Pediatrics International (2009) 51, 700–704 doi: 10.1111/j.1442-200X.2009.02835.

Original Article

Studies of anti-inflammatory effects of Rooibos tea in rats

Haruna Baba,1 Yoshikazu Ohtsuka,1 Hidenori Haruna,1 Tsubasa Lee,1 Satoru Nagata,2 Masato Maeda,1 Yuichiro Yamashiro2
and Toshiaki Shimizu1
1
Department of Pediatrics and Adolescent Medicine and 2Division of Probiotics Research, Juntendo University School of
Medicine, Tokyo, Japan

Abstract Background: Rooibos tea is known to be caffeine free with abundant flavonoids. Aspalathin and nothofagin, the main
flavonoids contained in Rooibos tea, have stronger anti-oxidative activity than other flavonoids.As oxidative stress can
induce inflammation, the anti-inflammatory effects of Rooibos tea were investigated using a rat colitis model.
Methods: Seven-week-old Wister rats were divided into two groups: one group given Rooibos tea, and one given water.
After four weeks of breeding, serum superoxide dismutase (SOD) levels were determined using the Electron Spin
Resonance analysis. Urine 8-hydroxy-2′-deoxyguanosine (8-OHdG) concentrations were also determined as reflections
of DNA damage using enzyme-linked immunosorbent assay. Furthermore, rats were administrated dextran sodium
sulfate (DSS), which is known to induce colitis in rodents, with or without Rooibos tea to evaluate its anti-inflammatory
activity. Clinical symptoms, hemoglobin, serum iron and SOD levels were compared between the groups.
Results: There were no significant differences in bodyweight gain or laboratory data between the groups. The serum
SOD levels were significantly increased, and urine 8-hydroxy-2′-deoxyguanosine levels were significantly decreased in
the Rooibos group compared with the controls (P < 0.05 in each). After DSS administration, the serum SOD levels were
significantly higher in the Rooibos group compared to the controls (P < 0.05). As a result, a decreased hemoglobin level,
observed in the control group, was prevented in the Rooibos group after the DSS challenge.
Conclusion: Rooibos tea may prevent DNA damage and inflammation by its anti-oxidative activity in vivo. As Rooibos
tea is free from caffeine, routine intake may be safe and useful in reducing oxidative stress in children.

Key words 8-hydroxy-2′-deoxyguanosine, colitis, dextran sodium sulfate, superoxide dismutase.

Rooibos tea (Aspalathus linearis) is a herbal tea that grows on the
slopes of the Cederberg mountain range in Cape Province,
Republic of South Africa. It has been consumed as a healthy
beverage for more than a century in the Republic of South Africa
and Europe. In South Africa, this tea has been said to have many
functions such as increasing appetite, improving bowel move-
ment, and controlling mental condition.1–3
Rooibos tea, unlike other teas such as green tea, contains no
caffeine, no alkaloids, and low contents of tannins.1,2 It contains
sodium, potassium, magnesium, calcium, and trace elements such
as zinc. It is known that abundant flavonoids are contained in
Rooibos tea, particularly, aspalathin, isoorientin, and nothofagin.4
Previous studies have shown its physiological and pharmaco-
logical actions. Nakano showed that the polysaccharide from
Aspalathus linearis inhibited the binding of HIV-1 to MT-4 cells,
which proved its anti-HIV activity.2 Komatsu showed the inhibi-
tory effects of Aspalathus linearis on X-ray-induced C3H10T1/2
cell transformation.5 Another study showed that Aspalathus
Correspondence: Yoshikazu Ohtsuka, MD, Department of Pediatrics
and Adolescent Medicine, Juntendo University School of Medicine,
2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Email: yohtsuka@
med.juntendo.ac.jp
Received 14 March 2008; revised 22 December 2008; accepted 9
January 2009.
linearis had an antifibrotic effect on CCl4-induced liver damage
in rats.6 These studies suggest that Rooibos tea may have anti-
inflammatory effects through its anti-oxidative activity.
In this study, we evaluated clinical symptoms such as body-
weight gain and the properties of stool, and biochemical param-
eters including hemoglobin, serum iron, cholesterol, triglyceride,
and alanine aminotransferase (ALT) in rats. We determined
serum superoxide dismutase (SOD) levels to evaluate their anti-
oxidative activity, and urine 8-hydroxy-2′-deoxyguanosine
(8-OHdG) concentrations as a reflection of DNA damage.
Furthermore, we investigated the effects of Rooibos tea on the
prevention of inflammation in dextran sodium sulfate (DSS)-
induced rat colitis, which resembles that in human ulcerative
colitis in both clinical and histopathological findings.7–10 In this
study, we examined their clinical symptoms, hemoglobin, serum
iron and SOD levels after DSS administration.
Methods
Plant material
The aqueous extract of Rooibos tea was prepared daily by boiling
1.6 g of unfermented leaves in 100 ml water at 92°C for 15 min,
and cooling down to room temperature. After separation of
insoluble residue, the solution was used for the experiments.

© 2009 Japan Pediatric Society

 Anti-inflammatory effects of Rooibos tea 701

Animals concentration was adjusted to the urinary concentration of crea-

Seven-week-old male Wister rats were prepared and maintained
under a 12 h light/dark cycle at a constant room temperature of
25°C. Twenty-four rats were divided into two groups: one group
given Rooibos tea, and one given water. Rats had free access to
standard food and tap water or Rooibos tea. Bodyweights were
measured three times a week.
Biochemical analysis
Blood samples were taken once a week. Hemoglobin and plasma
activities of iron, total cholesterol, triglyceride, and aminotrans-
ferases were determined by a standard automated technique.
Serum SOD
Each blood sample was centrifuged at 1500 rpm for 15 min at
4°C. The procedures of the Electron Spin Resonance (ESR)
analysis were referred to the method reported by Noda et al.11
Into a mixture containing 30 mL of 5,5-dimethyl pyrroline-N
oxide (DMPO), 50 mL of 4 mM hypoxanthine, 30 mL of dimeth-
ylsulfoxide (DMSO), 50 mL of serum sample, and 50 mL of xan-
thine oxidase (XOD) was added. After mixing well, the reaction
mixture was transferred into a flat cell, and the monitoring of the
ESR spectrum was started 1 min after the addition of XOD. The
ESR analysis was carried out using JES-FR30 free radical
monitor (JEOL, Tokyo, Japan). The instrumental conditions were
as follows: magnetic field, 335.5 1 5 mT; microwave power,
4 mW; sweep time, 1 min; modulation frequency, 100 kHz;
modulation width, 0.79 ¥ 0.1 mT; amplitude, 100; and time
constant, 0.03 s.
Urine 8-OHdG
Each urine sample was centrifuged at 1500 rpm for 5 min at 4°C.
The level of urine 8-hydroxy-2′-deoxyguanosine (8-OHdG) was
estimated by using a new 8-OHdG check enzyme-linked immu-
nosorbent assay (ELISA) (Japan Institute for the Control of
Aging, Shizuoka, Japan). The absorbance was measured
at 450 nm using a Biotrak II reader. The urinary 8-OHdG
tinine (mg 8-OHdG/ g creatinine) to control the variability in
urine dilution.
DSS-induced colitis rat model
After four weeks of breeding, rats were divided into four groups
of six rats each: one group given water, one given Rooibos, one
given water and DSS (MM 5000, Wako Jyunyaku, Tokyo, Japan),
and one given Rooibos and DSS. Four percent DSS was given
orally in drinking water for half a day. Clinical symptoms, such
as diarrhea and rectal bleeding, were recorded daily. We deter-
mined hemoglobin levels and serum iron levels on day 7 and
serum SOD on day 7, 9, 14, and 21.
Statistical analysis
Statistical analysis was performed using the Mann–Whitney
U-test for laboratory data and bodyweight gain. As for clinical
symptoms after DSS administration, Yates’s 2 ¥ 2 c2-test
was used for statistical analysis. P < 0.05 was considered as
statistically significant.
Results
There was no significant difference between the control group
and the Rooibos group in consumption of food and liquid. As for
their clinical symptoms, there was no difference in their general
condition, bodyweight gain, and the properties of stools among
each group. There was no difference between the two groups in
hemoglobin, serum iron, total cholesterol, triglyceride, or ALT
levels (Table 1).
After two weeks of the experiment, the serum SOD levels of
the Rooibos group were significantly increased compared with
those of controls (P < 0.05; Fig. 1). Urine 8-OHdG levels were
significantly decreased in the Rooibos group compared with the
controls (P < 0.05; Fig. 1).
After DSS administration, all rats had poor bodyweight gain
compared with the non-DSS-administered groups (P < 0.05).
Thus, there was no significant difference between the Rooibos

0.8 *
Urine 8 -OHdG (/Cr)

40
Serum SOD (U/ml)

*
* *
30
* 0.6 *

20 0.4

10 0.2

0 0
0 1 2 3 4 0 1 2 3 4

Fig. 1 Differences in the levels of serum superoxide dismutase (SOD), and urine 8-hydroxy-2′-deoxyguanosine (8-OHdG) between the
control (open column) and the Rooibos group (closed column) (n = 6 in each group). Values are expressed as means 1 standard deviation for
six rats. *Significant difference from the control group, P < 0.05 (Mann–Whitney U-test).

© 2009 Japan Pediatric Society

702 H Baba et al.

300 80 *
DSS

Serum SOD (U/ml)

60 *
Body weight (g)

200 *

40 *

Water
100 Rooibos 20
Water + DSS
Rooibos + DSS

0
0 1 2 3 4 5
Fig. 2 Bodyweight gaining after dextran sodium sulfate (DSS)
administration (n = 6 in each group). Open square represents the
bodyweight of the control group, closed square represents that of the
Rooibos group, open diamond represents that of control group with
DSS administration, and closed diamond represents that of the
Rooibos group.
group and controls in bodyweight gain/loss. The maximum body-
weight loss was 7.5 1 8.4 g and 7.3 1 8.8 g in the control group
and the Rooibos group, respectively (Fig. 2). There was no sig-
nificant difference in clinical symptoms between the two groups.
In the control group, both diarrhea and bloody stools appeared in
four rats, while diarrhea appeared in two rats and bloody stools in
four rats in the Rooibos group.
The hemoglobin levels were higher in the Rooibos group
(13.4 1 3.06 g/dl) compared with the controls (8.8 1 3.07 g/dl) on
day 7 (P < 0.05). The Rooibos group had a lower decrease in
hemoglobin levels compared to the control group (P < 0.05).
There was no significant difference in the serum iron level
between the control (315.3 1 114.9 mg/dl) and the Rooibos (273.1
1 111.9 mg/dl) groups, respectively (Fig. 3). Serum SOD levels of
0
0 7 9 14 21 (days)
6 7 8 (weeks) Fig. 4 Difference in serum superoxide dismutase (SOD) levels
between control (open column) and Rooibos group (closed column)
after dextran sodium sulfate administration (*P < 0.05; n = 6 in each
group).
the Rooibos group were significantly increased compared with
those of the control group throughout the experimental period
(P < 0.05; Fig. 4).
Discussion
Active oxygen and free radicals have been said to be involved in
aging and in diseases such as inflammation, cancer, and arterial
sclerosis. It is known that vitamin C, vitamin E, flavonoid, and
enzymes such as catalase, glutathione peroxidase (GPx), and
SOD have anti-oxidative activity to prevent these oxidative reac-
tions. Recently, there is a worldwide trend to seek safe and
effective anti-oxidants from natural sources.
In this study, serum SOD levels significantly increased in the
Rooibos group compared with the control group. The large
amount of flavonoids contained in Rooibos tea might play some
role in this mechanism. In addition to that, urine 8-OHdG levels
were significantly decreased in the Rooibos group compared with

a b

18 * 500
Hemoglobin (g/dl)

Serum Iron (mg/dl)

400
12
300
8
200
4
100

0 0
control Rooibos control Rooibos
Fig. 3 Differences in the levels of (a) hemoglobin and (b) serum iron between the control (open column) and the Rooibos group (closed
column) 7 days after dextran sodium sulfate (DSS) administration (*P < 0.05; n = 6 in each group).

© 2009 Japan Pediatric Society

 Anti-inflammatory effects of Rooibos tea 703

the controls. These results suggest that Rooibos tea has an effect

*Significant difference from the control group, P < 0.05 (Mann–Whitney U-test). 8-OHdG, 8-hydroxy-2′-deoxyguanosine; ALT, alanine aminotransferase; Hb, hemoglobin; SOD,
0.40 1 0.11*
340 1 45.3

61 1 19.8

27.0 1 6.1*
15.3 1 1.2

85 1 9.9

42.8 1 4.7
rooibos
to reduce DNA damage from oxidative reaction.
Table 1 Differences in the levels of hemoglobin, serum iron, total cholesterol, triglyceride, serum SOD, and urine 8-OHdG between control and Rooibos groups (n = 6 in each group)

There was no significant difference in clinical symptoms such

(P = 0.031)

(P = 0.010)
as appetite, defecation, or bodyweight gain in rats after Rooibos
4 weeks

administration. Furthermore, there was no difference in labora-

350 1 104.1 tory data between the two groups. Although most tea is known to
82 1 15.1
62 1 19.8

0.72 1 0.09
15.3 1 1.1

43.3 1 8.2
23.0 1 1.9
reduce iron absorption, there was no significant decrease in
water

hemoglobin or serum iron levels in the Rooibos group when

compared with the controls. A previous study in Africa showed
that Rooibos tea did not affect iron absorption, unlike ordinary
tea (Camellia sinensis).12 A higher amount of iron and low levels
355 1 109.5

0.34 1 0.05*
60 1 20.0

26.0 1 3.5*
15.2 1 1.2

78 1 9.9

41.3 1 6.1
rooibos

of tannins contained in Rooibos tea, compared with other tea,

might prevent the loss of iron.
(P = 0.032)

(P = 0.020)

In this study, we investigated whether Rooibos tea could

3 weeks

reduce oxidative stress and inflammation in DSS-induced colitis

rats. We could not find any statistical differences in clinical
365 1 54.1
81 1 13.9
52 1 19.2

0.46 1 0.12
15.3 1 1.3

41.8 1 6.0
20.0 1 2.7

symptoms such as bodyweight gain, diarrhea, or rectal bleeding

water

between the control and the Rooibos groups. Serum SOD levels
were significantly higher in the Rooibos group compared with
controls in all weeks. SOD is an antioxidant enzyme which is
increased by the upregulation of its scavenger 02-, and catalyze
0.34 1 0.04*
335 1 83.9
82 1 13.7
52 1 18.9

26.3 1 4.7*
15.5 1 1.2

39.0 1 6.1
rooibos

02- into less reactive or non-reactive products. Moreover, SOD is

known to exist in all kinds of tissues, and it enters the circulation
(P = 0.028)

(P = 0.021)

via tissue inflammation. From this point of view, the upregulation

2 weeks

of serum SOD in this study can be explained by not only the

effect of Rooibos tea but also the colic inflammation caused by
365 1 100.2

60 1 26.3

0.51 1 0.04
86 1 11.5
15.5 1 1.3

40.0 1 5.8
18.0 1 2.5

DSS. Two weeks after DSS administration, serum SOD levels

water

started to decrease in both groups. The improvement of colitis

may have downregulated the levels of SOD, and it also helped to
increase bodyweights (Figs 2,4).
In conclusion, our study suggests that Rooibos tea may reduce
285 1 75.4
79 1 13.9
63 1 19.9

0.41 1 0.08
15.3 1 1.3

40.0 1 5.7
22.5 1 1.2
rooibos

DNA damage from oxidation stress by its anti-oxidative activity

in vivo. As Rooibos tea is free from caffeine, routine intake may
be safe and useful in reducing oxidation stress for all ages.
1 week

Further experiments might widen the possibility of Rooibos tea

300 1 72.3
85 1 10.1
52 1 19.1

0.45 1 0.02

as a treatment of inflammatory bowel diseases.

15.4 1 1.2

41.0 1 5.2
18.5 1 4.6
water

Values are expressed as means 1 standard deviation for six rats.

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