Effect of Aqueous Extract of Carica papaya Dry Root Powder on Lactation of Albino Rats

Transcription

1 Effect of Aqueous Extrct of Cric ppy Dry Root Powder on Lcttion of Alino Rts G. Tosswnchuntr nd S. Aritjt Deprtment of Biology Fculty of Science Ching Mi University Ching Mi Thilnd Keywords: mmmry glnd, lkline phosphtse, oxytocin-receptor, ELISA, prolctin, Kjeldhl method, protein Astrct Effects of Cric ppy root (CP) on lcttion of rts were studied using the queous extrct from dry powder in vrious doses for 15, 21 nd 30 dys. The lctting rts were fed with distilled wter nd the queous extrct of CP twice dy t doses of 400, 500 nd 1,000 mg/kg/dy. The level of lkline phosphtse (ALP), prolctin nd totl protein in mmmry glnd were mesured. In cse of rts fed with CP for 15 dys, the verge weights of mmmry glnds of fed rts t ll doses were significntly higher (p<0.05) thn those of the control groups. The level of ALP in rts fed with CP t 500 mg/kg/dy ws highest while the level of prolctin in rts fed with CP t 400 mg/kg/dy ws significntly higher thn other groups. Rts fed with CP t 400 nd 1,000 mg/kg/dy contined higher protein content thn the control groups. As for rts fed with CP t 400 mg/kg/dy for 21 dys hd higher level of ALP nd the verge weight of their mmmry glnds ws significnt higher thn the control groups. The levels of prolctin in rts fed with CP t 400 nd 500 mg/kg/dy were significnt higher thn the control ones. The mmmry glnds of rt fed with CP t ll doses contined higher protein content thn the control groups. For rts fed with CP for 30 dys, there were no significnt differences in weight of mmmry glnd, ALP, prolctin nd protein contents mong the fed nd control rts. It ws concluded tht feeding lctting rts with CP for 15 nd 21 dys could ctivte higher lctting ility y incresing the weight of mmmry glnd, ALP, prolctin nd protein content. INTRODUCTION In Thilnd, ppy (Cric ppy) is widely grown nd hs herl properties. Mny Thi people found tht Cric ppy root hs effect on lcttion (Mudchchip, 1991). It is therefore very interesting to study the effect of CP on mmmry glnd. Primry study using the queous solution from dry powder fed to rts for 15 dys during lcttion, indicted tht the mmmry glnd weight of CP treted group ws significntly higher thn those of the control (Tosswnchuntr, 2000). This study ims to study the effects of Cric ppy root on lcttion of rts. Rts were fed with 3 doses of queous solution from dry CP powder for three different trils (15, 21 nd 30 dys). Oxytocin-receptor concentrtions in the rt mmmry glnd were determined y lkline phosphtse stining using histochemistry method (Suwnjrt, 1994). Oxytocin cuses milk ejection y mmmry glnd (Crowley nd Armstrong, 1992). Enzyme-linked immuno sorent ssy (ELISA) hve een used in immunology to mesure the quntity of prolctin (Signorell nd Hymer, 1984). Prolctin cused the development of mmmry glnd. Kjeldhl method ws used to determine the quntity of protein in mmmry glnd (Smsil, 2001). Proc. WOCMAP III. Vol. 4: Trgeted Screening of MAPs, Economics & Lw Eds. C. Frnz, Á. Máthé, L.E. Crker nd Z.E. Grdner Act Hort. 678, ISHS

2 MATERIALS AND METHODS Cric ppy Root Powder Cric ppy roots were incuted t 60 C until dry nd ground to powder. Dry powder ws dissolved in distilled wter to otin vrious concentrtions. Animl Wistr rts were used in this experiment. Vginl tissue ws tken y vginl smer to detect estrous cycle. Femle rts were mted with mle rts nd sperms in vgin were detected. If sperms were present, it ws counted s the first dy of pregnncy. After the dy of prturition, there would e one lctting rt per five pups. One hundred nd twenty rts were divided into 3 sets of 40 rts. Ech set ws divided into 4 groups of 10 rts. The four groups were fed with distilled wter, 400, 500 nd 1,000 mg/kg/dy of queous solution of dry powder of CP twice dy. The first, second nd third sets were fed for 15, 21 nd 30 dys respectively. Alkline Phosphtse Mmmry glnd ws sectioned to 0.5 x 0.5 cm. nd fixed with cetone t 4 C. Prfin sections were serilly cut t 7 µm thick. Alkline phosphtse ctivity ws detected y clcium colt method y the ppernce of lck precipittion of colt sulfide. Determintion of ALP ctivity ws done y rndom selection of 160 fields from 32 sections nd oserved the lck precipittion of colt sulfide t 4 level of intensities of lck colour of the precipittes. ELISA Blood smples were tken from the hert. They were left t room temperture for 1 hour nd centrifuged t 3,000 rpm for 30 minutes. Serum smples were ssyed for prolctin y competitive indirect ELISA method s descried y Signorell nd Hymer (1984). The ssy involved 8 steps: (1) Wells were coted with prolctin t the concentrtion of 20 ng/100 µl in coting uffer (t 4 C, 16 hours). (2) (2.1) Tues contining 125 µl ech of primry smple nd 125 µl ntiody (rit nti rt prolctin) dilution 1:10,000. (2.2) Stndrd curve: prolctin concentrtion 0, 10, 100, 1,000 nd 10,000 ng/ml 125 µl/tue nd 125 µl/tue rit nti rt prolctin (1:10,000) (t 4 C, 16 hours). (3) Wsh plte 4 times with 200 µl/well of wshing uffer. Add 100 µl/well 1.5% BSA into well (37 C, 1 hour) nd wsh 4 times. (4) Add 200 µl of the mixture from step 2 to well (step 3) (t room temperture, 4 hours) wsh 4 times. (5) Added 50 µl of conjugte (horserdish peroxidse conjugted got nti-rit immunogloulin (HRP), 1:1,000) (t room temperture, 2.5 hours) wsh 5 times. (6) Add 200 µl of sustrte OPD for HRP in the drk room temperture. (7) Terminte the rection y dding 50 µl of 4 N sulfuric cid to ech well. (8) Mesure the sornce t 492 nm. Kjeldhl Method Protein content ws estimted y Kheldhl method. One grm of mmmry glnd tissue ws digested y dding 10 ml concentrted sulfuric cid nd 5 g selenium rection mixture enzyme. The digestion ws done with digestion unit (BUCHI Model 426) t 110 C for 90 min. When the temperture of mixture reched the room temperture, 20 ml distilled wter nd 32% sodium hydroxide were dded. The rection mixture ws distillted using distilltion unit (BUCHI Model 323) for 5 min. Distilled solution ws dropped into 50 ml solution A (2% oric cid, 0.1% methyl red nd 0.025% methylene lue) nd titrted with stndrd 0.1 N hydrochloric cid until the color of regent ecme purple. Protein content ws clculted from the following formul: 86

3 Protein content (% in 100 g of mmmry glnd) = NV x 1.4 x 6.25 W Where N = concentrion of HCl (N) V = volume of HCl used for titrtion (ml) W = weight of mmmry glnd RESULTS AND DISCUSSION The verge weights of mmmry glnds in the 15-dy feeding group, the verge weight of mmmry glnd of rt fed with CP t every dose ws higher thn tht of the control group (p<0.05) (Fig. 1) which grees with our previous work (Tosswnchuntr, 2000). For the 21-dy group, only rt fed with CP t dose 400 mg/kg/dy hd higher mmmry glnd weight thn the control group (p<0.05). On the other hnd, there ws no significnt difference in mmmry glnd weight mong rt fed with distilled wter nd CP t every dose for 30 dys (Fig. 1). It ws concluded tht CP could e used for incresing weight of mmmry glnd during the first 21 dys of lcttion. Alkline Phosphtse Tle 1 shows the level of lkline phosphtse (ALP) in mmmry glnd of rt fed with CP t vrious doses nd periods. It could e noticed tht mmmry glnds of rts fed with CP for 15 nd 21 dys hd higher ALP thn those of the control groups, especilly the groups tht were fed with CP t 500 mg/kg/dy for 15 dys nd t 400 mg/kg/dy for 21 dys. For the rts fed with CP for 30 dys, their mmmry glnds contined slightly higher level of ALP thn those of the control group. The increse of ALP indicted tht feeding the rt with CP for 15 nd 21 dys could ctivte lcttion. The level of ALP ws directly relted to the increse of oxytoxin-receptor in mmmry glnd which plys n importnt role in lcttion (Soloff nd Weider, 1983). Prolctin In the 15-dy group, rts fed with CP t 400 mg/kg/dy hd serum with higher mount of prolctin thn tht of the control group (p<0.05). In the 21-dy group, serum of rts fed with CP t doses of 400 nd 500 mg/kg/dy contined higher mount of prolctin thn tht of the control (p<0.05). However, in the 30-dy group, serum of rts fed with CP t 400 mg/kg/dy showed only slightly higher mount of prolctin (Fig. 2). This increse of prolctin, the key sustnce in milk iosynthesis during lcttion (Flint nd Grdner, 1994), indicted tht feeding with CP for 15 nd 21 dys ws le to increse the degree of lcttion. Protein In the 15-dy group, rts fed with CP t 400 nd 1000 mg/kg/dy hd mmmry glnds with higher mount of protein thn tht of the control group (p<0.05) (Fig. 3). Mmmry glnds of rts fed with CP t every dose contined higher mount of protein thn of control (p<0.05). For the 30-dy group, there ws no significnt difference in protein content in mmmry glnds of the control nd CP-fed rts. However, the incresing trend of protein content in CP-fed rts could e noticed. The incresing protein content in the mmmry glnd during lcttion is the index for the incresing degree of lcttion (Greenum nd Slter, 1956). The increse of protein content in this experiment greed with the increse of mmmry glnd weight nd prolctin mount. It could e concluded tht CP ws le to ctivte the mmmry glnd of lctting rt to promote higher qulity nd mount of lcttion. CONCLUSION The queous solution from dry powder of CP given to the rts for 15 nd 21 dys could improve milk production nd secretion. The mmmry glnd weight, ALP, prolctin nd protein in the mmmry glnd lso incresed. It tended to improve 87

4 mmmry glnd ctivity. However, in 30 dy group the result ws not ovious. ACKNOWLEDGEMENTS The uthors wish to thnk Deprtment of Biology, Fculty of Science for lortory equipments, nimls nd fcilities, Associte Professor Peti Pongpichn, Deprtment of Animl Science, Fculty of Agriculture for ELISA technique, Dr. A.F. Prlow NIDDK Ntionl Hormone & Peptide Progrm for hormone nd prolctin ntiody. Literture Cited Crowley, W.R. nd Armstrong, W.E Neurochemicl regultion of oxytocin secretion in lcttion. Endocrine Rev. 13: Flint, D.J. nd Grdner, M Evidence tht growth hormone stimultes milk synthesis y direct ction on mmmry glnd nd tht prolctin exerts effects on milk secretion y mintennce of mmmry deoxyrionucleic cid content nd tight junction sttus. Endocrinology 135: Greenum, A.L. nd Slter, T.F Study on the prticulte components of rt mmmry glnd : 1. A method for determining the composition of the retined fluid. Biochem. J. 66: Mudchchip, S Medicicl plnt. Prwity Press, Bngkok. Smsil, P Effect of Goniothlmus loticus Finet et Ggnep qeous extrct on milk content nd lkline phosphtse in mmmry glnd of lino rt. Specil Project, Ching Mi University, Ching Mi. Signorell, A.P. nd Hymer, W.C An enzyme linked immunosorent ssy for rt prolctin. Anl. Bichem. 136: Soloff, M.S. nd Weider, M.H Oxytocin receptors in rt involuting mmmry glnd. Cn. J. Biochem. Cell Biol. 61:631. Suwnjrt, J Structure of histology nd histochemistry of Ltes clcrifer (Bloch) testes. J. Songhlnklin Univ. 2:16. Tosswnchuntr, G Effect of queous extrcts of Euphoi hirt Linn. nd Cric ppy on lcttion rts. Independent Study, Ching Mi University, Ching Mi. 88

5 Tles Tle 1. Level of lkline phosphtse (ALP) in mmmry glnd of rt fed with distilled wter (control) nd the queous extrct of Cric ppy (CP) t vrious doses for 15, 21 nd 30 dys. Feeding Period (dys) CP Dose (verge from 10 rts) Level of lkline phosphtse 15 control mg/kg/dy mg/kg/dy +++ 1,000 mg/kg/dy control mg/kg/dy mg/kg/dy ++ 1,000 mg/kg/dy control mg/kg/dy mg/kg/dy ++ 1,000 mg/kg/dy men highest level of lkline phosphtse Figures Weight of mmmry glnd (g/g ody weight) dy 21 dy 30 dy control 400 mg/kg/dy 500 mg/kg/dy 1,000 mg/kg/dy Fig. 1. Weight of mmmry glnd of rt fed with distilled wter (control) nd the queous extrct of Cric ppy (CP) t vrious doses for 15, 21 nd 30 dys. 89

6 Protein content (g/100g of mmmry glnd) c 15 dy c 21 dy 30 dy control 400 mg/kg/dy 500 mg/kg/dy 1,000 mg/kg/dy Fig. 2. Amount of prolctin in mmmry glnd of rt fed with distilled wter (control) nd the queous extrct of Cric ppy (CP) t vrious doses for 15, 21 nd 30 dys. Prolctin (ng/ml) dy 21 dy 30 dy control 400 mg/kg/dy 500 mg/kg/dy 1,000 mg/kg/dy Fig. 3. Protein content of mmmry glnd of rt fed with distilled wter (control) nd queous extrct of Cric ppy (CP) t vrious doses for 15, 21 nd 30 dys. 90