.6, and CT24.2) had increased by 53.41%, 83.19%, and 111.33%, respectively, compared to their initial FBG levels, while the FBG levels of the diabetic control group and acarbose group increased by 234.91% and 150.76%. Of the three doses of Cistanche tubulosa tested, only CT120.9 led to consistently lower FBG levels during the entire experiment. CT72.6 and CT24.2 were less effective at reducing the FBG level than CT120.9 (Table 2, Supplementary Fig. 1). The FBG levels of the CT120.9 group only increased from day 30, while the FBG levels of the other two Cistanche tubulosa groups and the acarbose group increased more rapidly. From day 10 to 40, the FBG levels of the three Cistanche tubulosa groups were lower or similar to the acarbose group (19.5 mg/kg BW); these differences were not statistically significant (p40.05).

On day 40, CT120.9 significantly suppressed the elevation in the postprandial blood glucose level ( p o0.05, Table 2) 2 h after a single oral dose of glucose (2 g/kg). The effect of CT120.9 on the postprandial blood glucose level in db /db mice was comparable with 19.5 mg/kg acarbose ( p 40.05). The effect of CT72.9 and CT24.2 were less effective than rosiglitazone ( p o0.001).

3.4.3. Effect of Cistanche tubulosa on fasting serum insulin level and inter-peritoneal insulin tolerance

Results of the FINS showed that after oral administration for 45 days, CT120.9 insignificantly elevated the serum insulin level ( p 40.05) in db /db mice by 40.73%, whereas rosiglitazone significantly elevated the serum insulin level by 58.90% ( p o0.05 vs. diabetic control; Table 3).

HOMA-IR index was calculated to quantitatively assess the contributions of insulin resistance and deficient β-cell function to the fasting hyperglycemia. Compared to the diabetic control group, CT120.9 insignificantly improved the HOMA-IR systemic insulin sensitivity index by 15.30% ( p 40.05), whereas rosiglitazone significantly improved the HOMA-IR index by 35.30% (PO 0.001 vs. diabetic control; Table 3). The ITT was used to evaluate the effect of Cistanche tubulosa on insulin sensitivity after exogenous insulin injection. Results showed that CT120.9 improved the insulin response in diabetic db /db mice. The effect of CT120.9 was comparable with rosiglitazone and was superior to acarbose. CT120.9 and rosiglitazone significantly reduced the blood glucose levels at 0 min (before insulin injection, p o0.001 vs. diabetic control) and 30 min ( p o0.01 for CT120.9, p o0.001 for rosiglitazone) after the injection of insulin (Fig. 4). CT120.9 and rosiglitazone decreased the AUCinsulin by 27.92% and 43.89% compared with diabetic control. There was no significant difference between blood glucose levels in the CT120.9 and rosiglitazone groups at any time point during the ITT. The blood glucose levels of the CT120.9 group were significantly lower than acarbose at 0 min (before insulin injection, p o0.01: CT120.9 vs. acarbose), 30 min ( p o0.01: CT120.9 vs. acarbose), and 60 min ( p o0.05: CT120.9 vs. acarbose) after the injection of insulin (Fig. 4). CT120.9 decreased the AUCinsulin by 28.79% compared with acarbose. Acarbose, CT72.6, and CT24.2 had no significant effects on the AUCinsulin or blood glucose levels at any time point of the ITT. Both the FIN and ITT results of the normal control group were significantly lower than that of the diabetic control group ( p o0.05: FIN; p o0.001: ITT).

3.4.4. Effect of Cistanche tubulosa on liver and skeletal muscle glycogen levels

The glycogen levels in various tissues are a direct reflection of insulin sensitivity, as insulin promotes intracellular glycogen deposition. Rosiglitazone significantly lowered the liver glycogen level by 59.61% (po0.001), and increased the muscle glycogen level by 33.93% ( p o0.05 vs. diabetic control). CT24.2 lowered the liver glycogen level by 30.41% ( p o0.05 vs. diabetic control). The lower doses of Cistanche tubulosa had no significant impact on either liver or skeletal muscle glycogen levels.

3.5. Hypolipidemic effects of Cistanche tubulosa in db/db mice

To evaluate the effects of Cistanche tubulosa on dyslipidemia, we determined the levels of total cholesterol, TG, HDL-c, LDL-c, and VLDL-c in db /db mice. Our results demonstrate that Cistanche tubulosa can improve dyslipidemia by increasing HDL-c levels, reducing TG, LDL-c, and VLDL-c levels in addition to reducing cholesterol levels in db/db mice. CT120.9 (po0.05) and CT24.2 (po0.001) significantly decreased the TC level in diabetic db/db mice (Table 3) compared to diabetic control. Of all experimental groups, only CT24.2 significantly decreased the levels of TG (po0.05) and VLDL-c (po0.05) compared to diabetic control. All three doses of Cistanche tubulosa significantly lowered the LDL-c level (po0.01 for CT120.9, po0.05 for CT72.6, po0.001 for CT24.2, vs. diabetic control). All three doses of Cistanche tubulosa(CT120.9, CT72.6, and CT24.2) insignificantly elevated the HDL-c level by 9.78%, 7.32%, and 7.78% (p40.05 vs. diabetic control).

Of these lipid parameters, a low HDL-c/TC level is the most significant independent risk factor for coronary artery disease. CT120.9 (po0.05) and CT24.2 (po0.001 vs. diabetic control) significantly elevated the HDL-c/TC level. The atherogenic index, which is a risk factor for coronary artery disease, was decreased by all three doses of Cistanche tubulosa (po0.001 for CT120.9 and CT24.2, po0.01 for CT72.6 vs. diabetic control).

In the positive control group, rosiglitazone significantly elevated the levels of TC (po0.001) and LDL-c (po0.01) compared to diabetic control. Rosiglitazone also significantly elevated the HDLc level by 57.32% (po0.001) but had no beneficial effect on the HDL-c/TC level or atherogenic index. Acarbose did not have any significant effect on the serum lipid indexes in db/db mice.

4. Discussion

The present study was designed to investigate the potential antihyperglycemic and hypolipidemic activity of Cistanche tubulosa.

The results of the in vitro assay indicated that Cistanchetubulosa contains an appreciated amount of phenolic and polysaccharides, and displays good anti-oxidant activity. The total phenolic content of Cistanche tubulosa is lower than the total phenolic content of tea (Ankolekar et al., 2011) and coffee (Chu et al., 2011), and higher than many other hyperglycemic herbs such as cranberry (Pinto et al., 2010), seaweed (Nwosu et al., 2011), chickpea (Sreerama et al., 2012) and some of the traditional medicinal plants used in the management of type 2 DM (Gulati et al., 2012). The polysaccharide content of Cistanche tubulosa is lower than the polysaccharide content of Panax ginseng (Wang et al., 2011) and tea (Chu et al., 2011), and higher than many other anti-hyperglycemic herbs such as Aloe vera L. (Hu et al., 2003), jujuba (Sun et al., 2011), Polyporus umbellatus (Tian et al., 2007), Rheum palmatum (Ni et al., 2007) and some of the traditional medicinal plants used in the management of type 2 DM (Gulati et al., 2012). Plants with a high content of antioxidants, such as polyphenols and phenylethanoid glycosides (PhGs), are frequently identified to possess anti-diabetes activity, which is due at least partially to their ability to decrease oxidative stress (Pinto et al., 2010; Saltiel, 2001; Gulati et al., 2012). Our anti-oxidative assay result is consistent with previous reports (Arthur et al., 2011; anti-Bao et al., 2010; Funes et al., 2009) which indicated that Cistanchetubulosa possesses significant antioxidant properties. The TEAC value and FRAP value of Cistanche tubulosa were higher than green cabbage, carrot, cucumber, green lettuce, celery (Pellegrini et al., 2003), and some other plants with the good anti-oxidant effects that used to treat diabetes (Rahimi et al., 2005), and was much higher than acarbose and rosiglitazone. Previous clinical trials have demonstrated that α-lipoic acid (Konrad et al., 1999) or other antioxidants (Paolisso et al., 1992) could improve insulin sensitivity in insulin-resistant and/or diabetic patients. Thus, Cistanche tubulosa is a source of antioxidants that may exert protective effects against diabetes and long-term diabetes complications.

Cistanche species have been used as tonic food or medicine for hundreds of years, suggesting that they are safe to take orally. A previous report of 14 days acute toxicity test showed Cistanchetubulosa extract belongs to actual nontoxic: the Maximum Tolerated Dose (MTD) of the Cistanche tubulosa extract was found to be 410 g/kg in mice. No mortality was recorded throughout 14 days' monitoring, and tested mice of each group did not show any overt signs of toxicity during 24 h and 14 days observation (Zhang et al., 2012). The highest dose in our experiment, 5.084 g CTAE /kg BW/ day, which is equivalent to 9.10 g crude powder /kg in mice, is much lower than the MTD. Thus, the dose is safe in our experiment.

Type 2 DM is one of the most prevalent and serious metabolic diseases in the world and is mainly characterized by hyperglycemia. In this study, we adopted BKS.Cg-Dock7m þ/þ Leprdb/J (db/db) mice as a model of type 2 DM to investigate the effects ofCistanche tubulosa on glucose and lipid metabolism in vivo. db/db mice are a well-accepted genetic rodent model of lifestyle-related metabolic diseases, which is especially useful for evaluating anti-diabetic and anti-obesity agents. This strain of mice carries a mutation in the leptin receptor gene, displays hyperglycemia, hyperlipidemia, insulin resistance, and obesity by 6–7 weeks of age (Hummel et al., 1966; Nishina et al., 1994; Uchida et al., 2005; Srinivasan and Ramarao, 2007).

With the progression of diabetes, the slight decrease in body weight of the diabetic control group (Supplementary Table 1) might be due to the decreased plasma insulin level which occurs in diabetic db/db mice aged 10–12 weeks (Hummel et al., 1966) and increased metabolic efficiency in diabetic db/db mice (Trayhurn, 1979). The reduced rate of weight loss in all three doses of Cistanche tubules may be due to the fact that Cistanche tubulosa also elevated the serum insulin levels (Table 3), which is beneficial for DM. Rosiglitazone led to a significant increase in body weight, which is a known side effect of rosiglitazone. The significantly increased liver weight and the liver/body weight ratio in the rosiglitazone group (po0.001) indicated that rosiglitazone may induce lipid accumulation and functional changes in the liver.

The results of the in vivo assay showed that Cistanche tubules could suppress the elevated fasting blood glucose levels and elevated postprandial blood glucose levels, and increased insulin sensitivity of db/db mice. Generally, Cistanche tubulosa was more effective in decreasing hyperglycemia and dyslipidemia and improving insulin resistance than acarbose during the experiment.

After 20 days oral administration, Cistanche tubulosa significantly inhibited the increasing tendencies of FBG level and FBG-increase % value on db/db mice constantly. At the end of the experiment, Cistanche tubulosa showed its anti-hyperglycemic effect in a dose-dependent manner. These results indicate that Cistanche tubulosa can potentially act as a potential blood glucose-lowering agent when administered at the appropriate dose. Most glucose disposal occurs in skeletal muscle after a meal, whereas the FBG levels are primarily determined by the liver glucose output. The FBG results showed that Cistanche tubulosa significantly improved the hepatic glucose output of db/db mice; poor hepatic glucose output is one of the three major metabolic defects in type 2 DM that contribute to hyperglycemia (Bavenholm et al., 2001). On day 30, the FBG-increase % values of the diabetic control group were lower than the expected levels, which might explain the fact that only CT72.6 (po0.01), acarbose (po0.05), and rosiglitazone (po0.001) significantly decreased the increase in FBG compared to the diabetic control group.

Collectively, the results in Table 2 indicate that Cistanche tubulosa effectively controlled the elevated postprandial blood glucose level after glucose loading, suggesting that Cistanche tubulosa is a candidate agent for alleviating postprandial hyperglycemia. A high post-prandial blood glucose level is an independent risk factor for the cardiovascular complications associated with type 2 DM (Ceriello, 2005; Blaak et al., 2012). CT120.9 significantly suppressed the elevation in the postprandial blood glucose level, suggesting that CT120.9 improved the impaired glucose tolerance of diabetic db/db mice, and the effect was comparable with 19.5 mg/kg acarbose. However, the anti-hyperglycemic effect of CT120.9 was less intensive than that of rosiglitazone.

Deficient insulin secretion and insulin resistance are two of the three major metabolic defects in type 2 DM that contribute to hyperglycemia (Bavenholm et al., 2001). Our analysis result of the FINS showed that a high dose of Cistanche tubulosa restored deficient insulin secretion in db/db mice to some extent. Therefore, Cistanche tubulosa effectively suppressed the elevated fasting and postprandial blood glucose levels (Table 2) partially by stimulating insulin secretion. Hyperglycemia and hyperlipidemia exert additional oxidative stress on beta-cells and mitochondria besides the primary diabetes pathogenesis. There is in vitro and in vivo evidence that chronic exposure to high levels of blood glucose and lipids are harmful to beta-cells, causing deficient insulin secretion, impaired insulin gene expression, and induction of cell death by apoptosis (Pitocco et al., 2010). As antioxidants could protect beta-cell lines and isolated islets against adverse effects from exposure to high glucose concentrations (Kaneto et al., 1996, Tajiri et al., 1997), Cistanche tubulosa may improve insulin secretion through its anti-oxidative effect.

The severely impaired insulin tolerance of the diabetic control group confirmed the insulin-resistant state of the db/db mice. The ITT results showed that the high dose of Cistanche tubulosa improved insulin resistance in db/db mice, which was comparable with rosiglitazone and superior to acarbose, suggesting that Cistanche tubulosa possesses potent insulin-sensitizing properties.

Glucose homeostasis is mainly regulated by the liver and skeletal muscle. Most glucose disposal occurs in the liver and skeletal muscle, with glycogen as the primary intracellular form of storable glucose (Saltiel, 2001). The glycogen levels in various tissues are a direct reflection of insulin sensitivity, as insulin promotes intracellular glycogen deposition. The insulin-stimulated glycogen content is markedly reduced in animals with insulin resistance, in proportion to the degree of insulin deficiency (Chandramohan et al., 2008). However, Cistanche tubulosa had no beneficial effects on glycogen storage in the liver or skeletal muscle.

The metabolic profile of type 2 DM is characterized by impaired glucose metabolism and insulin resistance, frequently combined with dyslipidemia (Cha et al., 2005). Dyslipidemia is one of the major risk factors for cardiovascular disease in DM. The characteristic features of diabetic dyslipidemia are a high plasma triglyceride (TG) concentration, low high-density lipoprotein cholesterol (HDL-c) concentration, increased low-density lipoprotein cholesterol (LDL-c) concentration, and increased very-low-density lipoprotein cholesterol (VLDL-c) concentration (Mooradian, 2009). After being treated with Cistanche tubulosa for 45 days, levels of total cholesterol, TG, LDL-c, and VLDL-c in db/db mice were decreased while HDL-c levels were increased, indicating that Cistanche tubulosa had a beneficial effect on dyslipidemia, and may potently decrease the risk of atherosclerosis, coronary heart disease and diabetic complications (Table 3). Cistanche tubulosa significantly decreased the TC level, which is consistent with a previous report (Shimoda et al., 2009). The mechanism by which Cistanche tubulosa improves dyslipidemia may be related to the mediation of hepatic mRNA expression related to cholesterol transport and metabolism (Shimoda et al., 2009). Although the prevalence of hypercholesterolemia is not increased in DM, lowering the levels of cholesterol can reduce the relative risk of cardiovascular disease in patients with DM (Mooradian, 2009). Cistanche tubulosa significantly elevated the HDL-c/TC level and decreased atherogenic index, which are risk factors for coronary artery disease, indicating that Cistanche tubulosa is beneficial for the management of both diabetes and cardiovascular disease. The positive control drug rosiglitazone significantly elevated the TC level, which is relevant to an increased risk of the progression of diabetes and cardiovascular disease.

5. Conclusion

In conclusion, we provide the first demonstration of the antihyperglycemic and hypolipidemic effects of Cistanche tubulosa in type 2 diabetic db/db mice. Cistanche tubulosa normalized hyperglycemia restored insulin sensitivity and reduced dyslipidemia. To some extent, Cistanche tubulosa improved all three of the major metabolic defects that contribute to hyperglycemia in type 2 DM. Thus, Cistanche tubulosa demonstrated a high potential for preventing the progression of type 2 DM and decreasing the risk of atherosclerosis, coronary heart disease, and diabetic complications. As a traditional Chinese medicine and tonic food, Cistanche tubulosa could be further developed into drugs, pharmaceutical foods, or dietary supplements in the future. However, further research is required to identify the bioactive component and elucidate the mechanisms responsible for the anti-hyperglycemic and hypolipidemic effects of Cistanche tubulosa.

Acknowledgments

This work was supported by Grants from the National Key Technology R&D Program in the 11th Five-Year Plan of China (2007BAI32B06) and the State Council Leading Group Office of Poverty Alleviation and Development (2008-3A).

Appendix A. Supplementary material

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2013.09.027.

From: ' Anti-hyperglycemic and hypolipidemic effects of Cistanche tubulosa' by Wen-Ting Xiong, et al

---W.-T. Xiong et al. / Journal of Ethnopharmacology 150 (2013) 935–945