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Assessment of the Antidiabetic Potential of an Aqueous Extract of Honeybush (Cyclopia intermedia) in Streptozotocin and Obese Insulin Resistant Wistar Rats

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Assessment of the Antidiabetic Potential of an Aqueous Extract of Honeybush (Cyclopia intermedia) in Streptozotocin and Obese Insulin Resistant Wistar Rats

13
Assessment of the Antidiabetic Potential
of an Aqueous Extract of Honeybush
(Cyclopia intermedia) in Streptozotocin and
Obese Insulin Resistant Wistar Rats
Christo J.F. Muller1, Elizabeth Joubert2,3, Kwazi Gabuza1,
Dalene de Beer2, Stephen J. Fey4 and Johan Louw1
1Diabetes Discovery Platform, Medical Research Council (MRC), Cape Town,
2Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC),
Infruitec-Nietvoorbij, Stellenbosch,
3Department of Food Science, Stellenbosch University, Stellenbosch,
4Department of Biochemistry and Molecular Biology,
University of Southern Denmark, Odense,
1,2,3South Africa
4Denmark
1. Introduction
It has been estimated that diabetes will affect 439 million adults by 2030, with the major
increase occurring in developing countries (Shaw et al., 2010). It is projected that it will rank
as the 9th leading cause of death in low-income countries (Mathers & Loncar, 2006). There
are two major types of diabetes, i.e. type 1 (T1D) or insulin dependent diabetes and type 2
(T2D) or non-insulin dependent diabetes. The incidence of T2D is reaching epidemic
proportions and has been associated with an increase in obesity (Venables & Jeukendrup,
2009). According to the World Health Organisation (WHO, 2011) the main complications
associated with diabetes are cardiovascular disease and renal failure. Although genetic
factors may play a role, life-style factors, such as reduced exercise and poor diet, specifically
a high carbohydrate, high fat diet devoid or low in fruit and vegetables, have been shown to
increase the risk of diabetes (Astrup, 2001).
Medicinal plants have been used in folk medicine and traditional healing systems such as
Ayurveda and Traditional Chinese Medicine (TCM) for the treatment of diabetes (T.S.C. Li,
2003; Modak et al., 2007; Singh et al., 2009; Yen et al., 2003). On the African continent as
many as 90% of the populations of some countries relies on plants as the principal source of
medicine for the treatment of different diseases, including diabetes (Hostettman et al., 2000),
as they provide an affordable alternative to drugs. In South Africa a large number of plants,
belonging to plant families such as the Asteraceae and Lamiaceae, amongst others, have
been traditionally used for the treatment of diabetes (Deutschländer et al., 2009; Erasto et al.,
2005; Thring & Weitz, 2006).
Phytochemicals – Bioactivities and Impact on Health
314
Globally, there is a movement towards alternatives to single chemical entities as favoured
by the pharmaceutical industry. These alternatives are rationally selected, carefully
standardised, synergistic traditional herbal formulations and botanical drug products which
are supported by robust scientific evidence (Patwardhan & Mashelkar, 2009). In many
instances the value of herbal and medicinal plant extracts lies not in a single compound, but
in their complex phytochemical nature. These complex mixtures of often unspecified
compounds are able to modulate multiple targets (Y. Li et al., 2008). Antidiabetic phenolic
compounds in the extracts also have the ability to ameliorate oxidative stress (Han et al.,
2007), an underlying mechanism to the pathogenesis of diabetes (Ceriello & Motz, 2004).
One such compound is the xanthone C-glucoside, mangiferin (1,3,6,7-tetrahydroxy-xanthone-
C2-β-D-glucoside), demonstrating antihyperlipidaemic, antihyperglycaemic and antioxidant
properties (Wauthoz et al., 2007). Mangiferin (Fig. 1) was shown to protect against
streptozotocin (STZ)-induced oxidative damage to cardiac and renal tissues in Wistar rats
(Muruganandan et al., 2002). Its presence in the endemic South African Cyclopia spp. (family
Fabaceae; tribe Podalyrieae) suggested the potential use of these plant species as antidiabetic
nutraceuticals or even phytopharmaceuticals. Hesperidin (Fig. 1), another antioxidant and
compound demonstrating hypoglycaemic properties in rodents (Akiyama et al., 2010; Jung et
al., 2004), is also one of the major monomeric polyphenols present in Cyclopia spp. (Joubert et
al., 2003). A decoction of Cyclopia spp. was used in the past as a restorative and as an
expectorant in chronic catarrh and pulmonary tuberculosis. However, it is as the herbal tea,
honeybush, that Cyclopia spp. are increasingly appreciated by consumers world-wide. The tea
is primarily exported to the Netherlands, Germany, United Kingdom and United States of
America. It is even exported to traditional tea-drinking countries such as Sri Lanka, India,
Japan and China. Commercial herbal tea production comprised three main species, viz. C.
genistoides, C. intermedia and C. subternata. Of these, C. intermedia, harvested almost exclusively
from the wild, provides the bulk of honeybush production.
O
O
R3
R4
R2
R1
2
, mangiferin
(R
1
= H; R
2
=
C
-
-D-glucoside)
3
, isomangiferin
(R
1
=
C
-
-D-glucoside; R
2
= H)
4,
eriodictyol-glucoside
(R
1
, R
2
, R
3
, R
4
= OH;
O
- or
C
-
-D-glucosyl in unknown position)
5,
eriocitrin
(R
1
=
O
-
-D-glucosyl; R
2
, R
3
, R
4
= OH)
6,
hesperidin
(R
1
=
O-
-D-glucosyl; R
2
, R
4
= OH;
R
3
= OCH
3
)
8,
hesperetin
(R
1
, R
2
, R
4
= OH; R
3
= OCH
3
)
O
O
OH
OH
OH
OH
7
, luteolin
R2
OOH
OH
OH
OH
O
R1
Fig. 1. Structures of phenolic compounds of C. intermedia extract.
Assessment of the Antidiabetic Potential of an Aqueous Extract
of Honeybush (Cyclopia intermedia) in Streptozotocin and Obese Insulin Resistant Wistar Rats
315
A contributing factor to its growing popularity is the body of scientific evidence that
honeybush has potential health benefits, including antioxidant, anticancer and
phytoestrogenic properties (Joubert et al., 2008a). The greatest demand is for the traditional
product, which is ‘fermented’ (oxidised) to form the characteristic dark-brown colour and
sweet flavour. However, this is accompanied by a substantial reduction in the phenolic
content of the plant material and extract, as well as a decrease in antioxidant activity
(Joubert et al., 2008b), justifying investigating the benefits of unfermented C. intermedia for
human health.
With T2D being the most common form of diabetes, representing more than 90% of all cases
(WHO, 2011), the antidiabetic potential of unfermented C. intermedia was investigated using
a diet-induced obese insulin resistant (OBIR) rat model. Feeding rats a high fat diet induces
a state of insulin resistance associated with impaired insulin-stimulated glycolysis and
glycogen synthesis (Kim et al., 2000). The STZ-induced diabetic rat model, following
pancreatic β-cell destruction and resulting in insulin deficiency rather than insulin resistance
(OBIR model), was used to establish the optimal acute glucose lowering dose of a C.
intermedia extract. The investigation focused on a hot water extract of C. intermedia as it
represents normal preparation of the herbal tea, albeit under more severe extraction
conditions, but without introducing qualitative changes to composition as is the case for
organic solvent extraction.
2. Material and methods
2.1 Chemicals
General analytical grade laboratory reagents were purchased from Sigma-Aldrich (St. Louis,
USA) and Merck (Darmstadt, Germany). Authentic reference standards were obtained from
Sigma-Aldrich (mangiferin, hesperidin) and Extrasynthese (Genay, France; eriocitrin,
luteolin). Isomangiferin was isolated from C. subternata (De Beer et al., 2009). Acetonitrile for
HPLC analysis was gradient grade for liquid chromatography (Merck, Darmstadt,
Germany). HPLC grade water was prepared by purifying laboratory grade water
(Continental Water Systems Corp., San Antonio, USA) with a Milli-Q 185 Académic Plus
water purification system (Millipore, Bedford, USA).
STZ, fluothane, 50% dextrose solution, metformin hydrochloride and rosiglitazone maleate
(Avandia®) were obtained from Sigma-Aldrich, AstraZeneca Pharmaceuticals
(Johannesburg, South Africa), Intramed (Johannesburg, South Africa), Rolab (Johannesburg,
South Africa) and GlaxoSmithKline (Bryanston, South Africa), respectively.
2.2 Plant material and extract preparation
Cyclopia intermedia shoots (ca 220 kg) were harvested according to normal practice by cutting
the shoots sprouting from the rootstock directly above soil level. All the plant material was
harvested from a natural stand on Nooitgedacht Farm in the Langkloof area, South Africa. The
part of the stem without leaves or with only a few leaves (Fig. 2) was removed before further
processing, entailing cutting of the shoots into small pieces (<4 mm) and mechanical drying at
40 °C to less than 10% moisture content (Joubert et al., 2008b), giving ca 50 kg dried plant
material. The plant material was pulverised with a Retsch rotary mill before pilot plant
extraction. Water heated to >95 °C was added to the pulverised plant material in a 1:10 (m/v)
Phytochemicals – Bioactivities and Impact on Health
316
ratio and continuously stirred for 30 min. (final extract temperature was 70 °C), whereafter the
extract was pumped to an in-line continuous centrifuge for removal of the insoluble matter.
Following centrifugation, the extract, containing 2.69 g soluble matter/100 mL, was cooled to
ca 20 °C using a tubular heat exchanger and aliquots bottled for daily feeding of OBIR rats.
The aliquots were stored at -20 °C until use. An aliquot (ca 1000 mL) was freeze-dried in an
Edwards Modulyo bench-top freeze-drier (Edwards High Vacuum Ltd, Crowley, UK) for
acute dosing of STZ-induced diabetic rats and for phenolic content analysis.
(a) (b)
Fig. 2. (a) Cyclopia intermedia plant showing aerial parts (top section of a typical shoot with thin
stems and leaves). (b) Harvested shoots before removal of the stem parts without leaves.
2.3 HPLC analysis of extract
HPLC analysis was performed using an Agilent 1200 series HPLC system consisting of a
quaternary pump, autosampler, in-line degasser, column oven and diode-array detector
(Agilent Technologies Inc., Santa Clara, USA) controlled with Chemstation 3D LC software.
Separation was performed on a Zorbax Eclipse XDB-C18 column (150 x 4.6 mm, 5 m
particle size, 80 Å pore size) from Agilent Technologies protected by a guard column with
the same stationary phase. The separation was achieved with mobile phases consisting of
0.1% formic acid and acetonitrile using the solvent gradient reported previously (De Beer &
Joubert, 2010). The flow-rate and column temperature were maintained at 1 mL/min and 30
°C, respectively. The freeze-dried extract was dissolved in 16% DMSO (ca 5 mg/mL) and
filtered using a 33 mm Millex-HV PVDF syringe filter unit with 0.45 m pore size
(Millipore) before injection (20 L). Compound identification was based on retention times
and UV-Vis spectra of authentic standards where available (mangiferin, isomangiferin,
eriocitrin, hesperidin, luteolin and hesperetin). An additional peak was identified as an
Assessment of the Antidiabetic Potential of an Aqueous Extract
of Honeybush (Cyclopia intermedia) in Streptozotocin and Obese Insulin Resistant Wistar Rats
317
eriodictyol-glucoside based on similarity of its retention time and UV-Vis spectrum with a
compound identified previously using liquid chromatography with mass spectrometric
detection (De Beer & Joubert, 2010). An unidentified compound was also observed, which
had retention time and UV-Vis characteristics similar to a compound previously detected in
several Cyclopia spp. (De Beer & Joubert, 2010). Mangiferin, isomangiferin and luteolin were
quantified using the peak areas at 320 nm, while the eriodictyol-glucoside, eriocitrin,
hesperidin, hesperetin and the unidentified compound were quantified using the peak areas
at 288 nm. A calibration series consisting of mangiferin (0.05-2.5 µg injected), isomangiferin
(0.05-2.5 µg injected), eriocitrin (0.01-0.8 µg injected), hesperidin (0.01–1.5 µg injected),
hesperetin (0.003-0.25 μg injected) and luteolin (0.05-0.35 µg injected) was used for external
calibration. The eriodictyol-glucoside was quantified in terms of eriocitrin equivalents,
while the unidentified compound was quantified in terms of hesperidin equivalents.
2.4 Animal study
Ethical approval was obtained from the Ethics Committee for Research on Animals (ECRA)
of the Medical Research Council of South Africa.
Male Wistar rats, obtained from the Primate Unit of the Medical Research Council
(Tygerberg, South Africa), were used throughout the study. The rats were housed
individually in wired top and bottom cages, fitted with PerspexTM houses and kept in a
controlled environment of 23–24 °C, 50% humidity and a 12 h light/dark cycle.
2.4.1 STZ-induced diabetic rats
Adult male Wistar rats (200-250 g) were injected intramuscularly with freshly prepared STZ
[35 mg/kg body weight (BW)] in 0.1 M citrate buffer (pH 4.5) to induce stable non-
ketoacidotic diabetic rats. Blood samples were taken from the tail tip 72 hrs after STZ
injection and the plasma glucose concentrations determined using a glucometer (Precision
Q.I.D, Abbott Laboratories, Johannesburg, South Africa). Rats with fasting blood glucose
levels of >15.0 mmol/L were considered diabetic and were selected for the acute dose
finding study of the extract.
2.4.2 Acute dosing of diabetic STZ rats with honeybush extract
The efficacy of a single dose of honeybush extract was determined by administering
different doses of freeze-dried extract to STZ diabetic rats. Following a 3 hr fast, baseline
plasma glucose concentrations of diabetic STZ rats were determined. The freeze-dried
honeybush extract (reconstituted in a fixed volume of distilled water yielding e.g. 5 mg/mL
for the 5 mg/kg BW dose, etc.) was administered by oral gavage under light anaesthesia (by
inhalation of 2% fluothane with 98% oxygen) to four experimental groups of five STZ rats
each at doses of 0 (vehicle control), 5, 25 and 50 mg/kg BW. Plasma glucose concentrations
were determined hourly over a 6 hr period.
2.5 Chronic treatment of OBIR rats with honeybush extract
The efficacy of honeybush extract to ameliorate diet-induced insulin resistance, characterised by
hyperglycaemia, hyperinsulinaemia, hyperglucagonaemia and dyslipidaemia was investigated
using OBIR Wistar rats chronically exposed to the extract for 12 wks (described below).
Phytochemicals – Bioactivities and Impact on Health
318
2.5.1 Inducing insulin resistance in Wistar rats
Three-week old weanling Wistar rats (male) were fed a 40% high fat diet (Table 1) and 30%
sucrose in their drinking water ad libitum for 9 wks. The high fat diet in combination with
sucrose induces insulin resistance and obesity with slightly elevated fasting glucose
concentrations (Hallfrisch et al., 1981; Krygsman et al., 2010). After 9 wks on the high fat and
sucrose diet, blood was collected for baseline glucose and insulin determination. Thereafter
the rats were allocated into experimental groups and maintained on the high fat and sucrose
diet during the subsequent 12-wk treatment.
2.5.2 Experimental groups
The untreated control consisted of six 12-wk old OBIR rats that were randomly assigned to
the control group.
Groups E1 – E5 (honeybush extract treated groups) consisted of five groups of ten OBIR rats
each, receiving 538, 1075, 1792, 2150 or 2688 mg/100 mL honeybush extract (hot water
soluble solids), respectively, as their drinking fluid, which also contained 30% sucrose
(Table 2). The daily fluid intake was measured for each rat and the average amount of liquid
Nutrients % Energy
Protein 15.09
Fat 40.17
Saturated fatty acids 18.27
Monounsaturated fatty acids 11.45
Polyunsaturated fatty acids 5.75
Carbohydrate 44.73
Kcal/g of food 2.06
Kcal/g of sucrose 0.60
Total energy of diet (Kcal/g) 2.66
Table 1. High fat diet (HFD) macronutrient and calorific composition.
Group Weight Treatment
concentration
Fluid
intake
Treatment
intake/day
Mangiferin
intake/day
Hesperidin
intake/day
Control 476 ± 20 30 ± 2
E1 441 ± 12 538 33 ± 4 77.2 ± 9.6 4.47 ± 0.55 0.27 ± 0.03
E2 456 ± 20 1075 42 ± 5 206.9 ± 27.5 11.99 ± 1.60 0.73 ± 0.10
E3 442 ± 25 1792 31 ± 4 255.1 ± 64.6 14.79 ± 3.74 0.65 ± 0.23
E4 438 ± 21 2150 32 ± 2 299.1 ± 23.5 17.33 ± 1.36 1.06 ± 0.08
E5 457 ± 36 2688 43 ± 2 531.3 ± 61.1 30.79 ± 3.54 1.88 ± 0.22
Met 489 ± 22 31 ± 4 22.0
Rosi 479 ± 26 29 ± 2 4.0
Table 2. OBIR rat body weight (g), extract concentration (mg/100 mL), fluid intake (mL),
treatment intake (mg/kg BW), i.e. C. intermedia extract (E1-5), metformin (Met) or
rosiglitazone (Rosi), as well as equivalent intake of mangiferin (mg/kg BW) and hesperidin
(mg/kg BW), for chronic treatment experiment.
Assessment of the Antidiabetic Potential of an Aqueous Extract
of Honeybush (Cyclopia intermedia) in Streptozotocin and Obese Insulin Resistant Wistar Rats
319
consumed per week for each rat was calculated (Table 2). Aliquots of the aqueous
honeybush extract were defrosted daily. In the case of E1 to E4 the extract was diluted with
water to give the required concentration of hot water soluble solids, while E5 represented
the undiluted extract.
The metformin and rosiglitazone treated groups consisted of three OBIR rats each that
received metformin hydrochloride or rosiglitazone maleate at dosages of 22 and 4 mg/kg
BW, respectively, in distilled water.
2.5.3 Blood parameters
Determination of fasting plasma glucose. The STZ-induced diabetic and OBIR rats were fasted
for 3 hrs and overnight, respectively, prior to determining their fasting plasma glucose
concentrations. A drop of blood was collected from the tail tip and the plasma glucose
concentration determined.
Determination of fasting serum insulin. Rats were anaesthetised by 2% fluothane inhalation
with 98% oxygen. Blood was collected from the tail tip into Eppendorf tubes and stored on
ice until centrifuged at 2500 rpm for 15 min. at 4 °C. Following centrifugation, the serum
samples were stored at -20 °C until analysis. The serum insulin concentration was
determined by radioimmunoassay using a rat insulin measurement kit from Linco®
Research (St. Charles, USA).
Intravenous glucose tolerance test (IVGTT). Rats fasted overnight were anaesthetised as
described above and a drop of blood obtained from the tail tips. This was used for
measuring baseline glucose. Glucose (50% dextrose solution), at a dose of 0.5 mg/kg BW,
was injected intravenously over 20 sec and glucose measurements taken at 5, 10, 20, 30, 40,
50 and 60 min.
Determination of fasting plasma cholesterol. Rats were anaesthetised as described above and
blood, collected from the tail tips, was prepared and stored at -20 °C for analysis. Total
cholesterol concentrations were determined by Pathcare Laboratories (Cape Town, South
Africa), using a Bayer-Technicon RA 1000 auto-analyser.
2.5.4 Immunocytochemistry and image analysis of the pancreata
Harvesting of pancreata. After 12 wks of treatment the rats were euthanised by exsanguination
under sodium barbital anaesthesia and pancreata harvested. The whole pancreas was
removed, fixed overnight in 4% buffered formaldehyde (pH 7.5) and processed into paraffin
wax by standard histological methods. Serial 4 μm thick sections were cut for
immunocytochemistry.
Immunocytochemistry. Serial wax sections attached onto silane coated slides were de-waxed
with xylene and hydrated through descending grades of ethanol into water. Slides were rinsed
in 50 mM Tris–buffered-saline (pH 7.4) and double immuno-stained, using anti-insulin and
anti-glucagon primary antibodies. Primary antibody binding to insulin or glucagon was
detected by avidin D-biotinylated horseradish peroxidase or streptavidin-biotin-
complex/alkaline phosphatase conjugated link antibodies. Insulin positive labeling (β-cells)
was visualised with fuchsin red and α-cells with diaminobenzidine tetrahydrochloride.
Method controls involved omission of the primary antibody (anti-insulin or anti-glucagon).
Phytochemicals – Bioactivities and Impact on Health
320
Image analysis. Both β- and α-cell areas were measured on each section (minimum of ten
sections per group). Computer-assisted measurements were taken with a Canon Powershot
S40 digital camera (Tochigi, Japan) mounted on an Olympus BX60 light microscope (Tokyo,
Japan), attached to a personal computer to capture images. The acquired images were
transferred to the computer using remote capture software from Canon. Image analysis was
performed with Leica Qwin Plus Software (Cambridge, UK). The ratio of either the β-cell
positive or the α-cell positive area to the total pancreas area was calculated. β-Cell and α-cell
sizes were calculated by dividing the total area of each of the cell types by the number of
nuclei counted.
2.6. Statistical analysis
Results were entered into an Excel spreadsheet and statistically analysed. For the acute STZ
rat experiment hourly (t = 1–6) glucose concentrations were compared against baseline (t =
0) and the control for each corresponding time point. For the chronic OBIR rat experiments
each data point was compared to the corresponding control value using ANOVA with a
Dunnet post-hoc test (Prism version 5, Graphpad software®). The values presented are the
mean ± SE.
3. Results
3.1 Extract composition
The phenolic compound structures for the major compounds of the C. intermedia extract are
shown in Fig. 1, the plant shoots in Fig. 2 and the extract HPLC profile and quantitative data
in Fig. 3. The major compounds detected included the xanthone isomers, mangiferin and
isomangiferin, the flavanone glycoside, hesperidin (hesperetin-7-O-rutinoside), and an
unidentified compound previously detected in several Cyclopia spp. (De Beer & Joubert,
2010). Additionally, a flavone, luteolin, and three flavanones, namely an eriodictyol-
Fig. 3. Phenolic profile and phenolic composition (g/100 g) of C. intermedia extract (1,
unidentified compound; 2, mangiferin; 3, isomangiferin; 4, eriodictyol-glucoside; 5,
eriocitrin; 6, hesperidin; 7, luteolin; 8, hesperetin).
0
50
100
150
200
250
300
0 5 10 15 20 25 30
Absorbance at 288 nm (mAU)
Time (min)
1
6
5
4
3
2
78
Compound Content
(g/100 g)
1 1.105
2 5.796
3 1.567
4 0.176
5 0.037
6 0.354
70.023
8 0.068
Assessment of the Antidiabetic Potential of an Aqueous Extract
of Honeybush (Cyclopia intermedia) in Streptozotocin and Obese Insulin Resistant Wistar Rats
321
glucoside, eriocitrin (eriodictyol-7-O-rutinoside) and hesperetin, the aglycone of hesperidin,
were also detected in small quantities.
3.2 Animal study
Results of the intake data are summarised in Table 2. The average intake of honeybush
extract by the various treatment groups varied from 77 to 531 mg/kg BW. This equals 4.5 to
30.8 mg/kg BW of the major xanthone, mangiferin, and 0.3 to 1.9 mg/kg BW of the major
flavanone, hesperidin.
Intramuscular injection of Wistar rats with STZ (35 mg/kg BW) induced diabetes in the rats
at an average fasting plasma glucose concentration of 27.8 ± 1.0 mmol/L (data not shown).
The acute effects of administering the honeybush extract by oral gavage under light
anaesthesia at doses of 0 (vehicle control), 5, 25 and 50 mg/kg BW are shown in Fig 4. The
optimal acute oral glucose lowering dose for the honeybush extract in STZ-induced diabetic
rats for the dose range tested was 50 mg/kg BW. This was the only acute dose of the extract
that significantly reduced the mean blood glucose concentrations relative to the baseline
fasting blood glucose concentration. Reductions of 33.5 ± 1.7% (p<0.05), 34.3 ± 3.6% (p<0.05)
and 35.6 ± 3.5% (p<0.01) were observed after 4, 5, and 6 hrs, respectively (Fig. 4).
After a chronic 3-month treatment of OBIR rats with aqueous honeybush extract their
hyperglycaemic fasting blood glucose concentrations were reduced to normoglycaemic
values. In other words, the honeybush extract reduced the fasting blood glucose levels from
12.2 mmol/L of the control to 4.8 – 5.4 mmol/L (p<0.001). All extract concentrations were
effective. Metformin and rosiglitazone had very similar effects and reduced the fasting
glucose to 6.3 mmol/L (p<0.001) and 5.6 mmol/L (p<0.001), respectively (Fig. 5).
Untreated control OBIR rats had IVGTT peak glucose concentrations of 18.2 ± 1.74 mmol/L.
Treatment with the honeybush extract reduced this value to 14.9 ± 0.7 mmol/L (treatment
E2, p<0.05), 14.8 ± 0.9 mmol/L (treatment E3, p<0.05) and 13.5 ± 1.2 mmol/L (treatment E4,
-50
-40
-30
-20
-10
0
10
01234567
% Reduction
Time (hrs)
Control 5 mg/kg 25 mg/kg 50 mg/kg
*
***
Fig. 4. Acute glucose lowering effect of C. intermedia extract in STZ diabetic rats (n=5).
Significant differences from baseline indicated with * (p<0.05) or ** (p<0.01).
Phytochemicals – Bioactivities and Impact on Health
322
0
2
4
6
8
10
12
14
16
ControlE1E2E3E4E5MetRosi
Glucose (mmol/L)
*** ***
***
***
***
*** ***
Fig. 5. The effect of chronic treatment with C. intermedia extract, metformin and rosiglitazone
(see Table 2 for treatment codes and dosage) on fasting plasma glucose concentration of
OBIR rats (n=10). Significant differences from control indicated with *** (p<0.001).
p<0.05), respectively (only E3 is shown for clarity; Fig. 6). The IVGTT area under the curve
values were reduced from 670 ± 9 for the control to 474 ± 15 (p<0.05) and 496 ± 13 (p<0.05)
for E2 and E5, respectively (data not shown). E1 (the lowest dose), as well as the known
drugs, metformin and rosiglitazone, had no significant effect (p0.05) (Fig. 6).
0
5
10
15
20
25
0 102030405060
Glucose (mmol/L)
Time (min)
Control E3 Met Rosi
*
Fig. 6. The effect of C. intermedia extract, metformin (Met) and rosiglitazone (Rosi) treatment
on intravenous glucose tolerance in OBIR rats (n=10) (see Table 2 for treatment codes and
dosage). E3 differs significantly from the control as indicated with * (p<0.05).
Treatment of OBIR rats with honeybush extracts, E1 to E5, for 3 months lowered the total
plasma cholesterol concentration compared to the untreated rats (2.9 ± 0.3 mmol/L) by 31.6 -
39.1% (Fig. 7). The effect of metformin and rosiglitazone on total cholesterol was not
determined.
Assessment of the Antidiabetic Potential of an Aqueous Extract
of Honeybush (Cyclopia intermedia) in Streptozotocin and Obese Insulin Resistant Wistar Rats
323
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
ControlE1E2E3E4E5
Cholesterol (mmol/L)
*** ** ** **
Fig. 7. The effect of chronic treatment with C. intermedia extract (see Table 2 for treatment
codes and dosage) on fasting total cholesterol concentrations in OBIR rats (n=10). Significant
differences from control indicated with * (p<0.05) or ** (p<0.01).
The average α-cell size in untreated OBIR rats was 106 ± 13.6 μm2. Treatment with the
honeybush extracts, E1 to E4, reduced the α-cell size to about half, i.e. to 48.4 – 54.9 μm2
(p<0.01). Metformin and rosiglitazone had similar effects and also reduced the average α-
cell size to 41.2 ± 1.9 μm2 and 44.0 41.2 ± 1.9 μm2, respectively (p<0.01) (Fig. 8). No data were
available for E5. These changes in the α-cell size were also reflected in the decreased α-cell to
β-cell ratio for all treatments (Fig. 9).
0
20
40
60
80
100
120
140
ControlE1E2E3E4MetRosi
Mean cell size (m
2
)
**
**
**
**
** **
Fig. 8. The effect of chronic treatment with C. intermedia extract, metformin and rosiglitazone
(see Table 2 for treatment codes and dosage) on mean α-cell size in the pancreata of OBIR
rats (n=10). Significant differences from control indicated with ** (p<0.01).
Phytochemicals – Bioactivities and Impact on Health
324
0
0.1
0.2
0.3
0.4
0.5
ControlE1E2E3E4MetRosi
Ratio
**
**
***
** ***
Fig. 9. The effect of chronic treatment with C. intermedia extract, metformin and rosiglitazone
(see Table 2 for treatment codes and dosage) on α- to -cell ratio in the pancreata of OBIR
rats (n=10). Significant differences from control indicated with * (p<0.05), ** (p<0.01) or ***
(p<0.001).
4. Discussion
The content of mangiferin, isomangiferin and the unidentified compound was higher than
the average for unfermented C. intermedia extracts previously analysed (De Beer & Joubert,
2010; Joubert et al., 2008b), while the hesperidin and eriocitrin contents of the present extract
were lower. De Beer & Joubert (2010) detected luteolin only in trace amounts, while no
hesperetin was detected. Joubert et al. (2003) found that the plant material (dry mass basis)
contain 1.69% mangiferin, while isomangiferin and hesperidin, respectively, comprised 0.22
and 1.76% of the dry plant material. Other phenolic compounds of C. intermedia include
flavones, isoflavones, other flavanones and coumestans (Ferreira et al., 1998; Kamara et al.,
2003). Quantitative differences between the present extract and aqueous hot water extracts
analysed previously (De Beer & Joubert, 2010; Joubert et al., 2008b) could be attributed to
natural variation and/or selective use of the upper part of the shoot. This has implications
for standardisation and efficacy. Testing of more C. intermedia extracts, specifically for their
efficacy as antidiabetic extracts, is required before more comprehensive claims can be made.
STZ was originally developed as an antibiotic derived from Streptomyces achromogenes but it
is toxic to pancreatic β-cells. It selectively enters insulin producing β-cells via their GLUT2
glucose transporter proteins, inducing irreparable DNA damage and death of β-cells in a
dose dependent manner (Lenzen, 2008). In the Wistar rat, intramuscular injection of STZ (35
mg/kg BW) increased fasting plasma glucose concentrations by ca 500% from the average
normoglyceamic concentration of 5.3 mmol/L, resulting in stable non-ketoacidotic T1D
diabetic rats. An acute 50 mg/kg BW dose of the aqueous honeybush extract induced a
sustained glucose lowering effect from 3 to at least 6 hrs in these STZ-induced diabetic rats.
These results are comparable to that of Miura et al. (2001) who assessed the acute
antidiabetogenic effect of an extract of Anemarrhena aspholoides in a hyperglycaemic KK-Ay
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diabetic mouse model. This plant, which contains the xanthones, mangiferin and a 7-
glucoside of mangiferin, is used as an Oriential medicine for the treatment of diabetes. After
oral treatment the maximal glucose lowering effect of the aqueous Anemarrhena aspholoides
extract was achieved after 7 hrs. Mangiferin and its glucoside showed similar activity at a
dose of 90 mg/kg BW. Mangiferin administered intraperitoneally for 30 days to mildly
hyperglycaemic STZ-induced rats at doses of 10 and 20 mg/kg BW ameliorated the diabetic
effects including weight loss, hyperglycaemia and hypercholesterolaemia (Dineshkumar et
al., 2010). Prior to the latter study, the antidiabetic effects of mangiferin in STZ-induced
diabetic Wistar rats were shown at the same doses after chronic treatment for 14 and 28 days
(Muruganandan et al., 2005).
In the present study 50 mg/kg BW of honeybush extract, equalling a dose of 2.90 mg
mangiferin, was effective at reducing plasma glucose concentrations in our STZ-induced
diabetic rat model. Other compounds in the extract could also contribute to the observed
hypoglycaemic effect through synergistic or additive effects. Hesperidin, in particular,
comprising 0.35% of the honeybush extract, was shown to have a hypoglycaemic effect in
marginally hyperglycaemic Wistar rats normalising their blood glucose concentrations after
16 days (Akiyama et al., 2010). Jung et al. (2004), using a spontaneously diabetic C57BL/KsJ-
db/db mouse model, showed that a 5-wk supplementation of the diet with 0.02%
hesperidin ameliorated the development of hyperglycaemia in these mice. Both Akiyama et
al. (2010) and Jung et al. (2004) attributed the hypoglycaemic effect of hesperidin to
upregulation of glucose regulating enzymes, in particular glucokinase, which enhances
glycolysis and increases glycogen synthesis.
Confirmation of the antidiabetic potential of C. intermedia extract in the STZ-induced
diabetic rat model was followed up with a chronic study in the high fat diet-induced OBIR
rat model. Feeding non-predisposed Wistar rats a high fat (Table 1) and sucrose diet from
weanling for three months induces obesity and glucose intolerance. Although these obese
rats maintain near normal fasting glucose levels they present with hyperinsulinaemia,
hyperglucagonaemia and dyslipidaemia (Buettner et al., 2006; Chalkley et al., 2002;
Kamgang et al., 2005). The addition of refined sugars, i.e. sucrose, exacerbates the metabolic
aberrations by the disruption of fatty acid metabolism resulting in hepatic and subsequent
muscle insulin resistance (Fukuchi et al., 2004). The chronic hyperinsulinaemia associated
with insulin resistance increases hepatic lipogenesis due to increased expression of the
major lipogenesis gene sterol response element-binding protein 1c (SREBP1c). In these obese
rats increasing levels of glucagon fail to regulate the insulin action, leading to increases in
hepatic fat accumulation and hypertriglyceridaemia (Buettner et al., 2006). Interestingly,
whereas the insulin sensitivity of the SREBP1c pathway is retained, the forkhead box protein
O1 (FOXO1) pathway becomes insulin resistant, resulting in decreased hepatic glucose
uptake and increased gluconeogenesis. Together the increase in fatty acid synthesis and
hepatic glucose release worsens muscle and pancreatic islet insulin resistance (Liu et al.,
2011). Ultimately, high fat feeding is directly associated with pancreatic endocrine
dysfunction and subsequent morphological changes of the pancreatic islets’ endocrine cell
ratios and cell sizes. The compensatory response to the increased demand for insulin results
in pancreatic β-cell hypertrophy and an increase in α-cell number and volume (Buettner et
al. (2007). As the diabetic pathogenesis worsens the β-cell mass declines and the α- to β-cell
ratio increases (Liu et al., 2011). Feeding the same 40% high fat diet to pregnant Wistar dams
during gestation resulted in offspring with similar increases in α-cell volume, while β-cell
Phytochemicals – Bioactivities and Impact on Health
326
volume decreased (Cerf et al., 2005). In human T2D pancreatic islets, larger islets with an
increased proportion of pancreatic α-cells have been shown to have impaired function
(Deng et al., 2004). Despite the diet-induced metabolic and morphological aberrations these
rats only develop slight hyperglycaemia over time without progressing to overt T2D. The
similarities of the diet-induced OBIR rat to the pathophysiology of human obesity and the
metabolic syndrome are well established (Buettner et al., 2007) and this animal model was
therefore selected to test the possible ameliorating effects of honeybush extract on these
metabolic aberrations.
Inclusion of the honeybush extract in an otherwise diabetogenic diet for OBIR rats resulted
in normalisation of the pre-existing hyperglycaemia over a wide range of dosages. This
confirms that the extract has glucose lowering potential without causing hypoglycaemia.
The latter is a potentially undesirable side effect of some T2D agents, specifically the
sulfonylureas and meglitinides (Bennett et al., 2011). In addition, the normoglycaemic effect
of the honeybush extract was achieved without dietary intervention. The extract proved to
be as effective as metformin and rosiglitazone, which are regarded as the gold standards for
treating human T2D. The efficacy of the extract at all doses and the lack of a clear dose
response could be contributed to the length of the treatment (12 wks) and relative high
doses even at the lowest dosage level. The reduction in IVGTT peak values and the area
under the curve values in OBIR rats treated with the honeybush extract clearly indicated
improved glucose homeostasis. Further, the improvement in glucose control was not
associated with significantly increased fasting insulin concentrations. This may suggest that
the mechanism whereby the extract elicits its effect on glucose metabolism is independent of
insulin, reflecting adaptive mechanisms in the fasted state. This would imply that, due to the
low fasting glucose concentrations, β-cells need to release less insulin to maintain glucose
homeostasis.
Plasma cholesterol levels of honeybush extract treated OBIR rats were significantly reduced
when compared with the plasma cholesterol levels of the untreated control rats. Mangiferin
and hesperidin have been shown to lower plasma cholesterol levels of diabetic rats
(Akiyama et al., 2010; Dineshkumar et al., 2010; Muruganandan et al., 2005). Mangiferin,
apart from lowering total cholesterol, also increases high-density lipoprotein-cholesterol
levels and therefore decreases the atherogenic index in diabetic rats (Muruganandan et al.,
2005). The hypolipidaemic effect of hesperidin has been attributed to the inhibition of
hepatic 3-hydroxy-3-methyl-glutaryl-CoA reductase and acyl CoA:cholesterolacyl
transferase, key enzymes in cholesterol synthesis and cholesterol esterification, resulting in
the reduction of plasma cholesterol (Bok et al., 1999; Jung et al., 2006). Finally, a potential
role of some minor compounds such as the inositol, (+)-pinitol, shown to be present in C.
intermedia plant material (Ferreira et al., 1998), in the glucose- and cholesterol-lowering
effects (Bates et al., 2000; Choi et al., 2009) elicited by the honeybush extract cannot be
excluded.
World-wide over 18 million people have died of cardiovascular disease in 2005. WHO has
identified high total cholesterol as a major contributing risk factor which is modifiable and
thereby could potentially reduce the incidence of cardiovascular disease (Rodgers et al.,
2004; Roth et al., 2011). Polyphenols have anti-atherosclerotic properties and are protective
against cardiovascular disease. Apart from their plasma cholesterol lowering effects,
polyphenols are associated with improved endothelial function and oxidative status
Assessment of the Antidiabetic Potential of an Aqueous Extract
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327
(Badimon et al., 2010). The protective effects of dietary flavonol intake against coronary
heart disease mortality in humans was highlighted in a meta-analysis (Huxley & Neil, 2003),
indicating a 20% lower risk in individuals with the highest consumption of dietary
flavonols. In another study, Peters et al. (2001) suggested drinking three cups of tea (Camellia
sinensis) a day could reduce the risk of myocardial infarction by 11%. In human trials
coronary artery disease patients have shown improved endothelial function and coronary
microcirculation following polyphenolic treatment (Hozumi et al., 2006).
The main morphometrical finding following honeybush treatment for 12 wks was a
decrease in the α-cell area, and subsequently by inference, total pancreas α-cell volume. The
role of glucagon in the pathogenesis of T2D has been largely neglected by researchers. In
mice, persistent hyperglucagonaemia induced by glucagon-producing glucagonomas results
in T2D (Y. Li et al., 2008). Glucagon secretion by the α-cell is tightly regulated by insulin via
insulin receptors at the surface of α-cells. This ensures that normoglycaemia is maintained.
However, as insulin resistance increases, as is the case with the OBIR rat model,
dysregulation of glucagon secretion by insulin occurs, resulting in persistent
hyperglucagonaemia. Morphologically, this results in increased α-cell numbers, α-cell
volume and glucagon secretion that worsen the insulin resistance of the liver causing more
hyperglycaemia and T2D (Liu et al., 2011). Honeybush extract treatment reduced the
average α-cell size and subsequently the total α-cell area leading to an improved α- to β-cell
ratio in OBIR rats. The reduction of α-cell size and therefore glucagon secretion can have
profound effects on glucagon induced insulin secretion and thereby alleviate the increased
stress of hypersecretion on the β-cells (Liu et al., 2011). Similar to our findings, treatment of
a high fat diet/STZ T2D mouse model with sitagliptin, a dipeptidyl peptidase-4 inhibitor
and a new generation of antidiabetic drug, reversed the increased proportion and
distribution of pancreatic α-cells and thereby restored the α- to β-cell ratio following chronic
11 week treatment. As with our study the reduction of the α- to β-cell ratio reflected the
overall improvement of the glucose and lipid metabolism (Mu et al., 2006). The fact that
metformin and rosiglitazone had a similar beneficial effect on the pancreatic α- to β-cell ratio
as the honeybush extract is not surprising. Both metformin and rosiglitazone are established
oral drugs for the treatment of T2D. Metformin, an activator of AMP-activated protein
kinase (AMPK), not only ameliorates hyperglycaemia but has been shown to have
additional beneficial effects on lipid metabolism. Activation of AMPK by metformin
increases fatty acid oxidation by inactivation of acetyl-CoA carboxylase and suppression of
lipogenesis by inhibiting SREBP-1 expression (Zhou et al., 2001). Rosiglitazone, a
peroxisome proliferator activated receptors-γ agonist, has been shown to improve insulin
resistance and to have a positive effect on lipid metabolism in pre-diabetic animals by
inhibiting malonyl-CoA and thereby increasing fatty acid oxidation in skeletal muscle and
liver (Zhao et al., 2009). In addition, rosiglitazone has also been demonstrated to activate
AMPK but via a different pathway from metformin (Fryer et al., 2002). Taken together, the
positive morphological changes observed support the improved metabolic changes induced
by chronic ingestion of aqueous honeybush extract.
5. Conclusion
The efficacy of an aqueous hot water honeybush extract in reducing hyperglycaemia was
demonstrated in two different diabetic rodent models (STZ-induced T1D and diet-induced
Phytochemicals – Bioactivities and Impact on Health
328
T2D OBIR rat models). Furthermore, the extract promoted normoglycaemia in the diet-
induced T2D OBIR rat model and improved other metabolic aberrations associated with
T2D. The high concentration of mangiferin and hesperidin in the extract could be partially
responsible for the observed effects.
6. Acknowledgment
The authors wish to thank the National Research Foundation of South Africa for funding
(Grant GUN 2053476 to EJ) and Q. Nortje of Nooitgedacht Farm, Langkloof area, South
Africa, for supplying the plant material.
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... This suggests that rooibos may improve glucose metabolism by enhancing glucose uptake and restoring normal insulin signalling. Studies have also highlighted the anti-diabetic potential of honeybush [42][43][44][45][46] and Sutherlandia [47][48][49] in both in vitro and in vivo experiments. All these studies showcasing the hypoglycaemic effects of rooibos, honeybush and Sutherlandia were carried out in models of cardiovascular and other systemic diseases. ...
... The potential hypoglycaemic effects of rooibos, honeybush and Sutherlandia extracts have been widely investigated in both in vivo 35,42,47 and in vitro studies. 49,59,60 The mechanisms through which this occur have been mostly determined in in vitro studies using cell lines, such as adipocytes, cardiomyocytes, fibroblast, myotubes/myoblast, and etcetera. ...
... 41 The decrease in the FBG of up 23.6% observed in the DHB animals of the current study concurs with Muller et al, who reported reduced FBG levels in obese insulin-resistant rats after administration of honeybush extract. 42 Additionally, the decrease in FBG levels of DSL animals in the current study is supported by Chadwick et al who showed diabetic rats receiving Sutherlandia displayed lower blood glucose as evidenced by normoinsulinaemic levels and increased glucose uptake. 47 The SS content (mg/mL) of rooibos was lower than that of honeybush and Sutherlandia (13.4±0.003; ...
Article
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Background: Testicular insulin signalling is altered in diabetic (DM) males. While unravelling the mechanism through which DM exert these detrimental effects, studies have shown the importance of insulin regulation in glucose homeostasis, and how a lack in insulin secretion indirectly led to reduced male fertility. The current study aimed to investigate the role of rooibos, honeybush and Sutherlandia on insulin signalling in the testicular tissue of type I diabetic rats. Methods: Animals (n=60) were randomly divided into six groups. The groups include a control group, a vehicle group, and diabetes was induced in the remainder of animals via a single intraperitoneal injection of STZ at 45mg/kg. The remaining four groups included a diabetic control (DC), diabetic + rooibos (DRF), diabetic + honeybush (DHB) and diabetic + Sutherlandia group (DSL). Animals were sacrificed after seven weeks of treatment, and blood and testes were collected. Results: All diabetic groups (DC, DRF, DHB, DSL) presented with a significant increase in blood glucose levels after diabetes induction compared to the control and vehicle (p<0.001). The DC animals presented with decreased testicular protein expression of IRS-1, PkB/Akt and GLUT4 compared to controls. DRF and DHB animals displayed an acute upregulation in IRS-1, while the DSL group showed improvement in IRS-2 compared to DC. Although, DRF animals presented with a decrease in PkB/Akt, DHB and DSL animals displayed upregulation (22.3%, 48%) compared to controls, respectively. Conclusion: The results taken together, it can be suggested that these infusions may enhance insulin signalling through diverse pathways.
... STZ-induced diabetic Wistar rats were treated with hot water extracts from C. intermedia, C. maculata and C. subternata in three separate studies. Acute doses of aqueous extracts of unfermented C. intermedia and C. subternata reduced the plasma glucose concentration of STZ-induced diabetic male Wistar rats over a six-hour period (Muller et al., 2011;Schulze et al., 2016). Obese, insulin-resistant Wistar rats, on a high fat and sucrose diet, chronically treated with the same C. intermedia extract for a 3-month period, were reported to have lowered fasting blood glucose concentrations, lowered total plasma cholesterol concentrations and decreased α-cell size (Muller et al., 2011). ...
... Acute doses of aqueous extracts of unfermented C. intermedia and C. subternata reduced the plasma glucose concentration of STZ-induced diabetic male Wistar rats over a six-hour period (Muller et al., 2011;Schulze et al., 2016). Obese, insulin-resistant Wistar rats, on a high fat and sucrose diet, chronically treated with the same C. intermedia extract for a 3-month period, were reported to have lowered fasting blood glucose concentrations, lowered total plasma cholesterol concentrations and decreased α-cell size (Muller et al., 2011). Chellan et al. (2014) showed that an aqueous extract of unfermented C. maculata ameliorated STZ-induced diabetes in Wistar rats, as well as protected against STZinduced diabetes, by prior treatment with the extract. ...
Article
The formal honeybush tea industry has reached a 20-year milestone on 24 February 2019, giving an opportunity to reflect on the research targeting phenolic composition, quality and bioactivity, since a previous review published in 2008. This research underpins ongoing endeavours to unlock the potential of this indigenous agricultural commodity. The current review illustrates that the bulk of the research followed a clear ‘vision’ to enhance consumer confidence in the product and by exploring value-adding opportunities. Gaps in knowledge were highlighted that could drive future research directions.
... Natural products are usually considered safer and more socially acceptable than conventional drugs, and in the case of food, consumed as part of the diet. Amongst these, extracts of Cyclopia, an endemic South African plant commonly consumed as a herbal tea (honeybush), has been shown to possess ameliorative properties against oxidative stress, inflammation, hyperglycemia, and obesity [10][11][12][13][14][15][16][17][18]. Likewise, a number of studies have reported that major phenolic compounds present in Cyclopia spp. ...
... Mounting scientific evidence demonstrates that extracts of Cyclopia spp. exhibit potential anti-diabetic [10,15], anti-oxidant [12,18], anti-mutagenic [71], anti-cancer [72], phytoestrogenic [73], anti-osteoclastogenic [74], anti-allergic [75] and anti-inflammatory [18] activities, in in vitro and in vivo models. Amongst the many phenolic compounds present in Cyclopia spp., high quantities of the xanthones, mangiferin (PubChem CID: 5281647) and its isomer, isomangiferin (PubChem CID: 5318597), the flavanone, hesperidin (PubChem CID: 10621), as well as benzophenone glucosides and dihydrochalcone glycosides have been detected [76]. ...
Article
Obesity is a significant contributor to increased morbidity and premature mortality due to increasing the risk of many chronic metabolic diseases such as type 2 diabetes, cardiovascular disease and certain types of cancer. Lifestyle modifications such as energy restriction and increased physical activity are highly effective first-line treatment strategies used in the management of obesity. However, adherence to these behavioral changes is poor, with an increased reliance on synthetic drugs, which unfortunately are plagued by adverse effects. The identification of new and safer anti-obesity agents is thus of significant interest. In recent years, plants and their phenolic constituents have attracted increased attention due to their health-promoting properties. Amongst these, Cyclopia, an endemic South African plant commonly consumed as a herbal tea (honeybush), has been shown to possess modulating properties against oxidative stress, hyperglycemia, and obesity. Likewise, several studies have reported that some of the major phenolic compounds present in Cyclopia spp. exhibit anti-obesity effects, particularly by targeting adipose tissue. These phenolic compounds belong to the xanthone, flavonoid and benzophenone classes. The aim of this review is to assess the potential of Cyclopia extracts as an anti-obesity nutraceutical as underpinned by in vitro and in vivo studies and the underlying cellular mechanisms and biological pathways regulated by their phenolic compounds.
... One of the species, Cyclopia genistoides, also called "Cape tea," has been recorded for traditional medicinal use [5]. Several studies, since commercialization of Cyclopia spp. in the 1990s, have provided scientific evidence of their health-promoting properties, including anti-diabetic [6,7], anti-oxidant [8], anti-mutagenic [9], anti-cancer [10], phytoestrogenic [11], and anti-osteoclastogenic [12] effects. The anti-obesity properties of Cyclopia was first demonstrated by Dudhia et al. [13], who reported that hot water extracts of Cyclopia maculata and Cyclopia subternata inhibit lipid accumulation in 3T3-L1 pre-adipocytes. ...
... Cyclopia spp. have attracted interest due to their reported beneficial health effects that include anti-obesity [13 -15] and anti-diabetic [6,7,19] effects. In the current study, CPEF displaying antiobesity potential [15], was fractionated using HPCCC. ...
Article
Cyclopia species are increasingly investigated as sources of phenolic compounds with potential as therapeutic agents. Recently, we demonstrated that a crude polyphenol-enriched organic fraction (CPEF) of Cyclopia intermedia, currently forming the bulk of commercial production, decreased lipid content in 3T3-L1 adipocytes and inhibited body weight gain in obese db/db mice. The aim of the present study was to determine whether a more effective product and/or one with higher specificity could be obtained by fractionation of the CPEF by purposely increasing xanthone and benzophenone levels. Fractionation of the CPEF using high performance counter-current chromatography (HPCCC) resulted in four fractions (F1–F4), predominantly containing iriflophenone-3-C-β-D-glucoside-4-O-β-D-glucoside (benzophenone: F1), hesperidin (flavanone: F2), mangiferin (xanthone: F3), and neoponcirin (flavone: F4), as quantified by high-performance liquid chromatography with diode array detection (HPLC-DAD), and confirmed by LC-DAD with mass spectrometric (MS) and tandem MS (MSE) detection. All fractions inhibited lipid accumulation in 3T3-L1 pre-adipocytes and decreased lipid content in mature 3T3-L1 adipocytes, although their effects were concentration-dependent. F1–F3 stimulated lipolysis in mature adipocytes. Treatment of mature adipocytes with F1 and F2 increased the messenger RNA expression of hormone sensitive lipase, while treatment with F1 and F4 increased uncoupling protein 3 expression. In conclusion, HPCCC resulted in fractions with different phenolic compounds and varying anti-obesity effects. The activities of fractions were lower than the CPEF; thus, fractionation did not enhance activity within a single fraction worthwhile for exploitation as a nutraceutical product, which illustrates the importance of considering synergistic effects in plant extracts.
... The genus Cyclopia, an indigenous South African fynbos plant, has attracted interest since its commercialisation as honeybush herbal tea and several studies provided scientific evidence of various health promoting properties (Marnewick et al., 2009;Muller et al., 2011;Dudhia et al., 2013;Pheiffer et al., 2013;Visser et al., 2013). Aqueous extracts of two species, C. maculata and C. subternata, inhibited adipogenesis and stimulated lipolysis in 3T3-L1 adipocytes Pheiffer et al., 2013). ...
... Several studies have reported that obesity increases the susceptibility to develop T2D (Pi-Sunyer, 2009). Muller et al. (2011) reported that a hot water extract of C. intermedia decreased blood glucose concentrations in streptozotocin (STZ)-induced diabetic rats, and blood glucose and cholesterol concentrations in diet-induced obese insulin resistant rats, while Chellan et al. (2014) demonstrated that C. maculata ameliorated STZ-induced diabetes in male Wistar rats, possibly by decreasing pancreatic β-cell cytotoxicity. Further, Schulze et al. (2016) reported that a hot water extract of C. subternata improved glucose tolerance in STZ-induced diabetic rats. ...
Article
Extracts of Cyclopia species, indigenous South African fynbos plants used for the production of honeybush tea, have potential as anti-obesity nutraceutical ingredients. Previously, we demonstrated that aqueous extracts of C. maculata and C. subternata exhibited anti-obesity effects in 3T3-L1 adipocytes. In this study, we further explored these anti-obesity effects of C. maculata and C. subternata as well as C. intermedia for the first time. Extracts were prepared using a 40% methanol–water mixture (40% MeOH) in order to enhance the polyphenolic content of the extracts. Moreover, these extracts were separated into aqueous and organic fractions using liquid–liquid partitioning with n-butanol and water to further enrich the polyphenol content of the organic fractions. Extracts of all three Cyclopia species decreased the lipid content in 3T3-L1 adipocytes, although differences in bioactivity of their aqueous and organic fractions were observed. The organic fraction of C. intermedia was further investigated. This fraction dose-dependently decreased the lipid content in 3T3-L1 adipocytes without affecting cell viability, while increasing mRNA expression of HSL (1.57-fold, P < 0.05), SIRT1 (1.5-fold, P = 0.07), UCP3 (1.5-fold, P < 0.05) and PPARγ (1.29-fold, P < 0.05). Daily treatment of obese db/db mice with 351.5 mg/kg bodyweight of the organic C. intermedia fraction for 28 days decreased bodyweight gain by 21% (P < 0.05) without any effect on food or water consumption. The organic fraction was enriched in phenolic content relative to the extract with neoponcirin, a flavanone not previously identified in Cyclopia species, mangiferin, isomangiferin and hesperidin comprising 17.37% of the organic fraction of C. intermedia compared to 4.96% of its “large scale” prepared 40% MeOH extract. Their specific roles as anti-obesity agents in these models needs to be studied to guide product development.
... It specifically interacts with the insulin-responsive glucose transporter (GLUT4) in rat adipocytes, which is suggested to play a key role in its recruitment to the plasma membrane (Park et al., 2004). The down-regulation of Ehd2 expression is therefore of interest in the anti-diabetic effects of honeybush, demonstrated for C. intermedia (Muller et al., 2011) and C. subternata (Schulze et al., 2016), meriting further investigations. Expression of peroxidase, Ptgs1, was up-regulated, while serpinb1b and Kif 9 were down-regulated by both honeybush PEEs. ...
... Therefore, down-regulation of Txnip expression by PECsub and PECgen is likely to play a key role in the anti-diabetic effects of honeybush when considering the effects on oxidative stress. Mangiferin, consumed at high levels in the current study, decreases insulin resistance in mice (Ichiki et al., 1998;Miura et al., 2001) and is implicated in the anti-diabetic properties of "unfermented" C. intermedia (Muller et al., 2011). Txnip is also associated with antiproliferative effects and inhibits tumor cell growth, while it is downregulated in many cancer cell lines and tissues (Pang et al., 2009). ...
Article
Full-text available
Interest in Cyclopia spp. (honeybush) as a source material for production of polyphenol-enriched extracts (PEEs) for the food ingredient and nutraceutical markets requires investigation of their safety. PEEs of Cyclopia subternata (PECsub) and Cyclopia genistoides (PECgen) were fed (2.5 g/kg feed) to male Fischer rats for 28 days, while PECsub, having the highest total polyphenol content and antioxidant activity, was also fed for 90 days. Their dietary intake did not significantly (P ≥ 0.05) affect body weight gain or relative liver and kidney weight. PECsub resulted in a significant (P < 0.05) increase in the total serum bilirubin after 28 days, while serum alkaline phosphatase levels were only significantly (P < 0.05) increased after 90 days, suggesting alterations in the biliary system. No effect on serum iron levels was noticed after 28 days, but PECsub significantly (P < 0.05) reduced serum iron after 90 days. Both PECsub and PECgen increased glutathione reductase activity in the liver significantly (P < 0.05) after 28 days. Reduced glutathione (GSH) content was significantly (P < 0.05) decreased after 90 days by PECsub resulting in a marked, but not significant (P ≥ 0.05) decrease in the GSH/GSSG ratio. Considering the expression of oxidative stress and antioxidant defense-related genes after 28 days, the expression of eleven genes was altered by PECsub and PECgen. Seven genes, mutually affected by PECsub and PECgen, included (i) antioxidant-related genes, prostaglandin-endoperoxide synthase 1 (Ptgs1), kinesin family member 9 (Kif9) and serine (or cysteine) peptidase inhibitor clade B member 1b (Serpinb1b); (ii) genes involved in reactive oxygen species (ROS) metabolism, xeroderma pigmentosum complementation group A (Xpa) and thioredoxin interacting protein (Txnip) as well as (iii) oxygen transporter-related genes, Fanconi anemia complementation group C (Fancc) and vimentin (Vim). The expression of NADPH oxidase organiser 1 (Noxo1) increased 19.97-fold by PECsub and the thyroid peroxidase (Tpo) 372-fold by PECgen. Changes in the expression of the oxidative stress and antioxidant defense-related genes could be indicative of an underlying stress effected by the honeybush PEEs in the liver. Differential gene expression could likely be attributed to the differences in phenolic composition of the extracts. The daily dietary intake of total polyphenol and individual phenolic constituents, recalculated to a human equivalent dose, was much higher, especially for C. subternata, than normally consumed when habitually drinking “fermented” honeybush herbal tea. Subsequent studies verifying the protein-associated changes governing the oxidative status in the liver is required.
... FRAP assay was performed according to the method (Muller et al., 2011) with slight modification. Different concentration of methanolic extract of Spondias mombin leaves at 5, 2.5, 1.25, 0.625, 0.312, 0.156 mg/ml were prepared each at volume of 100 µL and was mixed with 4.5 ml of FRAP reagent in test tubes, thoroughly mixed by vortexing and were incubated in water bath for 30 min at 37°C. ...
Article
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Objective: Spondias mombin is a plant that reported to have anticonvulsant, antimicrobial, antioxidant, antiulcer, antiasthmatic, and wound healing activities. Diabetes dyslipidemic effect of Spondias mombin leaves is not clear. Hence, current study planned to evaluate the antidiabetic and antihyperlipidemic effects of methanolic extract of leaves of Spondias mombin (MESM) in streptozotocin (STZ) induced diabetic rats. Methods: Phytochemicals were determined by standard method and antioxidant activity was determined by DPPH free radical scavenging and FRAP assay. Diabetes was induced by injecting a single dose of STZ (55 mg/kg) into female sprague dawley rats. After 3 days of induction of diabetes, the diabetic animals were treated for 28 days with MESM (125, 250, and 500 mg/kg) and glibenclamide (20 mg/kg) orally. The body weight of rats and blood glucose levels were monitored at regular intervals during the experiment. At the end of study, blood sample was collected from all the animals and subjected to biochemical, lipid profile, and they were sacrificed and their organs such as pancreas, liver and kidney were used for histopathological analysis. Results: Quantitative analysis of MESM showed the presence of anthraquinone, tannins, saponins, steroid, phenols, flavonoids, alkaloids, and reducing sugars. Reduction in body weight and elevated blood glucose were observed in diabetic rats. Treatment with MESM in a concentration of 125, 250, and 500 mg/kg significantly reversed the elevated levels of blood glucose, reduced aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, urea, creatinine, total serum cholesterol (TC), serum triglyceride (TG), low-density lipoprotein (LDL), Very low-density lipoprotein (VLDL), and increased plasma insulin, total protein, albumin, globulin, A/G ratio, and high-density lipoprotein (HDL). Conclusion: MESM exhibited a significant antidiabetic and antihyperlipidemic activities against STZ-induced diabetes in rats.
... Hesperidin improved antioxidant capacity, reduced oxidative DNA damage and lipid peroxidation, but showed no effect on fasting blood glucose and insulin resistance [128] Muller et al. [113] were the first to report the antidiabetic effect of honeybush herbal tea when they demonstrated the efficacy of a hot water extract of Cyclopia intermedia in ameliorating hyperglycemia using two diabetic rodent models. In the study, an acute hot water extract of fermented Cyclopia intermedia at a dose of 50 mg/kg body weight (equaling a dose of 2.90 mg mangiferin) was able to induce a sustained reduction in fasting blood glucose concentration from at least 3 to 6 h in STZ-induced diabetic rats. ...
Article
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Diabetes mellitus is a metabolic disease that can lead to high morbidity, mortality and long-term complications. Available treatment strategies, which are mainly based on treating hyperglycemia, with insulin and other pharmacological agents are not completely efficient and can even lead to development of unwanted side effects. Scientific evidence suggests that bioactive compounds from teas and other plant-based foods, which are known source of natural antioxidants, could be an attractive strategy to preferentially treat and manage type 2 diabetes mellitus (T2DM) and thus, have significant therapeutic implications. In this review, we attempt an in-depth analysis and discussion of the current progress in our understanding of the antidiabetic potential of two commercialized South Africa herbal tisanes-Rooibos and Honeybush and their polyphenols.
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Diabetes mellitus (DM) is a chronic disease which has clinched the world. More than 300 million people of the world are suffering from this disease and the number is still increasing at a rapid rate as modern medical science has no permanent solution for the disease. Current scenario of the nutraceuticals has increased patient’s faith on the traditional medicinal system and world nutraceutical industry is estimated to reach $285.0 billion by 2021. The increasing trend of nutraceuticals in diabetes treatment makes it important to collect the traditional knowledge of medicines under one heading as it can help researchers to formulate new functional foods and nutraceuticals which can either lower down the risk or cure DM. In addition, the discussion of market available food products, their active components and possible health benefits can help the patients to understand the herbal medicines in a better way.
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Type 2 diabetes mellitus (T2DM) results from insulin resistance and β-cell dysfunction, in the setting of hyperglucagonemia. Glucagon is a 29 amino acid peptide hormone, which is secreted from pancreatic α cells: excessively high circulating levels of glucagon lead to excessive hepatic glucose output. We investigated if α-cell numbers increase in T2DM and what factor (s) regulate α-cell turnover. Lepr(db)/Lepr(db) (db/db) mice were used as a T2DM model and αTC1 cells were used to study potential α-cell trophic factors. Here, we demonstrate that in db/db mice α-cell number and plasma glucagon levels increased as diabetes progressed. Insulin treatment (EC50 = 2 nM) of α cells significantly increased α-cell proliferation in a concentration-dependent manner compared to non-insulin-treated α cells. Insulin up-regulated α-cell proliferation through the IR/IRS2/AKT/mTOR signaling pathway, and increased insulin-mediated proliferation was prevented by pretreatment with rapamycin, a specific mTOR inhibitor. GcgR antagonism resulted in reduced rates of cell proliferation in αTC1 cells. In addition, blockade of GcgRs in db/db mice improved glucose homeostasis, lessened α-cell proliferation, and increased intra-islet insulin content in β cells in db/db mice. These studies illustrate that pancreatic α-cell proliferation increases as diabetes develops, resulting in elevated plasma glucagon levels, and both insulin and glucagon are trophic factors to α-cells. Our current findings suggest that new therapeutic strategies for the treatment of T2DM may include targeting α cells and glucagon.
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Diabetes mellitus is one of the commonest diseases affecting the citizens of both developed and poor countries. In South Africa, the number of people suffering from diabetes is believed to be rising steadily. An ethnobotanical study of plants used by the traditional healers, herbalists and rural dwellers for the treatment of diabetes mellitus was conducted in the Eastern Cape Province. The study revealed 14 plant species belonging to six families namely; Asteraceae, Hypoxidaceae, Apocynaceae, Asphodelaceae, Apiaceae and Buddlejaceae. The use of infusions from plant leaves and roots was the commonest method of herbal preparation. In all cases, the treatment involved drinking the extracts for a long period of time. There was a general belief on the efficacy of the prepared extracts.
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African plants have long been the source of important products with nutritional and therapeutical value. Coffee originates from Ethiopia, Strophanthus species are strong arrow poisons and supply cardenolides for use against cardiac insufficiency, the Catharanthus roseus alkaloids are well-known antileukaemic agents - just to mention a few examples. Research is continuing on the vegetable material from this continent in an endeavour to find new compounds of therapeutic interest. An outline is presented here covering the results obtained by the Institute of Pharmacognosy and Phytochemistry of the University of Lausanne during 15 years work on African plants. The strategy employed for the study of these plants is outlined, covering all aspects from the selection of plant material to the isolation of pure natural products. Different bioactivities have been investigated: the search for new antifungal, molluscicidal and larvicidal agents has been the most important axis. Results are also included for antibacterial, cytotoxicity, anti-inflammatory testing.
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This review details the vernacular names, origin, distribution, taxonomy and variety of Mangifera indica L. (Anacardiaceae), a medicinal plant traditionally used in tropical regions. Mangiferin, a major C-glucosylxanthone from M. indica stem bark, leaves, heartwood, roots and fruits occurs widely among different angiosperm families and ferns. The reported pharmacological activities of mangiferin include antioxidant, radioprotective, antitumor, immunomodulatory, anti-allergic, anti-inflammatory, antidiabetic, lipolytic, antibone resorption, monoamine oxidase inhibiting, antiviral, antifungal antibacterial and antiparasitic properties, which may support the numerous traditional uses of the plant.
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Context: Mangifera indica (Anacardiaceae) stem bark contains a rich content of mangiferin and is used traditionally in Indian Ayurvedic system to treat diabetes. Purpose of the study: To investigate anti-diabetic and hypolipidemic effects of mangiferin in type 1 and type 2 diabetic rats models. Streptozotocin was used to induce type 1 and type 2 diabetic rats. Mangiferin (at a dose 10 and 20mg/kg) was administrated intra-peritoneally in type 1 and type 2 diabetic rats daily up to 30 days. Biochemical parameters notably fasting blood sugar, total cholesterol, triglycerides, low-density lipoprotein, very low-density lipoprotein and high-density lipoprotein were estimated. In addition, in vitro alpha amylase and alpha glucosidase inhibitory effects of mangiferin were performed and IC 50 values were determined. Results: Mangiferin exhibited significant (P<0.05) anti-diabetic as well as hypolipidemic effects by lowering FBS, TC, TG, LDL, and VLDL levels; but also with elevation of HDL level in type 2 diabetic model rats. In addition, mangiferin showed appreciable alpha amylase inhibitory effect (IC 50 value 74.35±1.9µg/ml) and alpha glucosidase inhibitory effect (IC 50 41.88±3.9µg/ml) when compared with standard drug acarbose (IC 50 83.33±1.2µg/ml). Conclusions: Mangiferin showed anti-diabetic as well as hypolipidemic potentials in type 2 diabetic model rats. Therefore, mangiferin possess beneficial effects in the management of type 2 diabetes with hyperlipidemia.
Book
Covering 1800 species of Chinese herbs and 700 related North American species, Chinese and Related North American Herbs: Phytopharmacology and Therapeutic Values provides clearly formatted tables that give you quick and easy access to information gathered from a wide variety of sources, both eastern and western, including those not generally available through usual searches. Written by the author of Medicinal Plants this book: Presents the major constituents and therapeutic values of Chinese medicinal herbs Contains data on toxicity, major chemical components and their therapeutic values Provides Latin, Chinese, and English names for more than 1800 species. Includes three appendices that provide handy cross-references: Chinese and scientific names; major chemical components of Chinese herbs, and major chemical components of related North American herbs Highlights the relationship between Chinese and North American medicinal herbs and possible replacements for Chinese with North American herbs Compares active ingredients and claimed therapeutic values The wealth of information, attention to detail, and extensive research and references presented by Chinese and Related North America Herb: Phytopharmacology and Therapeutic Values makes it the resource to have on your shelf.
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The processed leaves and stems of Cyclopia intermedia contain 4-hydroxycinnamic acid, the isoflavones formononetin, afrormosin, calycosin; pseudobaptigen, and fujikinetin, the flavanones naringenin, eriodictyol, hesperitin, and hesperidin, the coumestans medicagol, flemichapparin, and sophoracoumestan B, the xanthones mangiferin and isomangiferin, the flavone luteolin, and the inositol (+)-pinitol.
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Rooibos (Aspalathus linearis (Brum.f) Dahlg.) and honeybush (Cyclopia Vent. species) are popular indigenous South African herbal teas enjoyed for their taste and aroma. Traditional medicinal uses of rooibos in South Africa include alleviation of infantile colic, allergies, asthma and dermatological problems, while a decoction of honeybush was used as a restorative and as an expectorant in chronic catarrh and pulmonary tuberculosis. Traditional medicinal uses of Athrixia phylicoides DC., or bush tea, another indigenous South African plant with very limited localised use as herbal tea, include treatment of boils, acne, infected wounds and infected throats. Currently rooibos and honeybush are produced for the herbal tea market, while bush tea has potential for commercialisation. A summary of the historical and modern uses, botany, distribution, industry and chemical composition of these herbal teas is presented. A comprehensive discussion of in vitro, ex vivo and in vivo biological properties, required to expand their applications as nutraceutical and cosmeceutical products, is included, with the main emphasis on rooibos. Future research needs include more comprehensive chemical characterisation of extracts, identification of marker compounds for extract standardisation and quality control, bioavailability and identification of bio-markers of dietary exposure, investigation of possible herb–drug interactions and plant improvement with regards to composition and bioactivity.
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Cyclopia species are used to manufacture a herbal infusion called honeybush tea. The pleasant sweet, honey-like flavour and anecdotal evidence of the beneficial qualities of honeybush tea consumption make this herbal infusion a healthy alternative to coffee and black tea. Honeybush tea has been known to South Africans for centuries and, until recently, was processed only on a small-scale, mainly for home consumption. Growing interest in herbal teas, both locally and overseas, has resulted in a revival of the honeybush tea industry. Research addressed the lack of a standardized processing method, which in turn lead to the production of poor and inconsistent quality tea.