the most common underlying mechanism of bioactive peptides has been studied in angiotensin - converting enzyme ( ace ) inhibition [ 24 ] .
although the ace inhibitor effects of bioactive peptides are most often studied , the vasorelaxant activity has also shown an important evidence for lowering blood pressure .
it is well established that dietary protein found in soy protein , milk casein , and sweet potato   and its hydrolysates are capable of reducing blood pressure and modulating vascular activity .
recently , there are many reports that dietary - protein - derived peptides lower blood pressure and modulate vasodilation from several sources including egg white , soy protein , and -casein .
moreover , silk fibroin , a core protein of silk fiber , has been shown to have potential hypotensive and antioxidant actions by fibroin hydrolysate .
silk - fibroin - derived dipeptides , such as glycine - tyrosine , also produce an antihypertensive effect through angiotensin - converting enzyme ( ace ) inhibition .
sericin is a glue protein consisting of 20%30% of the total cocoon weight which is synthesized in the middle gland of the bombyx mori silkworm .
sericin is composed of 18 amino acids and contains protein in a wide range of molecular weights from 10 to over 300  kda .
sericin has shown numerous bioactivities , such as antioxidant , antitumor , antiproliferation , and anticholesterolemic properties .
however , sericin and its hydrolysates have not been reported for vasorelaxation and blood pressure lowering .
therefore , in the present study , we investigated the possible mechanism involved in the blood pressure - lowering and vasomodulating effects of sericin - derived oligopeptides .
the sericin - rich protein solution was filtered through a cheese cloth to separate the extracted cocoons from the liquid part .
the sericin solution obtained was subjected to enzymatic hydrolysis by protease ( from bacillus species , 16  unit / g , ec no .
one ml of protease enzyme solution ( 0.01  unit / ml protease enzyme in 0.036  m cacl2 solution at a 1  :  1 volumetric ration ) was added to 300  ml of the obtained sericin solution and incubated under shaking conditions at 37c for 1  h. the solution was then heated to 90c for 15  min to stop the enzymatic activity and cooled to room temperature before centrifugation at 9500  g for 15  min at 4c to separate the solid portions .
oligopeptides with a molecular weight lower than 5  kda were separated from larger oligopeptides by ultra membrane filtration using a hollow fiber membrane with 5000  mwco ( molecular weight cutoff ) ( ge healthcare bio - sciences ab , uppsala , sweden ) .
the oligopeptides solution obtained was freeze - dried and kept in a sealed container at room temperature until use .
male wistar rats ( 200250  g ) were obtained from the national laboratory animal center , mahidol university , salaya , nakornpathom , thailand .
all animals were housed under a 12:12  h light - dark cycle conditions , with maintained temperature ( 24  1c ) .
the experiment protocol was approved by the animal ethics committee in accordance with the guide for the care and use of laboratory animals prepared by chiang mai university .
the rats were anesthetized by intraperitoneal injection of sodium pentobarbital ( 50  mg / kg  bw ) .
the femoral artery was cannulated with polyethylene tubing 50 ( clay - adams pe-50 ) filled with 100  iu of heparin / ml connected to a pressure transducer to measure blood pressure .
the blood pressure signal was amplified and converted to a digital signal by a bridge amplifier coupled with powerlab ( adinstruments , sydney , australia ) .
systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , and heart rate ( hr ) were recorded with labchart 7 software ( adinstruments , sydney , australia ) .
oligopeptides were administered via a cannula inserted into the femoral vein with similar tubing to facilitate the intravenous injection of oligopeptides ( 0.1  g1000  g / kg  bw ) .
animals were allowed to equilibrate at least 20  min before the administration of any drugs .
arterial pressure was allowed to return to the baseline level before the subsequent injections were conducted .  
the rats were anesthetized by intraperitoneal injection of sodium pentobarbital ( 50  mg / kg  bw ) . the thoracic aorta was immediately removed and cleaned from the connective tissues and fat .
aortic rings ( 35  mm ) were obtained and immediately immersed into ice - cold kreb 's solution ( composition in mm : nacl 122 ; kcl 5 ; ( hepes ) 10 ; kh2po4 0.5 ; nah2po4 0.5 ; mgcl2 1 ; glucose 11 ; cacl2 1.8 ; ph 7.4 ) until use .
the endothelium - denuded rings were obtained by mechanically removing the endothelial layer by gentle rubbing around the internal vascular surface .
the aortic rings were mounted between a pair of platinum wires in an organ bath containing krebs ' solution , maintained at 37c and continuously bubbled with air .
a resting tension of 1 gram was applied to each tissue and allowed to equilibrate at least 1  h. the endothelium integrity was verified by relaxation to ach ( 1  m ) in rings precontracted by pe ( 1  m ) .
the vascular endothelium was considered intact when aortic rings were relaxed 90% of the pe - induced precontractions , whereas the endothelium - denuded ring was confirmed with an absence of vasorelaxation .
changes in isometric tension were recorded and analyzed through a force transducer ( iworx systems , inc .
, nh , usa ) , coupled with bridge amplifier ( adinstruments , sydney , australia ) , powerlab ( adinstruments , sydney , australia ) , and signal virtualization by labchart 7 software ( adinstruments , sydney , australia ) .
vasorelaxant effects of oligopeptides were investigated in both endothelium - intact and endothelium - denuded aortic rings .
after the ring was preequilibrated , the aortic ring was precontracted with pe ( 1  m ) or kcl ( 80  mm ) until the stability of tension was developed and followed by cumulative exposure to oligopeptides at the concentration of 0.00110  mg / ml .
the extent of relaxation was expressed as the percentage of pe- or kcl - induced contraction . to investigate the mechanism responsible for oligopeptides - induced vasorelaxation
, the endothelium - intact rings were precontracted with pe ( 1  m ) for 30  min after being exposed to either l - name ( 100  m ) , an inhibitor of the no synthase ( nos ) , indomethacin ( 10  m ) , an inhibitor of the cyclooxygenase ( cox ) , or l - name plus indomethacin . and ,
then , vasorelaxation was carried out by the cumulative exposure to oligopeptides at the concentrations of 0.00110  mg / ml . to examine the role of k channels involvement in vasorelaxation , the endothelium - denuded ring was used for this determination by preincubation with one of the following k channel blockers : tetraethylammonium , a nonselective inhibitor of k channels ( tea , 5  mm ) , 4-aminopyridine , an inhibitor of voltage - operated k channel ( 4-ap , 1  mm ) , glibenclamide , an inhibitor of k channels activated by adenosine triphosphate ( 10  m ) , and bacl2 ( 1  mm ) for 30  min before pe ( 1  m ) precontraction .
then , the cumulative concentration response of oligopeptides at the concentrations of 0.00110  mg / ml was directly added . to evaluate whether oligopeptides possessed vasorelaxation via the sgc activation , the endothelium - intact ring was incubated with 1h - oxadiazolo[4,3-]quinoxalin-1-one , a selective inhibitor of the guanylyl cyclase enzyme ( odq , 1  m ) for 30 min before contraction with pe ( 1  m ) treatment .
the ability of vasorelaxation was compared in absence ( control ) and presence of odq . to investigate the effects of oligopeptides on ca channel ,
the endothelium - denuded aortic ring was placed under ca - free krebs ' solution for 20  min and then exposed for an additional 10  min to k ( 80  mm ) , ca - free solution for complete smooth muscle cell depolarization to open a voltage - operated ca channel ( vocc ) .
the cumulative concentration - response curve of cacl2 ( ranging from 10  m to 10  mm ) was obtained .
after the maximal response was performed , the rings were washed out and replaced with ca - free solution for 20  min .
the ca - free 80  mm k was reexposed to flowing with preincubation of either oligopeptides ( 1 , 3 , 5 , and 10  mg / ml ) or nifedipine , l - type ca channel blocker ( 1  m ) for 20  min . the maximal contraction obtained with the control concentration response curve to cacl2 was taken as 100% , and all values were calculated as a percentage of the maximal response . to investigate the effect of oligopeptides on pe- or caffeine - sensitive intracellular calcium stores , endothelium - denuded rings were bathed in ca - free kreb 's solution containing 1  mm egta after incubation with 80  mm kcl for successful ca loading into sr .
the transient contractions were obtained in aortic rings by pe ( 1  m ) or caffeine ( 20  mm ) in ca - free solution before and after being incubated with oligopeptides ( 1 , 5 , and 10  mg / ml ) .
the results were expressed as percentage of the response induced by pe or caffeine alone .
statistical analysis was performed using student 's t - test or one - way analysis of variance ( anova ) followed by tukey 's post hoc test .
concentration - response curves were plotted , and experimental data were obtained by using nonlinear curves fit program ( graphpad prism 5 ) .
the baselines of sbp , dbp , and hr were 122.8  1.64  mmhg , 107  3.74  mmhg , and 366  2.67  bpm , respectively .
intravenous administration of oligopeptides dose dependently decreased sbp and dbp in rats ( figures 1(a ) and 1(b ) ) .
the hypotensive response in each dose of oligopeptides was completely recovered to the baseline within a few minutes . in addition
, oligopeptides at all doses did not show any obvious effect on hr ( figure 1(c ) ) .
oligopeptides significantly relaxed pe- and 80  mm kcl - precontracted aortic rings in a concentration - dependent manner . however , oligopeptides completely relaxed in pe- ( emax = 95.46  1.86% ) induced contractions in the aortic rings and partially relaxed in kcl- ( 80  mm , emax = 55.22  7.56% ) induced contractions ( figures 2(a ) and 2(b ) ) .
the endothelium - denuded ring significantly decreased the vasodilator effects only on the pe precontraction ( figure 2(a ) ) , while , for 80  mm kcl , precontraction had no effects ( figure 2(b ) ) .
no and prostacyclin are known to be vasorelaxing mediators derived from endothelium . in endothelium - intact preparation , oligopeptides relaxed the pe- ( 1  m ) induced contractions with an ec50 value of 1.86  0.16  mg / ml ( figure 3 ) . in the presence of l - name and l - name plus indomethacin , the relaxant effect of oligopeptides against pe- ( 1  m ) induced contraction was markedly attenuated with an ec50 value of 13.49  0.18 and 8.71  0.15  mg / ml , respectively ( figure 3 ) .
by contrast , treatment of the aortic ring with indomethacin showed no modification in endothelium - intact rings ( figure 3 ) . to verify the role of k channel in oligopeptides - induced vasorelaxation , the endothelium - denuded ring was preincubated with different potassium channel inhibitors : tetraethylammonium ( 5  mm ) , 4-aminopyridine ( 1  mm ) , glibenclamide ( 10  m ) , or bacl2 ( 1  mm ) before the ring was contracted with pe .
all inhibitors did not alter the concentration - response curve for oligopeptides as shown in figures 4(a ) and 4(b ) .
however , the maximal relaxant activity of oligopeptides which were preincubated with tetraethylammonium was markedly reduced ( emax = 54.82  2.63% ) compared to the control ( emax = 83.74  5.61% ) .
odq ( 1  m ) significantly abolished the oligopeptides - induced vasorelaxation in the endothelium - intact ring ( figure 5 ) .
it markedly decreased both the potency ( ec50 = 11.22  0.91  mg / ml ) and maximal relaxation ( emax = 12.09  4.63% ) when compared to untreated control values ( ec50  1.86  0.79  mg / ml , and emax = 95.03  1.86% ) . the concentration - response curves of cacl2 ( 10  m10  mm ) were performed in the 80  mm kcl ca - free solution .
the percentage of maximal response ( emax ) of cacl2 alone ( 100% ) was attenuated in the presence of oligopeptides ( 1 , 3 , 5 , and 10  mg / ml ) in a concentration - dependent manner ( 83.02  6.78% , 54.00  1.98% , 39.52  2.28% , and 31.61  2.6% , resp . ) , while ec50 values were similar ( figure 6 ) .
in addition , nifedipine ( 1  m ) also abolished the contraction of cacl2 .
preincubation of endothelium - denuded rings with oligopeptides at the concentrations of 1 , 5 , and 10  mg / ml dose dependently decreased the transient contractions induced by pe ( 10  m ) in ca - free krebs ' solution containing egta ( 1  mm ) ( figure 7(a ) ) .
by contrast , oligopeptides produced no significant effect on the transient contractions induced by caffeine ( 20  mm ) in a similar condition ( figure 7(b ) ) .
sericin is a protein derived from the silk cocoon that appears as waste in silk processing .
sericin is a mixture of polypeptides of molecular mass varying between 10 and 300  kda   and has high serine contents ( 30%33% ) . in the past decade , various biological activities regarding sericin have been reported in gastric injuries   and protection against tumorigenesis , antioxidation , inhibition of tyrosinase , alcohol - induced liver , and uv - induced keratinocyte apoptosis . in the present study ,
sericin was hydrolyzed , and it generated the bioactive peptides with low - molecular- ( 5  kda ) sized peptides .
the sericin - derived oligopeptides exerted vasorelaxant activities in rats , which explain their hypotensive effects .
the reduction of blood pressure was transient , and it recovered to baseline level in a few minutes by dose - dependent intravenous administration of oligopeptides .
although the reduction of blood pressure occurred , the level of blood pressure was still within normotensive limits , whereas heart rate was unaffected .
oligopeptides induced a concentration - dependent vasorelaxation on pe - induced precontractions in endothelium - intact and -denuded aortic rings , and this relaxation was attenuated by endothelium denudation .
these results suggested that oligopeptides acted to cause vasorelaxation in endothelium - dependent aortic rings .
the vascular endothelial cells pivotally acted in vascular homeostasis by modulating the vascular smooth muscle tone .
thus , indomethacin , a cyclooxygenase inhibitor , and l - name , an inhibitor of no synthase , were employed to investigate the involvement of prostacyclin and no in vasodilation effect .
the results showed that oligopeptides - induced vasorelaxation was markedly suppressed by l - name , but not by indomethacin , suggesting the participation of no in the vasorelaxation effect of oligopeptides .
furthermore , l - name plus indomethacin revealed no further inhibition than that observed with l - name alone .
this showed that no is a powerful relaxant mediator involvement in oligopeptides - induced vasorelaxation .  
it is well known that the no / sgc pathway is one of the most common potential mechanisms that induce vascular smooth muscle relaxation through activation of guanylyl cyclase , leading to the accumulation of cyclic gmp that inhibits ca influx .
odq , a soluble guanyly cyclase inhibitor , was used to confirm the relationship of the vasorelaxant response of oligopeptides .
this finding showed the significant inhibitory effects of odq in oligopeptides - induced endothelium - dependent vasorelaxation , which confirmed that the vasorelaxation of the aorta elicited by oligopeptides was mediated in the endothelium - dependent
however , the vasorelaxation effect of oligopeptides was still observed in aortic endothelium - denuded rings , suggesting that oligopeptides have a direct effect on vascular smooth muscle cells .
the opening of k channels or ca channel blocker in the vascular smooth muscle cells provides an important mechanism to dilate arteries .
the vasorelaxant effect of oligopeptides was partially inhibited by the tea ( ca - activated k channel blocker ) .
other k blockers , glibenclamide ( atp - sensitive k channel blocker ) , 4-ap ( voltage - activated k channel blocker ) , and bacl2 ( inwardly rectifying k channel blocker ) had no effects , suggesting that the relaxant response of oligopeptides was involved in the role of k channel opening of ca - activated k channel in vascular smooth muscle cells .
influx of extracellular ca through voltage and/or receptor - operated calcium channels ( voccs and/or roccs ) plays an important role in vascular smooth muscle contraction .
we note that oligopeptides induced vasorelaxation in aortic rings precontracted with kcl or phenylephrine . from this result , it can be concluded that oligopeptides induced vasorelaxation via different pathways .
it is well known that kcl induces smooth muscle contraction through the activation of voltage - dependent calcium channels and subsequent release of calcium from sarcoplasmic reticulum , whereas phenylephrine - induced vasoconstriction is mediated by the stimulation of g - proteins coupled to alpha - adrenoceptors .
we then investigated the mechanism for the vasodilator action of oligopeptides that could directly inhibit the ca influx in the vascular smooth muscle cells .
oligopeptides significantly reduced the contractile responseinduced by cacl2 in the endothelium - denuded ring , in concentration - dependent manner under a depolarizing solution .
the contraction was abolished by nifedipine , a typical l - type voltage - operated calcium channel blocker , confirming the involvement of l - type voltage - operated calcium channels in the contractile response .
the concentration - response curve of cacl2 decreased in emax , while the ec50 values were unchanged , suggesting that oligopeptides could act as a calcium channel blocker that interferes with ca influx through l - type ca channel of aorta smooth muscle membrane .
the influences of oligopeptides in ca released from intracellular stores were sensitive to phenylephrine , and caffeine was also determined .
the oligopeptides markedly decreased the contractions induced by phenylephrine , which stimulates ip3-dependent ca release from the intracellular store .
by contrast , caffeine - induced contractions , which release ca from intracellular stores by ip3-independence , were not altered .
thus , it seems likely that the vascular effects of oligopeptides involved a reduction of ip3-dependent ca releases form sr sensitive to phenylephrine [ 27 , 28 ] .   our results demonstrated that bioactive oligopeptides ( 5  kda ) fractions obtained from silk sericin lower blood pressure by a direct effect on both endothelium and vascular smooth muscle leading to vasodilation .
results from this study have also supported us to further identify and characterize bioactive peptides .
this study demonstrated the hypotensive effect and vasorelaxant effect of silk sericin - derived oligopeptides on isolated rat aorta and the possible mechanisms .
the results suggested that oligopeptides have a dose - dependent relaxing effect on the isolated rat aorta .
the relaxing effect of oligopeptides is mediated through ca antagonism and the no / sgc / cgmp pathway , which possibly explains the fall in bp .
these findings provide scientific evidence supporting the therapeutic uses of sericin - derived oligopeptides as vascular modulators .
 sericin - derived oligopeptides obtained from silk cocoons were investigated for the in vivo hypotensive effect and investigated for the underlying mechanism involved in vasodilation in isolated rat thoracic aorta . in normotensive anesthetized rats , oligopeptides induced an immediate and transient hypotensive activity . in rat aortic rings , oligopeptides induced a concentration - dependent vasorelaxation in vessels precontracted with both kcl and phenylephrine ( pe ) with endothelium - intact or endothelium - denuded rings . in endothelium - intact rings , pretreatment with n-nitro - l - arginine methyl ester hydrochloride ( l - name , 100  m ) , an inhibitor of the no synthase ( nos ) or 1h-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one ( odq , 1  m ) , a selective inhibitor of the guanylyl cyclase enzyme , significantly reduced the relaxant effect of oligopeptides . 
 however , indomethacin , an inhibitor of the cyclooxygenase , had no effect on oligopeptides - induced relaxation . 
 in addition , pretreatment with tetraethylammonium ( tea , 5  mm ) reduced the maximal relaxant effect induced by oligopeptides . by contrast , relaxation was not affected by 4-aminopyridine ( 4-ap , 1  mm ) , glibenclamide ( 10  m ) , or barium chloride ( bacl2 , 1  mm ) . in depolarization 
 ca2 + -free solution , oligopeptides inhibited calcium chloride- ( cacl2- ) induced contraction in endothelium - denuded rings in a concentration - dependent manner . 
 nevertheless , oligopeptides attenuated transient contractions in ca2 + -free medium containing egta ( 1  mm ) induced by 1  m pe , but they were not affected by 20  mm caffeine . it is obvious that potent vasodilation effect of oligopeptides is mediated through both the endothelium and the vascular smooth muscle . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions