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Antidiabetic effects of the Cimicifuga racemosa extract Ze 450 in vitro and in vivo in ob/ob mice.

ABSTRACT

Introduction: It was the aim of the present experiments to examine potential antidiabetic effects of the Cimicifuga racemosa extract Ze 450.

Methods: Ze 450 and some of its components (23-epi-26-deoxyactein, protopine and cimiracemoside C) were investigated in vitro for their effects on AMP-activated protein kinase (AMPK) compared to metformin in HepaRG cells. Ze 450 (given orally (PO) and intraperitonally (IP)), metformin (PO) and controls were given over 7 days to 68 male ob/ob mice. Glucose and insulin concentrations were measured at baseline and during an oral glucose tolerance test (OGTT).

Results: Ze 450 and its components activated AMPK to the same extent as metformin. In mice, Ze 450 (PO/IP) decreased significantly average daily and cumulative weight gain, average daily food and water intake, while metformin had no effect. In contrast to metformin, PO Ze 450 virtually did not change maximum glucose levels during OGTT, however, prolonged elimination. Ze 450 administered PO and IP decreased significantly post-stimulated insulin, whereas metformin did not. HOMA-IR index of insulin resistance improved significantly after IP and PO Ze 450 and slightly after metformin. In summary, the results demonstrate that Ze 450 reduced significantly body weight, plasma glucose, improved glucose metabolism and insulin sensitivity in diabetic ob/ob mice. In vitro experiments suggest that part of the effects may be related to AMPK activation.

Conclusions: Ze 450 may have utility in the treatment of type 2 diabetes. However, longer term studies in additional animal models or patients with disturbed glucose tolerance or diabetes may be of use to investigate this further.

Keywords:

Cimicifuga racemosa Ze 450

In vitro

In vivo

ob/ob mice

Diabetes mellitus

Introduction

Cimicifuga racemosa extracts (CRE) have been traditionally used in folk medicine for the treatment of postmenopausal (climacteric) complaints. They are described for this indication in a monograph of the European Scientific Cooperative on Phytotherapy (ESCOP) as pharmacologically active treatment (ESCOP, 2003). Several randomized controlled clinical trials have shown clinically significant effects of CRE in postmenopausal women with climacteric symptoms (Bai et al., 2007; Liske et al., 2002; Osmers et al., 2005; Stoll, 1987; Wuttke et al., 2003). In 2010, the community herbal monograph of the Committee on Herbal Medicinal Products (HMPC) of the European Medicines Agency (EMEA, 2007a,b, 2010) granted CRE with a well-established use status.

Menopausal transition is often accompanied with a significant weight gain (Polotsky and Polotsky, 2010). In an American survey about 66% of the women between 40 and 59 years were overweight or obese (Ogden et al., 2006) posing them to an increased risk for cardiovascular complications and diabetes mellitus (DM). In postmenopausal women increased central abdominal fat apposition was associated with impaired glucose metabolism (Barrett-Connor et al., 1996) and in particular inversely and independently related to insulin sensitivity (Sites et al., 2000). However, it is still controversial, whether menopausal status directly influences the risk to DM (Szmuilowicz et al., 2009). Obviously, a treatment would be desirable which efficiently treats both the climacteric symptoms and metabolic complications.

Recently, beneficial effects of CRE were reported in ovariectomized rats, an animal model of postmenopausal status, where they diminished abdominal fat apposition and metabolic syndrome (Seidlova-Wuttke et al., 2012). Therefore, it was the aim of the present investigations to determine potential beneficial effects of Cimicifuga racemosa on carbohydrate metabolism in an animal model without impaired sexual hormone status.

Due to its efficacy and safety (e.g. low risk of hypoglycemia and weight gain) several guidelines recommend metformin as first-line oral treatment of type II DM (Nathan et al., 2009; NICE, 2009; Rodbard et al., 2009). The principal mechanisms of metformin actions are related to reduced oxygen consumption, inhibition of gluconeogenesis and lipid synthesis in the liver, enhancement of glucose uptake in the muscle and sensitizing effects of the insulin receptor (Rena et al., 2013). Most of the effects are related to the activation of the AMP-activated protein kinase (AMPK), a critical cellular energy sensor and regulator of energy homeostasis (Hardie et al., 2012; Musi and Goodyear, 2006). It was, therefore, the aim of the present experiments to examine, whether the CRE Ze 450 showed in vitro evidence of AMPK activating activities and if these effects may translate to clinically beneficial effects in vivo.

For the assessment of activation effects on the AMPK, HepaRG cells were used. HepaRG cells are an immortalized hepatic cell line that retains many characteristics of primary human hepatocytes (Antherieu et al., 2010). The cells were incubated with different concentrations of Ze 450 and with some of its selected pure components. The AMPK activity was measured with an ELISA assay.

The anti-diabetic effects of different compounds were assessed in the ob/ob mouse model (Herberg and Coleman, 1977; Zhang et al., 1994). These animals are devoid of intact leptin genes. They are obese due to unrestricted eating and suffer from type II diabetes with insulin resistance. The model has been proven to be a well-established animal model for type II DM.

Materials and methods

Reagents

23-Epi-26-deoxyactein was obtained from PhytoLab (Vestenbergsgreuth, Germany), protopine hydrochloride was obtained from Extrasynthese (Z.I Lyon Nord, Genay Cedex, France) and cimiracemoside C was obtained from LGC Standards (Teddington, Middlesex, UK). Metformin and Tween 80 were obtained from Sigma-Aldrich (Sigma, St. Louis, MO, USA). D-Glucose and polyethylene glycol 300 or 400 (PEG300 or PEG400) were from Fisher Scientific. All chemicals were obtained in the highest grade.

Cells

Fully differentiated HepaRG cells were obtained from Invitrogen (Life Technologies, Ltd., Paisley, UK). Cells were thawed and seeded in Williams' E medium containing 2 mM GlutaMax and HepaRG[TM] thaw, plate, and general purpose medium supplement. All media and supplements were purchased from Gibco (Life Technologies, Ltd., Paisley, UK).

Drug

The ethanolic (v/v) Cimicifuga racemosa dry extract Ze 450 was manufactured of dried rhizomes and roots and obtained from Max Zeller Soehne AG (Romanshorn, Switzerland). The drug extract ratio was 7.3:1, the content of active triterpene glycosides was 8.4%. The extract has been registered for the treatment of postmenopausal complaints in several European (including Germany and Switzerland) and other countries. HPLC fingerprints of two extract batches of Ze 450 have been recently published in this journal (Drewe et al., 2013).

Sample preparation for in vitro experiments

Ze 450 was dissolved in EtOH 60% (v/v) and diluted with [H.sub.2]O u.p. The pure substance protopine hydrochloride was dissolved in [H.sub.2]O u.p., and 23-epi-26-deoxyactein and cimiracemoside C were dissolved in EtOH abs. The final solvent concentration was 1% or less in each case.

AMPK assay

HepaRG cells were seeded in collagen coated (5 [micro]g/[cm.sup.2]) 12-well cell culture plates (1.2 x [10.sup.6] cells/well) and grown for 4h. Afterwards, the medium was changed to serum-free medium, but with 30 mM glucose. Cells were preincubated overnight. Afterwards, they were incubated with the samples and the positive control metformin (2 mM) dissolved in the same medium for 24 h. At the end of incubation the cells were washed with phosphate buffered saline and lysed with cell extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM [Na.sub.4][P.sub.2][O.sub.7], 2 mM [Na.sub.3]V[O.sub.4], 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM PMSF and protease inhibitor cocktail (2 mM [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], 14 [micro]M (N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide], 130 [micro]M bestatin, 0.9 [micro]M leupeptin, 0.3 [micro]M aprotinin, and 1 mM EDTA)).

AMPK[alpha] [pT172] protein was measured by an ELISA technique (Life Technologies, Ltd., Paisley, UK) according the manufacturer's protocol. Protein determination was carried out using a kit from Cayman (Cayman Chemical Company, Ann Arbor, MI, USA) based on the Bradford method according to the manufacturer's protocol.

The selected metformin concentration of 2 mM was chosen as described by others (Zang et al., 2004; Zhou et al., 2001). The clinically optimal dose in humans is 2g/day, resulting in maximum plasma levels of 4 [micro]g/ml and steady state values of 1 [micro]g/ml (6 mM), respectively. Ze 450 was tested at concentrations of 1, 10, and 100 [micro]g/ml, the pure components protopine hydrochloride, 23-epi-26-deoxyactein and cimiracemoside C were tested at concentrations of 0.3 and 3 [micro]M. The latter concentrations were the same for each component and were chosen to match the concentrations of 23-epi-26-deoxyactein in 10 and 100 [micro]g/ml Ze 450.

Animals

Sixty-eight male ob/ob mice (7-8 weeks of age upon arrival) were obtained from Janvier, France. Mice were singly housed in polypropylene cages with free access to a standard diet (Harlan Teklad Global 2018 diet) and tap water at all times. All animals were maintained at 24 [+ or -] 2[degrees]C and 55 [+ or -] 20% humidity on a reverse phase 16 h on/8 h off light dark cycle (lights on approx. 17:30-09:30 h). All work detailed in this manuscript was performed under the Animals Scientific Procedures Act 1986 UK.

Ze 450 was stored refrigerated. A factor of 1.25 was applied to all Ze 450 dosing solutions to compensate for formulation excipients. The compound was formulated in a vehicle of 5% Tween 80, 5% PEG400 and 90% saline (v/v/v). Metformin was dissolved in de-ionized water. All dosing solutions of Ze 450 were prepared fresh daily prior to dosing and dosed as clear solutions. D-Glucose was dissolved in de-ionized water. All dosing was in the volume of 3 ml/kg by the PO or IP route.

Animal experiments

Animals were acclimatized to the animal facility for 1 week. Afterwards, all animals began a baseline protocol where they were dosed with water (n = 17) or PEG300 vehicle orally (PO; n = 33) or intraperitoneally (IP; n = 17) once daily for a 7-day baseline period (Day -6 to 0). Prior to the start of baseline dosing animals were allocated into the groups on the basis of BW. Dosing began at approximately 08:45 each day. BW and food and water intake were recorded daily. Due to some animals showing transient reductions in activity after IP administration of PEG300, this vehicle was switched to 5% Tween 80, 5% PEG400 and 90% saline (v/v/v) from Day -5 onwards.

Blood sample analysis

On Day -4 of the baseline phase animals underwent blood sampling in the fed state at 16:00. All blood samples were spun in a centrifuge immediately after collection and the plasma fraction stored frozen (-80[degrees]C) prior to determination of plasma glucose (in duplicate) and insulin (single replicate) using commercially available kits and reagents: Thermo Scientific Infinity glucose reagent TR15498, Alpco mouse ultrasensitive insulin kit 80-1NSMSU-E10. Toward the end of the baseline phase, animals were allocated into eight treatment groups (n = 8, each) on the basis of BW, baseline food and water intake, and plasma glucose and insulin.

For dosing, Ze 450 was dissolved in vehicle and metformin in water. Mice were dosed once daily PO (per gavage) or IP for 7 days as follows--A: water PO, B: metformin (200 mg/kg PO; water), C: vehicle PO, D: Ze 450 (10 mg/kg PO), E: Ze 450 (30 mg/kg PO), F: Ze 450 (90 mg/kg PO), G: Vehicle IP, and H: Ze 450 (30 mg/kg IP).

At the completion of dosing, animals were examined and any overt behavior was recorded. On Day 7, mice underwent an oral glucose tolerance test (OGTT) under fasting conditions. Animals were dosed -60 min (Predose) with treatments and after 60 min (0 min, [C.sub.0]) with D-glucose (2g/kg PO) immediately after baseline blood samples. Further blood samples were taken 15, 30, 45 and 60 min later. All blood samples (30 [micro]l) were taken from the tail vein and plasma was frozen (-80[degrees]C) pending analysis for glucose and insulin.

Statistical analysis

All statistical analyses were performed using SAS 9.1.3 or IBM SPSS version 20.0. After all analyses, multiple comparisons against the appropriate vehicle group were done by Williams' test for Ze 450 PO, and the multiple t tests for metformin and Ze 450 IP. Level of significance was p = 0.05. All tests were carried out as two-sided tests.

Animals in groups A-B, C-F and G-H were treated differently during the baseline phase, it was not appropriate to analyze all groups in the same analysis with baseline as a covariate, in case the different vehicle treatments had different effects on baseline BW, food and water intake and Day -4 glucose and insulin. Therefore three separate analyses were performed, one for each vehicle/route.

BW and overall BW gains (g) were analyzed by analysis of covariance with Day 1 BW as a covariate. Food and cumulative food intake and water intake (g) were analyzed by analysis of covariance with average daily food or water intake during the baseline phase (Days -6 to 0) as a covariate.

Plasma glucose and insulin analysis

On Day 7, glucose and insulin concentrations were tested under fasting conditions immediately before the last dosing (T = -60 min; [C.sub.Predose]), immediately before the glucose administration in the OGTT (T = 0 min; [C.sub.0]), and 15, 30,45 and 60 min later. For the OGTT, the treatment groups were compared at [C.sub.0] as well as at each post-dose time and by the area under the curve from 0 to 60 min AUC (0-60 min). AUC was calculated as by linear trapezoidal route related to the baseline (e.g. [C.sub.0]). The shape of the glucose and insulin concentration/time curve during OGTT was further analyzed by fitting a one-compartment model (absorption and elimination) for each animal and maximum concentrations ([C.sub.max]) and the time point, when it was reached ([T.sub.max]) as well as elimination half-life ([T.sub.1/2]) were estimated.

The homeostasis model assessment of insulin resistance (HOMA-IR index) was calculated from fasting glucose and insulin values as (insulin ([micro]U/ml) x glucose (mmol/l))/22.5 as described in Matthews et al. (1985). This was log transformed and analyzed by general linear model with treatment and assay day as factors, Day 1 BW, bleeding order. Day -4 glucose and log(Day -4 insulin) as covariates.

A log transformation was used for plasma insulin, apart from the AUC (0-60 min), which can be negative, so this was not transformed and [C.sub.max] (glucose and insulin). Analysis of each time, AUC and [C.sub.max] was by robust regression model using M estimation, Huber weighting, using the default parameter c = 1.345. The model included treatment and assay day as factors and bleeding order, Day 1 BW and Day -4 (unfasted) baseline plasma glucose or insulin (log transformed, if appropriate) as covariates.

For [T.sub.max] and [T.sub.1/2], no covariates were relevant, so all eight groups were included in the same analysis and analysis was by robust regression of log transformed data with treatment and assay day as factors.

Results

AMPK assay

The effects of Ze 450 and its components were assessed in three independent experiments (n = 4) using medium and 2 mM metformin as negative and positive control, respectively, in each of the experiments. The results are summarized in Table 1.

Ze 450 dose-dependently activates AMPK. At the highest concentration this activation was even slightly higher than the positive control metformin. 23-Epi-26-deoxyactein showed in both tested concentrations a comparable or slightly elevated activation of AMPK compared with metformin. For protopine and cimiracemoside C, the activation at the lowest concentration (0.3 [micro]M) was higher than that of metformin. In the highest concentration, a fold activation of 5.91 for protopine and 3.36 for cimiracemoside C was observed.

In vivo experiments in ob/ob mice

With the exception of some animals not tolerating PEG300 when dosed by the intraperitoneal route (prior to switching to vehicle), all mice were in good condition throughout the duration of the study with no adverse events evident.

With the exception of the animals treated with Ze 450 by the IP route, animals tended to exhibit a daily increase in BW over the study duration. When Ze 450 (PO) BW gain decreased dose-dependently with significant reductions from control evident at the 90 mg/kg PO groups (p = 0.002) (Fig. la). In the IP group there was an absolute reduction in weight (p< 0.001) and compared to Day 1, a small increase in BW was observed on Day 6 in the vehicle control for metformin of 0.84 [+ or -] 0.40 g (SEM).

The effects on BW may partly be explained by the effects of the treatments on food intake. Metformin did not significantly affect food intake, although the 10% reduction did approach statistical significance (p = 0.054). In contrast, Ze 450 significantly decreased average daily food intake in a dose-dependent manner after PO administration (30 mg/kg and 90 mg/kg PO groups) as well as after IP administration (p < 0.001) (Fig. 1b). Accordingly, with the exception of the 10 mg/kg PO dose, Ze 450 significantly reduced cumulative food intake compared to relevant controls (Fig. 1c and d).

Daily water intake was stable during the baseline period and animals drank approximately 10 g of water each day. Once-daily IP administration of 30 mg/kg Ze 450 tended to reduce daily water intake compared to controls and a significant reduction in average daily water intake (p = 0.002, Fig. 2). When dosed by the PO route, Ze 450 also significantly reduced average daily water intake (at doses [greater than or equal to] 30 mg/kg). Metformin significantly reduced average daily water intake as well (p = 0.002; Fig. 2).

No significantly different fasting glucose levels were observed in the metformin or PO Ze 450 groups. However, in the Ze 450 IP group fasting glucose was significantly lower than that of the control (p = 0.034, Fig. 3a and b). Compared to their vehicle control, no significantly different fasting insulin levels were in the metformin group. In contrast to metformin, PO Ze 450 administration decreased slightly fasting insulin levels from 10.56 ng/ml to 7.35ng/ml for Ze 450 (10 mg/kg), p = 0.059 and further to 6.08ng/ml for the higher doses of Ze 450 (30 mg/kg: p = 0.007 and to 6.06 ng/ml for 90mg/kg; p = 0.006). IP administration of Ze 450 (30 mg/kg) decreased fasting insulin concentration compared to their vehicle control from 10.98ng/ml to 4.63 ng/ml; p = 0.002 (Fig. 4a and b).

Effect of administration of Ze 450 and metformin on glucose tolerance

In the OGTT, after the glucose load glucose [C.sub.max] values decreased significantly (p = 0.014) from 43.47 nmol/l (vehicle) to 30.56 mmol/l for the metformin treatment. Compared to their respective control glucose [C.sub.max] decreased (although non-significantly; p = 0.075) after IP administration of Ze 450 from 35.62 mmol/l to 26.21 mmol/l. No significant changes in glucose [C.sub.max] were observed after PO administration of Ze 450, however, [T.sub.max] was significantly (p < 0.05) increased after Ze 450 PO and IP administrations. Compared with their respective controls, metformin and Ze 450 administrations showed a clearly evident change in shape of the concentration/time curve, as indicated by a significantly prolonged half-life (Table 2). This effect was most prominent after IP administration of Ze 450. No significant changes were observed in glucose AUC (0-60 min) after Ze 450 after PO and IP administration in contrast to metformin, where AUC (0-60 min) significantly (p = 0.003) decreased.

Metformin decreased insulin [C.sub.max] from 39.25 ng/ml (control) to 24.20 ng/ml, however, this change was not statistically significant. In contrast, PO administration of Ze 450 decreased significantly and dose-dependently insulin [C.sub.max] from 51.01 ng/ml (control) to 33.73 ng/ml (10 mg/kg; p = 0.029) to 27.72 ng/ml (30 mg/kg; p = 0.002) and 25.01 ng/ml (90 mg/kg; p < 0.001). IP administration of Ze 450 decreased insulin [C.sub.max] significantly (p = 0.001) to a similar extent from 53.48 ng/ml (control) to 27.76 ng/ml. Insulin [T.sub.max] did not change in the metformin or Ze 450 PO groups, however, after IP administration of Ze 450, it was significantly decreased (p < 0.001). Insulin AUC (0-60 min) was significantly decreased after both PO and IP administration of Ze 450. In addition, the insulin concentration/time profiles were significantly prolonged after PO and IP administration of Ze 450 (Table 2).

HOMA-IR index of insulin resistance improved for metformin by 38.2% from the control treatment, however this was not significant. In contrast, for Ze 450 PO an improvement of 55.9% (10 mg/kg; n.s.), 48.6% (30 mg/kg; p = 0.005) and 47.9% (90 mg/kg; p = 0.005) was observed. The greatest improvement was observed after Ze 450 IP administration of 59.3% (p = 0.024; Table 3).

Discussion

The aim of the study was to investigate whether administration of the Cimicifuga racemosa extract Ze 450 may lead to an improvement in carbohydrate metabolism and glucose homeostasis. Therefore, in vitro the effect on the human AMP-activated protein kinase (AMPK) was investigated, that is an important anti-diabetic target.

The results of the AMPK assay showed the potential of Ze 450 and some of its pure components to increase AMPK activity. Ze 450 and its components 23-epi-26-deoxyactein, protopine hydrochloride showed a significant AMPK activation. These data demonstrate that Ze 450 effect was at least of the same magnitude or even higher than after the administration of clinically relevant concentration of metformin (a standard drug for oral antidiabetic treatment). These experiments suggest that Ze 450 may have potentially beneficial effects in diabetic subjects. Therefore, in a second experiment, Ze 450 was evaluated in a relevant animal model for insulin resistance and diabetes, the double leptin receptor deficient ob/ob mouse (Herberg and Coleman, 1977; Tschop and Heiman, 2001; Zhang et al., 1994).

The principal finding of the study was that sub-chronic administration of Ze 450 improved insulin sensitivity in the animals as assessed in the OGTT. Specifically, although the plasma glucose excursion post oral glucose load was not markedly improved (i.e. there were no significant reductions in the AUC compared to controls), there were significant improvements in plasma insulin. This corresponded to a significant reduction in the HOMA-IR index of insulin resistance. In addition, when given by the IP route (presumably a route that led to improved compound exposure at target receptors), treatment with Ze 450 significantly reduced freely feeding and fasted levels of plasma glucose in the diabetic animals. If such data extend to the clinic then the present results suggest that Ze 450 may have utility in the treatment of type 2 DM. However longer-term studies in other diabetic animal models (e.g. the ZDF rat) with the additional monitoring of HbA1c or finally in patients with disturbed blood sugar control or DM may be warranted to investigate this effect further.

Ze 450 was associated with dose-dependent and statistically significant reductions in food intake. Reduced food intake and BW are typically associated with improvements in insulin sensitivity (Jung et al., 2013). Accordingly it is likely that some of the effects of Ze 450 to improve insulin sensitivity in the present study are attributable, at least in part, to the effect of the compound to reduce food intake and BW. The reduction in food intake may theoretically result from local irritation of the gastrointestinal tract and a secondary food avoidance behavior. However, the largest reduction of food intake has been observed after IP administration of Ze 450 making this explanation more unlikely and suggesting rather central nervous system effects of Ze 450 on food regulation. CRE interact with brain with some serotonergic (Burdette et al., 2003; Powell et al., 2008) and central opioid receptors (Reame et al., 2008) that may among others be involved in appetite regulation (Halford et al., 2010, 2011; Janssen et al., 2011).

Once daily treatment with metformin led to significant improvements in glucose control in the animals and this action is consistent with a huge scientific literature and the status of metformin as the first-line drug treatment for overweight patients with type 2 DM (Anon, 1998). Unlike Ze 450, sub-chronic treatment with metformin significantly improved the plasma glucose excursion subsequent to the glucose load. This was associated with a reduced plasma insulin concentration at each time point compared to vehicle-treated controls. Accordingly, like Ze 450, metformin appeared to improve insulin sensitivity in the animals.

Metformin did not reduce BW or overall average daily food intake in the present study. In the clinic, metformin treatment is typically associated with a reduction in BW (Chaudhry et al., 2006; Mauras et al., 2012). This is often regarded as a major advantage of the drug since it improves glycemic control but with significantly less weight gain than when other anti-diabetic drug classes (e.g. sulfonylureas) are used (Baptista et al., 2007; Makimattila et al., 1999).

Ze 450 significantly reduced average daily water intake over the study duration at doses of 30 mg/kg and above, irrespective of route. The reason for this may be twofold. Firstly, reductions in drinking are likely to be attributable to the drug treatment improving the glucose control of the animals and so reducing polyuria and glycosuria. If, after drug therapy, less urine is expelled then it would be expected that water intake would reduce accordingly. Indeed, normal lean mice would be expected to drink approximately 3 ml daily in contrast to the control ob/ob mice in the present study which drank approximately 9 ml. Secondly, drinking is often prandial in nature. Since Ze 450 significantly reduced food intake in the study, it is possible that the effect of the treatment on water intake is associated with this reduction in eating behavior. These two factors may be related. Specifically, if Ze 450 is successfully treating the diabetes, then glucose is getting to the target tissues more readily than in controls. Accordingly the need to ingest further glucose (i.e. eat) is not as great in animals treated with Ze 450.

Metformin administration was associated with a statistically significant reduction in average daily water intake in the absence of a change in BW. This reduction in water consumption is likely to be attributable to the improved glycemic status of the animals, as discussed earlier.

In conclusion, the present data demonstrate that the novel test compound, Ze 450, led to statistically significant reductions in BW, freely feeding levels of plasma glucose and improvements in insulin sensitivity in male, diabetic, ob/ob mice at the doses tested. The present data suggest that Ze 450 may have utility in the treatment of type 2 DM. However, longer term studies in additional animal models or patients with disturbed glucose tolerance or diabetes may be of use to investigate this further.

Conflict of interest

CM and JD are employees of Max Zeller Soehne AG. SPV, RB and SCC have received a research grant from Maz Zeller Soehne AG.

ARTICLE INFO

Article history:

Received 4 March 2014

Received in revised form 10 April 2014

Accepted 6 June 2014

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C. Moser (a), S.P. Vickers (b), R. Brammer (b), S.C. Cheetham (b), J. Drewe (a),*

(a) Preclinical Research, Max Zeller Soehne AC, Romanshom, Switzerland

(b) RenaSci Ltd., Nottingham, UK

* Corresponding author at: Seeblickstr. 4, CH-8950 Romanshorn, Switzerland.

Tel.: +41 71 466 0500: fax: +41 71 466 0707.

E-mail address: juergen.drewe@zellerag.ch (J. Drewe).

http://dx.doi.org/10.1016/j.phymed.2014.06.002

Table 1
AMPK activation of Ze 450 and its components related to the
medium control.

                                    AMPK activation
                                    factor (mean
Sample             Concentration    [+ or -] SEM)

Experiment 1

Metformin          2mM              2.48 [+ or -] 0.28
Ze 450             1 [micro]g/ml    1.24 [+ or -] 0.11
                   10 [micro]/ml    1.43 [+ or -] 0.14
                   100 [micro]/ml   2.63 [+ or -] 0.17

Experiment 2

Metformin          2mM              2.35 [+ or -] 0.22
23-Epi-26-
  deoxyactein      0.3 [micro]M     2.28 [+ or -] 0.45
                   3 [micro]M       2.59 [+ or -] 0.35

Experiment 3

Metformin          2mM              1.86 [+ or -] 0.61
Protopine          0.3 [micro]M     2.47 [+ or -] 0.61
hydrochloride      3 [micro]M       5.91 [+ or -] 0.53
Cimiracemoside C   0.3 [micro]M     2.70 [+ or -] 0.69
                   3 [micro]M       3.36 [+ or -] 0.66

                   p values vs   p values vs
Sample             control       metformin

Experiment 1

Metformin          <0.001        --
Ze 450             NS            0.006
                   NS            0.02
                   <0.001

Experiment 2

Metformin          0.009         --
23-Epi-26-
  deoxyactein      0.067         NS
                   0.03          NS

Experiment 3

Metformin          NS            --
Protopine          NS            NS
hydrochloride      0.006         0.005
Cimiracemoside C   NS            NS
                   NS            NS

Unpaired t-test with Bonferroni's correction.

Table 2
Glucose and insulin parameters on Day 7 (mean [+ or -] SEM).

                       [C.sub.predose]       [C.sub.0]
Glucose                (mmol/l)              (mmol/l)

Vehicle metformin      18.37 [+ or -] 2.46   14.20 [+ or -] 3.99
Metformin              15.54 [+ or -] 1.42   11.59 [+ or -] 0.37

Vehicle PO Ze 450      13.71 [+ or -] 0.49   12.99 [+ or -] 0.42
Ze 450 PO 10 mg/kg     14.64 [+ or -] 0.61   14.33 [+ or -] 1.45

Ze 450 PO 10 mg/kg     12.30 [+ or -] 0.65   13.14 [+ or -]0.89

Ze 450 PO 10 mg/kg     13.34 [+ or -] 0.75   12.82 [+ or -] 0.97

Vehicle IP Ze 450      15.45 [+ or -] 0.76   13.33 [+ or -] 0.76
Ze 450 IP 30 mg/kg     12.75 [+ or -] 0.70   13.72 [+ or -] 1.10
                       (p = 0.034)

                       [C.sub.max]           [T.sub.max]
Glucose                (mmol/l)              (min)

Vehicle metformin      43.47 [+ or -] 2.81   20.7 [+ or -] 2.1
Metformin              30.56 [+ or -] 2.43   19.1 [+ or -] 10.2
                       (p = 0.014)
Vehicle PO Ze 450      38.53 [+ or -] 1.15   20.6 [+ or -] 0.6
Ze 450 PO 10 mg/kg     40.32 [+ or -] 1.74   27.7 [+ or -] 1.3
                                             (p < 0.05)
Ze 450 PO 10 mg/kg     34.27 [+ or -] 3.40   30.0 [+ or -] 3.7
                                             (p = 0.014)
Ze 450 PO 10 mg/kg     34.42 [+ or -] 1.33   29.7 [+ or -] 3.0
                                             (p = 0.014)
Vehicle IP Ze 450      35.62 [+ or -] 1.51   19.3 [+ or -] 4.3
Ze 450 IP 30 mg/kg     26.21 [+ or -] 3.21   26.9 [+ or -] 7.9
                       (p = 0.075)           (p = 0.027)

                       AUC (0-60 min)        Half-life
Glucose                (mmol x h/l)          (min)

Vehicle metformin      16.89 [+ or -] 0.72   14.7 [+ or -] 1.6
Metformin              10.06 [+ or -] 1.92   33.2 [+ or -] 13.1
                       (p = 0.003)           (p = 0.007)
Vehicle PO Ze 450      17.44 [+ or -] 0.71   14.3 [+ or -] 0.7
Ze 450 PO 10 mg/kg     20.09 [+ or -] 0.43   26.0 [+ or -] 6.9
                                             (p < 0.05)
Ze 450 PO 10 mg/kg     16.15 [+ or -] 1.92   29.1 [+ or -] 11.1
                                             (p = 0.019)
Ze 450 PO 10 mg/kg     16.90 [+ or -] 1.11   29.4 [+ or -] 11.6
                                             (p = 0.018)
Vehicle IP Ze 450      16.75 [+ or -] 0.79   15.5 [+ or -] 2.6
Ze 450 IP 30 mg/kg     14.66 [+ or -] 1.76   207.4 [+ or -] 152.9
                                             (p = 0.027)

                       [C.sub.predose]       [C.sub.0]
Insulin                (mmol/l)              (mmol/l)

Vehicle metformin      8.98 [+ or -] 1.82    8.64 [+ or -] 1.46
Metformin              5.70 [+ or -] 0.85    6.14 [+ or -] 0.67
Vehicle PO Ze 450      10.56 [+ or -] 1.51   10.08 [+ or -] 1.31
Ze 450 PO 10 mg/kg     7.35 [+ or -] 1.22    8.21 [+ or -] 1.30
                       (p = 0.059)
Ze450 PO 10 mg/kg      6.08 [+ or -] 0.52    9.59 [+ or -] 0.81
                       (p = 0.007)
Ze450 PO 10 mg/kg      6.06 [+ or -] 0.47    7.74 [+ or -] 0.61
                       (p = 0.006)
Vehicle lP Ze 450      10.98 [+ or -] 1.76   8.62 [+ or -] 0.77
Ze 450 IP 30 mg/kg     4.63 [+ or -] 0.61    5.20 [+ or -] 1.14
                       (p = 0.033)

                       [C.sub.max]           [T.sub.max]
Insulin                (mmol/l)              (min)

Vehicle metformin      39.25 [+ or -] 5.00   8.87 [+ or -] 0.56
Metformin              24.20 [+ or -] 4.13   8.87 [+ or -] 0.18
Vehicle PO Ze 450      51.01 [+ or -] 5.88   9.33 [+ or -] 0.22
Ze 450 PO 10 mg/kg     33.73 [+ or -] 2.73   8.31 [+ or -] 3.42
                       (p = 0.029)
Ze450 PO 10 mg/kg      27.72 [+ or -] 2.05   8.78 [+ or -] 3.36
                       (p = 0.002)
Ze450 PO 10 mg/kg      25.01 [+ or -] 3.61   8.74 [+ or -] 4.45
                       (p < 0.001)
Vehicle lP Ze 450      53.48 [+ or -] 4.06   9.34 [+ or -] 2.72
Ze 450 IP 30 mg/kg     27.76 [+ or -] 2.22   1.20 [+ or -] 0.51
                       (p = 0.001)           (p < 0.001)

                       AUC (0-60 min)        Half-life
Insulin                (ng x h/ml)           (min)

Vehicle metformin      11.70 [+ or -] 1.84   8.12 [+ or -] 1.65
Metformin              8.55 [+ or -] 1.39    10.91 [+ or -] 1.69
Vehicle PO Ze 450      15.28 [+ or -] 1.58   9.28 [+ or -] 1.51
Ze 450 PO 10 mg/kg     10.38 [+ or -] 1.11   20.89 [+ or -] 4.15
                       (p = 0.006)           (P = 0.01)
Ze450 PO 10 mg/kg      8.48 [+ or -] 1.07    18.23 [+ or -] 4.78
                       (p < 0.001)           (P = 0.01)
Ze450 PO 10 mg/kg      7.93 [+ or -] 2.07    24.66 [+ or -] 4.85
                       (p < 0.001)           (p < 0.001)
Vehicle lP Ze 450      17.39 [+ or -] 1.67   11.06 [+ or -] 1.18
Ze 450 IP 30 mg/kg     10.26 + 1.31          37.45 [+ or -] 11.76
                       (p = 0.04)            (p < 0.001)

n = 8, Statistical comparisons were always performed for verum
treatment vs their respective vehicles were performed by Williams'
test or multiple f tests, as appropriate.

Table 3
HOMA-IR index of insulin resistance.

Treatment              n   Mean    SEM    % of vehicle   p values

Vehicle metformin      8   139.8   29.7
Metformin              8    86.4   13.0   61.8            0.178
Vehicle PO Ze 450      8   146.3   22.8
Ze 450 (10 mg/kg PO)   8   108.5   18.5   74.1            0.161
Ze450 (30 mg/kg PO)    8    75.3    6.7   51.4           <0.005
Ze 450 (90 mg/kg PO)   8    76.2    7.1   52.1           <0.005
Vehicle PO Ze 450      8   141.8   26.0
Ze 450 (30 mg/kg IP)   7    57.7    7.4   40.7            0.024

Multiple comparisons against the appropriate vehicle group were by
Williams' test for Ze 450 PO, the multiple t test for metformin and
Ze 450 IP.
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Author:Moser, C.; Vickers, S.P.; Brammer, R.; Cheetham, S.C.; Drewe, J.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Sep 25, 2014
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