Biological Evaluation of Aqueous Herbal Extracts and Stirred Yoghurt Filtrate Mixture Against Alloxan-Induced Oxidative Stress and Diabetes in Rats

MATERIALS AND METHODS

Milk samples: Fresh cow milk samples (3.10% fat) were purchased from healthy animals in private farms (Saudi Arabia).

Yoghurt cultures: Starter cultures of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus were obtained from Chr. Hansen`s Lab., Copenhagen Denemark.

Medicinal plant samples: Plants used in this study are showed in Table 1. Fenugreek, greater burdock, goat`s rue, colocynth, chicory and lupine were collected from local

Table 1:	English, botanical and family name of plants used in the experiments

market, Qassim, Saudi Arabia. This study was conducted during the period extending between March, 2006 and September, 2007.

Preparation of traditional yoghurt and its filtrate: Traditional yoghurt milk was prepared according the method described by Tamime and Robinson (1999) and Al-Wabel et al. (2007) with slight modifications. Yoghurt containing probiotic strains (1.0x10⁹) of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus strains were stirred in electric blender and filtered through filter paper (Whatman No. 1) and stored at 4 °C for mixing with aqueous extracts of medicinal plants (fenugreek, greater burdock, goat`s rue, colocynth, chicory and lupine) to be used in the experiments for feeding.

Preparation of aqueous extracts of fenugreek, greater burdock, goat`s rue, colocynth, chicory and lupine: Aqueous extraction of fenugreek, greater burdock, goat`s rue, colocynth and lupine were carried out according to methods described by Melendeza and Caprilesa (2006). The extracts were then filtered and preserved in sterile dark bottles (500 mL) in a cool environment (4 °C) until further mixing with yoghurt filtrate before feeding.

Animals: Thirty-six Male albino Wistar rats weighting about 100-120 ± 19.8 g were randomly divided into three-test groups (each containing 18 rats). Animals were placed in cages and were given initially the basal diet.

Basal diet: The composition of basal diets used in this study is as follows: milk protein (12%), sucrose (5%), fat (10%), vitamin mixtures (1%), salt mixtures (4%), fiber (4%) and starch (64%).

Treatments: The first group (G1) was kept as untreated, control group fed on the basal diet only (negative control).

The other two groups (G2, G3) were both treated with alloxan as inducer of diabetes. Group (G2) was fed on the basal diet only and used as a positive control.

The third group (G3) was fed on basal diet + aqueous herbal extracts mixed with stirred yoghurt filtrate in drinking bottles.

The feeding experiment was continued for 6 weeks and at the end of the experiment, animals were anesthetized by exposure to an atmosphere of 100% diethyl ether and then they were killed by decapitation. Blood was collected in dry tube and left to clot, then centrifuged at 3000 rpm for 10 min. Serum was separated and stored frozen at -20 °C for subsequent analyses.

Biochemical analysis: At the end of the experimental period, animals were anesthetized by exposure to an atmosphere of 100% diethyl ether then they were killed by decapitation. Blood samples were taken into plain tubes. Serum was harvested after centrifugation (3000 rpm) for 15 min frozen at -20 °C for subsequent analyses.

Blood glucose concentration was determined by the Haemo-Glukotest 20-800-R and measurements read with a Reflolux test (Boehringer-Mannheim) (Brodrick et al., 1987).

Determination of Glutathione-S-Transferase (GST) in serum: The enzyme activity of Glutathione-S-Transferase (GST) was determined according to Habig etal. (1974).

Determination of ALT and AST activities: ALT and AST activities were determined according to the method described by Reitman and Frankel (1957).

Statistical analysis: Mean and standard Error of the obtained data from each different experimental group were calculated and analyzed.

Chemical compositions analysis were carried out according to the official method AOAC (2000) and sensory evaluation was adopted from NASA (1999).

RESULTS AND DISCUSSION

The results of this study indicated that feeding aqueous herbal extracts of six medicinal plants-fenugreek, greater burdock, goat`s rue, colocynth, chicory and lupine mixed with stirred yoghurt filtrate against alloxan-induced oxidative stress and diabetes in rats resulted in beneficial effects. In this study the chemical composition of the tested yoghurt was investigated. It has been found that the total solid, carbohydrate, fat, protein and ash values were 13.2, 5.5, 3, 1, 3.80 and 0.85%, respectively. Also, the sensory evaluation properties were found to have good scores and were acceptable for consumers.

The blood glucose level in diabetic rats was found to be 246 mg/100 mL. Treatment with herbal extracts mixed

Table 2:	Comparison of serum glutathione-s-transferase activities between control and Herbal extracts with yoghurt filtrate

Table 3:	Comparison of serum AST activity between control and herbal extracts with yoghurt filtrate (U L-1)

Table 4:	Comparison of serum ALT activity between control and Herbal extracts and yoghurt filtrate (U L-1)

with yoghurt filtrate lowered serum glucose level in diabetic rats from 246 to 150 mg/100 mL indicating hypoglycemic effect.

The activities of the liver enzymes (AST and ALT) were decreased and the activity of serum GST increased due to the treatment (Table 2-4).

Diabetic patients were found to exhibit abnormal antioxidant status due to auto oxidation of glucose and excess glycosylated proteins (Ceriello et al., 1991; Mak et al., 1996). OS in diabetes leads to tissue damage and complications including retinopathy, nephropathy and coronary heart disease (Jennings et al., 1991; Lyons, 1991).

Oxidative stress is an important factor that may induces liver fibrosis and represents a key feature of hepatitis induced by various conditions (Poli et al., 1987; Wasmuth et al., 2003). In this study, treatment with herbal plants and yogurt markedly reduced the activities of AST and ALT and increased the activity of GST after diabetes induction by alloxan. The herbal plants used in this study included burdock root and chemicals in this root were found to help the body to regulate the blood sugar and/or the release of insulin from the pancreas (Silver and Krantz, 1931; Swanston-Flatt et al., 1990). Inulin is a helpful type of sugar, beneficial to diabetics and have hypoglycemic effect because it does not trigger quick insulin production from the pancreas. This root contains numerous other constituents (chemicals) which either directly or indirectly affect the blood sugar level (Brinker, 2001; Swanston-Flat et al., 1990; Lapinina and Sisoeva, 1964; Silver and Krantz, 1931).

The phytochemical galegin present in Galega officinalis lowers the blood sugar level and this explains why goat`s rue is used in treatment of diabetes mellitus (Bailey and Day, 1989). Studies have shown that extracts from goat`s rue inhibit the transport of glucose into cells. These studies do not recommend the use of goat`s rue for self medication of diabetes because diabetes is a serious condition and it is difficult to standardize the strength of the active component levels (UKPDS Group, 1989).

Chicory (Cichorium intybus) is mainly cultivated for its roots that contain high amounts of inulin (a frupolymer) (Westerdijk, 2000). The chicory components were found to positively affect various physiological functions. They affect the bioavailability of minerals, reduce the risk of intestinal infectious diseases, cardiovascular diseases, non-insulin-dependant diabetes, obesity and osteoporosis.

Glutathione depletion in liver and kidneys is seen during severe oxidative stress in goats induced by lead administration (Mousa et al., 2002) and in diabetes. The depletion of GSH content also may lower GST enzyme, because GSH is required as a substrate for GST activity (Rathore et al., 2000) Glutathione is a tripeptide synthesized in the body from free amino acids, cysteine, glutamic acid and glycine. In many cancer epidemiological studies, it has been found that the major milk proteins caseins and whey have protective effect against cancer (McIntosh, 1993). In this connection we can assume that milk proteins plus microbial proteins that are synthesized by probiotics can enrich amino acids pool in the body and consequently increase glutathione, main bio-antioxidant and other protein precursors and synthesis. Similar assumption were proposed by McIntosh (1993).

It can be concluded that feeding diabetic rats aqueous herbal extracts and stirred yoghurt filtrate mixture have beneficial effects as manifested by lowering of blood glucose level and enhancement of the antioxidant status. This protocol can be used as a new nutritional and therapeutic products that may be administered orally and replace the laborious administration of insulin.