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Marrubiin, a constituent of Leonotis leonurus, alleviates diabetic symptoms.

ABSTRACT

Aims: Marrubiin and an organic extract of Leonotis leonurus were tested in vitro and in vivo for their antidiabetic and anti-inflammatory activities.

Materials and methods: INS-1 cells were cultured under normo-and hyperglycemic conditions conditions. An in vivo animal model confirmed the biological activities of marrubiin and the organic extract observed in the studies in vitro.

Results: The stimulatory index of INS-1 cells cultured under hyperglycemic conditions was significantly increased in cells exposed to the organic extract and marrubiin, relative to the hyperglycaemic conditions. Insulin and glucose transporter-2 gene expressions were significantly increased by the organic extract and marrubiin. Similarly, the extract and marrubiin resulted in an increase in respiratory rate and mitochondria! membrane potential under hyperglycaemic conditions. Marrubiin increased insulin secretion, HDL-cholesterol, while it normalized total cholesterol, LDL-cholesterol, atherogenic index, IL-1[beta] and IL-6 levels in an obese rat model.

Conclusion: The results provide evidence that marrubiin, a constituent of Leonotis leonurus, alleviates diabetic symptoms.

[c] 2012 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords: Leonotis leonurus Marubiin Hyperglycemia Insulin Antidiabetic Anti-inflammatory

Introduction

Pancreatic 13-cells secrete insulin which is the primary hormone that maintains physiological glucose homeostasis. Hyperglycemia and/or free fatty acids (FFAs) have been shown to trigger insulin deficiency in type 2 diabetes (T2D). In T2D a deficiency of insulin is caused by the loss of (3[beta]-cells function and mass, as they fail to compensate against insulin resistance (IR) (Choi et al. 2007). Obesity is closely associated with IR which is a pre-diabetic state that often results in T2D. Exposure to FFAs for a long time could hinder glucose-stimulated insulin secretion (GSIS), insulin gene expression and cause apoptosis of cultured [beta]-cells or isolated islets (Choi et al. 2007). GSIS relies heavily on mitochondria) metabolism since glucose oxidation is tightly associated with ATP production (Affourtit and Brand 2008). An increase in cytosolic [[Ca.sup.2+]] occurs when blood glucose levels increase, as a result mitochondria! [[Ca.sup.2+]]increases, leading to the activation of the respiratory chain through the stimulation of [Ca.sup.2+]-sensitive NADH-generating dehydrogenases. The resulting NADH and FAD[H.sub.2] are fed into the electron transport chain, thereby increasing ATP production through an increase of the mitochondrial membrane potential (Affourtit and Brand 2008).

Leonotis leonurus (Lamiaceae) R.Br a plant indigenous to South Africa contains the diterpenoid labdane lactones premarrubiin and marrubiin (M) (Fig. 1). Previous studies have shown that the leaf extract of this plant possessed hypoglycaemic effects in a streptozotocin (STZ)-induced diabetic rat model (Ojewole 2005; Scott et al. 2004). L. leonurus has been reported to be traditionally used to treat hypertension (Van Wyk et al. 2000). Ojewole (2003) evaluated the aqueous extract for its cardiovascular and hypotensive effects in rats and observed that the arterial blood pressures and heart rates of normal, anaesthetized spontaneously hypertensive rats were significantly reduced (Ojewole 2003). This study was undertaken to investigate the mechanism of the hypoglycaemic activity of L leonurus extracts and to determine if M is one of the compounds responsible for the biological effects observed.

Methods and materials

All the reagents used were of an analytical grade.

Ethical clearance

Ethical clearance for this study was approved by the Nelson Mandela Metropolitan University (NMMU) Animal Ethics Committee (Ref: A09-SCI-BCM-001).

Plant material

L. leonurus was collected at NMMU, South campus and examined by Mr C. Weatherall-Thomas (the curator of the Herbarium of the Department of Botany, Nelson Mandela Metropolitan University) with reference number 19302.

Plant extraction and marrubiin quantification

The extraction procedure used is a modified version described by Mnonopi et al. (2011), Rivett (1964) and Knoss et al. (1997) where they isolated and quantified M from L. leonurus and Marrubium vulgare L. (Lamiaceae), respectively. M isolated from the L. leonurus leaf organic (OL) extract was quantified as described by Mnonopi et al. (2011). For some experiments completed OL1 = 2.5 [micro]g/ml, OL2 = 5[micro]g/ml, OL3=10[micro]g/ml M1 =125 ng/ml, M2 = 250 ng/ml, and M3 = 500 ng/ml.

Cell culture and GSIS

INS-I rat insulinoma cells were maintained at 37[degrees]C, in a humidified atmosphere supplemented with 10% [CO.sub.2] in RPMI 1640 media containing glutamax, 5% FBS, 10 mM Hepes, 50 mM 2-mercaptoethanol and I mM sodium pyruvate. The INS-I cells were exposed to OL (2.5-10[micro]g/m1) and M (125-500 ng/ml) in 11.1 mM glucose RPMI (normoglycaemic conditions) and 33.3 mM glucose (hyperglycaemic conditions) RPMI for 48 h. GSIS was conducted thereafter, and chronic, basal, stimulated, and insulin content samples were collected (Maedler et al. 2001). Insulin content was determined using the insulin radioimmunoassay (RIA) kit (Linco Research), which was evaluated using a liquid scintillation analyser (TRI-CARB 2300TR).

Cell respiration

INS-1 cells were treated with the OL3 extract and M3 in RPM! for 48 h under both normo-and hyperglycaemic conditions, respectively. After the treatment, a cell count was determined using Trypan Blue and a haemocytometer. Cell respiration rates were determined using the polarographic method. RPMI (1ml) was used to set the oxygen limit (to 100%), while aliquots of treated cells (1ml, [10.sub.6] cells/ml) were placed in fresh media within the 37[degrees]C incubation chamber. The oxygen consumption of these cells was measured over 500 s, and captured and analyzed using LabChart 6. Oxygen consumption rates were calculated from the initial steady traces of oxygen uptake and expressed per [10.sub.6] viable cells.Oligomycin (10011M) was used as a negative control (NC).

Mitochondrial membrane potential

The mitochondria! membrane potential (MMP) in INS-1 cells was determined by using the MMP detection kit (BD Biosciences).Cells were exposed to experimental conditions as indicated in section 2.5. Camptothecin (100[micro]M) was used as the NC. After treatment, cells were trypsinized, and resuspended in culture media (1ml). Cells (20 000 cells/ml) from each experimental condition were dyed using JC-1 dye, and quantified using the flow cytometry (Beckman Coultier, Cytomics FC 500).

qPCR

INS-1 cells were exposed to the OL extract (10[micro]g/m1) or M (500 ng/ml). RNA was isolated for qPCR using an RNeasy Mini kit (Qiagen). Total RNA was quantified spectrophotometrically and cDNA synthesized using a QuantiTect Reverse Transcription kit. qPCR was performed using the iQ SYBR Green Supermix (B10-RAD) and the BIO-RAD iQ detection system. GeNorm was used to determine the stability of reference genes tubulin, GAPDH and cyclophilin A (M value all less than 1.5), and generate normalization factors across all experimental conditions, relative to the target genes, glucose transporter (Glut)-2 and insulin.

Obesity animal model

Two-week-old male Wistar rats, weighing an average 69.34 [+ or -] 5.9 g were obtained from the University of Cape Town Animal Unit and housed in pairs/triplicates in standard animal cages. They were exposed to a 12L:12D photoperiod cycle. Animals were randomly divided into 7 groups, containing 6 rats each: lean control (LC) group, the obese control (0C) group, the obese metformin (MET) group, the obese sulfonylurea (SU) group, the obese aspirin (ASP) group, the obese OL treatment group and the obese marrubiin (M) treatment group (Ashour et al. 2009).The lean rats were fed rat chow and the obese rats were fed on a cafeteria diet as described by Mnonopi et al. (2011). After a treatment period of 2 weeks, intraperitoneal glucose tolerance test (1PGTT), intraperitoneal insulin tolerance test (IPITT), fasting insulin-, triglyceride-, cholesterol levels and pro-inflammatory markers (IL-1[beta]and IL-6) were evaluated.

Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT)

After the treatment period, rats were fasted for 15 h. Rats were injected intraperitoneally with either glucose (2 g/kg body weight) or insulin (1 unit/kg body weight). After administration of either glucose or insulin, blood glucose levels were determined from the tail vein at 0, 30, 60 and 120 min. Blood glucose was determined using a OneTouch Select AW 06505401A (Lifescan glucometer). Area under curve (AUC) of glucose (AU[Csub.g]) and insulin (AUC;) were calculated using the following formula: AUC = ([concentration.sub.0][degrees] + [concentration.sub.1]])/2 x [time.sub.1_0] (Chiou 1978).

Plasma insulin, triglycerides and cholesterol levels

Fasting plasma insulin concentrations were determined using a insulin RIA kit (Linco Research). Arterial blood was collected using a 1 ml syringe that contained 100 [micro]l of 0.105 M sodium citrate, and centrifuged at 300 x g for 15 min to obtain plasma. Post-experimental plasma triglyceride levels were determined with the GPO-PAP kit (Roche Diagnostics) and plasma cholesterol using the CHOD-PAP kit (Roche Diagnostics).

Plasma levels of 1L-1[beta]and 1L-6

The pro-inflammatory chemokines interleukin (IL)-1[beta] and IL-6 were determined across all experimental groups using ELISA kits (eBioscience, UK).

Statistical analysis Results are expressed as mean [+ or -] SEM or SD, unless otherwise indicated. Statistical significance was determined using the Student's t-test. All experiments were compared to their respective controls unless otherwise indicated.

Results

OL extract qua nti fication yielded a 5% marrubiin content (results not shown). Therefore, for all experiments M concentration was 5% of the OL extract concentration. INS-1 cells cultured under hyperglycaemic conditions increased chronic insulin secretion 1.5-fold, relative to the normoglycaemic control (NGC) cells (p < 0.05) (Fig. 2A). For chronic samples, exposure of INS-1 cells to M improved insulin secretion in a dose-dependent manner compared to NGC and hyperglycaemic control (HGC). Most noticeably, GSIS (Fig. 2B) and the stimulatory indices (Fig. 2D) significantly decreased insulin secretion at normoglycaemic conditions, but increased the levels at hyperglycaemic conditions with OL and M treatment, relative to the respective controls. The extract and M had a similar trend, significantly decreasing the stimulatory effect relative to NGC (Fig. 2B). A similar trend was observed for the stimulatory indices (Fig. 2D). Insulin content (Fig. 2C) was significantly increased in a dose-dependent manner by the extract and M under hyperglycaemic conditions.

Oxygen consumption was enhanced by OL and M treatments within the INS-1 cells under both the normo-and hyperglycaemic conditions (Fig. 3A). Both OL3 and M3 showed increased oxygen consumption rates under normoglycemic (1.6-fold, p < 0.05) and hyperglycaemic (1.7-fold, p < 0.05) conditions, relative to NGC and HGC, respectively. M3 continued to increase oxygen consumption, after the addition of oligomycin under both normo-and hyperglycaemic conditions to 1.8-and 1.7-fold (p < 0.05), respectively, relative to the respective control cells.Under similar conditions, 013 maintained the oxygen consumption rate relatively similar to that of the NGC cells, and increased the oxygen consumption rate to 1.7-fold (p <0.05) relative to the HGC. Hyperglycaemia conditions increased oxygen consumption by 1.5-fold (p <0.05) relative to NGC cells, while oligomycin treatment reduced oxygen consumption rate by 1.4-fold and 1.5-fold (p < 0.05) in NGC and HGC, respectively, relative to their respective controls.

Under normoglycemic conditions, the extract and M significantly increased MMP to 1.8 (p < 0.05) and 2.9-fold (p < 0.01), respectively (Fig. 3B). Under hyperglycemic conditions, both OL3 and M3 significantly increased MMP by 2.0-fold (p < 0.01) relative to the HGC cells. Camptothecin (NC) significantly decreased MMP of untreated INS-1 cells under hyperglycemic conditions by 2.0-fold (p < 0.01), relative to the HGC cells, however, this effect was not observed under normoglycemic conditions.

Under normoglycemic conditions, M3 significantly decreased Glut-2 expression, while a slight increase in expression was induced by OL treatment. Glut-2 expression was up-regulated by both OL3 (11-fold, p < 0.01) and M3 (12-fold, p < 0.01) under hyperglycemic conditions (Fig. 3C). Both the OL3 and M3 decreased insulin expression under normoglycemic conditions by 1.5-and 1.3-fold (p < 0.05), relative to control cells, supporting the GSIS results (Fig. 3D). Insulin expression was up-regulated in INS-1 cells exposed to OL3 (3.7-fold, p < 0.01) or M3 (4.5-fold, p < 0.01) under hyperglycemic conditions.

Table 1 summarizes the AUC obtained from the IPGTT and IPITT data for the obese rat model. The AU[C.sub.g] associated with IPGTT for the OC group was 1.5-fold higher than the LC group (p <0.05). Metformin, sulfonylurea and M decreased the AU[C.sub.g] by 1.5-fold relative to the OC group, which was comparable to the LCgroup. Aspirin had an AU[C.sub.g] similar to the OC group, where the OL extract decreased the AU[C.sub.g] by 1.4-fold relative to the OC group.

Table 1 The total AUC forlPCTTand IPlTT(n = 6)

Group        AU[C.sub.g]           AU[C.sub.1]
        (lPGTT)(mmol/(min1))       (IPITT)
                               (mmol/(min 1))

LC          912 [+ or -] 2.38    428 [+ or -]
                                         8.54

OC     1380 [+ or -] 5.62 (a)    499 [+ or -]
                                         8.71

MET     931 [+ or -] 5.90 (c)    273 [+ or -]
                                 2.31(a), (c)

SU      947 [+ or -] 8.42 (c)    489 [+ or -]
                                         6.71

ASP        1286 [+ or -] 9.87    411 [+ or -]
                                         6.43

OL         994 [+ or -] 11.54    549 [+ or -]
                                        12.34

M       932 [+ or -] 8.43 (c)    496 [+ or -]
                                         6.17

(a) p<0.05: relative to LC.
(c) p<0.05; relative to OC


The AUC, of the OC group was increased 1.2-fold relative to the LC group. Metformin and sulfonylurea decreased the AU[C.sub.i], by 1.8-and 1.1-fold, respectively, relative to the OC group. Aspirin decreased the AU[C.sub.i], by 1.2-fold, similar to the OC and LC groups, respectively. The AU[C.sub.i] of the OC and M groups were similar, and increased the AU[C.sub.i] to 1.2-fold relative to the LC group. The OL extract increased the AU[C.sub.i] to 1.1-and 1.3-fold, respectively, relative to the OC and LC groups.

Plasma insulin levels in OC rats were 1.3-fold higher, relative to LC rats (Table 2). The insulin levels of MET, ASP, SU and the OC groups were relatively similar, while all 3 groups increased insulin levels relative to the LC group. The OL extract increased insulin secretion to 4.0-and 5.0-fold (p < 0.01) while M increased insulin secretion to 2.2-and 2.8-fold (p < 0.01) relative to the OC and LC groups, respectively. Table 3 indicates that the triglyceride levels were very high (1.8-fold increase) in the OC group as compared to the LC group. While all treatments decreased triglycerides, total cholesterol, LDL cholesterol and increased HDL, the most noticeable effect was observed on M-treated rats. As a result M maintained an AI similar to LC.

Table 2 The effect of various treatments on insulin
secretion in cafeteriadiet induced obesity (n-6).

 Groitp         [Insulin] ng/M1

LC                 0.96 [+ or -] 0.01
OC                 1.24 [+ or -] 0.02
MET                 1.4 [+ or -] 0.01
SU              1.6 [+ or -] O.Ol (a)
ASP                 1.4 [+ or -] 0.02
OL        4.75 [+ or -] 0.01 (b), (d)
M           2.75 [+ or -] 003 (b),(d)

(a) p<0.05: relative to LC.
(b) p<0.01: relative to LC.
(d) p<0.01: relative to OC.

Table 3 The effect of metformin, sulfonylurea, aspirin, OLand
M treatment on triglycerides, total cholesterol.
HDL-cholesterol. LDL-cholesterol and Al (n = 7).


Group   Triglycerides      Total     HDL-choIesterol
       (mmol/1)        cholesterol
                       (mmol/l)

LC      2.1 [+ or - ]   145.6 [+ or  120.2 [+ or - ]
                 0.03      - ] 0.02             0.12

OC      3.8 [+ or - ]   245.0 [+ or  78.00 [+ or - ]
             0.02 (a)      - ] 0.11        0.004 (a)
                                (b)

MET     2.6 [+ or - ]   164.0 [+ or  131.0 [+ or - ]
             0.50 (c)      - ] 0.13        0.007 (c)
                                (c)

SU      2.3 [+ or - ]   163.0 [+ or  135.0 [+ or - ]
             0.30 (c)      - ] 0.03        0.006 (c)
                                (c)

ASP     2.9 [+ or - ]   176.0 [+ or  120.0 [+ or - ]
             0.16 (c)      - ] 0.12         0.07 (c)
                                (c)

OL      2.9 [+ or - ]   200.0 [+ or  150.0 [+ or - ]
             0.07 (c)      - ] 0.07         0.O5 (c)

M       2.0 [+ or - ]   145.0 [+ or  123.0 [+ or - ]
             0.01 (c)      - ] 0.01         0.02 (c)
                                (c)

Group   LDL-cholesterol   AL

LC       25.4 [+ or - ]   0.2
                   0.02

OC       67.1 [+ or - ]   2.1
               0.07 (b)   (b)

MET      33.3 [+ or - ]   0.3
              0.006 (b)   (d)

SU       28.2 [+ or - ]   0.2
               0.01 (b)   (d)

ASP      56.2 [+ or - ]   0.5
          0.07 (a), (b)  (a),
                          (d)

OL       50.3 [+ or - ]   0.3
          0.02 (a), (b)   (d)

M        22.1 [+ or - ]   0.2
               0.01 (b)   (d)

(a) p<0.05: relative to LC.
(b) p<0.07: relative to LC.
(c) p<0.05: relative to OC.
(d) p<0.01: relative toOC.


IL-1[beta] levels of the OC group were 1.2-fold higher than that of the LC group (p < 0.05), Fig. 4. All the treatments maintained levels similar to that of the LC. Similar results were observed for IL-6 secretion, where all treatments normalized levels similar to LC, while OC had elevated IL-6 levels by 1.4-fold relative to LC.

Discussion and conclusion

Control of insulin secretion by pancreatic 13-cells is critical for glucose homeostasis. Drugs currently available for diabetes management have certain drawbacks (Kaleem et al. 2006). Recently, there has been great concern and withdrawal of thiazolidinediones (TZDs) as oral drugs that enhance insulin sensitivity in T2D patients (Saraogi et al. 2011). Rosiglitazone use has been found to increase the risk of congestive heart failure in T2D patients (Saraogi et al. 2011). Therefore, research needs to continue to find alternative more effective and safer treatments to manage T2D. This study was undertaken to investigate the mechanism of the hypoglycaemic and cardioprotective effects of L leonurus extracts and of a terpenoid, marrubiin. GSIS studies indicated that the OL extract and M induce insulin secretion under hyperglycaemic conditions. This was linked with mitochondrial metabolism as ATP production is stimulated by an increase in membrane potential (Zhang et al. 2006) and oxygen consumption via the respiratory chain. Both the OL and M increased oxygen consumption and the expression of Glut-2 and insulin genes. Where equivalent concentrations of M were maintained in the OL extract these effects could be attributed to M in the plant extract.

A cafeteria diet was selected to mimic the common practises of a Western style diet which is rich in carbohydrates and fats, leading to hyperphagia and weight gain. It is widely used to induce experimental obesity (Sciafani and Springer 1976). The rats that were on the cafeteria diet (OC group) were hyperglycemic relative to the LC group. The OC group failed to clear glucose levels after 2 h (IPGTT).Although the insulin levels of OC and LC were similar, the OC group failed to improve glucose uptake. MET treatment is associated with increase glucose utilization and decreases hepatic glucose production (Yoshida et al. 2009). In this study, 2 weeks treatment with MET improved IPGTT and IPITT as expected. SU improved IPGTT and IPITT through increasing insulin secretion. Although ASP has the potential to increased insulin sensitivity (IPITT), it should be noted that this is dose dependent. The dose administered in this study was too low. M significantly lowered the fasting blood glucose levels in this model. The effect of the OL extract on IPGTT was not as significant as that exhibited by M. Ojewole (2005) had shown that the aqueous extract of L leonurus exhibited hypoglycaemic effects on STZ-induced diabetic rats, and further concluded that terpenoids present in this plant could potentially play a role in improving glucose levels (Ojewole 2005). It had been reported that the mechanism of action of terpenoids and steroids are to stimulate insulin secretion thereby increasing the sensitivity of islets to promote glucose uptake in pancreatic [beta]-cells (Li et al. 2004). A study using Tectona grandis (Verbenaceae), a medicinal plant used to treat diabetes mellitus (DM) revealed that it contained terpenoids amongst other compounds, also supporting these findings (Ghaisas et al. 2009). Ethanolic extracts of this plant reduced plasma glucose levels and triglycerides improving glucose uptake and decreased IR.These findings support our proposal that M, a diterpenoid present in the L. leonurus extract is responsible for the stimulation of insulin secretion in INS-1 cells and in the obese rat model. M enhanced insulin secretion, relative to the OC group, and correlated with GSIS where the stimulatory index was increased relative to the HGC. M also enhanced insulin secretion relative to the LC group. This could possibly be due to the cells being directly exposed to M, while the in vivo studies possibly resulted in metabolites of M stimulating the release of insulin under normoglycaemic conditions (LC group). M did not significantly improve IPITT, relative to both controls (LC and OC groups), therefore it could be eliciting its effects directly on islet cells, causing secretion of insulin. This, however, requires future investigation. The OL extract had the most prominent effect on insulin secretion, which can be possibly attributed to other phytochemical compounds present in the extract or the synergy of the compounds.

The OC group had significantly high levels of plasma cholesterol, triglycerides and LDL with a significant decrease in HDL levels. Based on the epidemiological data, there are 3 risk categories for Al, low <0.11, intermediate 0.11-0.21 and high >0.21 (Holmes et al. 2008).The atherogenic index indicates cardiovascular risk (Holmes et al. 2008). The Al of the OC group was 2.1 indicative of a high risk category which was normalized through treatment with MET, M and SU. Metformin has been reported to reduce circulating levels of triglycerides, FFAs, and LDL cholesterol, while it increases the levels of HDL cholesterol (Nathanson and Nystrom 2009). ASP is commonly recommended for CVD patients. However, in this study the dose of aspirin utilized, showed the least effect on Al profile (0.5). M decreased triglyceride, cholesterol and LDL levels, while it significantly increased HDL levels. Although OL also improved the lipid profile of the OC rats, it was not as significant as M.M maintained the HDL, LDL, total cholesterol and the Al levels relative to the LC group, placing this group in the intermediate risk category. In a study conducted on M. vulgare, it significantly decreased cholesterol and triglyceride levels in human T2D patients by 4.16 and 5.78%, respectively (Herrera-Arellano et al. 2004). Ichnocarpus frutescence (L.) R. Br. (Apocynaceae) and Tectona grandis studies also observed decrease triglyceride, LDL cholesterol levels, and increased HDL levels (Ghaisas et al. 2009; Subash-Babu et al. 2008). The presence of terpenoids in these two plants is proposed to be responsible for insulin secretion and the anti-atherogenic properties, correlating to our findings. IL-1[beta] is the key regulator of the inflammatory response (Maedler et al. 2011). T2D patients have been found to have elevated levels of IL-1 p and it sets on and regulates inflammatory response which has a negative effect on [beta]-cell mass and function. Several studies have shown that treatment of T2D patients with IL-1 antagonist reduces hyperglycaemia while improving [beta]-cell function (Ehses et al. 2009). Higher circulating levels of IL-6 are associated with IR in skeletal muscle and liver (Alarcon-Aguilar et al. 2010). M and L. leonurus reduced the levels of these pro-inflammatory markers. M also has been found to significantly reduce TNF-cx plasma levels in cafeteria diet inducedobesity (Mnonopi et al. 2011), where TNF-[alpha] is associated with hinderance of Glut-4 translocation in skeletal muscle.

A multi-model therapeutic approach is required for the treatment of T2D since it is a multifactorial pathogenetic disease.Medicinal plants offer a large scope for combating the threat of the diabetic pandemic (Tiwari and Rao 2002). In this study, M was found to stimulate insulin secretion in vitro and in vivo. The increase in MMI, and respiration correlated with the increase in GS1S. The in vivo animal model was utilized to evaluate the effect of M in a physiological environment. The terpenoid M, therefore presents as a lead compound that could be used in the treatment of T2D patients who are at risk of CVD, because of its antidiabetic, anti-atherogenic and anti-inflammatory properties.

Acknowledgements

The authors wish to acknowledge the support of this study by the National Research Foundation (NRF) of South Africa and the International Foundation of Science (IFS). We wish to thank Martin Jastroch for his assistance with oxygen consumption experiments.

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N. Mnonopi (a), R.-A. Levendal (a), N. Mzilikazi (b), C.L. Frosta (a.)*

(a.) Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, P.O. Box 77000, Port Elizabeth 6031, South Africa

(b.) Centre for African Conservation Ecology, Department of Zoology, Nelson Mandela Metropolitan University, P.O. Box 77000, Port Elizabeth 6031, South Africa

Abbreviations: Al, atherogenic index; ASP, aspirin; AUC, area under the curve; C, control; CVD, cardiovascular diseases; DM, diabetes mellitus; FFAs, free fatty acids; Glut, glucose transporter; GSIS. glucose-stimulated insulin secretion; IL, interleukin; IPGTT, intraperitoneal glucose tolerance test; HGC, hyperglycaemic control; WITT, intraperitoneal insulin tolerance test; IR, insulin resistance; LC, lean control; M, marrubiin; MET, metformin; MMP, mitochondria! membrane potential; NMMU, Nelson Mandela Metropolitan University; NC, negative control; NGC, normoglycaemic control; OC, obese control; 01, organic extract; PFP. pentafluorophenyl; RIA, radioimmunoassay; STZ, streptozotocin; SU, sulfonylurea; T2D, type 2 diabetes; TZDs, thiazolidinediones.

* Corresponding author. Tel.: +27 41 504 4123; fax: +27 41 504 2814.

E-mail address: carminita.frost@nmmu.ac.za (C.L. Frost).

doi: 10.1016/j.phymed.2011.12.008
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Author:Mnonopi, N.; Levendal, R.-A.; Mzilikazi, N.; Frost, C.L.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:6SOUT
Date:Apr 15, 2012
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