Hypoglycemic effect of Hibiscus rosa sinensis L. flower extract in Alloxan induced hyperglycemic rats --------------------------------------------------------
A Research Paper Presented to the Faculty of Daao !edical "chool Foundation# $nc. %ollege of !edicine# !edical "chool Drie# &a'ada# Daao %ity ((((((((((((((( (((((((((((((((((((((( (((((((( ( $n Partial Fulfillment of the Re)uirements $n Pharmacology ((((((((((((((( ((((((((((((((((((((((( ((((((((
"ubmitted by*
Nadera, Milliza Jane Nasiluan, Joriel Neco Nawal, Norhaiah Naparan, Fiel Ninal, Kryscel Czarinah Nuval, Clarence Ong, April Joy Orongan, Rachel Pacatang, Christine Pacatang, Jenelle Palaca, eo Paolo Pa!a, "vonne Panto#an, Marie Parada, $rvin %antos, Jose Rigel +roup , !ed &
%hapter $ntroduction
&ac'ground o( the %tudy People who are diagnosed with having ia)etes type * can achieve and !aintain a nor!al )lood sugar level through diet and e+ercise )ut so!e, despite the change in li(estyle, still need (urther interventions rug therapy is an e((ective !ean o( lowering the )lood sugar level granted that the patient is co!pliant One o( the drugs prescri)ed (irst when patient is diagnosed with dia)etes type * is Met(or!in -.lucophage, .lu!etza/ 0his drug i!proves the sensitivity o( the )ody tissue to insulin so that the )ody !ay ta'e in insulin !ore e((ectively Another drug with the si!ilar action as Met(or!in is 0hiazolidinediones )ut this drug is lin'ed to serious side e((ects such as weight gain, increased ris' o( heart (ailure and (ractures %ul(onylureas and Meglitinides act )y helping the )ody secrete !ore insulin )ut unli'e the (or!er, the drug action o( the latter is !uch (aster, they do not stay in the )ody very long and they have decrease ris' o( producing co!plications Possi)le side e((ects o( %ul(onylureas are low )lood sugar and a nd weight gain As (or the Meglitinides, side e((ects include low )lood sugar levels PP12 inhi)itors and .3P14 receptor agonists are )eing used also to lower down )lood sugar levels )ut are not as e((e e((ecti ctive ve %.3 %.30* inhi inhi)i )ito tors rs are are the the newes newestt dia) dia)et etes es drug drug in the the !ar' !ar'et et 0hey 0hey prev prevent ent rea)sorption o( glucose in the )lood )y the 'idneys and (acilitating e+cretion o( the glucose throug through h the urine urine Anoth Another er e((ect e((ective ive therapy therapy )ut is 5uite 5uite invasiv invasivee is $nsuli $nsulin n therapy therapy 0his 0his intervention is used as the last resort to control )lood sugar levels rug interventions are popular !ediu!s nowadays to lower down )lood sugar levels )ut )e(ore the discovery o( hypoglycae!ic drugs, insulin in#ections, and other arti(icial !ethods
%hapter $ntroduction
&ac'ground o( the %tudy People who are diagnosed with having ia)etes type * can achieve and !aintain a nor!al )lood sugar level through diet and e+ercise )ut so!e, despite the change in li(estyle, still need (urther interventions rug therapy is an e((ective !ean o( lowering the )lood sugar level granted that the patient is co!pliant One o( the drugs prescri)ed (irst when patient is diagnosed with dia)etes type * is Met(or!in -.lucophage, .lu!etza/ 0his drug i!proves the sensitivity o( the )ody tissue to insulin so that the )ody !ay ta'e in insulin !ore e((ectively Another drug with the si!ilar action as Met(or!in is 0hiazolidinediones )ut this drug is lin'ed to serious side e((ects such as weight gain, increased ris' o( heart (ailure and (ractures %ul(onylureas and Meglitinides act )y helping the )ody secrete !ore insulin )ut unli'e the (or!er, the drug action o( the latter is !uch (aster, they do not stay in the )ody very long and they have decrease ris' o( producing co!plications Possi)le side e((ects o( %ul(onylureas are low )lood sugar and a nd weight gain As (or the Meglitinides, side e((ects include low )lood sugar levels PP12 inhi)itors and .3P14 receptor agonists are )eing used also to lower down )lood sugar levels )ut are not as e((e e((ecti ctive ve %.3 %.30* inhi inhi)i )ito tors rs are are the the newes newestt dia) dia)et etes es drug drug in the the !ar' !ar'et et 0hey 0hey prev prevent ent rea)sorption o( glucose in the )lood )y the 'idneys and (acilitating e+cretion o( the glucose throug through h the urine urine Anoth Another er e((ect e((ective ive therapy therapy )ut is 5uite 5uite invasiv invasivee is $nsuli $nsulin n therapy therapy 0his 0his intervention is used as the last resort to control )lood sugar levels rug interventions are popular !ediu!s nowadays to lower down )lood sugar levels )ut )e(ore the discovery o( hypoglycae!ic drugs, insulin in#ections, and other arti(icial !ethods
used used to to lower lower down down )lood )lood glucos glucose, e, healers healers in the past past -and -and so!e so!e stil stilll prac practic ticee it it
today/ today/
relied relied
on the use o( her)s $n the !odern ti!es such as today, a lot o( !edicinal plants were !ade su)#ect (or e+peri!ents with the goal o( discovering the hidden )ene(its they possess A!ong the possi)le )ene(its, researchers were relentless to discover what plants have hypoglycae!ic e((ects Hibiscus rosa sinensis or sinensis or also co!!only 'nown here in the Philippines as 6.u!a!ela7 have a wide range o( )ene(icial e((ects and included with its e((ects is hypoglycae!ia 0his study ai!s to !easure the hypoglycae!ic e((ect o( Hibiscus o( Hibiscus rosa sinensi. sinensi. O)#ectives o( the study 0he study ai!s to deter!ine the hypoglyce!ic h ypoglyce!ic e((ect o( 8i)iscus rosa sinensis (lower e+tract containing (lavonoid to the )lood sugar o( la)oratory induced dia)etes al)ino !ice
%peci(ically, the study ai!s9 4 0o e+tract (lavonoid (ro! the 8i)iscus rosa sinensis (lower * 0o deter!ine i( the (lavonoid o( 8i)iscus rosa sinensis (lower has hypoglyce!ic e((ect : 0o deter!ine deter!ine the )aseline )lood sugar o( the la) induced dia)etic !ice 2 0o deter!ine deter!ine the )lood sugar o( the !ice a(ter the e+peri!ent using the (lavonoid and a hypoglyce!ic drug ; 0o deter!ine i( there is signi(icant di((erence )etween the hypoglyce!ic e((ect o( (lavonoid and the hypoglyce!ic drug
Hypothesis
0here is a signi(icant relationship )etween the gu!a!ela plant e+tract to the )lood glucose levels o( a rat
/ull Hypothesis
0here is no signi(icant relationship )etween the gu!a!ela plant e+tract to the )lood glucose levels o( a rat "ignificance of the "tudy
0his research is signi(icant to the (ollowing9
%ociety 0his study ai!s to provide the society with a possi)le solution to dia)etes that is
natural, widely availa)le and has no to+ic e((ect Researchers. 0his study allows the researcher to understand and e+perience the phases o( !a'ing !edicine Allowing the! to apply their 'nowledge in phar!acology and use their
0
'nowledge in developing a naturally occurring cure (or dia)etes Future Researchers 0his study will )e a good start (or the 5uest (or a natural cure (or dia)etes that could )e used to the ne+t phase o( drug develop!ent
"cope and Limitations
0his study was li!ited within the school la)oratory o( M%F 0he su)#ects are al)ino rats which were weighed and !easured prior to the e+peri!ent Only the (lower o( the gu!a!ela will )e used in the study 0he study will (ocused on the e((ect o( gu!a!ela e+tract in lowering the su)#ect
Flavonoid
upta'e in peripheral tissues, regulation o( the e+pression o( the rate1li!iting enzy!es involve in car)ohydrate !eta)olis! pathway and acting as insulin secretagogues or !i!etics, pro)a)ly, )y in(luencing the pleiotropic !echanis!s o( insulin signaling -Cazarolli, *==>/ %everal studies have proven the e((ect o( (lavonoids on )lood glucose levels in !ice A study )y ?hou in *==@ concluded that (lavonoids e+tracted (ro! lotus should )e evaluated as a candidate (or (uture studies on dia)etes !ellitus 0he e((ect o( (lavonoids hesperidin and naringin was investigated to signi(icantly increase the gluco'inase !RNA level in type1* dia)etic !ice -Jung, *==;/
%3/%2P16AL FRA!243R5 $ndependent 7ariable .u!a!ela Flower +tract
Dependent 7ariable &lood glucose levels
%hapter Reiew of Related Literature Hibiscus rosa-sinensis Linn.
%ommon /ame* .u!a!ela, China Rose &otanical /ame* Hibiscus rosa-sinensis L.
Kingdo!9 Plantae %u)'ingdo!9 Tracheobionta – (Vascular plants) %uper division9 Spermatophyta – (Seed plants) ivision9 Magnoliophyta – (Flowering plants)
Class9 Magnoliopsida – (icotyledons) %u)class9 illeniidae Order9 Mal!ales Fa!ily9 Mal!aceae .enus9 Hibiscus %pecies9 Hibiscus rosa"sinensis
Plant Description
Hibiscus rosa"sinensis are native to tropical and su)tropical regions such as Asia Bith its wide ranging colors, it is used as an orna!ental plant grown in al!ost all houses i((erent parts o( the plant have )een studied these include the roots, leaves and (lowers 0he roots are cylindrical !easuring appro+i!ately ;14;c! in length and *c! in dia!eter, with a sweet and !ucilaginous taste 0he leaves are also !ucilaginous, shaped as ovate or ovate1lanceolate 0he (lowers are penta!erous with corolla consisting o( (ive petals, usually red in color and a)out : inches long -Ku!ar, *=4*/ Dariations in color are widespread
%onstituents
0he edi)le portion o( the (lower -E4E/ was reported to have the (ollowing nutrient co!position -per 4== g/9 !oisture >@>, nitrogen ==E2, (at =:E, crude (i)er 4;E, calciu! 2=2!g, phosphorous *EE>!g, iron 4E@!g, thia!in ==:4!g, ri)o(lavin ==2>!g, niacin =E4!g, and ascor)ic acid 24E!g Petals o( Hibiscus rosa sinensis were reported to contain 5uercetin1:1di1O1&11 glucoside
5uercetin1:1G1di1O1&11glucoside
5uercetin1:1O1&11sophorotrioside
and
'ae!p(erol and 'ae!p(erol1:1O1&11glucoside 0he !a#or anthrocyanin contained in the red (lowers was cyaniding1:1sophoroside Red1petalled varieties were (ound to have !ore nu!)er o( anthrocyanin )ands co!pared to that o)served in yellow Hyellow orange varieties 0he varieties in the di((erent colored groups di((ered in the 5uantitative distri)ution o( anthrocyanins, leucoanthrocyanins, (lavonol and carotenoids Flavonoid aglycones (ound in the (lowers -per g (resh tissues/ included 5uercetin G !g, and cyaniding :E !g 0he (lowers were also reported to contain the (ollowing (lavones9 5uercetin1:,;1 diglucoside 5uercetin1:,G1diglucoside cyaniding1:,;1diglucoside, and cyaniding1:1sophoroside (ro! the deep yellow and white (lowers and (ro! ivoery white (lowers is 'ae!p(erol1:1 +ylosylglucoside Five co!pounds were e+tracted (ro! the chloro(or! e+tract o( the (lower, n1nonacosa1 4:1one, n1triacontane, n1dotetracontane, n1nonacosan121ol14>1one, and n1hentriacontan121one1 4=1ol and (ive co!pounds were isolated (ro! the hydroalcoholic (lower e+tract, n1docosane, henicos1441ene1@1one, stig!ast1;1ene1:&, 2a1diol, stig!ast1;1ene1:&1)enozylo+y14*&1ol, and n1 pentacos121en1:1one14>, *:1diol 0he (ollowing che!icals were isolated (ro! the ethanol e+tract o( the (lowers9 he+adecanoic acid, he+anedioic acid and s5ualene Polyphenolic co!pound isolated (ro! the (lowers
included
5uercetin1G1O1galactoside
'ae!p(erol1G1O14E1O1p1hydro+y)enzoyl1&11
glucosyl1-41IE/1&11glucopyranoside, and scutellarein1E1O1a131rha!nopyranoside1>1C1&11 glucopyranoside 0he (lowers were reported to contain c yclopeptide al'aloids as well -3i!/ 3eaves and ste!s contain 1sitosterol, stig!asterol, tara+eryl acetate and three cyclopropane co!pounds and their derivatives Flowers contain cyanidin diglucoside, (lavonoids and vita!ins, thia!ine, ri)o(lavin, niacin and ascor)ic acid -.hani, *==:/ uercetin1:1
diglucoside, :,G1diglucoside, cyanidin1 :,;1diglucoside and cyanidin1:1sophoroside1;1 glucoside have )een isolated (ro! deep yellow (lowers all a)ove co!pounds and 'ae!p(erol1:1 +ylosylglucoside have )een isolated (ro! ovary white (lowers -Rastogi L Mehrotra, 4@@:/ 0he phytoche!ical analysis o( 8i)iscus rosasinensis leaves e+tract has a positive response (or the presence o( al'aloids, (lavonoids, tannins, saponins, steroids, and terpenoids Phytoche!icals are naturally occurring )iologically active che!ical co!pounds in plants Phytoche!icals are 'nown to have protective and disease preventing properties particularly (ree radical !ediated diseases such as dia)etes, cancer etc Certain phytoche!icals are al!ost structurally identical to insulin and act as an insulin !i!ic7 that helps in the re!edy o( dia)etes Most plants with antidia)etic properties have )een (ound to contain secondary !eta)olites such as glycosides, al'aloids and (lavonoids:: $t has )een shown that !any plants e((icient antio+idant properties owing to their phenolic constituents $t has )een reported that 8i)iscus rosasinensis leaves contain )ioactive co!pouds such as anthocyanins, and (lavonoids al'aloids and vita!ins 8ence, co!pounds which can scavenge the e+cess o( (ree radicals (or!ed or that inhi)it their production are o( wide therapeutic value 0he presence o( )iologically active ingredients in the e+tract !ay account (or the phar!acological activity -Anandhi, *=4:/
6ses
$t is considered as an e!ollient, e!!enagogue, anodyne, e+pectorant, re(rigerant and can act as anti1in(ectious, antispas!odic
Flaonoid
anthel!intic,
anti1in(la!!atory,
diuretic,
antipyretic,
hypotensive,
and
Flavonoid/ %everal studies have proven the e((ect o( (lavonoids on )lood glucose levels in !ice A study )y ?hou in *==@ concluded that (lavonoids e+tracted (ro! lotus should )e evaluated as a candidate (or (uture studies on dia)etes !ellitus 0he e((ect o( (lavonoids hesperidin and naringin was investigated to signi(icantly increase the gluco'inase !RNA level in type1* dia)etic !ice -Jung, *==;/ Diabetes !ellitus
ia)etes !ellitus is a group o( !eta)olic diseases characterized )y h yperglyce!ia resulting (ro! de(ects in insulin secretion, insulin action, or )oth 0he chronic h yperglyce!ia o( dia)etes is associated with long1ter! da!age, dys(unction, and (ailure o( various organs, especially the eyes, 'idneys, nerves, heart, and )lood vessels -A!erican ia)etes Association, *==2/
%riteria for the Diagnosis of Diabetes !ellitus
+libenclamide !echanism of Action
0he drug wor's )y )inding to and activating the A0P1sensitive potassiu! channels -KA0P/ inhi)itory regulatory su)unit sul(onylurea receptor 4 -%R4/ -%errano1Martin et al, *==E/ in pancreatic )eta cells 0his inhi)ition causes cell !e!)rane depolarization, opening voltage1dependent calciu! channels 0his results in an increase in intracellular calciu! in the )eta cell and su)se5uent sti!ulation o( insulin release $ts action in dia)etic patients is there(ore dependent upon the presence o( (unctioning 1cells in the islets o( 3angerhans
A(ter a cere)ral ische!ic insult, the )loodH)rain )arrier is )ro'en and gli)encla!ide can reach the central nervous syste! .li)encla!ide has )een shown to )ind !ore e((iciently to the ische!ic he!isphere Moreover, under ische!ic conditions %R4, the regulatory su)unit o( the KA0P1 and the NCCa1A0P1channels, is e+pressed in neurons, astrocytes, oligodendrocytes, endothelial cells -%i!ard et al, *=4*/ and ) y reactive !icroglia -Ortega et al, *=4*/
Dose# Route# 8 !ode of Administration
#ral route$ %mg&'g o body weight For su)stances )eing tested (or sa(ety, oral dosing !i!ics the !ost co!!only used !ode o( ad!inistration o( su)stances to hu!ans Bhen placing su)stances directly into the !outh, it is i!portant to ensure that ta)lets or gelatin capsules containing test !aterial are placed (ar )ac' in the !outh and that the ani!al swallows, to ensure receipt o( the (ull dose 0he nu!)er and size o( capsules or ta)lets ad!inistered should )e proportional to the size o( the ani!al )eing dosed,
to !ini!ize regurgitation .avage -esophageal or gastric/ is o(ten used in research settings, instead o( !i+ing su)stances in water or (ood, to ensure precise and accurate dosing o( ani!als sing the s!allest volu!e possi)le is reco!!ended (or the oral route o( ad!inistration, opti!ally ; !3'g (or all species -&rown et al, *===/ Related "tudies
$n a study )y %achdewa A et al -*==4/, *;= !g'g Hibiscus rosa sinensis alcoholic lea( e+tract ad!inistered orally everyday (or seven days showed signi(icant i!prove!ents in the study ani!als< a)ility to use the e+ternal glucose load, with an average o( :: )lood glucose lowering e((ect $n another study -%achdewa, *===/, the sa!e ad!inistration signi(icantly i!prove glucose tolerance o( rats $n an article pu)lished in an $ndian national newspaper, Narendran -*=4:/ states that there is an antidia)etic properties o( hi)iscus rosa sinensis 0he epart!ent o( &ioche!istry o( the niversity o( Kerala induced dia)etes in rats using a drug 0he rats were given e+tract o( the (lower petals and o)served 0he e((ect o( the petal e+tract was co!pared with the e((ect o( a standard drug, !et(or!in, used (or controlling dia)etes $t was (ound that not only was hi)iscus e+tract e((ective as an anti1dia)etic agent )ut the overall e((ect was !uch )etter than !et(or!in treat!ent 0he histopathological analysis o( the hearts o( the ani!als also showed that the petal e+tract had a protective e((ect Non1o)ese dia)etic !ice were rando!ly divided into E groups and were su)#ected to the e((ects o( 8i)iscus rosa sinensis leaves on the )lood glucose 0he e+peri!ent was conducted (or (our wee's %eru! glucose levels were esti!ated using a gluco!eter every wee' in every group 0he result showed decrease in )lood glucose levels in the group that used the e+tract o( 8i)iscus rosa sinensis -Mo5)el, F,et al, *=44/
A study conducted )y %undarra#an et al in *=44 ai!ed to deter!ine the antidia)etic e((ect o( 8i)iscus canna)inus lea( e+tracts on Al)ino wistar rats o( !ale se+ weighing *==1*;= g!s %treptozotocin was in#ected intra1peritonially to induce the increase in )lood glucose levels 0he e+peri!ental design were co!posed o( 2 groups, each group contained E !ice .roup 4 was the negative control .roup * was the dia)etic control group
.roup : was
co!posed o( dia)etic rats receiving !ethanolic e+tract o( 8i)iscus canna)inus .roup 2 contained dia)etic reats ad!inistered with .li)encla!ide &lood e+traction was done on the or)ital sinus o( the eye on the ;th day, 4;th day and *=th day post induction &lood glucose was !easured )y .O1PO 'it !ethod 0he statistical tools used were ANODA (ollowed )y unnet
0he statistical tool used was %tudentQs t1test in order to
deter!ine signi(icance )etween the groups A p ==; was considered to )e signi(icant A study done )y Mandade L
%reenivas in *=44 ai!ed to deter!ine the antidia)etic
e((ects o( the thanolic +tract o( 8i)iscus rosasinensis 3 on %treptozotocin1$nduced ia)etic Rats Possi)le Morphologic Changes in the 3iver and Kidney were also o)served 0he aerial
part o( 8i)iscus rosa sinensis was used in creating the a5ueous1ethanol e+tract Phytoche!ical studies were done on the plant e+tract in order to chec' i( sterols, car)ohydrates and glycosides were present 0he ani!als used in this study are Bistar al)ino rats o( either se+ weighing )etween E=1>= g %treptozotocin was used to induce hyperglyce!ia 0he e+tract was ad!inistered the rats (or 4; days A(ter ad!inistration, the livers and 'idneys o( the rats were ta'en in order to )e e+a!ined )y a pathologist 0he study concluded that 8 rosa sinensis aerial e+tract was a)le to lower the )lood glucose o( the test ani!als An increase in the plas!a insulin and C1peptide levels were also o)served $t was also concluded that the e+tract can protect liver and Kidney (ro! da!age due to dia)etes A study )y %har!a et al in *==G loo'ed into the hypoglyce!ic and hypolipide!ic e((ect o( Aegle !ar!elos -3/ lea( e+tract on %treptozotocin induced dia)etic !ice 0he leaves were lyophilized into powder 0he ani!als used in the study were 4; !atured al)ino !ice (ive to si+ wee's o( age, weighing :; g %treptozotocin was used to induce hyperglyce!ia 0he study (ound out that the Aegle !ar!elos lea( e+tract could have hypoglyce!ic, anti1hyperglyce!ic and hypolipide!ic properties A study conducted )y Mishra et al in *=4: ai!ed to co!pare the antidia)etic pro(ile o( Ayurvedic her)o1!ineral (or!ulation and its constituents on nor!al and streptozotocin1induced dia)etic rats 0he co!position o( the Ayurvedic polyher)o1!ineral preparation are9 ymnema syl!estre$ Momordica charantia$ Sy*ygium cumini$ +*adirachta indica$ ,ithania somniera$ Hibiscus rosa"sinensis$ #cimum sanctum$ Pterocarpus marsupium and -occinia indica and a her)o!ineral su)stance %hila#eet<
%treptozotocin
was used to induce the hyperglyce!ia
Results o( the study showed that the levels o( )lood glucose and glycosylated he!oglo)in -8)A4c/ were lowered
%elvara# et al in *=4: conducted a study a)out how (ractions o( Tinospora cordiolia ste! e+tract can de!onstrate insulin secretion in ia)etes $nduced Bistar Rats %treptozotocin was used to induce hyperglyce!ia 0wo (ractions o( the alcoholic e+tract were used, the thyl acetate solu)le (raction -F4/ and )asic (raction -F2/ Results showed that )oth (ractions showed signi(icant hypoglyce!ic e((ect &ut a!ong the * (ractions, the F2 was the one that was a)le to enhance insulin secretion A study done )y elaviz et al in *=44 evaluated the Anti1dia)etic e(( ects o( an alcoholic e+tract o( uglans regia in an ani!al !odel Four groups o( ani!als were e!ployed Nor!al diet was given to the group 4 ani!als %treptozotocin was ad!inistered to groups *, : and 2 .roups : and 2 were treated with the . regia lea( e+tract in dosages o( *== and 2== !g'g )ody weight (or *> days 0he results showed that there were signi(icant decreases in in )lood glucose, glycosylated he!oglo)in, 33, triglyceride, and total cholesterol $t was also noted that there was a signi(icant increase in the insulin and 83 levels r Richard A Anderson and his colleagues at the 8u!an Nutrition Research Center o( the % epart!ent o( Agriculture screened e+tracts o( a nu!)er o( co!!only consu!ed plants to see how well they could !i!ic the e((ects o( insulin, a protein hor!one that is responsi)le (or regulating our )lood sugar levels Fro! a selection o( 2@ culinary and !edicinal plants, they (ound in la)oratory tests that cinna!on was (ar !ore e((ective than any other plant in (ul(illing insulin
(or
its
insulin1li'e
activity
is
a
water1solu)le
che!ical
co!pound
called !ethylhydro+ychalcone poly!er, or M8CP 0hey (ound that M8CP was highly e((ective, providing essentially the sa!e )iological activity as insulin itsel( $t was e((ective not only in increasing the upta'e o( glucose -)lood sugar/ )y cells, )ut also o( sti!ulating the synthesis o(
glycogen, a poly!eric (or! o( glucose that is stored pri!arily in the liver and !uscle tissues (or use at ti!es o( pea' energy de!and, such as e+ercise And M8CP turned out to )e synergistic with insulin in these actions, providing a net e((ect greater than the su! o( its parts + study was conducted on . syl!estre leaves e+tract 0he study (ocused on the hypoglyce!ic e((ets o( . syl!estre 0he present study de!onstrates the e((ect o( . syl!estre leaves e+tract -4> !g'g )ody weight/ on several etiological (actors o( %0?1dia)etic rats including hyperglyce!ia, hypoinsuline!ia, hyperlipide!ia and o+idative stress at the sa!e ti!e 0his study showed a signi(icant decrease in plas!a glucose level -*=*=/ in dia)etic rats treated with . syl!estre leaves e+tract -4> !g'g )ody weight/ co!pared to that o( untreated dia)etic rats %igni(icant increase in dia)etic rats< insulin level post treated with . syl!estre leaves e+tract con(ir!ed the result Moreover, glucose level !ight )e decreased in treated dia)etic rats as a result o( decreasing gluconeogenesis that was indicated )y low levels o( A30 and A%0 in treated dia)etic rats co!pared to untreated dia)etic rats Ra#ase'aran et al in *==E studied the oral ad!inistration o( +loe !era gel e+tract at a dose o( :== !g'g )ody weight per day to %0?1induced dia)etic rats (or a period o( *4 days 0hey (ound that it resulted in a signi(icant reduction in (asting )lood glucose, hepatic transa!inases -A%0 and A30/, plas!a and tissue -liver/ cholesterol, triglycerides, (ree (atty acids and phospholipids and a signi(icant i!prove!ent in plas!a insulin $n addition, the decreased plas!a levels o( high1density lipoprotein1cholesterol and increased plas!a levels o( low1density lipoprotein1 and very low1density lipoprotein1cholesterol in dia)etic rats were restored to near nor!al levels (ollowing treat!ent with the e+tract Further!ore, they analyzed the (atty acid co!position o( the liver and 'idney and (ound that the altered (atty acid
co!position in the liver and 'idney o( dia)etic rats was restored (ollowing treat!ent with the e+tract 0hey reco!!ended use o( +loe !era as an anti1dia)etic agent
Chapter : Methodology 2xtraction of Plant "ample*
4 0he chopped sa!ple was placed in a ;==!3 rlen!eyer (las' weighing ;== g
* %u((icient @; ethanol is poured in 0his is done to !acerate the solution
: 0he !i+ture is le(t (or 2> hours
2 0he product was (iltered and discarded the re!aining plant !aterials
; 0he e+tract is su)#ected to rotatory evaporization
E 0he concentrated e+tract was o)tained
Administration of 2xtract*
All rats are induced with Allo+an 0he rats will )e grouped into three co!prising o( si+ rats9 +roup 9 Negative control, dia)etic !ice with water +roup * ia)etic !ice treated with Hibiscus rosasinensis corolla ethanol e+tract -*;= !g'g/ +roup 0* Positive control, dia)etic !ice treated with .li)encla!ide -;!g'g/
&e(ore giving the e+tract and the drug, each !ouse !ust )e weigh to calculate the right dosage and to o)serve any untoward e((ects !ethod of Administration of Hibiscus rosasinensis
4 Prepare 8i)iscus rosa sinensis e+tract using an e!pty syringe with the calculated dose o( *;=!g'g * 8old the !ouse properly and place the nozzle o( the syringe into its !outh care(ully and slowly allowing the !ouse to swallow
: Repeat steps 4 and * to the other !ouse using di((erent syringe 2 O)serve the !ice (or :=!ins (or any untoward e((ect ; Record
!ethod of Administration of +libenclamide
4 isperse the gli)encla!ide ta)let in water * Prepare the dissolved drug using an e!pty syringe with the calculated dose o( ;!g'g : 8old the !ouse properly and place the nozzle o( the syringe into its !outh care(ully and slowly allowing the !ouse to swallow 2 Repeat steps 4 to : to other !ouse using di((erent syringe ; O)serve the !ouse (or :=!ins (or any adverse e((ect E Record Allo+an1 $nducer o( hyperglyce!ia
Allo+an -*,2,;,E1tetrao+ypyri!idine ;,E1dio+yuracil/ has )een co!!only utilized to induce hyperglyce!ia to ani!al !odels such as rats, !ice, and ra))its According to %z'udels'i , 0 -*==4/, one o( the actions o( allo+an on the pancreas is the rapid upta'e )y insulin1secreting cells
Dosage
According to %z'udels'i, 0 -*==4/, the dosage that should )e given rat !odels to induce hyperglyce!ia depends on the rat
su)cutaneously, the e((ective dose !ust )e *1: ti!es higher or it !ay )e insu((icient i( given )elow 4;=!g'g-%z'udels'i, 0, *==4/
Route of administration
Allo+an can )e e((ective when given parenterally9 intravenously, intraperitoneally, or su)cutaneously -%z'udels'i, 0 ,*==4/
!echanism of action
Allo+an1induced dia)etes has )een co!!only used (or e+peri!ental !odel o( insulin dependent dia)etes !ellitus $n an article written )y Rohilla, A and Ali, % -*==4/, they e+plained the !echanis! o( action allo+an on rat !odels $t is de!onstrated that allo+an can induce a sudden rise o( insulin1secreting cells with or without glucose A(ter a release o( insulin, it will ) e (ollowed )y a co!plete inhi)ition o( the islets to respond to high levels o( glucose One o( the !ost i!portant actions o( allo+an in the pancreas is the rapid upta'e )y pancreatic )eta cells that gave an i!portant (eature deter!ining allo+an dia)etogenicity Reduction process (ollows due to the presence o( di((erent reducing agents such as reduc ed glutathione -.%8/, cysteine, ascor)ate and protein1)ound sul(hydryl -1%8/ groups $n the sugar )inding site o( gluco'inase, allo+an reacts with two1%8 groups resulting in the (or!ation o( disul(ide )ond and inactivation o( the enzy!e 0he !ain product o( allo+an reduction is dialuric acid that is then re1o+idized )ac' to allo+an to esta)lish a redo+ cycle (or the generation o( reactive o+ygen species -RO%/ and supero+ide radicals 0hese superio+ide radicals release (erric ions (ro! (erritin and reduce the! to (errous and (erric ions Also, to yield hydrogen pero+ide, supero+ide under go dis!utation ,in the presence o( supero+ide dis!utase As a result in the presence o( (errous and 8*O* according
to Fenton reaction, highly reactive hydro+yl radical are (or!ed $n addition, one o( the e((ect o( the RO% to the NA o( pancreatic cells is the (rag!entation in the )eta cells, that causes NA da!age and sti!ulates AP1ri)osylation, a process in NA repair -Rohilla, A and Ali, %, *==4/
&lood extraction
$n !easuring the glucose level o( the !ice, the study will utilize the !ethod o( choice which is o)taining )lood (ro! the tip o( the tail %a!pling (ro! the tail tip is a si!ple procedure that can )e carried out in any la)oratory As proposed )y Mouse Meta)olic Phenotyping Center -MMPC/ Consortiu!, standard procedures were !ade (or assessing !eta)olic diseases o( !ice particularly dia)etes and o)esity
Collection o( )lood re5uires 4H* !! o( tissue cut (ro! the tail tip distal to the )one with sharp scissors or a scalpel, and then )lood is o)tained )y direct (low or )y gently !assaging the tail and collecting the )lood in a capillary tu)e or other container Following the initial cut, a *1hour recovery period is reco!!ended prior to o)taining the (irst test sa!ple %u)se5uent sa!ples are o)tained )y gently re!oving the sca) and repeating the !assaging procedure Dein is easily accessed and !oderate 5uantities o( )lood can )e o)tained (ro! this procedure Assess!ent o( glucose !eta)olis! should not )e per(or!ed on anesthetized !ice due to its hyperglycae!ic e((ect
Multiple sa!ples will )e ta'en and a !a+i!u! o( the )lood volu!e to )e drawn is no !ore than 4; o( the total )lood volu!e o( the !ice in *2 hours A )aseline pre1procedure (asting glucose level o( !ice, an induced state o( hyperglyce!ia, and a post1procedure glucose level will )e !easured
1esting for blood sugar
.lucose concentration can )e o)tained (ro! whole1)lood sa!ples or (ro! plas!a or seru! sa!ples $deally, glucose levels should )e !easured (ro! plas!a sa!ples
0he study will utilize co!!ercially availa)le instru!ent such as plas!a glucose ana lyzer or enzy!atic spectrophoto!etric or (luoro!etric assay (ollowing co!pletion o( glucose !easure!ent level $( not availa)le, whole1)lood glucose !onitors will )e used $t is !ore co!!only used and has the advantage o( re5uiring only s!all )lood volu!e 0he !achine (ollows enzy!atic type o( assay
Re(erences 9
epart!ent o( 3a)oratory Ani!al Resources niversity o( 0oledo -*=44/ .uidelines (or )lood collection9 Rodents and Ra))its pp :1;
Ayala, J, %a!uel, D, Basser!an, , tal, -*=4=/ %tandard operating procedures (or descri)ing and per(or!ing !eta)olic tests o( glucose ho!eostasis in !ice
Rats weighing *==1*:= g were used in one study Another study utilised rats weighing 4G;1*;= g Rats that weighed *;=1:== g were also used in one study 0hose that weigh around 4;=1*;= g !ay also )e utilised One study stated the use o( rats weighing *==1*2= g, while another stated the use o( those weighing 42=14E= g
Rats are (ed nor!al pellet diets and had access to water ad li)itu! 0he pelleted (eed is supplied as regular, )reeder, certi(ied, irradiated, or autoclava)le 0hey e at 4=1:= gday, and drin' *=1;= !3day o( water
Rats share @= o( their geno!es with hu!ans Al!ost all disease1lin'ed hu!an genes have e5uivalent genes within their geno!es, !a'ing the ! a suita)le research tool Male al)ino rats, speci(ically Bistar rats, !ay )e used in this study as they are usually the species )eing utilised when conducting invasive e+peri!ents 0hey are a lso the (irst rat strain developed to serve as !odel ani!als, !a'ing the! ideal (or the study %prague1awley rats were also derived (ro! this strain, and thus !ay also )e used i( Bistar rats are not availa)le $ts !ain advantage is it cal!ness and ease o( handling Males are pre(erred over (e!ales due to the (act that (e!ale rats are s!aller and considera)ly !ore active, and there(ore !ay )e a little di((icult to handle co!pared to that o( !ales
Rats are pre(era)ly 'ept steel cages Cages !ade with wood are unaccepta)le the urine o( rats !ay soa' in to the wood, causing a!!onia )uildup, which !ay lead to respiratory disease, or worse, e+acer)ate it 0hey !ay also chew through the wood, allowing the! to escape Bood !ay also have splinters, !a'ing it unsa(e (or the rats Cage (loors should )e solid, not wired Bired (loors !ay cause chilling even in a war! roo!, and this !ay i!pact the rats in a )iologically a)nor!ally !anner and also cause unnecessary disco!(ort or pain 0hey tend to pile up in heaps when resting on grids or wired (loors, while those on solid (looring spread out 5uite co!(orta)ly on the )edding $( solid PDC (loors are chosen however, the claws o( the rats !ust )e clipped to avoid considera)le disco!(ort, given that the sur(ace is not a)rasive ust1(ree woodchip )eddings are also reco!!ended Polypropylene cages !ay also )e used
Rat roo!s are usually !aintained at :=1G= relative hu !idity and a te!perature o( 4>1*E degree Celsius, with a 4*1hour lightdar' cycle
References
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