Expression of Aspergillus nidulans phy gene in Nicotiana benthamiana produces active phytase with broad specificities.

A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.

Antioxidant effect of dietary supplement Withania somnifera L. reduce blood glucose levels in alloxan-induced diabetic rats.

The phenolic compounds and flavonoids were determined from the extracts of Withania somnifera root (WSREt) and leaf (WSLEt). The WSREt has 28.26 mg/g total phenolic compounds and 17.32 mg/g flavonoids, whereas WSLEt has 5.4 mg/g total phenolic compounds and 5.1 mg/g flavonoids. The WSREt, WSLEt and glibenclamide were orally administered daily to diabetic rats for 8 weeks. After the treatment, the levels of urine sugar, blood glucose, liver glycogen, and antioxidants like vitamin C and E in plasma and superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and reduced glutathione (GSH) in liver, kidney and heart were determined. Diabetic rats showed a significant (p < 0.05) elevation in glucose and TBARS and a significant (p < 0.05) reduction in glycogen, vitamin C and E, SOD, CAT, GPx, GST, and GSH levels when compared to normal control rats. Administration of WSREt, WSLEt and glibenclamide to diabetic rats restored the levels to normal. In the light of aforesaid facts, it is suggested that the presence of phenolic compounds including flavonoids in W. somnifera root and leaf extracts and their antioxidant activity may play a vital role in reduction of blood glucose level in alloxan-induced diabetic rats.

Hypoglycaemic and hypolipidaemic effects of Withania somnifera root and leaf extracts on alloxan-induced diabetic rats.

Withania somnifera is an important medicinal plant, which is used in traditional medicine to cure many diseases. Flavonoids were determined in the extracts of W. somnifera root (WSREt) and leaf (WSLEt). The amounts of total flavonoids found in WSREt and WSLEt were 530 and 520 mg/100 g dry weight (DW), respectively. Hypoglycaemic and hypolipidaemic effects of WSREt and WSLEt were also investigated in alloxan-induced diabetic rats. WSREt and WSLEt and the standard drug glibenclamide were orally administered daily to diabetic rats for eight weeks. After the treatment period, urine sugar, blood glucose, haemoglobin (Hb), glycosylated haemoglobin (HbA1C), liver glycogen, serum and tissues lipids, serum and tissues proteins, liver glucose-6-phosphatase (G6P) and serum enzymes like aspartate transaminase (AST), alanine transaminase (ALT), acid phosphatase (ACP) and alkaline phosphatase (ALP) levels were determined. The levels of urine sugar, blood glucose, HbA1C, G6P, AST, ALT, ACP, ALP, serum lipids except high density lipoprotein-bound cholesterol (HDL-c) and tissues like liver, kidney and heart lipids were significantly (p < 0.05) increased, however Hb, total protein, albumin, albumin:globulin (A:G) ratio, tissues protein and glycogen were significantly (p < 0.05) decreased in alloxan-induced diabetic rats. Treatment of the diabetic rats with WSREt, WSLEt and glibenclamide restored the changes of the above parameters to their normal level after eight weeks of treatment, indicating that WSREt and WSLEt possess hypoglycaemic and hypolipidaemic activities in alloxan-induced diabetes mellitus (DM) rats.

Determination of mineral content in methanolic safflower (Carthamus tinctorius L.) seed extract and its effect on osteoblast markers.

Safflower (Carthamus tinctorius L.) seeds are used as a folk medicine to enhance bone formation or to prevent osteoporosis in Korea. Therefore, the methanolic extract of safflower seeds (MESS) containing high mineral content, such as calcium (Ca), potassium (K) and phosphorous (P), was evaluated for the role on osteoblast (Ob) markers of Sprague-Dawley rats. In serum of 3 to 11 weeks (wks) old rats, both osteocalcin (OC) content and bone-specific alkaline phosphatase (B-ALP) activity increased to their maximum levels in 4-7 wks. Hence, 3 wks old rats were selected for 8 wks oral treatment of MESS, resulted in the significant increase of Ob markers in serum such as OC content (4-8 wks), B-ALP activity (1-2 wks) and insulin-like growth factor I (IGF-I) level (1 wk), and the growth parameter such as the length of femur (2-8 wks) and tibia (4 wks). On the basis of Pearson's correlation coefficient, there were a moderate correlation between OC and B-ALP at 8 wks, a low correlation between OC and IGF-I at 1, 4 and 8 wks, a moderate correlation between OC and femur length at 1, 2 and 8 wks, and a moderate correlations between OC and tibia length at 1 and 8 wks of MESS-treated groups. The result reveals that the changes of OC correlated at low to moderate level with the changes of B-ALP activity, IGF-I content and femur and tibia length in the MESS-treatment period. On the other hand, there were a strong correlation between IGF-I and femur length at 2 wks and moderate correlation between IGF-I and tibia length at 1, 2 and 8 wks of MESS-treated groups. Therefore, the effect of MESS on bone formation likely appears to be mediated by IGF-I at the early stage of treatment.

Comparison between ethanolic and aqueous extracts from Chinese juniper berries for hypoglycaemic and hypolipidemic effects in alloxan-induced diabetic rats.

AIM OF THE STUDY: Hypoglycaemic and hypolipidemic properties of the ethanolic and aqueous extracts, respectively, from Chinese juniper (Juniperus chinensis L.) berries were investigated in alloxan-induced diabetic rats. MATERIALS AND METHODS: After oral administration of each extract singly or repeatedly to alloxan-induced diabetic rats, the blood glucose, glutamate-pyruvate transferase (GPT), glutamate-oxaloacetate transaminase (GOT), total cholesterol (TC) and triglyceride (TG) levels were assayed. RESULTS: The blood glucose levels after a single oral administration of the ethanolic extract significantly reduced in a time-dependent manner, which is much faster and more than that of glibenclamide. The blood glucose levels of alloxan-induced diabetic rats treated with the ethanolic extract were reduced to 94, 81%, 66%, 45% and 40% at 1, 3, 5, 7 and 9h, respectively (p<0.05), while the aqueous extract had no effect at all. Repeated oral administration of the ethanolic extract also effectively reduced the GPT value to 58% of the diabetic rats, but slightly reduced the GOT value to 87% of the diabetic rats (p<0.05). On the other hand, the repeated oral administration of aqueous extract effectively reduced the GOT value to 43% of the diabetic rats, without affecting the GPT level. Effects of both extracts on the TC and TG levels were different. There was no significant difference in the TC and TG levels between diabetic control and diabetic groups when repeatedly administered orally with ethanolic extract. On the other hand, the aqueous extract brought down the TC value to 57% and the TG value to 37% of the diabetic control rats (p<0.05). CONCLUSIONS: The results suggested that the ethanolic extract of Chinese juniper berries possesses a potential hypoglycaemic effect while the aqueous extract has a potential hypolipidemic effect.