CORRECTING EFFECT OF AQUEOUS EXTRACT FROM DUCKWEED LEMNA MINOR FROND ON MORPHOLOGICAL STATE OF THYROID GLAND IN RATS WITH HYPOTHYROIDISM

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1 CORRECTING EFFECT OF AQUEOUS EXTRACT FROM DUCKWEED LEMNA MINOR FROND ON MORPHOLOGICAL STATE OF THYROID GLAND IN RATS WITH HYPOTHYROIDISM Alevtyn Kononenko Deprtment of Physiology nd Humn Antomy Ntionl University of Phrmcy 53 Pushkinsky str., Khrkov, Ukrine Ver Krvchenko Deprtment of Physiology nd Humn Antomy Ntionl University of Phrmcy 53 Pushkinsky str., Khrkov, Ukrine Astrct The present work studied the effect of queous extrct from duckweed Lemn minor frond on morphologicl stte of the thyroid glnd in the experimentl hypothyroidism. Helthy lino rts weighing etween 120 g nd 150 g were used. The nimlswere rndomly llotted into four groups, ech contining ten rts respectively. Three of the groups (II, III nd IV) induced with hypothyroidism y 1 % solution of sodium perchlorte with drinking wter for 20 dys. Control (vehicle) rts were given norml sline. After 20 dys hypothyroid groups (III nd IV) of rts treted with queous extrct from duckweed Lemn minor frond t dose 0.5 ml/100 g of ody weight nd with Iodomrin 200 (reference drug) t dose 12 µg/kg dily orlly for 21 dys. Results otined from the study showed tht the introduction of sodium perchlorte leds to morphologicl nd functionl chnges in the structure of the thyroid glnd in rts, ccompnied y sings of hyperplstic tissue prolifertion nd corresponds with hypofunctionl stte. It ws estlished tht introduction of queous extrct from duckweed Lemn minor frond hs positive effect on the restructuring of the thyroid glnd in rts with hypothyroidism, incresing its functionl ctivity. There is decrese of prolifertive processes y extrfolliсulr epithelium nd thyrocytes in the follicles. The outer dimeter of follicles decresed y 7.65 % (P>0.05), the height nd size of thyrocytes in the wll of the follicle were significntly decresed y % nd %, respectively, compred to untreted hypothyroid group. The otined results hve prcticl significnce for the development of drugs with thyroid-stimulting ction for preventing or delying the development of hypothyroidism nd its complictions. Keywords: queous extrct, Lemn minor frond, experimentl hypothyroidism, sodium perchlorte, morphology. DOI: / Alevtyn Kononenko, Ver Krvchenko 1. Introduction Hypothyroidism is one of the most common dysfunction of the thyroid glnd, ccompnied y inhiition of mny functions of orgns nd systems nd led to restructuring thyroid [1 5]. According to some epidemiologicl studies in some popultions, the prevlence of hypothyroidism reches % [1, 6 8]. For the tretment of hypothyroid sttes of thyroid glnd the replcement therpy is minly used thyroid hormones or therpy imed t restoring the deficiency of iodine [9 11]. Tody, ccording to the vrious estimtes of significnt shre of the Europen nd world phrmceuticl mrketoccupied y herl products, this shre is pproximtely % [2, 10]. Arsenl of thyroid-stimulting drugs in Ukrine is chrcterized y uniformity of dosge forms, the predominnce of imported producers, nd the lck of herl drugs [12, 13]. Therefore, there is need to develop phrmceuticl drugswith thyroid-stimulting effect sed on medicinl plnts. One of the hers used in trditionl nd folk medicine to tret nd prevent hypothyroidism is Lemn minor. The results of phytochemicl reserch showed the presence of iodine nd 14 elements 3

2 (clcium, potssium, silicon, sodium, etc.) in duckweed Lemn minor frond. There were identified 32 iologiclly ctive sustnces of different chemicl groups (phytosterols, sturted hydrocrons, ldehydes nd ketones, mino cids, ftty cids, etc.) [14, 15]. The im of our reserch ws to prove the effect of queous extrct from duckweed Lemn minor frond on morphologicl stte of the thyroid glnd t experimentl hypothyroidism [16]. 2. Mterils nd Methods Aqueous extrct from duckweed Lemn minor frond otined t the Deprtment of Qulity, Stndrdiztion nd Certifiction of drugs (Ntionl University of Phrmcy) nd stndrdized in ccordnce with the requirements of the Stte Phrmcopoei of Ukrinin nd Europen Phrmcopoei [12]. (Iodomrin 200 (Berlin-Сhemie AG/Menrini Group, Germny), sodium perchlorte nd other chemicls otined commercilly Experimentl Animls Fortylinomle rts weighing etween 120 to 150 g otined nd housed in the Centrl Scientific Reserch Lortory, Ntionl University of Phrmcy, Ukrine. The norml stndrd rt chow nd tp wter provided d liitum during the experiment. Animls were stilized to cclimtize to niml house environment for one week efore commencement of the experiment. The study protocol ws pproved y Bioethics Commission of the Ntionl University of Phrmcynd the Generl ethicl niml experimenttion (Kyiv, 2001), consistent with the provisions of the Europen Convention for the Protection of Verterte Animls used for Experimentl nd Other Scientific Purposes (Strsourg, 1986) Experimentl procedures Induction of Hypothyroidism Experimentl hypothyroidism induced y sodium perchlorte in distilled drinking wter (1 g per liter) for 20 dys. Control (vehicle) rtsreceived equl volume of drinking wter [17]. Twenty dys fter induction of hypothyroidism tretment of nimls ws strting Experimentl Design Norml helthy rts used s norml control nd hypothyroidism-induced rts were rndomly llotted into four groups (n=10): Group 1: Norml untreted rts were given the norml sline 0.5 ml/100 g of ody weight dily orlly for 21 dys. Group 2: Hypothyroid untreted rts were given the norml sline 0.5 ml/100 g of ody weight dily orlly for 21 dys. Group 3: Hypothyroid rts were treted with queous extrct from duckweed Lemn minorfrond 0.5 ml/100 g of ody weight dily orlly for 21 dys. Group 4: Hypothyroid rts were treted with Iodomrin µg/kg dily orlly for 21 dys Histologicl nlysis After the experiment, nimls were scrificed y immedite decpittion nd the thyroid glnd removed. The loes of thyroid glnds fixed in 10 % formlin solution, desiccted in lcohols with incresing concentrtion nd emedded in prffin-celloidine. Sections 5 6 microns thick stined with hemtoxylin nd eosin [18].Algorithms of histologicl description ccording to O. K. Khmelnytsky were used for pthomorphologicl nlysis of thyroid glnd. To unify histopthologicl studies nd ssy chngesthe quntittive nlysis (height of folliculr epithelium nd folliculr outer dimeter, numer of thyrocytes in the folliculr wll) of glndulr tissuews conducted. Height of folliculr epithelium nd folliculr outer dimeter (µm) ws mesured using Toupcm Grnum progrm. Microphotogrphy of imges ws crried out using digitl video cmer Grnum DSM 310. Photos ws processed on computer Pentium 2,4 GHz using ToupView. 4

3 2. 3. Sttisticl Anlysis All dt expressed s Men±SEM were entered nd nlyzed using «Sttistic 6.0» sttisticl pckge with multiple comprisons. The Newmn-Keylstest ws used to determine difference etween the groups. Vlues of P<0.05 were considered s sttisticlly significnt [19, 20]. 3. Results nd discussion The results of the present study supply morphologicl nd quntittive dt out structure of thyroid glnd in norml rts nd out how its chnges fter sodium perchlorte induced hypothyroidism nd tretment with queous extrct from duckweed Lemn minor frond nd Iodomrin 200. Thus, thyroid glnd of control rts hs typicl folliculr structure. Sections of tissue were seprted from ech other y thin connective sept. Thyroid follicles hve vrious sizes, mostly round or slightly ovl shpe, with cler contours. The follicles on the periphery re lrger thn centrl ones. Intrcellulr spce of follicles is filled with moderte density, uniform oxyphilic colloid. Typiclly, colloid fill the entire cvity of the follicle, sometimes ws little crcked or ehind the wlls of the follicles. Thyroid epithelium lined wll folliclesis mostly cuic shpe, in lrge follicles modertely flttened. Folliculr thyrocytesis plced on the sement memrne in one row. The nuclei of cells re thick, round, centrlly locted. Extrfolliculr epithelium looks s the islnds nd it is locted etween follicles. Perifolliculr cpillry net hs norml lood supply (Fig. 1). Fig. 1. Thyroid glnd of control rts: norml folliculr structure of glndulr tissue ( 100); thyrocytes in the wll of the follicle hve cuic shpe, locted in one row; colloid fills the entire cvity, densely. Hemtoxylin-eosin The descried morphologicl structure of thyroid glnd corresponds to the norml, vile, modertely ctive tissue tht is confirmed y morphometric prmeters (Tle 1). The dt demonstrted tht fter 20 dys introduction of sodium perchlorte with drinking wter the distinct chnges in the structure of the thyroid glnd could e found. Thesechnges hve diffuse chrcter nd hyperplstic processes oserved in ll rts. Most nimls (67 %) follicles in different res were monomorphicnd densely djcent to ech other. Colloid in the folliculr lumen is sent. Thyrocytes incresed in sizewith shded cell oundries. The lumen of follicles is curved, reduced or sent. The nuclei of mny thyrocytes hve typicl shpe, the hypochromic one. The cytoplsm of the vst numer of cells is vcuolted. In the wlls of mny follicles re seen the vrying degrees of forming Sndersoncushions. Extrfollicul re pit helium hs sings of prolifertion, often with the formtion of smll nd micro-follicles (Fig. 2, Fig. 3,, ). The remining nimls restructuring process nd hyperplsi lso hs diffuse chrcter, ut ws mrked y certin vriility in size nd shpe follicles (Fig. 4). 5

4 Fig. 2. The thyroid glnd of rts fter dministrtion of sodium perchlorte: monomorphic follicles; dystrophy, multi rowed folliculr thyrocytes, the sence of colloid in the lumen of the follicles ( 200). Hemtoxylin-eosin Fig. 3. The thyroid glnd of rts fter dministrtion of sodium perchlorte, Snderson cushion ws formed in the wll of the follicle, mitosis of thyrocyte s nuclei ( 400); moderte focl sclerosis ( 200). Hemtoxylin-eosin Fig. 4. Thyroid glnd of rts fter dministrtion of sodium perchlorte: some moderte vriility in size nd shpe of follicles ( 100); loulr cpillry hyperemi of perifolliculr wll ( 200). Hemtoxylin-eosin Visul chnges in the stte of follicles correlted with chnges in their morphometric chrcteristics. The verge outside dimeter of follicles incresed y % (P<0.05), thyrocytes height incresed in 2.13 times (P<0.05), nd numer of thyrocytes in the wll of the follicle ecme more in 1.72 times (P<0.05) (Tle 1). 6

5 Thus, the long-term introduction of sodium perchlorte in rts resulted in significnt morphologicl nd morphometric chnges of the thyroid glnd of nimls tht correspond to the hypothyroid stte with n evident goitriferous effect. Tle 1 Morphometric prmeters chrcterizing the functionl stte of the thyroid glnd of rts, µm Group The outer dimeter of the follicle Prmeters Thyrocyte height Numer of thyrocytes in the wll of the follicle Control 37,33±1,74 5,51±0,25 9,06±0,26 Hypothyroid 47,96±1,87* 11,72±0,10* 15,56±0,80* Treted with AEDF 44,56±2,07* 7,41±0,73*/** 13,14±0,73*/** Treted with Iodomrin 44,91±1,75* 8,86±0,49*/** 13,62±0,53*/** Note: AEDF queous extrct from duckweed Lemn minor frond. Vlues re men ±SEM, n=10. * P<0.05 versus control group; ** P<0.05 versus hypothyroid group Structurl orgniztion of the thyroid glnd of 50 % of the nimls treted with queous extrct from duckweed Lemn minor frond showed its significnt functionl ctivity. Follicles, thyrocytes, colloid, extr folliculr epithelium y morphologicl chrcteristics reching the control group (Fig. 5, c, d). Morphometric prmeters chrcterizing the functionl stte of the thyroid glnd, in the whole group decresed, the outer dimeter of follicles y 7.65 % (P>0.05), ut the height nd size of thyrocytes in the wll of the follicle significntly decresed y % (P<0.05) nd % (P<0.05) (Tle 1). The rest 50 % of the rts retined signs of hypothyroidism nd goitriferous effect, though not of such severity s untreted group. Overll, the follicles in shpe nd size ecome more typicl, ut mny of them hd hypertrophy nd hyperplsi of folliculr thyrocytes, cells dystrophy (Fig. 5,, ). c Fig. 5. Thyroid glnd of rts treted with queous extrct from duckweed Lemn minor,, visile signs of hypothyroidism nd goitriferous effect ( 100, 200); c, d norml structure ( 100, 250). Hemtoxylin-eosin 7 d

6 Microscopic picture of the thyroid glnd in nimls treted with drug Iodomrin 200 in the course of sodium perchlorte introduction showed n increse of its functionl ctivity in 67 % of rts compred to untreted group. Most follicles rnged in size within cceptle stndrds, shpews typicl. Colloid ws not determined in ll follicles nd hd the different view. Folliculr thyrocytes in the wll of the follicles hd mostly high cuic shpe, rrnged in row. Dystrophy, expressive hypertrophy nd hyperplsi of the thyroid epithelium were oserved in reltively smll numer of follicles. Hyperplstic sings of extr folliculr epithelium reduced nd were focl (Fig. 6,, ). In 33 % of the rts, microscopic picture of the structure of thyroid glnd visully unchnged compred to the hypothyroid group: the sme diffuse monomorphic follicles, sence of colloid; dystrophy, hypertrophy nd hyperplsi of thyrocytes, focl hyperemi of perifolliculr cpillry net (Fig. 7,, ). Fig. 6. Thyroid glnd of rts treted with Iodomrin 200, glnd structure is close to norml ( 100); folliculr thyrocytes of most follicles hve typiclview, colloid density ( 200). Hemtoxylin-eosin Fig. 7. The thyroid glnd of rts treted with Iodomrin 200; diffuse monomorphic follicles ( 100); dystrophy, hypertrophy nd hyperplsi of folliculr thyrocytes, the sence of colloid in the lumen of the follicles ( 200). Hemtoxylin-eosin Such differences in the structurl orgniztion of the thyroid glnd ffected the verges prmeters tht chrcterized its functionl stte inside the group. The outer dimeter of the follicles reduced compred to control y only 6.79 % (P>0.05), the numer of folliculr thyrocytes in the wll of the follicles significntly decresed y % (P<0.05), the height of the cells lso significntly reduced y % (P<0.05) (Tle 1). 4. Conclusions In conclusion, the dt otined in this study suggest tht sodium perchlorte mrkedly inhiits the functionl ctivity of the thyroid glnds in rts, cusing morphologicl fetures inherent in the iodine 8

7 deficiency stte. Tretment of rts in experimentl hypothyroidism with queous extrct from duckweed Lemn minor frond showed corrective effects on the thyroid glnd structure, whichresulted in the decrese of the externl dimeter of the follicle, height nd numer of the wll of the follicle. Therefore, it my e useful for preventing or delying the development of hypothyroidism nd its complictions. Acknowledgments The uthors wish to thnk the stff of the Centrl Scientific Reserch Lortory of the Ntionl University of Phrmcy for the cre of the experimentl nimls throughout the period of this reserch work. References [1] Fdeev, V. V. (2012). Prolems of sustitution tretment of hypothyroidism: present nd prospects. Clinicl nd Experimentl Thyroidology, 8 (3), [2] Mncini, A., Rimondo, S., Di, C., Persno, M., Pontecorvi, A. (2013). Non-Thyroidl Illness: Physiopthology nd Clinicl Implictions. Current Topics in Hypothyroidism with Focus on Development. doi: /55644 [3] Ozpinr, A., Golu, M. S., Poppeng, R. H., Blount, B. C., Gillespie, J. R. (2011). Thyroid sttus of femle rhesus monkeys nd preliminry informtion on impct of perchlorte dministrtion. Lortory Animls, 45 (3), doi: /l [4] Pnkiv, V. І. (2012). Generl Thyroidology. Inter Journl of Endocrinology, 2 (42). Aville t: [5] Volkov, V. P. (2014). A new pproch to the evlution of the morphofunctionl stte of the endocrine glnds.universum, 9 (10). Aville t: [6] Bjj, J. K., Slwn, P., Slwn, S. (2016). Vrious possile toxicnts involved in thyroid dysfunction: A Review. Journl of Clinicl nd Dignostic Reserch, 10 (01), FE01 FE03.doi: /jcdr/2016/ [7] Krvchenko, V. I., Postol, S. V. (2011). Dynmics of the incidence of thyroid pthology in Ukrine. Interntionl Journl of Endocrinology, 3 (35), [8] Leedynets, N. V., Pruoch, O. M. (2012). Modern spects of the dynmics of endocrine pthology of the child popultion. Environment Helth, 3, [9] Filtov, S. V. (2010). Thyroid disese tretment trditionl nd non-trditionl wys.rypol Clssic, 256. [10] Jved, Z.,Sthypln, T. (2015). Levothyroxine tretment of mild suclinicl hypothyroidism: review of potentil risks nd enefits. Therpeutic Advnces in Endocrinology nd Metolism, 7 (1), doi: / [11] Mngge, H. (2014). Antioxidnts, inflmmtion nd crdiovsculr disese.world Journl of Crdiology, 6(6), 462.doi: /wjc.v6.i6.462 [12] Vldimirov, I. M. (2014). The stndrdiztion of pproches to trgeted serch of herl medicines for the tretment of thyroid disese.khrkiv, 440. [13] Vldimirov, I. M., Georgiynts, V. A. (2010). Anlyticl review of current thyroid-stimulting gents.phrmceuticl journl, 4, [14] Nikiphorov, L. A., Belousov, M. V., Furs, N. S. (2011). Study of mino cid composition of duckweed (Lemn minor L.).BulletenSiirskoyMedicini, 5, [15] Vldimirov, I. M., Georgiynts, V. A. (2013). Biologicl ctive sustnces of Lemn minor S. F. gry. Phrmceuticl Chemistry Journl, 47(11), [16] Mohmed, D. A. (2015). Histologicl Study of Thyroid Glnd fter Experimentl Exposure to Low Frequency Electromgnetic Fields in Adult Mle Alino Rt nd Possile Protective Role of Vitmin E. Journl of Cytology & Histology, 06 (06). doi: / [17] Yu, K. (2002). The Phrmcokinetics of Perchlorte nd Its Effect on the Hypothlmus Pituitry Thyroid Axis in the Mle Rt. Toxicology nd Applied Phrmcology, 182(2), doi: /tp [18] Merkulov, G. A. (1969). Course of pthologic-histologicl technology. Moscow: Medicine, 424. [19] Khlphyn, A. A.(2007). STATISTICA 6: Sttisticl nlysis of dt. Moscow:Binom-Press, 512. [20] Lpch, S. N., Chuenko, A. V., Bich, P. N. (2001). Sttisticl methods in iomedicl reserch using Excel.Kyiv: Morion,