• The Journal of Korean Medicine
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• v.31 no.6
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• pp.47-57
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• 2010

Objective: The present study investigated the effect of ethanol extract of Cynanchum wilfordii (ECW) on vascular relaxation and vascular inflammation in rat artery isolated from rats and anti-inflammatory activity in human aortic smooth muscle cells (HASMC). Methods: Vascular tone and guanosine 3',5'-cyclic monophosphate (cGMP) production were examined in rat artery isolated from Sprague Dawley rats, in the presence of ECW. HASMC were incubated with tumor necrosis factor-alpha (TNF-${\alpha}$) or Angiotensin II for 24 h. Matrix metalloproteinase (MMP)-2 and anti-oxidant activity of ECW was investigated by pretreatment with ECW in HASMC. Results: Cumulative treatment of ECW relaxed aortic smooth muscles of rats in a dose-dependent manner. ECW-induced vasorelaxation was significantly decreased by pretreatment of L-arginine methyl ester (L-NAME) or oxadiazolo-quinoxalinone (ODQ). Furthermore, ECW treatment of thoracic aorta significantly increased cGMP production. Incubation of ECW with ODQ or L-NAME markedly decreased ECW-induced cGMP production. ECW treatment dose-dependently suppressed TNF-${\alpha}$- or Angiotensin II-induced increase in matrix metalloproteinase-2 expression in HASMC. Also, ECW exhibited 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity in vitro and reduced TNF-${\alpha}$-induced increase in reactive oxygen species production in a dose-dependent manner. Conclusions: Taken together, the results suggest that ECW exerts vascular relaxation via NO/cGMP signaling pathway and decreases MMP-2 expression via anti-oxidant activity.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.11
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• pp.1570-1574
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• 2004

A flock of AA breed chickens were reared in peterstme brood-vait chamber and were provided with high energy pelleted feed. At 14 d of age, a total of 350 birds were randomly divided into 3 groups as follows: 100 birds were exposed to normal ambient temperature of 20$^{\circ}C$ for control group; 150 birds were exposed to lower ambient temperature of 11$^{\circ}C$ to induce ascites (treatment I); and another group of 100 birds were exposed to lower ambient temperature of 11$^{\circ}C$ and fed diet containing 1% L-arginine for ascitic prophylactic treatment (treatment II). Samples were collected from blood and abdominal fluid of chicken at 3, 4, 5, 6 and 7 wk of age subsequently, to analysis the contents of plasma endothelin (ET-1), angiotensin II (Ang II), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP). The results indicated that the contents of cAMP, cGMP, and Ang II in reatment I and ascitic broilers were higher than the corresponding control group (p<0.01, p<0.05), ET-1 of preascitic broilers were control group (p<0.05), while there was an insignificant difference with later ascitic broilers. The contents of cAMP and cGMP in treatment II were higher than the treatment I and control groups (p<0.01, p<0.05), whereas, the contents of Ang II were gradually decreased compared to the control group (p<0.05), the contents of ET-1 were insignificantly different. On further analysis, the increased plasma Ang II at low ambient temperature condition in broilers made endothelium cell secretion of increased ET-1, cAMP, cGMP and decreased NO. Therefore, low temperature accelerated ascites syndrome in broilers. Supplemently L-arginine can decrease ET-1, and increase cAMP and cGMP. It is concluded that cAMP mediated in broilers pulmonary hypertension syndrome.

• BMB Reports
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• v.40 no.5
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• pp.678-683
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• 2007

Extracts from the leaves of the Ginkgo biloba are becoming increasingly popular as a treatment that is claimed to reduce atherosclerosis, coronary artery disease, and thrombosis. In this study, the effect of ginkgolide B (GB) from Ginkgo biloba leaves in collagen (10 ${\mu}g/ml$)-stimulated platelet aggregation was investigated. It has been known that human platelets release matrix metallo-proteinase-9 (MMP-9), and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GB to form an MMP-9 (86-kDa) on gelatinolytic activities. And then, activated MMP-9 by GB dose-dependently inhibited platelet aggregation, intracellular $Ca^{2+}$ mobilization, and thromboxane $A_2$ ($TXA_2$) formation in collagen-stimulated platelets. Activated MMP-9 by GB directly affects down-regulations of cyclooxygenase-1 (COX-1) or $TXA_2$ synthase in a cell free system. In addition, activated MMP-9 significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have the anti-platelet function in resting and collagen-stimulated platelets. Therefore, we suggest that activated MMP-9 by GB may increase the intracellular cAMP and cGMP production, inhibit the intracellular $Ca^{2+}$ mobilization and $TXA_2$ production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that activated MMP-9 is a potent inhibitor of collagen-stimulated platelet aggregation. It may act a crucial role as a negative regulator during platelet activation.

• Journal of Dairy Science and Biotechnology
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• v.34 no.2
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• pp.117-135
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• 2016

The present study was performed to develop a functional raw food material from hydrolyzed whey protein powder (23%-GNANA) medication containing sialic acid as a marker compound that is naturally occurring at 7% concentration in GMP (glycomacropeptide). GMP is used worldwide in foodstuffs for babies and infants and is obtained from the milk protein as safe food. While the purpose of our detailed evaluation was aimed to assess preliminary NOAEL values for and above 2,000 mg/kg/day, a clinical dose allowance for 23%-GNANA (as per characteristic of a functional health product, a highly refined test substance of 23% (v/v) sialic acid combined in GMP), at the same time we also wanted to assess the safety of GMP hydrolyzate lacking sialic acid but with identical properties as GMP. Animal safety evaluation was conducted using 23%-GNANA as the test substance, produced from hydrolyzed whey protein powder (product name: HELICOBACTROL-23; provided by Medinutrol Inc. [Korea]; composed of 23% sialic acid and GMP protein) after isolating the sialic acid using enzymes approved as food additives, with GMP as a raw material, and subsequently increasing the content of xx up to 23% through 80% (v/v) ethanol soaking and concentrating, in accordance with GLP Guideline. The animal safety evaluation mentioned above was made on the basis of toxicity in SPF Sprague-Dawley female and male rats dosed with 10 mL of the test substance diluted to 0, 1,250, 2,500, and 5,000 mg/kg directly into their stomachs for 90 d. This was determined in terms of the general symptoms and animal viability, weight and amount of feed intake, eye examination, uracrasia tests, hematological and blood biochemical disorder tests, blood coagulation test, abnormal intestine weight, abnormalities during postmortem and histopathological examinations. Statistical significance was set at P<0.05. Based on the toxicity determination, a certain minor effect associated with the test substance was observed in male rats with no major effects of the tested substance, in comparison with the control group dosed with sterilized water. Nevertheless, the NOAEL value, evaluated as per toxicity criteria, was verified as 5,000 mg/kg/day (P<0.05). Similarly, for female rats, a certain minor effect associated with the test substance was observed in 5,000 mg/kg/day dosed group, with no major effect, yet the NOAEL value (as assessed as per toxicity criteria) was determined to be 5,000 mg/kg/day (P<0.05), which was the same as for male rats. Accordingly, the NOAEL values of the test substances for all female and male rats were finally verified as 5,000 mg/kg/day (P<0.05). In conclusion, it was determined that the 23%-GNANA test substance exceeds 2,000 mg/kg/day, the clinical allowance characteristic for functional health food, and was finally evaluated to cause no safety concerns when used as a raw material in functional health food production, which was the ultimate goal of the present study.

• Korean Journal of Pharmacognosy
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• v.37 no.2 s.145
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• pp.85-91
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• 2006

The present study was undertaken to evaluate the effect of evening primrose oil (EPO) on the male sexual functions. EPO (daily 0.5 ml/mouse) was orally intubated for 28 consecutive days to experimental ICR mice, and same vol. of vehicle to control mice. On the 14th and 28th experimental day, the testis weight, number of complete intromissions and mating, serum testosterone and cGMP levels, prostaglandin leveIs of penile corpus cavernosum smooth muscle cells, and NO-productive activity of endothelial cells were determined. The weight of body and testis, the number of complete intromissions during the 3hour period were somewhat increased in EPO treated mice than those of control. The number of sperm-positive females and testosterone level in serum were increased in experimental groups. The serum cGMP level was significantly increased but the NO production of ionomycin-stimulated HUVEC cells was not affected when EPO was added into cultures. These results suggest that oral administration of EPO enhanced the sexual functions of male mice, and EPO could be developed as a tonic improving sexual functions.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.6
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• pp.337-341
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• 2006

The present study was designed to investigate the protein expression of nitric oxide synthase (NOS) and guanylyl cyclase (GC) activity in ischemia/perfusion (I/R) renal injury in rats. Renal I/R injury was experimentally induced by clamping the both renal pedicle for 40 min in Sprague-Dawley male rats. The renal expression of NOS isoforms was determined by Western blot analysis, and the activity of guanylyl cyclase was determined by the amount of guanosine 3', 5'-cyclic monophosphate (cGMP) formed in response to sodium nitroprusside (SNP), NO donor. I/R injury resulted in renal failure associated with decreased urine osmolality. The expression of inducible NOS (iNOS) was increased in I/R injury rats compared with controls, while endothelial NOS (eNOS) and neuronal NOS (nNOS) expression was decreased. The urinary excretion of NO metabolites was decreased in I/R injury rats. The cGMP production provoked by SNP was decreased in the papilla, but not in glomerulus. These results indicate an altered regulation of NOS expression and guanylyl cyclase activity in I/R-induced nephropathy.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.4
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• pp.343-351
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• 2001

This study was undertaken to investigate the mechanism of lipopolysaccharide (LPS) and nitric oxide (NO) as a regulator of vascular smooth muscle cell (VSMC) proliferation. VSMC was primarily cultured from rat aorta and confirmed by the immunocytochemistry with anti-smooth muscle myosin antibody. The number of viable VSMCs were counted, and lactate dehydrogenase (LDH) activity was measured to assess the degree of cell death. Concentrations of nitrite in the culture medium were measured as an indicator of NO production. LPS was introduced into the medium to induce the inducible nitric oxide synthase (iNOS) in VSMC, and Western blot for iNOS protein and RT-PCR for iNOS mRNA were performed to confirm the presence of iNOS. Inhibitors of iNOS and soluble guanylate cyclase (sGC), sodium nitroprusside (SNP) and L-arginine were employed to observe the action of LPS on the iNOS-NO-cGMP signalling pathway. LPS and SNP decreased number of VSMCs and increased the nitrite concentration in the culture medium, but there was no significant change in LDH activity. A cell permeable cGMP derivative, 8-Bromo-cGMP, decreased the number of VSMCs with no significant change in LDH activity. L-arginine, an NO substrate, alone tended to reduce cell count without affecting nitrite concentration or LDH level. Aminoguanidine, an iNOS specific inhibitor, inhibited LPS-induced reduction of cell numbers and reduced the nitrite concentration in the culture medium. LY 83583, a guanylate cyclase inhibitor, suppressed the inhibitory actions of LPS and SNP on VSMC proliferation. LPS increased amounts of iNOS protein and iNOS mRNA in a concentration-dependent manner. These results suggest that LPS inhibits the VSMC proliferation via production of NO by inducing iNOS gene expression. The cGMP which is produced by subsequent activation of guanylate cyclase would be a major mediator in the inhibitory action of iNOS-NO signalling on VSMC proliferation.

• Journal of Yeungnam Medical Science
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• v.14 no.2
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• pp.337-349
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• 1997

This study was undertaken to examine the intensity of involvement of inducible nitric oxide synthase (iNOS) and cyclic GMP signal transduction pathway as one of the mechanisms of vaso-relaxative action of bacterial lipopolysaccharide (LPS) on the canine femoral artery strips. Canine femoral arteries were isolated and spiral strips of 10 mm long and 2 mm wide were made in the Tyrode solution of $0-4^{\circ}C$. The strips were prepared for isometric myography in Biancani's isolated muscle chamber containing 1 ml of Tyrode solution, which was maintained with pH 7.4 by aeration with 95% $O_2$/5% $CO_2$ at $37^{\circ}C$ and nitric oxide (NO) production was measured simulltaneously with isolated nitric oxide meter. LPS induced NO production, suppressed the phenylephrine (PE) induced contraction and enhanced the acetylcholine (ACh) induced relaxation. $N^G$-nitro-L-arginine methyl ester (L-NAME), an NOS inhibitor, methylene blue, a guanylyl cyclase inhibitor, potentiated PE induced contraction and suppressed ACh induced relaxation on the LPS treated strips. The inhibitory potency of methylene blue for LPS induced vascular hyporesponsiveness was stronger than that of L-NAME. These results suggest that in canine femoral artery, both iNOS and cyclic GMP signal trnasduction pathway are related with LPS induced vascular hyporeponsiveness, but in minor with iNOS and in major with cyclic GMP signal trnasduction pathway.

• Journal of Physiology & Pathology in Korean Medicine
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• v.29 no.6
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• pp.492-497
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• 2015

In the present study, vasorelaxant effect of the extract of seeds of Oenothera odorata (SOO) and its possible mechanism responsible for this effect were examined in vascular tissues isolated from rats. Changes in vascular tension, 3',5'-cyclic monophosphate (cGMP) levels were measured in thoracic aorta rings from rats. Methanol extract of seeds of Oenothera odorata relaxed endothelium-intact thoracic aorta in a dose-dependent manner. A dose-dependent vascular relaxation was also revealed by treatment of ethylacetate, n-butanol, and H₂O (aqua extract of seeds of Oenothera odorata , ASOO) extracts partitioned from methanol, but not by hexane extract. However, the vascular relaxation induced by ASOO were abolished by removal of endothelium of aortic tissues. Pretreatment of the endothelium-intact vascular tissues with N^G-nitro-L-arginine methyl ester (L-NAME) or ¹H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1- one (ODQ) significantly inhibited vascular relaxation induced by ASOO. Moreover, incubation of endothelium-intact aortic rings with ASOO increased the production of cGMP. However, ASOO-induced increases in cGMP production were blocked by pretreatment with L-NAME or ODQ. The vasorelaxant effect of ASOO was attenuated by tetraethylammonium (TEA), 4-aminopyridine, and glibenclamide attenuated. On the other hand, the ASOO-induced vasorelaxation was not blocked by verapamil, and diltiazem. Taken together, the present study demonstrates that ASOO dilate vascular smooth muscle via endothelium-dependent NO-cGMP signaling pathway, which may be closely related with the function of K⁺ channels.

• Journal of Physiology & Pathology in Korean Medicine
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• v.31 no.1
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• pp.52-58
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• 2017

This study was aimed to examine relaxing effects of Acanthopanacis cortex(AC) through nitric oxide(NO) production and phosphodiesterase type 5(PDE-5) inhibition in corpus cavernosum. In order to define the relaxation effects of AC extract, rabbit corpus cavernous tissues were prepared in $2{\times}2{\times}8mm$ sized strip. AC extract ($0.01-3.0mg/m{\ell}$) were treated in contracted strips induced by phenylephrine(PE) and $N{\omega}$-nitro-L-arginine (L-NNA) was treated before AC extract-treated. And calcium chloride($Ca^{2+}$) 1 mM was infused into precontracted strips after pretreatment of AC extract in $Ca^{2+}-free$ krebs-ringer solution. When AC extract was applied to human umbilical vein endothelial cell(HUVEC), cell viability was measured by MTT assay, and NO concentration was measured by Griess reagent system. Ratio of smooth muscles to collagen fibers and eNOS, PDE-5 positive reaction were measured by histochemical and immunohistochemical process on mice corpus cavernosum. AC extract significantly affected relaxion of the cavernous strips, and the pretreatment of L-NNA inhibited AC extract-induced relaxation. Contraction induced by the addition of $Ca^{2+}$ was inhibited by treatment with the AC extract in $Ca^{2+}-free$ solution. In AC group, NO concentration, ratio of smooth muscle to collagen fibers, and eNOS positive reaction were increased, PDE-5 positive reaction was decreased compared to PE group. As a result of the above experiment, it was thought that AC extract inhibits the inflow of extracellular $Ca^{2+}$ by activating cGMP through the increase of eNOS / NO and the decrease of PDE-5 which inhibits cGMP activity, in the corpus cavernosum.