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Article
A Beneficial Role of Rooibos in Diabetes Mellitus:
A Systematic Review and Meta-Analysis
Moe Sasaki 1, Nami Nishida 2and Masako Shimada 1, 2, *ID
1
Graduate School of Nutritional Science, Sagami Women’s University, 2-1-1 Bunkyo, Minami-ku, Sagamihara,
Kanagawa 252-0383, Japan; s1771101@st.sagami-wu.ac.jp
2Faculty of Nutritional Science, Sagami Women’s University, 2-1-1 Bunkyo, Minami-ku, Sagamihara,
Kanagawa 252-0383, Japan; ntake39928@yahoo.co.jp
*Correspondence: shimada_masako@isc.sagami-wu.ac.jp; Tel.: +81-42-742-1927
Academic Editor: Francesca Giampieri
Received: 14 March 2018; Accepted: 5 April 2018; Published: 6 April 2018
Abstract:
In a rapid increase in cases of diabetes mellitus worldwide, there has been interested
in the use of plant-derived polyphenols as nutraceuticals to prevent the onset and progression
of diabetes mellitus and its associated complications. Aspalathus linearis, commonly known
as rooibos, is a rich source of uncommon glycosylated plant polyphenols with various critical
health-promoting properties, including the prevention and treatment of diabetes mellitus (DM).
This study aimed to examine these effects by meta-analyzing the current evidence in diabetic
rodent models. Peer-reviewed studies written in English from two databases, PubMed and Embase,
were searched up to 28 February 2018. Studies reporting blood glucose levels in diabetic rodents
with and without receiving rooibos extracts or their major phenolic compounds are included. Twelve
studies enrolling 88 diabetic rodents treated with rooibos extracts or their polyphenols and 85 diabetic
control males reported blood glucose levels. The pooled effect size was
−
0.89 (95% CI:
−
1.44 to
−
0.35) with a substantial heterogeneity (I
2
= 67.0%). This effect was likely to be modified by type of
rooibos extracts and their polyphenols and treatment period. Blood glucose levels were significantly
lower in diabetic rodent models treated with the phenolic compound rich in rooibos extracts, PPAG.
Keywords: rooibos extracts; diabetic rodent models; blood glucose levels; meta-analysis
1. Introduction
The number of individuals living with diabetes mellitus (DM) was estimated to be 425 million
in 2017 and this figure is expected to reach 629 million by the year 2045 [
1
]. Type 2 DM (T2DM) is a
chronic metabolic disorder that makes up about 90% of DM cases, and primarily occurs as a result of
obesity and lack of exercise. T2DM is characterized by high blood glucose levels, insulin resistance
in the muscle, liver and adipose tissues and relative deficiency of insulin secreted from the pancreas.
Moreover, patients with DM often develop various complications including triopathy, dyslipidemia
and cardiovascular diseases, which are the major causes of their mortality [
1
]. Therefore, it is critical to
explore new strategies to combat DM.
In recent years, there has been a growing interest in various natural products including herbal teas
and traditional Chinese medicinal or desert/semi-desert plants in prevention and treatment of diabetes
due to their natural origin and relatively less side effects than pharmaceuticals [
2
–
8
]. Aspalathus linearis,
rooibos, is usually grown in the Cederberg, a small mountainous area in the Western Cape province
region of South Africa [
9
]. Compared with green and black teas rooibos tea is a caffeine-free and
low-tannin beverage that contains various minerals and polyphenols, including dihydrochalcones
(aspalathin (ASP) and nothofagin), phenylpropenoids (phenylpyruvic acid-2-O-glucoside (PPAG)),
Molecules 2018,23, 839; doi:10.3390/molecules23040839 www.mdpi.com/journal/molecules
Molecules 2018,23, 839 2 of 15
flavones (isoorientin and orientin) and flavonols (quercetin-3-O-robinobioside). Because of its unique
properties, rooibos tea has gained popularity around the globe, particularly among people such as
expectant and nursing mothers who are encouraged to avoid caffeinated beverages.
Two forms of rooibos teas are available: fermented and unoxidized “green” tea. The fermented
rooibos extracts (FREs) contain high levels of major phenolic compounds, isoorientin (mean
11.8 ±2.6 mg/g
dry powder, 25.2% w/w), orientin (7.9
±
0.7 mg/g, 16.9%), quercetin-3-O-robinobioside
(7.4
±
2.9 mg/g, 15.8%), PPAG (6.7
±
0.4 mg/g, 14.3%) and ASP (3.7
±
0.0 mg/g, 8.1%) [
10
,
11
].
Other flavonoids including nothofagin, vitexin, isovitexin, hyperoside, rutin and isoquercitrin are
also found at a concentration of less than 3 mg/g dry powder of FREs or GREs. Water-based green
rooibos extracts (GREs) contain about three times higher levels of total phenolic compounds than
FREs (GREs, mean 160.6
±
22.7 vs. FREs, 46.7
±
8.0 mg/g dry powder). Quantitative composition
analysis of GREs showed that ASP is the major flavonoid (106.2
±
16.5 mg/g, 66.1% w/w), followed by
isoorientin (13.4
±
2.4 mg/g, 8.3%), nothofagin (11.7
±
2.5 mg/g, 7.3%), orientin (10.6
±
1.6 mg/g,
6.6%), queretin-3-O-robinobiside (6.4
±
0.6 mg/g, 4.0%) and PPAG (2.9
±
0.4 mg/g, 1.8%) [
11
–
13
].
ASP (C
12
H
24
O
11
, 452.4 Da) and nothofagin (C
21
H
24
O
10
, 436.4 Da) are two major active C-linked
dihydrochalcones uniquely found in rooibos tea. Orientin and isoorientin (C
21
H
20
O
11
, 448.4 Da) are
major C-glycosyl-containing flavones present widely in natural plants, including rooibos. The mean
content of PPAG is similar to or more than ASP in FREs [
10
,
11
]. PPAG concentration in GREs is
approximately 2%; however, that likely varies depending on batches and parts of plants, for example,
stems vs. leaves [11–13].
Growing volumes of
in vitro
and
in vivo
data have suggested the potentially beneficial roles
of rooibos tea extracts in glucose metabolism and associated complications, including oxidative
stress, insulin resistance and diabetic myopathy [
14
]. In human subjects, a single dose of rooibos tea
significantly reduced angiotensin-converting enzyme activity [
15
] and increased plasma antioxidant
capacity [
16
], while chronic consumption of fermented rooibos tea improved markers for blood lipid
levels and reduced those of oxidative stress [
17
]. However, no clinical trials have been performed
to examine the effect of rooibos on diabetic parameters. Therefore, the aim of this study was to
assess the effect of rooibos tea and associated polyphenols on blood glucose levels of diabetic rodent
models by meta-analyzing the currently available studies and attempting to sort out the potential
source of heterogeneity that may lead to the discrepancies in the current literature with subgroup and
meta-regression analyses.
2. Results
2.1. Search Results
The flowchart of our literature search is shown in Figure 1. It resulted in a total of 135 articles
(81 from Embase and 54 from PubMed). Upon removal of the duplicates and reviews of the titles and
abstracts, 27 articles moved on to a full-text assessment. Of these, the majority of articles were excluded
from the original meta-analysis because they failed to report blood glucose levels in DM rodent
models treated with rooibos tea extracts or their associated polyphenols. Therefore, 12 studies were
finally included in the meta-analyses [
13
,
18
–
28
]. There were no articles reporting the effect of either
nothofagin, orientin, or quercetin-3-O-robinobioside alone on blood glucose levels in DM rodents.
Molecules 2018,23, 839 3 of 15
Figure 1. Flow diagram of literature search and selection process.
2.2. Study Characteristics and Quality Assessment
The main characteristics of each included study are summarized in Table 1. Studies were
generally published since 2005. The sample sizes ranged from 10 to 20 in each study. Of the included
studies, animals are treated with FREs in two studies [
18
,
28
], GREs in one study [
13
], ASP in four
studies [19,21,22,26]
, isoorientin in one study [
25
] and PPAG in four studies [
20
,
23
,
24
,
27
]. db/db mice
were used in three studies, ob/ob mice in one study, diet-induced obese insulin-resistant (OBIR)
rodents (in two studies one in rats and one in mice) and KK-Ay mice in one study and streptozotocin
(STZ)-induced DM rodent models in five studies (three in rats and two in mice). Six studies used plasma
for blood glucose measurement, one used serum and five used whole blood samples. Nine studies
assessed glucose levels using fasting blood samples and three using non-fasting samples.
Table 1. Characteristics of included studies in the meta-analysis.
Authors (Year) Rooibos or
Poly-phenols Dose, Route Duration Animal
Models
Total n
(T/no-T)
Age or
Weight at a
Baseline
Diet Fasting or
ad Lib.
Blood
Sample
Ayeleso A et al.,
(2015) [18]FRE
2 g/100 mL
boiling water.
As drinking
water
7 w
STZ-induced
DM rats
(50 mg/kg
i.m.)
16 (8/8) 176–255 g Control Overnight
fasting Plasma
Dludla PV et al.,
(2017) [19]ASP (98%)
13 or
130 mg/kg BW
via daily oral
gavage
6 w db/db mice 12 (6/6) 9 w Control 16-h fasting Plasma
Himpe E et al.,
(2016) [20]PPAG (99%)
10 mg/kg BW
via daily oral
gavage.
11 d
STZ-induced
DM mice (200
mg/kg i.p.)
15 (8/7) 9–11 w,
approx. 25 g Control Ad lib. Whole
blood
Johnson R et al.,
(2017) [21]ASP (98%)
13 or
130 mg/kg/day
via daily oral
gavage
6 w db/db mice 12 (6/6) 9 w Control 4-h fasting Plasma
Kamakura R et al.,
(2015) [13]
GRE (6.62% ASP)
Add to diet at
0.3% and then
0.6%.
5 w KK-Ay mice 11 (5/6) 4 w Control 3-h fasting Whole
blood
Kawano A et al.,
(2009) [22]ASP (98.5%) Added to diet
at 0.2% 5 w db/db mice 10 (4/6) 6 w Control 4-h fasting Whole
blood
Molecules 2018,23, 839 4 of 15
Table 1. Cont.
Authors (Year) Rooibos or
Poly-phenols Dose, Route Duration Animal
Models
Total n
(T/no-T)
Age or
Weight at a
Baseline
Diet Fasting or
ad Lib.
Blood
Sample
Mathijs I et al.,
(2014) [23]PPAG (99%)
10 mg/kg BW
via daily oral
gavage
6 w OBIR mice 13 (7/6) 15 w
High fat
and
fructose
Fasting Whole
blood
Muller CJ et al.,
(2013) [24]PPAG (99%)
0.3–3 mg/kg
BW via daily
oral gavage
3 w OBIR rats 12 (7/5) 24 w
High fat
and
sucrose
4-h fasting Plasma
Sezik E et al., (2005)
[25]isoorientin
15 or 30 mg/kg
BW/d via daily
oral gavage
15 d
STZ-induced
DM rats
(55mg/kg i.p.)
12 (6/6) 200–250 g Control 18–20 h
fasting
Whole
blood
Son MJ et al., (2013)
[26]ASP 0.1% dietary
supplement 5 w ob/ob mice 20 (11/9) 6 w Control 3-h fasting Serum
Song I et al., (2017)
[27]PPAG
A dose of
10 mg/kg BW
via daily oral
gavage
4 d
STZ-induced
DM mice (200
mg/kg i.p.)
20
(10/10)
9–11 w,
approx. 25 g Control Ad lib. Whole
blood
Ulicna O et al.,
(2006) [28]FRE
2.5 g/1L of
boiling water,
5 mL/kg
BW/d via
gavage
9 w
STZ-induced
DM rats
(45 mg/kg i.v.)
20
(10/10) 290–340 g Control Ad lib. Plasma
FRE, fermented rooibos extract; BW, body weight; STZ, streptozotocin; GRE, green rooibos extract; ASP, aspalathin;
PPAG, phenylpyruvic acid-2-O-glucoside; i.v., intravenous, i.m., intramuscular; i.p., intraperitoneal. T, treatment;
no-T, non-treatment.
The detailed quality assessment of each study is shown in Table S1. The study quality was fair in
general with the risk of bias judged to be low to medium.
2.3. Effect of Rooibos Tea Extracts and Associated Polyphenols on Blood Glucose Levels in DM Rodent Models
Twelve studies from 12 articles enrolling 88 diabetic male rodents treated with rooibos extracts or
their major polyphenol compounds, ASP, PPAG and isoorientin and 85 diabetic control males treated
with vehicles reported their blood glucose levels and were included in this meta-analysis (Figure 2).
Four studies showed that treatment with polyphenols, ASP, PPAG and isoorientin, significantly
reduced blood glucose levels in DM rodent models [
24
–
27
]; eight studies did not observe any significant
effects. By pooling all those studies using a random-effects model, results revealed that rooibos tea
extracts or associated polyphenols reduce blood glucose levels in DM rodents (g =
−
0.89, 95% CI
−
1.44 to
−
0.35; I
2
= 67%, p< 0.001) (Figure 2). To determine the influence of each study on the overall
result, the stability of results was next evaluated using a leave-one-out strategy. Upon removal of
each individual study, all the re-pooled summary estimates remained unchanged compared with the
primary estimates with the effect sizes ranging from
−
1.00 (95% CI,
−
1.57 to
−
0.42) to
−
0.70 (95% CI,
−1.12 to −0.28).
Subgroup analyses suggested that PPAG and isoorientin have significantly reduced blood glucose
levels in DM rodents (PPAG, g =
−
1.35, 95% CI:
−
1.89 to
−
0.81. isoorientin, g =
−
5.63, 95% CI:
−
8.11
to
−
3.15); however, FRE, GRE and ASP have little, if any, effect (Figure 3). Blood glucose levels are not
influenced by type of rodent (mice vs. rats), blood sample (plasma, serum, vs. whole blood), or the
blood sampling time point (non-fasting vs. fasting) (Table 2).
Molecules 2018,23, 839 5 of 15
Figure 2.
Flow diagram of literature search and selection process. Meta-analysis of Hedges’ g of
blood glucose levels in DM rodents with and without treatment of rooibos extracts or associated
major phenolic compounds. Summary estimates were analyzed using a random-effects model. CI,
confidence interval.
Figure 3.
Subgroup analysis for Hedges’ g of blood glucose levels in DM rodents treated with various
types of rooibos extracts and major phenolic compounds or vehicles. Summary estimates were analyzed
using a random-effects model. CI, confidence interval.
Molecules 2018,23, 839 6 of 15
Table 2. Subgroup analyses.
Subgroups Effect Size Heterogeneity (I2)
No. of Studies g95% CI P-value
Rooibos and Polyphenols
FRE 2 0.05 −0.58 0.67 0.88 <0.001
GRE 1 −1.08 −2.25 0.10 0.07 <0.001
ASP 4 −0.46 −1.03 0.11 0.12 18.12
PPAG 4 −1.35 −1.89 −0.81 <0.001 <0.001
Isoorientin 1 −5.63 −8.11 −3.15 <0.001 <0.001
DM rodent models
db/db 3 −0.18 −0.80 0.45 0.02 <0.001
ob/ob 1 −1.08 −1.99 −0.17 0.58 <0.001
KK-Ay 1 −1.08 −2.25 0.10 0.07 <0.001
OBIR 2 −1.28 −2.10 −0.46 0.002 <0.001
STZ 5 −1.29 −2.54 −0.05 0.04 84.65
Rodent
Mice 8 −0.84 −1.28 −0.39 <0.001 30.09
Rats 4 −1.41 −3.03 0.22 0.09 86.70
Blood sample
Plasma 6 −0.54 −1.26 0.19 0.15 67.42
Serum 1 −1.08 −1.99 −0.17 0.02 <0.001
Whole blood 5 −1.43 −2.47 −0.39 0.01 70.58
Sampling time point
Non-fasting 3 −0.88 −2.03 0.27 0.134 77.02
Fasting (>3h) 9 −0.91 −1.58 −0.24 0.007 67.51
2.4. Meta-Regression Analyses
Univariate meta-regression analyses were performed next. Type of rooibos tea extracts and
polyphenols is found to be a significant covariate to explain approximately 100% of between-study
variance (R
2
= 1.0). The treatment period is also, at least in part, responsible for between-study variance
(coefficient = 0.28, 95% CI: 0.11 to 0.45, p< 0.002, R
2
= 0.64) (Figure 4). Moreover, the analyses also
showed that type of rodents, DM models, or blood samples and blood sampling time point do not
affect the variance (R2= 0.00), confirming the results of subgroup analyses described above (Table 2).
Figure 4.
Meta-regression analysis for Hedges’ g of blood glucose levels and treatment period in DM
rodents treated with or without rooibos extracts or major phenolic compounds. Summary estimates
were analyzed using a random-effects model.
Molecules 2018,23, 839 7 of 15
2.5. Publication Bias
No significant evidence of publication bias was observed in the analyses of the effect of rooibos tea
extracts and polyphenols on blood glucose levels as indicated by funnel plots. Moreover, there were
no imputed studies found in re-displayed funnel plots after Duval and Tweedie’s Trim and Fill
adjustment [29,30] (Figure 5).
Figure 5.
Funnel plots of standard error by Hedges’ g of blood glucose levels in DM rodents treated
with or without rooibos extracts or major phenolic compounds. Open and closed diamond indicates
the imputed summary estimates before and after Duval and Tweedie’s Trim and Fill adjustment
(random-effects models), respectively. No imputed studies were found in re-displayed funnel plots by
Duval and Tweedie’s Trim and Fill analysis.
3. Discussion
3.1. Main Findings
To the best of our knowledge, this study is the first meta-analysis that summarizes the evidence
for a possible beneficial role of rooibos extracts and associated polyphenols in blood glucose levels
of DM rodent models. We showed that elevated blood glucose levels of DM rodents are significantly
reduced by intake of FREs, GREs, and their major phenolic compounds compared with those of DM
controls. This association was largely influenced by type of rooibos extracts and their polyphenols
and, at least partly, by treatment period in the subgroup and meta-regression analyses, respectively.
PPAG significantly reduced blood glucose levels in DM mice or rats; however, FREs, GREs, or ASP
failed to demonstrate the similar effects. The chronical treatment with rooibos extracts or their
major polyphenols was thus likely to lose the beneficial effect on reduced glucose levels over time.
Furthermore, types of rodents (mice or rats), DM models (db/db, ob/ob, KK-Ay, OBIR, or STZ-induced)
and blood samples (plasma, serum, or whole blood), or blood sampling time point (fasting or
non-fasting) did not markedly influence the association.
3.2. Interpretation
3.2.1. Structures and Pharmacological Properties of Phenolic Compounds Rich in Rooibos Extracts
The structures of different classes of flavonoids present in the rooibos extracts are shown
in Figure 6.
Molecules 2018,23, 839 8 of 15
Figure 6.
Structures of major flavonoids in rooibos extracts [
14
] Z-2-(
β
-D-glucopyranosyloxy)-3-
phenylpropenoic acid, PPAG.
Two anti-diabetic pharmacological properties are known to be present in rooibos extracts. First,
α
-glucosidase inhibitors are oral anti-diabetic drugs used for treatment of T2DM by preventing glucose
absorption in intestine and thus postprandial hyperglycemia. Muller et al. detected stronger inhibition
of
α
-glucosidase activity in GREs. Further HPLC-based assay confirmed that
α
-glucosidase inhibitory
activity corresponded with the retention time of ASP (Figure 6, top) [
12
]. Moreover, various C-glucoside
flavones detected in rooibos extracts exhibited stronger
α
-glucosidase inhibition activity than acarbose,
one of the most potent
α
-glucosidase inhibitors; the inhibitory activity decreases in the order isoorientin
≥
orientin
≥
isovitrexin
≥
vitrexin. The C-3 hydroxylation of the B-ring of flavones was suggested
to be critical for the inhibition of
α
-glucosidase activity in flavones [
31
] (Figure 6, middle). Flavonols
in rooibos extracts, isoquercitrin and rutin, also showed similar but much weaker
α
-glucosidase
inhibitory activity than acarbose [
32
]. Thus, the glucose lowering effects of rooibos extracts could
be due, at least partly, to the
α
-glucosidase inhibition activity of their contained flavonoids. Second,
inhibition of glucose reabsorption in the kidney became a strategy for lowering blood glucose levels
in T2DM [
33
]. The renal glomerulus filters approximately 160 g of glucose per day, 98% of which is
then reabsorbed primarily in the proximal tubules of nephrons via sodium glucose co-transporters
(SGLTs) [
34
]. Among six known SGLTs in human, SGLT1 and 2 have been well studied. SGLT1 is
located in the small intestine, heart and kidney with an affinity for both glucose and galactose [
35
];
while, SGLT2 is localized only in the kidney with the high selectivity for glucose [
36
,
37
]. Therefore,
chemical compounds with selective inhibition of SGLT2 over SGLT1 and a Glut family, another family
of glucose transporters, would be ideal drug targets against T2DM. Phlorizin, a natural glucosylated
dihydrochalcone present in the bark of apple trees, is the first reported SGLT inhibitor [
38
]. In the
search of new selective drug targets for SGLT2, C-glucoside dihydrochalcones were examined; a line
of research demonstrated the anti-SGLT2 activity in ASP and nothofagin, two major C-glucoside
dihydrochalcone in rooibos [
39
,
40
]. Thus, the anti-SGLT2 property in ASP and nothofagin in rooibos
extracts might also play a critical role in their in vivo anti-diabetic action.
Molecules 2018,23, 839 9 of 15
3.2.2. FREs, GREs and Major Phenolic Compounds in Rooibos in DM Rodent Models
FREs were shown to reduce DM-mediated H
2
O
2
- and ischemia-induced oxidative stress in
STZ-injected DM rats. Aqueous and alkaline extracts of fermented rooibos tea were reported to
significantly lower levels of oxidative stress markers, advanced glycation end products (AGEs) in
plasma and advanced lipoxidation end products, malondialdehyde, in plasma, liver and lends of
STZ-induced DM rats; the extracts also slightly, but not significantly, reduced advanced oxidation
protein products [
28
]. Moreover, FREs increased oxygen radical absorbance capacity, superoxide
dismutase and thiobarbituric acid reactive substances in STZ-induced DM rats [
41
]. However,
FREs alone did not significantly improve plasma glucose and lipid profiles in DM rats [
28
]. Collectively,
FREs might reduce oxidative stress in STZ-induced DM rats. However, consistent with the present
meta-analysis, FREs alone are unlikely to significantly improve plasma glucose and lipid profiles,
at least, in STZ-induced DM rats as reported in control rats [18,42].
In our subgroup analysis which included the solo study using KK-A
y
mice, the chronic treatment
with GREs for more than 3 days showed a trend but failed to exhibit significant beneficial effects on their
blood glucose levels. However, it has been reported that acute and sub-chronical oral administration
of GREs significantly lowered glucose levels in some DM rodent models [
12
]. The underlying
mechanisms by which acute GREs reduce glucose levels could be that GREs stimulate glucose uptake
in muscle [
12
,
13
] and liver cells [
12
], at least in part, by increasing phosphorylation of 5
0
-adenosine
monophosphate-activated protein kinase (AMPK) and serine/threonine kinase (Akt), which then
promotes translocation of glucose transporter 4 (Glut4) to the plasma membrane [
13
]. GREs also
reduced AGE- and H
2
O
2
-induced oxidative stress in pancreatic
β
-cells [
13
]. Moreover, GREs showed
to reverse the palmitate-induced insulin resistance and suppress inflammatory pathway by inhibiting
palmitate-mediated nuclear factor-
κ
B activation in 3T3-L1 adipocytes [
43
]. In summary, GREs have
demonstrated more effective, yet not significant in this study, anti-diabetic potentials than FREs; GREs
might reduce plasma glucose levels in a DM rodent model likely through mechanisms involving
multi-organ systems such as liver, muscle, pancreas and adipose tissue.
The total antioxidant activity of GREs was positively associated with its ASP content [
44
];
therefore, several research examined how ASP alone could modulate glucose metabolism. A study
first compared effects of GREs and ASP alone on glucose metabolism in STZ-induced DM rats [
12
].
The result suggests that GREs are more effective than ASP alone in glucose lowering effect in the
DM mice, which is compatible with our subgroup analysis (ASP;
−
0.46 (
−
1.03 to 0.11) p= 0.12
vs. GRE;
−
1.08 (
−
2.25 to 0.10) p= 0.07). This was likely because GREs contain other polyphenols
such as rutin [
45
–
50
] and vitexin/isovitexin [
51
,
52
], which previously showed glucose lowering
effects in STZ-induced DM rats. ASP suppressed the increased fasting blood sugar levels and/or
improved glucose tolerance in two T2DM and obese mouse models, db/db [
22
] and ob/ob [
26
] mice.
The consequent
in vitro
studies demonstrated that these effects were presumably due, at least in part,
to an ASP-mediated dose-dependent increase in glucose uptake in the muscle cells; ASP enhanced
phosphorylation of AMPK and promoted Glut4 translocation to the plasma membrane [
13
,
22
,
26
].
ASP also increased insulin secretion [
22
] and reduced AGE-mediated rise in reactive oxygen species,
a marker for oxidative stress, [
26
] in pancreatic
β
-cells. Collectively, ASP exhibited two anti-diabetic
pharmacological properties, inhibition of both
α
-glucosidase and SGLT2 activities and glucose lowering
effects presumably targeting muscle cells in some T2DM mouse models, however, the present study
failed to display a marked reduction of blood glucose levels by the administration of ASP alone to
DM rodents.
Analysis of infusions prepared from various production batches of fermented rooibos
demonstrated that PPAG is one of the major monomeric phenolic compounds present at similar
concentrations to ASP (Figure 6, bottom) [
53
]. Biological studies showed that PPAG delayed the
onset of diabetes by modifying cell death and necrosis, but not by increasing cell proliferation or its
ability for DNA damage/repair, of pancreatic
β
-cells in STZ-induced DM mice [
20
]. Moreover, PPAG
has been recently shown to improve fasting blood glucose levels, glucose tolerance, insulin levels
Molecules 2018,23, 839 10 of 15
and insulin-resistance in OBIR rats [
24
]. The effects were presumably through increased expression
of glucokinase, Glut 1 and 2, insulin receptor, peroxisome proliferator-activated receptor
α
and
suppressor of cytokine signaling 3 in the liver and through reduced apoptosis or neogenesis of
pancreatic
β
-cells [
24
]. Therefore, these results suggest that PPAG could be a significant modulator
for glucose metabolism in the liver and pancreas in the rodent DM models and our meta-analysis
supports a significant role of PPAG in regulation of blood glucose levels.
The role of isoorientin in STZ-induced DM mice was investigated as the main constituent of
Cecropia obtusifolia or Gentiana olivieri, plants found in Central America (Columbia, Costa Rica, Mexico
and Panama) or Asia, respectively [
25
,
54
]. Aqueous extracts of Cecropia obtusifolia was described for
use of the treatment of diabetes in mice and rabbits [
55
,
56
]. Dried flowering herbs of Gentiana olivieri
in water was used to lower blood glucose levels of T2DM patients [
57
]. The present meta-analysis
based on a single study also suggested the significant glucose lowering effect of isoorientin in DM
rodent models; this could be due to strong pharmacological ability to inhibit
α
-glucosidase activity as
mentioned above. However, the strength of meta-analysis with one study using a dozen rats is quite
low and more future studies will be thus necessary to draw a certain conclusion regarding an effect of
isoorientin on blood glucose levels.
3.2.3. Strength and Limitations
The primary strength of this meta-analysis is the inclusion of relatively large number of DM
mouse and rat models and focusing on an effect of rooibos on their blood glucose levels. We also
systematically assessed various cofounding factors. This meta-analysis has also several limitations.
First, although a broad literature search was applied using two electronic databases, the number
of articles assessing an effect of GREs and isoorientin on blood glucose levels in DM rodents was
quite small and the language restriction and the exclusion of ambiguous literature might increase
the risk of publication bias. Second, some evidence of heterogeneity existed in our meta-analysis
although this could be mostly explained by the pre-specified variables, type of tested rooibos and
related polyphenols as well as treatment period. This heterogeneity may also potentially weaken
the robustness of our findings. Third, all the included studies used male rodents, the outcome could
be different when studies include females. Forth, STZ-induced DM rodent models were generated
after grouping for diet modulation in three included studies [
20
,
27
,
28
]. There exists, at least, a slight
possibility that the investigators’ technical skill for STZ injection could directly or indirectly influenced
the experimental outcome of blood glucose levels.
4. Materials and Methods
4.1. Data Sources and Search Strategies
A comprehensive literature search of the electronic databases PubMed and Embase for
the period between 1 January 1962 and 28 February 2018 was conducted using the keywords
(“Aspalathus linearis” or rooibos or aspalathin or nothofagin or PPAG or orientin or isoorientin or
quercetin-3-O-robinobioside) and (diabetes or “insulin resistance”). In addition, the reference lists of
the retrieved articles were manually searched to ensure that no relevant articles had been missed.
4.2. Inclusion and Exclusion Criteria
Peer-reviewed articles in the English language were eligible for inclusion when they fulfilled
the following inclusion criteria: (i) studies used diabetic rodent models with or without treatment
with fermented or green rooibos extracts (FRE and GRE, respectively), PPAG, ASP, nothofagin,
orientin, isoorientin, or quercetin-3-O-robinobioside for more than 3 days; (ii) they also reported
the blood glucose levels of the DM rodents at the end of the treatment period. As the polyphenols
associated with fermented and green rooibos teas, PPAG, ASP, nothofagin, orientin, isoorientin and
quercetin-3-O-robinobioside are included because they were previously reported to be contained
Molecules 2018,23, 839 11 of 15
more than 10% w/w of total phenolic compounds either in FREs or GREs. Studies were excluded if
they are reviews, commentaries, editorials, letters, conference abstracts, duplicates, not in English,
or not studied on blood glucose levels with the treatment for longer than 3 days in rodent DM models.
Unpublished research was not sought. This meta-analysis was strictly conducted according to the
PRISMA guidelines (Table S2).
4.3. Data Extraction and Quality Assessment
Titles and abstracts of retrieved publications were screened initially for potentially eligible
studies, which were subsequently evaluated by full-text review. Data were collected by three authors
(M.S., N.N. and M.S.) in an independent manner using pre-designed standardized data extraction
form; which includes type of rooibos extracts or associated compounds, dose, method and period of
administration, blood glucose levels, baseline age, sex, type of rodent DM models and their relevant
controls. Study quality was assessed by the Cochrane Collaboration “Risk of Bias” Tool [
58
]. The risk of
bias for each quality variable in each criterion was assessed by 2 authors in an independent manner and
judged as “low”, “unclear”, “high”, or “not applicable (N/A)” based on its description in each included
study. Any disagreements in any phase were resolved by discussion until consensus was achieved.
4.4. Data Synthesis and Analysis
Continuous variables were presented as means
±
standard deviation (SD). For studies reporting
the standard errors of means (SEs), the corresponding SDs were calculated by multiplying by the
square root of the respective sample size. For studies providing glucose levels in mmol/L, these levels
were converted into mg/dL using the conversion table offered by the Joslin Diabetic Center at
http://www.joslin.org/info/conversion_table_for_blood_glucose_monitoring.html. For studies
reporting more than one measure of blood glucose levels, the levels after the longest period of
treatment with rooibos extracts or associated compounds and at the treatment dose which gave the
most robust difference in blood glucose levels between the two DM rodent groups were selected and
included in the primary meta-analyses.
Standardized mean difference, Hedges’ g, transformation was used to calculate the related
statistics including variance and 95% CIs of each study and the summary effect size generated in the
meta-analyses and publication bias assessment. The random-effects model was chosen in this study
because it is more conservative and incorporates better between-study variability. Heterogeneity was
assessed using I
2
statistics with its value
≥
50% interpreted as evidence of substantial heterogeneity [
58
].
Subgroup and meta-regression analyses were performed based on type of rooibos extracts and
their major polyphenols (FRE, GRE, ASP, PPAG, or isoorientin), type of blood samples (whole blood,
plasma, or serum), blood sampling time point (fasting vs. ad libitum (ad lib.)), types of rodents
(mice vs. rats) and DM models (db/db, ob/ob, KK-Ay, or STZ-induced) and treatment period to
examine their influence to the outcome estimates. Sensitivity analyses were used to evaluate the
robustness of the outcome estimates mainly by removing one study at a time with a repeat of the
primary meta-analyses. Publication bias was assessed by funnel plots with Duval and Tweedie’s
Trim and Fill analysis (random-effect models). All the statistical analyses were carried out using
Comprehensive Meta-Analysis 3.0 (Biostat, Englewood, NJ, USA) and Review Manager (Version 5.3,
the Nordic Cochrane Center, Copenhagen, Denmark) software.
5. Conclusions
The present meta-analyses demonstrated that blood glucose levels were significantly reduced in
diabetic rodent models treated with PPAG, a rooibos-associated phenolic compound. Some sporadic
case studies reported severe yet reversible adverse effects of rooibos tea on liver in humans [
59
,
60
].
Thus, further clinical studies would be needed to establish the safe and practical use of the rooibos
tea for prevention and treatment of diabetes and its associated complications in humans in the future.
Finally, it would have a profound impact on an increasing number of pre-diabetic patients worldwide,
Molecules 2018,23, 839 12 of 15
in particular, if herbal teas such as rooibos could be developed as natural nutraceuticals for prevention
or delayed onset of diabetes.
Supplementary Materials:
The following are available online. Table S1: Risk of Bias; Table S2: PRISMA checklist.
Acknowledgments:
This research was partially supported by JSPS (Kaken-17K09870) and Sagami Women’s
University (the special research fund (B)) to M.S.
Author Contributions:
M.S., N.N. and M.S. designed the research content, collected, reviewed literature and
analyzed the data; M.S. and M.S. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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