Pediatrics International (2009) 51, 700–704

doi: 10.1111/j.1442-200X.2009.02835.x

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 movement, 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 pharmacological 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 inhibitory effects of Aspalathus linearis on X-ray-induced C3H10T1/2 cell transformation.5 Another study showed that Aspalathus

linearis had an antifibrotic effect on CCl4-induced liver damage in rats.6 These studies suggest that Rooibos tea may have antiinflammatory effects through its anti-oxidative activity. In this study, we evaluated clinical symptoms such as bodyweight gain and the properties of stool, and biochemical parameters including hemoglobin, serum iron, cholesterol, triglyceride, and alanine aminotransferase (ALT) in rats. We determined serum superoxide dismutase (SOD) levels to evaluate their antioxidative 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.

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.

Plant material

© 2009 Japan Pediatric Society

Methods 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.

Anti-inflammatory effects of Rooibos tea Animals

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 aminotransferases were determined by a standard automated technique.

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concentration was adjusted to the urinary concentration of creatinine (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 determined hemoglobin levels and serum iron levels on day 7 and serum SOD on day 7, 9, 14, and 21.

Serum SOD

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 immunosorbent 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

Serum SOD (U/ml)

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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

Urine 8 -OHdG (/Cr)

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 dimethylsulfoxide (DMSO), 50 mL of serum sample, and 50 mL of xanthine 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.

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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

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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 bodyweight 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 significant 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

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 reactions. 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

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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

0.41 1 0.08 0.45 1 0.02 0.40 1 0.04 0.40 1 0.02 Urine 8-OHdG (/Cr)

Values are expressed as means 1 standard deviation for six rats. *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, superoxide dismutase; T-cho, total cholesterol; TG, triglyceride.

water rooibos 15.3 1 1.1 15.3 1 1.2 350 1 104.1 340 1 45.3 82 1 15.1 85 1 9.9 62 1 19.8 61 1 19.8 43.3 1 8.2 42.8 1 4.7 23.0 1 1.9 27.0 1 6.1* (P = 0.031) 0.72 1 0.09 0.40 1 0.11* (P = 0.010) water rooibos 15.3 1 1.3 15.2 1 1.2 365 1 54.1 355 1 109.5 81 1 13.9 78 1 9.9 52 1 19.2 60 1 20.0 41.3 1 6.1 41.8 1 6.0 20.0 1 2.7 26.0 1 3.5* (P = 0.032) 0.46 1 0.12 0.34 1 0.05* (P = 0.020) water rooibos 15.5 1 1.3 15.5 1 1.2 365 1 100.2 335 1 83.9 86 1 11.5 82 1 13.7 60 1 26.3 52 1 18.9 40.0 1 5.8 39.0 1 6.1 18.0 1 2.5 26.3 1 4.7* (P = 0.028) 0.51 1 0.04 0.34 1 0.04* (P = 0.021) rooibos 15.3 1 1.3 285 1 75.4 79 1 13.9 63 1 19.9 40.0 1 5.7 22.5 1 1.2 water 15.4 1 1.2 300 1 72.3 85 1 10.1 52 1 19.1 41.0 1 5.2 18.5 1 4.6 rooibos 15.4 1 1.3 515 1 93.0 84 1 8.2 40 1 15.2 40.0 1 5.8 16.5 1 2.1 water 15.7 1 1.3 325 1 110.5 82 1 11.9 34 1 17.1 41.0 1 5.1 16.5 1 3.2 Hb (g/dl) Iron (mg/dl) T-cho (mg/dl) TG (mg/dl) ALT (mg/dl) Serum SOD (U/ml)

4 weeks 3 weeks 2 weeks 1 week 0 week

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)

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the controls. These results suggest that Rooibos tea has an effect to reduce DNA damage from oxidative reaction. There was no significant difference in clinical symptoms such as appetite, defecation, or bodyweight gain in rats after Rooibos administration. Furthermore, there was no difference in laboratory data between the two groups. Although most tea is known to reduce iron absorption, there was no significant decrease in 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 of tannins contained in Rooibos tea, compared with other tea, might prevent the loss of iron. In this study, we investigated whether Rooibos tea could reduce oxidative stress and inflammation in DSS-induced colitis rats. We could not find any statistical differences in clinical symptoms such as bodyweight gain, diarrhea, or rectal bleeding 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 02- into less reactive or non-reactive products. Moreover, SOD is known to exist in all kinds of tissues, and it enters the circulation via tissue inflammation. From this point of view, the upregulation of serum SOD in this study can be explained by not only the effect of Rooibos tea but also the colic inflammation caused by DSS. Two weeks after DSS administration, serum SOD levels 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 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. Further experiments might widen the possibility of Rooibos tea as a treatment of inflammatory bowel diseases.

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8 Takizawa H, Sasakawa T, Nakazawa T et al. Frontiers of mucosal immunology. Excerpta Med. (Amsterdam) 1991; 853–4. 9 Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 1990; 98: 694–702. 10 Shimizu T, Suzuki M, Fujimura J et al. The relationship between the concentration of dextran sodium sulfate and the degree of

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induced experimental colitis in weanling rats. J. Pediatr. Gastroenterol. Nutr. 2003; 37: 481–5. 11 Noda Y, Anzai K, Mori A, Kohno M, Shinmei M, Packer L. Hydroxyl and superoxide anion radical scavenging activities of natural source anti-oxidants using the computerized JES-FR30 ESR spectrometer system. Mol. Biol. Int. 1997; 42: 35–44. 12 Hesseling PB, Klopper JF, van Heerden PD. The effect of rooibos tea on iron absorption. S. Afr. Med. J. 1979; 14: 631–2 (inAfrikaans).