Effect of Mulberry (Morus alba L.) Leaves Infusion on the Reproductive Status of Chronic Diabetic Models

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1 J. Appl. Environ. Biol. Si., 5(5)14-18, , TextRo Pulition ISSN: Journl of Applie Environmentl n Biologil Sienes Effet of Mulerry (Morus l L.) Leves Infusion on the Reproutive Sttus of Chroni Dieti Moels Byyintul Muhtromh *, Romii, Roliyni E. Putri, Fri D. Nurini Deprtment of Biology, Fulty of Siene n Tehnology, Stte Islmi University of Mlng, Jln. Gjyn No 50 Mlng, Inonesi Reeive: Jnury 22, 2015 Aepte: Mrh 17, 2015 ABSTRACT Leves of mulerry (Morus l L.) is one of the meiinl plnts tht ontins ntihyperglyemi n ntioxint ompouns. These ompouns re known s gent tht oul erese the high loo gluose levels n improve orgn mge use y free rils in hyperglyemi onitions. This stuy ime to etermine the effet of mulerry leves infusion on reproution sttus of hroni ietes moel. This reserh ws n experimentl stuy using ompletely rnomize esign (CRD) with 6 tretments n 4 replitions. The tretments were C+ (positive ontrol), C- (negtive ontrol), T1 (400 mg/kgw), P2 (600 mg/kgw), P3 (800 mg/kgw) n P4 (1000 mg/kgw). 24 of wistr rts, mle, 1 month ol, n grms of oy weight were use s niml moels. Dt oserve in this stuy inlue the numer of germ ells (spermtogoni, primry spermtoytes, seonry spermtoytes, spermtis n spermtozo), sertoli ells, seminiferous tuules imeter, leiig ells, testes weight n testosteron levels. Dt were nlyze with One Wy Anov, n then followe y DMRT (1%) nlysis if there is signifint ifferenes etween tretments. The results showe tht the infusion of mulerry leves inrese the numer of germinl ells, sertoli ells, imeter of seminiferous tuules, leiig ell, testis weight, n testosteron levels of hroni ieti moel. The optimum infusion ose of mulerry leves ws T4 (1000 mg/kgw). KEYWORDS Chroni Dieti Moel, Histology Preview, Mulerry Leves (Morus l L.), Rt, Testosterone I. INTRODUCTION Dietes mellitus is hroni isese use y omplex metoli isorer ue to the reue insulin either solute or reltive, whih is hrterize y hyperglyemi n glyosuri n the evelopment of mrovsulr n mirovsulr omplitions [1]. One impt of the omplitions of ietes mellitus in hroni phse is the mle reproutive orgn ysfuntion. A omplition of hroni Dietes mellitus uses the eresing of LH, FSH n testosterone levels [2]. FSH, LH n testosterone hs n importnt role in spermtogenesis proess [3]. If the mount of these hormones reues, it will istur the proess of spermtogenesis, n the finl onsequeny will e followe y the eresing of germ ell numers s well s testiulr weight [4]. Reently, the utiliztion of synteti ntiieti meiine hve sie effets for the oy n reltively expensive. One lterntive to overome this prolem is to o reserh on plnts tht hve ntiieti effets ie mulerry (Morus l L.). This plnt is rih in hemil onstituents suh s lklois, flvonois, n polyphenols [5]. Approximtely fifteen polyhyroxylte lklois hve een isolte from the leves of mulerry, one of whih is 1-Deoxynojirimyin (DNJ), whih hs poteny to erese loo gluose y inhiiting lph-gluosise [3]. This enzyme tlyzes the hyrolysis of ons in mltose to proue two moleules of gluose [1]. Leves n roots extrt of mulerry ontin 0.24% DNJ ompouns [6]. Mulerry leves lso ontin severl hemil ompouns suh s eysterone [7] n sopoletin [6] whih lso ontriute for the eresing of loo gluose. In ition, mulerry leves lso ontin foli i n zin [7] tht re le to inrese the numer of sperm ells in men with infertility experiene. II. MATERIALS AND METHODS A. Preprtion for niml moel Before use for experimentl stuy, moel nimls were limtize for 2 weeks, fe n wtere liitum. Alloxn, ose of 100 mg/kg or s muh s 0,125 ml of intrvenous inution, ws ministrte to mke the onition of ietes mellitus in mie n given 1 time n h fste for 24 hours. Chroni omplitions use y hyperglyemi onitions within perio of 5-10 yers, if onverte into rt out 1 month, thus using the interruption of loo flow, whih n use omplitions to vrious orgns inluing the reproutive orgns. Therefore rts fter lloxn inue, left for 4 weeks n then trete with infusion of new mulerry leves. B. Mesurement of loo gluose Mesurement of loo gluose levels ws performe efore n fter tretment with lloxn s well s fter the ministrtion of mulerry leves infusion. Before tking the loo, the rts moel were fste for 16 hours. Mesurement of loo gluose levels ws performe y using glukotest, where the mesure loo is from the loo veins of rt til. *Corresponing Author: Byyintul Muhtromh, Deprtment of Biology, Fulty of Siene n Tehnology, Stte Islmi University of Mlng, Jln. Gjyn No 50 Mlng, Inonesi. E-mil: yyintul_uin@yhoo.o.i 14

2 Muhtromh et l.,2015 Hyperglyemi onition ours in rts when the loo gluose levels re more thn 200 mg/l in non-fsting or 126 mg/l in fsting onition [8]. C. Preprtion of mulerry lef infusion Preprtion of mulerry lef infusion ws performe y wshing mulerry leves with wter. After rying up t room temperture for 5 ys, the pulverize of mulerry leves ws then ooke for 15 minutes temperture of 90 C n ministrte s 0, 400, 600, 800 n 1000 mg /kgw/y of 2.5 ml for 30 ys. D. Group of tretment The group ws ivie s follows: Control (-): rt tht not infuse y oth lloxn n mulerry leves infusion, Control (+): rts inue y lloxn without mulerry leves infusion, T1: rts inue y lloxn + mulerry lef infusion of 400 mg/kgw, T2: rts inue y lloxn + mulerry lef infusion of 600 mg/kgw, T3: rts inue y lloxn + mulerry lef infusion of 800 mg/kgw, T4: rts inue y lloxn + mulerry lef infusion 1000 mg /kgw. Infusion leves of mulerry ws given for 30 ys orlly using stomh sone. At the en of the stuy, rt ws srifie n testis ws issete, weighe n use for preprtions. E. Determintion of testis weight Rt testes ws remove n lene from ft, weighe n then soke in formlin for histologil preprtions. The numer of leyig, sertoli, spermtogoni, primry spermtoyte, seonry spermtoyte n spermti ells were ounte from five fiels of view. For imeter of seminiferous tuules mesurement, three seminiferous tuules were onute on lmost equl size y mesuring the istne of the longest n the shortest istne from the seminiferous tuules whih re roun or spheril onsiere then verge. The histologil oservtion ws performe y using Olympus CX31 mirosope with 400 x mgnifition. F. Determintion of testosterone level Step of testosterone mesurement ws s follows: Prepre wsh solution y mens of iluting the wsh solution with eionize wter. Preprtion of stnr, stnr 5 (80 pg/ml) = 75 ml of wsh solution + 75 ml stok wsh solution; stnr 4 (40 pg/ml) = 67.5 ml of wsh solution + 75 ml stnr 5; stnr 3 (20 pg/ml) = 75 ml wsh solution + 75 ml stnr 4; stnr 2 (10 pg/ml) = 150 ml wsh solution + 75 ml stnr 3; stnr 1 (5 pg/ml) = 150 ml wsh solution + 75 ml stnr 2, 40 ml n 10 ml smple ilution of serum inlue in eppenorf, n mixe. 50 ml stnr n the smple were then introue into the well (exept lnk well), overe n inute for 30 min t 37 C, the memrne plte ws opene, the liqui ws sore y using tissue, then wshe with 100 ml wsh solution tht ws rete in point 1, shken, the flui ws sore y the tissue isre, wshe 3 times, inute for 30 min t 37 C, the mixture ws e 50 ml hromogen A n B into the well, inute for 15 min t 37 C, e 50 ml stok solution, the sorne re t 450 nm in n ELISA reer. G. Dt Anlysis Dt nlysis for ll tretment ws performe y One Wy ANOVA, n followe y Dunn test if F ount> F tle. III. RESULTS AND DISCUSSION Sttistil nlysis revele tht the numer of spermtogeni ells, sertoli ells, leiig ells, seminiferous tuule imeter n testes weight of hroni ieti mellitus rt moel in T1 (400 mg/kg w) re not signifintly ifferent with positive ontrol tretment (C+). However, in T2, T3 n T4, the numer of these ell re signifintly ifferent from (C+) n lose to norml onition (Tle 1). TABLE 1. NUMBER OF SPERMATOGENIC, SERTOLI AND LEIDIG CELLS, SEMINIFEROUS TUBULE DIAMETER AND TESTES WEIGHT OF CHRONIC DIABETIC MELLITUS RAT MODEL Dose Averges ± stnr evition mg/kg w Spg Spt 1 Spt 2 Spt Sptz Srt Ø Ts (µm) C+ (0) ± ± ± ± ±6.99 TI (400) ± ± ± ± ±1.19 T2 (600) ± ± ± ± ±1.22 T3(800) ± ± ± ± ±1.51 T4(1000) ± ± ± ± ± 3.06 C ± ± ±2.4 7.,75± ±2.67 Norml Testis weight (mg) 3.20± ± ± ± ± ±2.66 Spg = spermtogoni, spt 1 = primry spermtoyte, spt 2 = seonry spermtoyte, spt = spermtie, sptz = spermtozo, srt = sertoli, Ø Ts = imeter of the seminiferous tuule. 15

3 J. Appl. Environ. Biol. Si., 5(5)14-18, 2015 After mulerry leves infusion tretment, the histology of spermtogeni ells improve. The lyout of spermtogeni n sertoli ells on the seminiferous tuules is grully ense in C +, T1, T2, T3, T4. It is presumly ue to the ell regenertion uring the tretment n the numer of ells inrese in T4 tretment n lmost lose to norml onition (Fig.1). T4 (1000 mg/kgw) is the optiml ose in inresing the numer of spermtogeni ells n testiulr sertoli ells. FIGURE 1. Histologil piture of the testes on hroni ietes moel fter ministrtion of murei leve infusion. M: 400x. 1. Spermtogoni ell, 2.primry spermtoyte ell, 3. Seonry spermtoyte ell, 4.spermti ell, 5.spermtozo ell, 6. Sertoli ell Free rils in hroni ietes mellitus rts inrese ue to the hyperglyemi onitions. Hyperglyemi onitions use uto-oxition of gluose, glytion of proteins, n tivtion of the polyol metoli pthwy whih further elertes the formtion of retive oxygen speies. The formtion of retive oxygen speies inrese lipis, DNA, n proteins moifition in vrious tissues [8]. Moleulr moifition in vrious tissues resulte in n imlne etween protetive ntioxints n free ril proution. It ws the eginning of oxitive mge known s oxitive stress [9]. Oxitive stress will inrese the mount of ROS tht n mge ell memrnes, lipi peroxie or mlonilehye (MDA), the mount of exess ROS ffets the hypothlmi-pituitry xis thus inhiiting the seretion of FSH n LH. The eresing in FSH levels will use isruption of mitosis proess n prolifertion of spermtogoni [4]. While FSH n LH hormones hve n importnt funtion in the proess of spermtogenesis. LH stimultes the funtion of leyig ell to proue testosterone in testes, while FSH stimulte spermtogenesis proess whih unergo the hnge of spermtogoni into spermtis. In ition, FSH lso serves to stimulte the formtion of Sertoli ell nrogen-ining protein (ABP) in whih this protein ontriutes in trnsporting testosterone to the seminiferous tuules n epiiymis [3]. Hypothlmus seretes GnRH (gonotrophin relesing hormone) nterior pituitry to proue FSH n LH. These hormones ply mjor role in regulting mle sexul funtion. FSH is rrie through the loo to the testiles to initite the prolifertion proess of spermtogenesis. LH will stimulte leiig ell to proue testosterone to omplete the mturtion proess n the formtion of spermtozo. Disruption in FSH n LH seretion uses the proess of ell ivision n mturtion of spermtogeni eomes loke, n finlly the numer of spermtogeni ells ws lso reue [11]. The eresing in testiulr spermtis of rt s seminiferous tuules possily ours through severl mehnisms suh s isruption of sertoli ell funtion, whih uses the eresing of germ ells nutrients, ltte n pyruvte. Ltte n pyruvte re energy soures of the spermtis. The eresing of these energy soures might interferene the meiosis proess, the other possiility is euse in the erly spermiosis lrey impire. With the isruption of spermiogenesis erly the next proess will e impire [3]. Aministrtion of the mulerry lef infusion for 30 onseutive ys inrese in spermtogeni ells n Sertoli ells. In generl, n inrese in the numer of spermtogeni ells n Sertoli ells ours euse the mie were initilly experiening hyperglyemi onition fter eing fe mulerry leves infusion loo gluose levels to e own, so tht the free rils tht h inrese to prtiipte reue. Free rils re erese to reue tissue mge in the oy, inluing mge to the orgn-forming germ ells. Chemil onstituents of mulerry lef tht ply role in lowering high loo gluose levels is the DNJ. DNJ hs the potentil to inhiit lph-gluosise, n enzyme tht tlyzes the hyrolysis of ons in mltose to proue two moleules of gluose [12]. In ition, mulerry leves re le to repir tissue mge ue to their ntioxint ontent. One of ntioxint ontent of mulerry leves is vitmin C [10]. The role of sori i (vitmin C) for ietes is s lose reutse enzyme inhiitor [1], thus reuing the use of equivlent reue. The willingness of the reuing equivlent is useful for the onversion of oxiize glutthione (GSSG) to reue glutthione (GSH). It n further prevent the uilup of soritol in tissues [12]. In extrellulr, this ntioxint oul erese the superoxie rils whih is generte from the gluose utooxition n nitri oxie synthesis. If the superoxie rils re in exess onition, they will ret with nitri oxie n 16

4 Muhtromh et l.,2015 finlly proue peroxynitrite rils tht re ytotoxi. Inhiition of the formtion of peroxynitrite rils will keep vsoilttion funtion plye y nitri oxie. In enothelil ells, sori i ffets the enzyme nitri oxie synthse tht n suppress superoxie rils s result of nitri oxie formtion. Menwhile, foli i n zin ontine in mulerry leves re to e tough to e le to inrese the numer of sperm ells [13]. The inrese in weight of the testes of trete mie ompre to ontrol mie given positive fter infusion mulerry leves for 30 ys this hppens euse the numer of spermtogeni ells in the testes lso inrese. This is onsistent with the sttement of Zhng et l [3], tht the rih ontent of spermtogeni ells in the seminiferous tuules in the testes n lso inrese the weight of the testis itself lthough testiulr weight ws lso influene y other ftors. Testis weight is not only influene y the presene of germ ells n seminiferous tuule imeter, testis weight ws lso ffete y onnetive tissue n smooth musle ells [7]. This network serves to support the proess of spermtogenesis is one y the testes. So not only gine weight testiulr tuules ut lso supporte y network of onnetive tissue n loo vessels. The inresing of the testes weight in ieti rts fter mulerry leves infusion is ue to foli i n zin in mulerry leves tht finlly support the inresing the numer of spermtogeni ells [4]. In ition, the ntioxint ontent owne mulerry leves supposely le to suppress the inrese of free rils tht n interfere with spermtogenesis, so tht when the numer of ells inreses, testiulr spermtogeni will experiene weight gin Effet of mulerry leves infusion on histologil preview of leiig ell n testosteron level of hroni ieti moels From Figure 2 () n () the numer of leiig ells n testosterone levels signifintly inrese. Low level of leiig ell n testosterone ws foun in the C + n not signifintly ifferent from T1. While in T2 n T3, n T4, leiig ell numer n testosterone levels inrese up to norml onition. FIGURE 2. () Leiig ell numer n () testosteron level on hroni ieti moels fter tretment of mulery leve infusion. Oxitive stress ue to hyperglyemi will inrese the mount of ROS tht n mge ell memrnes, the lipi peroxie or Mlonilehye (MDA), whih ultimtely ffets the hypothlmi-pituitry xis work so tht seretion of LH n FSH erese. FSH levels will le to isruption the mitosis proess n spermtogoni prolifertion. Aoring Zhng [4] FSH n LH hormones hve n importnt role in the proess of spermtogenesis. LH stimultes the Leyig ell funtion to proue testosterone in testes. Furthermore, FSH oul stimulte spermtogenesis proess, the formtion of spermtogoni into spermtis. In ition, FSH lso serves to stimulte the formtion of Sertoli ell nrogen-ining protein (ABP) in whih this protein plys role in the trnsport of testosterone to the seminiferous tuules n epiiymis. The estrution of Leyig ells ue to the inhiition of the luteinizing hormone (LH) seretion in rts with ietes mellitus n e seen from the numer of Leyig ells n testosteron level tht erese s shown in Fig. 3. Mulerry leves infusion n improve the numer of leyig ells, euse mulerry leves ontin vitmin C s n ntioxint tht n ountert on free rils, in ition to other ompouns tht hve ntioxint funtions tht moretin, isoquersetin, eugenol, n rotene. IV. CONCLUSION Mulerry leves infusion n improve the reproutive sttus of the rt moel of hroni ietes mellitus hrterize y the inresing of spermtogeni n sertoli ells numer, seminiferous tuule imeter, testes weight, leyig ells n testosterone levels, where the optiml ose is 1000 mg / kg. It is suggeste to o further reserh on the effet of mulerry lef infusion on sperm qulity moels of hroni ietes mellitus. REFERENCES [1] J. Nowoot, P. Pnnngpeth, V. Kukongviriypn, U. Kukongviriypn, S. Nkmreong, n A. Ithrt, Mulerry lef extrt restores rteril pressure in streptozotoin-inue hroni ieti rts., Nutr. Res., vol. 29, no. 8, pp , Aug

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