Antioxidant effect of Kombucha tea during chemically induced hepatic injury
Zakhary NIa*, Morcos Nb, Hosny OMSb, Daba ASc, Anwar Nd, and Michaeal N
a. Cancer Biology Department, National Cancer Institute, Cairo University
b. Biochemistry Department, Faculty of Science, Ain Shams University
c. Pharmaceutical-Bioproducts, Mubarak City for Scientific Research and Technology Application, Alexandria.
d. Surgical Pathology Department, National Cancer Institute, Cairo University.
ABSTRACT
Background & Aim of Work: Kombucha(KT), a fermented blacktea, is known to have many beneficial properties, and few toxic effects. In the present study, the antioxidant valuable effect of KT has been investigated against experimentally induced hepatotoxicity.
Material and Methods: 120 female Western albino rats were divided into six groups treated with either: a control group; KT alone; diethyl nitrosamine (DEN) + carbon tetrachloride (CCl4); or KT + DEN + CCl4. The latter group was further divided into 3 subgroups according to KT administration; before, simultaneous, or posts DEN. Reduced glutathione, activities of glutathione reductase, superoxide dismutase, and total antioxidant capacity (TAC) were determined in liver homogenates, together with histopathological examination.
Results: KT did not cause any changes compared to controls. The DEN + CCl4 group showed elevations in all the studied parameters, except for TAC. Treatment with KT partially improved most of the investigated parameters, regardless of the mode of its administration. Histological examination confirmed the partial protective effects of KT.
Conclusion: The partial protective effect of KT against chemically induced liver injury could be through its antioxidant effect. Nevertheless, hepatic injury was not completely alleviated by KT.
Key wards: Kombucha tea, hepatotoxicity, antioxidants, detoxification, reduced glutathione, glutathione reductase, superoxide dismutase, and total antioxidant capacity.
* Mailing address:
INTRODUCTION
Disorders with persistent and chronic destruction of liver tissue, including chronic types of viral or alcoholic hepatitis, liver cirrhosis, and liver cancer, are leading causes of death worldwide (1). The human body constantly produces unstable molecules called oxidants, also commonly referred to as free radicals. To become stable, oxidants take electrons from other molecules and, during this process, damage cell proteins and genetic material. Antioxidants are substances that allow the human body to scavenge and seize oxidants. They are hypothesized to decrease liver injury and hepatocellular carcinoma risk by trapping free radicals and / or deactivating excited oxygen molecules, a by-product of many metabolic functions. The antioxidant defense system is altered in cancerous tissues and lead to disturbances in free radicals, which may be associated with increased risk of cancer. Administration of antioxidant may be of substantial value in treating cancer (2).
There has been a great deal of interest in the role of complementary and alternative medicines (CAM) for the treatment of various acute and chronic diseases (3). Accordingly, CAM is becoming popular among patients with liver disease (4). Tea showed many health promoting activities including chemo-preventive action during carcinogenesis due to the presence of antioxidative constituents, and was suggested to protect man against liver injury (5&6).
Kombucha tea is a highly efficient free radical scavenger and has potent antioxidant properties. It should be prepared with extreme care to avoid contamination with pathogenic bacteria (7). Kombucha tea has been proven to cure or protect from many different diseases including those of the liver (8) by detoxifying the xenobiotics and enhancing the metabolism that improves the defense capacity (9). Sai Ram et al. (10) attributed the protective effect of KT to be due to beneficial acids such as acetic, lactic, and glucuronic acids that can aid in healthy digestion and liver function. Some of these acids may combine with toxins in the digestive system, making those toxins more soluble for subsequent elimination from the body. It also contains vitamins B and C, which are believed to aid in prevention of different diseases.
Kombucha tea helps the liver work more efficiently and improves liver functions (11). It can remove the brown spots on the hands that are produced due to liver disorder. It can reduce liver disorders and increase the efficiency of the liver to detoxify xenobiotics. It can fight gallstones, high cholesterol, and multiple sclerosis (7).
In an experiment by Pauline et al. (12), hepatotoxicity was induced in animals by an acute oral dose of 1 gm/kg paracetamol. They determined the plasma levels of ALT, AST and malondialdehyde. Their results indicated that KT had no significant toxicity as revealed by various biochemical and histopathological parameters. It has also been found to decrease paracetamol induced hepatotoxicity significantly, indicating its hepato-protective effect. Similar results were reported by Chang et al. (8).
On the other hand, there is a great concern about the safety of KT, since serious health problems and toxic effects have been reported and attributed to drinking kombucha (13). There have been warnings of potential hepatotoxicity following the report of a man who developed a skin rash, hepatomegaly, and abnormal liver function tests after drinking the tea for one month (14). It is also, not recommended for pregnant or nursing mothers in large amounts because it is a fermented tea, and contains caffeine; however, it can be made with decaffeinated tea (15). In this respect, Srinivasan et al. (13) reported that KT may contain antibiotic substances and, theoretically, could cause antibiotic resistance. However, those who drink more than 4 ounces daily of kombucha tea frequently experience nausea, vomiting, and headaches, where there have been reports of allergic reactions, jaundice, and head and neck pain (1).
Likewise, Perron et al. (14) and Srinivasan et al. (13) reported that KT causes liver toxicity. There are case reports that suggest that improper preparations of KT might cause problems such as jaundice and liver inflammation.
AIM OF THE WORK
Due to the contradicting points of view discussed in the introduction, the present work was designed to study the antioxidative detoxifying effect of KT against chemically induced hepatic injury. These effects were determined by measuring changes in hepatic free radical scavenging compounds and enzymes such as reduced glutathione, superoxide dismutase, glutathione reductase, and the total antioxidant capacity. Liver enzymes were also determined to study the safety of KT on the liver. The biochemical results were backed by histopathological examination of liver tissues.
MATERIALS AND METHODS
I-Preparation of KT:
Kombucha colony was supplied from a commercial source at the USA (16). The tea was prepared according to the procedure described by Frank (15). In brief, the kombucha colony and 250 ml starter KT, prepared previously, were placed in a glass jar with 300 gm of sugar and five tea bags left for thirty minutes then removed. The volume was completed to one liter by chlorine free water. Kombucha prefers a temperature of around 22-27oC and out of direct sunlight. The fermenting container was covered with the cloth or paper and fastened with a rubber band. Rarely molds may form on the top of the culture that looks fuzzy like bread mold. Their color may be white, green, or black with a powdery appearance. If molds develop, it should be thrown all out and start over again with a completely new colony. After eight to ten days, the colony was harvested from the fermenting jar and placed into a glass pie-plate or container. Enough KT was poured in the container to cover the colony completely. The next batch of kombucha started right away. If needed to be stored for a while, it might be left in the brewing jar, at room temperature, in its own tea and covered with a cloth. The remaining liquid, which is the KT used in the present study, was poured into glass bottles, sealed and refrigerated.
II-Experimental animals
The work was conducted at the Cancer Biology Department, National Cancer Institute, Cairo University, and Mubark City for Scientific Research and Technologic Application. This study involved a total number of 120 female albino Western rats, weighing 80-90 gm each. All rats were fed a standard diet containing 20 % protein, 15 % corn oil, 55 % cornstarch, 5 % salt mixture and 5 % vitaminized starch. They were divided into the following six groups; each containing 20 rats divided in four cages.
1- Control group (G I): This group included 20 normal healthy untreated rats drinking tap water ad libitum. Animals were hosted for a period of 8 weeks.
2- KT group (G II): Rats were administered 10 % KT in their drinking water. They were hosted for a period of 8 weeks.
3- DEN + CCl4 (G III): Rats were injected ip. with a single dose of 200 mg DEN / kg body weight. Two weeks later, the rats were given a single oral dose of 2 ml CCl4 / kg body weight. The CCl4 was dissolved in corn oil 1: 1 (v:v) (17). They were hosted for a period of 8 weeks post DEN administration.
4- KT + DEN + CCl4 [a] (G IV): This group was designed to study the prophylactic effect of KT. Rats were administered 10 % KT in their drinking water for one month followed by ip. injection with 200 mg DEN / kg body weight. Two weeks later, the rats were given a single oral dose of 2 ml CCl4 / kg body weight. They were hosted for a period of 8 weeks post DEN administration.
5. KT + DEN + CCl4 [b] (G V): This group was designed to study the protective effect of KT. Rats were injected ip. with 200 mg DEN / kg body weight. They were administered 10 % KT in drinking at the same day. The 2 ml CCl4 / kg body weight were administered after 15 days. They were hosted for a period of 8 weeks post DEN administration.
6. KT + DEN + CCl4 [c] (G VI): This group was designed to study the therapeutic effect of KT. Rats were injected ip. with 200 mg DEN / kg body weight, followed by 2 ml CCl4 / kg body weight after 15 days. Three weeks later, rats were administered 10 % KT in their drinking water. They were hosted for a period of 8 weeks post DEN administration.
III-Parameters investigated:
1-Body weight: The changes in body weight for all rats were recorded weekly throughout the experimental period.
2- Biochemical parameters investigated in liver homogenate:
Rats were sacrificed by decapitation after an overnight fasting at the end of the experimental period. Liver was dissected, washed with saline, and dried between two filter papers. One gm from whole liver was excised and was homogenized in 10 ml saline and centrifuged for 15 min. The supernatant was separated and stored at -80 ˚C until used. The liver homogenate was subjected to the following investigations:
i. Determination of reduced Glutathione (GSH) was carried out enzymatically, according to the method proposed by Beutler et al. (18) using the kit supplied by Biodiagnostic Company (USA).
ii. Determination of glutathione reductase (GR) was carried out kinetically, according to the method proposed by Carlberg and Mannervik (19), using the kit supplied from Cayman Chemical Company, Ann Arbor (USA).
iii. Determination of superoxide dismutase (SOD) was carried out enzymatically, according to the method proposed by Nebot et al. (20), using kit supplied from Oxis Research (USA).
iv. Determination of the total antioxidant capacity (TAC) was carried out enzymatically, according to the method proposed by Beers and Sizer (21), using kit supplied from Immundiagnostik Company.
3- Biochemical parameters investigated in sera of rats:
Blood samples were collected and serum was separated, divided into aliquots; to avoid freezing and thawing, and stored at -80˚C for the estimation of following liver function tests:-
i. Determination of aspartate aminotransferase (AST) activity in serum was carried out spectro-photometric, using the kit supplied from Randox Laboratories LTD. (U.K) based on the method described by Reitman and Frankel (22).
ii. Determination of alanine aminotransferase (ALT) activity in serum was carried out spectro-photometric, using the kit supplied from Randox Laboratories LTD. (U.K) based on the method described by Reitman and Frankel (22).
iii. Determination alkaline phosphatase (ALP) activity in serum was carried out spectro-photometric, using the kit supplied from Cromatest Linear Chemicals SL. according to Luppa et al. (23).
iv.Determination of gamma glutamyl transferase (γ-GT) activity in serum was carried out kinetically, using kit supplied from Centronic Chemicals (GmbH, Germany) based on the method described by Szasz (24).
4- Histopathological examination:
Small sections of freshly excised rat liver, of all groups, were dissected and immediately fixed in 8% phosphate buffered formalin. After 24 h, the liver sections were dehydrated with successive dilutions of ethanol. The preserved tissue was embedded in paraffin blocks, which were sectioned at 5-µm thickness, stained with hematoxylin and eosin stain and then examined microscopically (25).
IV- Statistical analysis:
Results were statistically analyzed using the method of Saunders and Trapp (26), to determine the significance between the different investigated groups using SPSS (15). The ANOVA test was used for the comparison between the means of the different groups.
Results:
The present study revealed the following results:
I- Body weight
Figure (1) represents the percent of change in the mean body weight of rats in each group. An increase in the percent of change in the body weight for all groups is obvious. However; the rate of increase was not the same for all groups, since the normal control group G I showed the highest value, while G III group had the lowest one as compared with control group or groups administered KT; namely G II, G IV, G V and G VI.
II- Biochemical investigations:
The levels of reduced glutathione (GSH), as well as the activities of glutathione reductase (GR) and superoxide dismutase (SOD) were significantly elevated in G III, which is intoxicated with DEN and CCl4. However, they were all reduced in groups administered KT in addition to DEN and CCl4, but they did not retain their corresponding values in the control group or the group administered KT alone. On the other hand, G III exhibited a markedly reduced total antioxidant capacity. Administration of KT in addition to DEN and CCl4 significantly elevated the TAC to retain it around its control level (tables 1 and 2).
Regarding the liver enzymes, the aspartate aminotransferase (AST) was significantly increased in G III. Its activity was reduced in groups administered KT in addition to DEN and CCl4, especially in G V and G VI. The enzymatic activities of alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma glutamyl transferase (γ-GT) showed a more or less similar pattern of variation (tables 3 and 4).