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RESEARCH ARTICLE

SA JOURNAL OF DIABETES & VASCULAR DISEASE

62

VOLUME 13 NUMBER 2 • DECEMBER 2016

the Committee for the use of animals in research of the University

of Stellenbosch – numbers P05/11/013 and P07/11/020.

The

P glandulosa

plant material was originally obtained from

naturally growing plants. The material was handled according to a

patented and standardised procedure

8

and pre-packed in capsules

for human consumption, which we emptied and weighed. The

voucher specimen was reported previously.

8

Plasma glucose levels were determined in the fasting state. Blood

was obtained via a tail prick and glucose levels were determined

using a conventional glucometer (Cipla MedPro). Plasma was stored

at –80°C in a Snijders Scientific Ultracool (Tilburg, the Netherlands)

and insulin levels were determined using a coat-a-count assay

(Diagnostic Products).

Intra-peritoneal glucose tolerance curves (IPGTTs) were

generated in the animals after an 18-hour fast. Animals were

injected intra-peritoneally with 1 g/kg of a 50% sucrose solution

and blood glucose levels were monitored over a 120-min period.

After removal, the hearts were arrested in ice-cold Krebs Henseleit

(KH) medium (in mM: NaCl 119, NaHCO

3

25, KCl 4.75, KH

2

PO

4

1.2, MgSO

4

.7H

2

O 0.6, Na

2

SO

4

0.6, CaCl

2

.2H

2

O 1.25, glucose 10)

and immediately (within 30 sec) mounted onto the aortic cannula

of a perfusion rig. The pulmonary vein was connected to a second

cannula in order to perform perfusions in the working-heart mode

with a preload of 15 cm H

2

O and an afterload of 100 cm H

2

O, as

described previously.15 The perfusion medium was continuously

gassed with 95%O

2

/5%CO

2

. Hearts were fitted with a temperature

probe and the temperature was kept constant at 36.5–37°C.

After a stabilisation period of 30 min, rat hearts were subjected

to 35-min regional ischaemia by coronary artery ligation, followed

by reperfusion for one hour, as described previously.

15

Infarct size

was determined according to a well-established protocol,

15

followed

by planimetry, and expressed as a percentage of the area at risk.

Planimetry was performed blind by a third party.

Mouse hearts were perfused retrogradely, meaning via the

aorta without a connection to the pulmonary vein. After the

30-min stabilisation period, the hearts were subjected to 20-min

normothermic ischaemic cardiac arrest (NICA) by stopping all

perfusion. This was followed by one hour of reperfusion, after

which the infarct development through the whole heart was

determined as described above.

To measure blood pressure, rats were placed in restraining

holders with a dark nose cone to calm them. The restrainers were

placed on a heating pad (32 ± 2°C) to warm the rat and maintain

blood flow to the tail. Animals were placed in the restrainers for

at least five minutes before monitoring the blood pressure using

a computerised tail-cuff blood pressure monitor (Kent Scientific

Corporation, Connecticut, USA). Prior to commencement of the

experiment, rats were subjected to the above procedure daily for

at least a week to train the animals for the procedure and to avoid

stress in the rats during experimental determinations.

Animals were placed individually in metabolic cages and the

volume of urine was determined over a period of 24 hours.

Western blotting

Frozen tissues were pulverised with a liquid nitrogen pre-cooled

mortar and pestle and then extracted in lysis buffer containing

in mM: Tris-HCl 20 (pH 7.5), EGTA 1, EDTA 1, NaCl 150, Na

2

VO

3

1, beta-glycerophosphate 1, sodium-pyrophosphate 2.5, PMSF

0.3, Triton X-100 1% (v/v) plus 10 μg/ml leupeptin and aprotinin,

respectively, using a Polytron PT10 homogeniser, 2 × 4 sec, at

anti-infective and anti-parasitic compounds have also been isolated

from this plant.

9

In view of the hypoglycaemic effects of

P glandulosa

, as well as

its ability to partially restore the function of pancreatic tissue and

increase cardiomyocyte insulin sensitivity,

7

we set out to determine

the cardiovascular effects of treatment, using a wellcharacterised

rat model of obesity and pre-diabetes with known cardiovascular

insufficiency and endothelial dysfunction.

10,11

In addition, using a

rat model of high-fat feeding known to develop hypertension,

12

we determined whether

P glandulosa

had any effects on the

development of high blood pressure.

Models

Diet-induced obesity (DIO)

As described previously,

10

Wistar rats (180–200 g) were randomly

divided into a control and diet group. The DIO group was fed

a diet of normal rat chow supplemented with sucrose and

condensed milk for a basic period of eight weeks. From weeks

eight to 16 the rats were treated with

P glandulosa

(100 mg/kg/

day) set in jelly/gelatine blocks and given to each one individually

according to the weight of the animal.

8

This was done to ensure

absolute compliance and dose control. The dose of

P glandulosa

was calculated as previously described.

8

The diet to induce pre-diabetes in the animals was

based on hyperphagia.

13

Animals were anaesthetised with

sodium pentobarbital (160 mg/kg, intra-peritoneally) before

experimentation. At the time of sacrifice, their body weight and

the weight of the intra-peritoneal fat were noted and trunk blood

was collected for biochemical analyses. For Western blot analyses,

the hearts were removed, immediately snap-frozen in

liquid nitrogen and stored at –80°C.

High-fat diet (HFD)

To induce high blood pressure, the rats were fed a diet containing

the following per kg of food: cooking fat 400 g, fructose 100 g,

casein 100 g, cholesterol 10 g, and rat chow pellets 390 g. Blood

pressure was monitored on a weekly basis over 16 weeks. Treatment

with

P glandulosa

(100 mg/kg/day) given in jelly blocks was either

started at the onset of the diet to study the effect on prevention of

the development of hypertension, or after a period of 12 weeks of

the HF diet to study its anti-hypertensive effects. Rats treated with

captopril (50 mg/kg/day) from the onset of the diet were included

as a positive control. All animals were also placed individually in

metabolic cages in order to collect urine samples.

CIRKO mice

A mouse model of animals with a cardiac conditional ablation of

the insulin receptor was used in conjunction with their C57Bl6

littermates.

14

Mice were fed normal chow and treated with

P

glandulosa

at a similar dose to that of the rats for a period of eight

weeks before experimentation.

Methods

Animals had free access to food and water and were kept on a

12-hour day/night cycle in the Central Research Facility of the

Faculty of Health Sciences of the University of Stellenbosch. The

study conformed to the revised South African National Standard for

the Care and Use of Animals for Scientific Purposes (South African

Bureau of Standards, SANS 10386, 2008) and was registered with