Byoung Seob Ko

Central prolactin modulates insulin sensitivity and insulin secretion in diabetic rats.

Background: Prolactin secretion is self-regulating as it acts upon hypothalamic dopaminergic systems which inhibit prolactin release from the anterior pitutary. Circulating prolactin improves glucose homeostasis by increasing insulin action and secretion, but central prolactin effects on glucose homeostasis have not been examined. Here, we determined that chronic central infusion of prolactin modulates insulin resistance and β-cell function and mass in 90% of pancreatectomized diabetic male rats. Methods: Diabetic rats were divided into three groups according to the dose of intracerebroventricular infusion of prolactin during 4 weeks: (1) low-dose prolactin (Low-PRL; 0.1 µg/h), (2) high-dose prolactin (High-PRL; 1 µg/h) and (3) vehicle only (cerebrospinal fluid). Nondiabetic rats were centrally infused with the vehicle. Results: Chronic intracerebroventricular infusion of Low-PRL lowered body weight and epididymal fat pads by increasing hypothalamic dopamine levels that reduced serum prolactin levels and potentiated leptin signaling. However, High-PRL slightly exacerbated energy dysregulation, decreased hypothalamic dopamine levels, and elevated serum prolactin levels. Both dosages promoted β-cell mass but in a different manner: Low-PRL decreased β-cell apoptosis, whereas High-PRL increased its proliferation. However, only Low-PRL enhanced first-phase insulin secretion and improved insulin sensitivity at a hyperglycemic state in comparison to the control. Low-PRL also increased glucose infusion rates and decreased hepatic glucose output in hyperinsulinemic states, signifying an improvement in hepatic insulin sensitivity. However, High-PRL exacerbated hepatic insulin resistance compared with the control diabetic rats. Conclusions: In contrast to the exacerbation of insulin resistance caused by High-PRL, Low-PRL may improve energy and glucose metabolism by increasing hypothalamic dopamine levels in diabetic rats.

Monitoring and identification of Cynanchum wilfordii and Cynanchum auriculatum by using molecular markers and real-time polymerase chain reaction

Cynanchum wilfordii (Asclepiadaceae) is widely distributed throughout Korea, Japan, and China. Dried roots of this plant have been used as a tonic to promote renal function. Due to the morphological similarities of the dried roots of this plant to those of Cynanchum auriculatum, which is used as a substitute herbal medicine for C. wilfordii, distinguishing these two species is extremely difficult. The present study was conducted to develop molecular markers to distinguish C. wilfordii and C. auriculatum by using conventional polymerase chain reaction (PCR) and realtime PCR analyses. Comparative analysis based on the sequence of the trnL-trnF intergenic spacer revealed 4 base-pair variations, and the inter-individual sequences of the 2 species separately showed 100% homology. According to these results, the variations were divided into 2 groups. The 2 species were further distinguished using a sequence-characterized amplified region marker developed based on a randomly amplified polymorphic DNA-PCR product, and then a single nucleotide polymorphism marker was designed based on the trnL-trnF intergenic spacer for more efficient detection in real-time PCR. The results showed that speciesspecific molecular markers might allow accurate discrimination of C. wilfordii and C. auriculatum.

Discrimination of korean Rehmannia glutinosa from chinese Rehmannia glutinosa using sequence-characterized amplified region marker

Rehmanniae Radix, from the roots of Rehmannia glutinosa Libosch has been used in traditional herb medicine for the treatment of fever and strengthening liver function, among others. Information on the phylogenetic relationship is very limited in the region of its cultivation. It is very important to know the information of the close relatives of R. glutinosa Libosch and R. glutinosa Libosch. f. hueichingensis Hsiao, R. glutinosa produced in Wen County, Meng County, Bo’ai County, Qinyang County in Henan province, China. In this study, we examined the polymorphism analysis of Rehmanniae Radix originated from both Korea and China to compare the difference at the genomic DNA level. Results revealed that ITS and rps16 region sequences of R. glutinosa in Korea and R. glutinosa in China were correspond, while randomly amplified polymorphic DNA analysis showed a difference in UBC 301 primer. The specific primer designed was amplified at 334 bp for R. glutinosa originated from China. This primer (HRgF and HRgR) would be used efficiently to distinguish R. glutinosa from different sources.

Multiplex polymerase chain reaction (PCR) and fluorescence-based capillary electrophoresis for identification of deer species from antlers

Multiplex polymerase chain reaction (PCR) and fluorescence-based capillary electrophoresis (CE) of blood and tissue samples have been used to distinguish between deer species such as red deer, sika deer, wapiti and reindeer. We constructed 4 species-specific primers by using the D-loop of mitochondrial DNA and cytochrome b as an internal PCR control. The amplified species-specific product lengths of 199, 299, 245 and 375 bp for red deer, sika deer, wapiti subspecies and reindeer, respectively were detected from mitochondrial D-loop DNA fragments. The specificity was confirmed by analysis of blood and tissue samples of 4 deer antlers and 8 other samples of mammalian DNA (mouse, rat, pig, chicken, cat, cow, dog and human DNA). The specificity and accuracy of the multiple primers for identification were assessed at various concentrations and from mixed samples. In this study, we established a method for the identification of deer species using deer antlers. Key words: Deer antler, identification, mitochondrial DNA, multiplex polymerase chain reaction (PCR).

Phenols displaying tyrosinase inhibition from Humulus lupulus.

Abstract Tyrosinase is the rate-limiting enzyme for the production of melanin and other pigments via the oxidation of l-tyrosine. The methanol extract from Humulus lupulus showed potent inhibition against mushroom tyrosinase. The bioactivity-guided fractionation of this methanol extract resulted in the isolation of seven flavonoids (1–7), identified as xanthohumol (1), 4′-O-methylxanthohumol (2), xanthohumol C (3), flavokawain C (4), xanthoumol B (5), 6-prenylnaringenin (6) and isoxanthohumol (7). All isolated flavonoids (1–7) effectively inhibited the monophenolase (IC50s = 15.4–58.4 µM) and diphenolase (IC50s = 27.1–117.4 µM) activities of tyrosinase. Kinetic studies using Lineweaver–Burk and Dixon-plots revealed that chalcones (1–5) were competitive inhibitors, whereas flavanones (6 and 7) exhibited both mixed and non-competitive inhibitory characteristics. In conclusion, this study is the first to demonstrate that the phenolic phytochemicals of H. lupulus display potent inhibitory activities against tyrosinase.

Discrimination of Phellodendron amurense and P. chinense based on DNA analysis and the simultaneous analysis of alkaloids.

Phellodendri Cortex is the bark of the stems of Phellodendron amurense Ruprecht or P. chinense Schneider (Rutaceae), which is orginated from periderm. The internal transcribed spacer sequences of 20 originated plants and identified samples were analyzed. The result showed that the 99% of the base sequences of P. amurense were identical to that of P. chinense, but the differentiation of P. amurense and P. chinense was difficult. In addition, the ribulose-1, 5-bisphospate carboxylase large subunit (rbcL) intergenic spacer sequences of specific parts produced the same result. However, when the analysis was carried out by using the RAPD (randomly amplification polymorphism DNA) analysis method, which utilizes 48 randomly primers, it allowed us to confirm the polymorphism of P. amurense and P. chinense in 12 primers. A high-performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous quantitation of berberine, palmatine and jatrorrhizine in a traditional herbal drug, Phellodendri Cortex. The HPLC method was applied successfully to the quantification of three constituents in the extract of twenty Phellodendri Cortex. The results indicated that the established HPLC and RAPD methods are suitable for the quantitative analysis and the quality control multi-simultaneous discrimination in Phellodendri Cortex.

Effects of an Aqueous Extract of Dangguijagyagsan on Serum Lipid Levels and Blood Flow Improvement in Ovariectomized Rats

Dangguijagyagsan (DJS), a traditional herbal prescription, has long been used to treat menopause-related symptoms. We identified the cardioprotective effects of an aqueous extract of DJS using an ovariectomized (OVX) and ferric chloride- (FeCl-) induced carotid thrombosis rat model. Female Sprague-Dawley (SD) rats were ovariectomized or Sham-operated (Sham-control). The ovariectomized rats were divided into three groups: OVX with saline (OVX-control), aspirin 30 mg/kg/day (OVX-ASA), and DJS 100 mg/kg/day (OVX-DJS). The treatments were administered for 5 weeks. Then, blood samples were collected to analyze the serum lipid levels and platelet aggregation. The topical application of 40% FeCl3 induced intravascular thrombosis, which was used to test thrombotic occlusion and for histological examination. Body weight and the levels of total cholesterol (TC), triglyceride (TG), and LDL-cholesterol (LDL-C) increased in the OVX rats. These effects were reduced by ASA and DJS treatment. In addition, ASA and DJS treatment significantly inhibited platelet aggregation. These treatments also increased time to occlusion and decreased both thrombus size and the presence of collagen fibers in surrounding vessel walls compared with the Sham-control and OVX-control groups. These results suggest that DJS has beneficial effects in terms of preventing cardiovascular disease in menopausal woman because it can reduce the serum lipid levels and improve blood flow by inhibiting platelet aggregation and thrombus formation.

Tetragonia tetragonioides (Pall.) Kuntze Regulates Androgen Production in a Letrozole-Induced Polycystic Ovary Syndrome Model

Tetragonia tetragonioides (Pall.) Kuntze (TTK) is a medicinal plant traditionally used to treat various diseases such as diabetic, inflammatory, and female-related disorders. Polycystic ovary syndrome (PCOS) is a common endocrinological disorder in women of reproductive age, and hyperandrogenism is a prominent feature of PCOS resulting in anovulation and infertility. In this study, we investigated the effects of a TTK extract on androgen generation and regulation of steroidogenic enzymes in vitro and in vivo. Human adrenocortical NCI-H295R cells were used to assess the effects of TTK extract on production of dehydroepiandrosterone and testosterone, as well as the protein expression of steroidogenic enzymes. Further, a letrozole-induced PCOS rat model was used in vivo to assess whether dietary administration of TTK extract restores normal hormones and reduces PCOS symptoms. TTK extract significantly inhibited forskolin (FOR)-induced androgen production in NCI-H295R cells and serum luteinizing hormone, testosterone, and follicular cysts, but not estradiol, were reduced in letrozole-induced PCOS rats orally administered the TTK extract. In addition, TTK extract inhibits androgen biosynthesis through the ERK-CREB signaling pathway, which regulates CYP17A1 or HSD3B2 expression. TTK extract could be utilized for the prevention and treatment of hyperandrogenism and other types of PCOS.

Palmiwon attenuates hepatic lipid accumulation and hyperlipidemia in a menopausal rat model

ObjectiveWe examined the phytoestrogenic effects of palmiwon on breast carcinoma, lipid accumulation in methyl-&bgr;-cyclodextrin–induced HepG2 cells, and lipid-related diseases in a rat model of menopausal hyperlipidemia. MethodsE-Screen assay was used to screen for phytoestrogens, especially those with antiestrogenic activity, in MCF-7 cells. Oil Red O staining and intracellular cholesterol analyses were used to quantify cellular cholesterol levels. 3-Hydroxy-3-methyl glutaryl coenzyme A reductase assay was used to measure enzyme activity. The levels of phosphorylated adenosine monophosphate–activated protein kinases and products of genes involved in cholesterol synthesis were measured by Western blot analysis. Thirty rats were either ovariectomized or sham-operated and randomly assigned to four groups (n = 5)—Sham, OVX, OVX-SV, or OVX-PMW (50, 150, or 450 mg/kg) group—for 8 weeks. A number of targets associated with lipid-related diseases were examined to confirm the estrogenic effects of palmiwon. ResultsPalmiwon showed antiestrogenic activity in MCF-7 cells. Palmiwon decreased lipid accumulation, total cholesterol levels, and low-density lipoprotein/very-low-density lipoprotein levels in HepG2 cells. Moreover, palmiwon reversed the effects of methyl-&bgr;-cyclodextrin on cholesterol synthesis regulators and inhibited the activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase. Phosphorylation of adenosine monophosphate–activated protein kinase was stimulated by palmiwon. In ovariectomized rats, palmiwon reduced retroperitoneal and perirenal fat accumulation, serum lipids, atherogenic index, cardiac risk factor score, intima-media thickness, and nonalcoholic steatohepatitis scores. ConclusionsThese results indicate that palmiwon inhibits lipid accumulation without estrogenic activity in the breast. Therefore, palmiwon may have potential as a therapeutic agent for the treatment of hyperlipidemia in postmenopausal women.

Sagunja-Tang Improves Lipid Related Disease in a Postmenopausal Rat Model and HepG2 Cells

The present study was conducted to investigate the effect of Sagunja-tang on the lipid related disease in a rat model of menopausal hyperlipidemia and lipid accumulation in methyl-β-cyclodextrin-induced HepG2 cells. In in vivo study using menopausal hyperlipidemia rats, Sagunja-tang reduced retroperitoneal and perirenal fat, serum lipids, atherogenic index, cardiac risk factor, media thickness, and nonalcoholic steatohepatitis score, when compared to menopausal hyperlipidemia control rats. In HepG2 cells, Sagunja-tang significantly decreased the lipid accumulation, total cholesterol levels, and low-density/very-low-density lipoprotein levels. Moreover, Sagunja-tang reversed the methyl-β-cyclodextrin-induced decrease in the protein levels of critical molecule involved in cholesterol synthesis, sterol regulatory element binding protein-2, and low-density lipoprotein receptor and inhibited protein levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase as well as activity. Phosphorylation level of AMP-activated protein kinase was stimulated by Sagunja-tang. These results suggest that Sagunja-tang has effect on inhibiting hepatic lipid accumulation through regulation of cholesterol synthesis and AMPK activity in vitro. These observations support the idea that Sagunja-tang is bioavailable both in vivo and in vitro and could be developed as a preventive and therapeutic agent of hyperlipidemia in postmenopausal females.