Effect of Pectic Acid and?l-glucan on Prolactin Secretion by Ovine Pituitary Explants

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1 Iranian Int. J. Sci. () Effect of ectic Acid and?l-glucan on rolactin Secretion by Ovine ituitary Explants Houri Sepehri, oya Zorahi and Ali, Haeri ouhani Department of Bioloy, Faculty of Science, University of Tehran, Iran Abstract ecent studies have indicated that several plant extracts reputed to be lactoenic are capable of inducin -casein synthesis in mammary epithelial cells. This enhancement is mediated throuh a stimulation of prolactin release from lactotropes in anterior pituitary. Therefore it is admitted that these extracts are capable of stimulatin prolactin secretion from hypophysis. A partial purification of these extracts, revealed that their active compounds are pectin in most cases and - lucan in other cases. In this work, the effect of various concentrations of both pure pectic acid and -lucan on prolactin secretion from ewe hypophysis framents is investiated. It is shown that pectic acid in the 5-5?(l rane concentration and -lucan in the -4?(l rane concentration are capable of stimulatin prolactin secretion sinificantly (<.5, <.), from hypophysis explants. Keywords: ectic Acid,?W-Glucan, rolactin Secretion, Ovine ituitary Explants Introduction lant extracts are traditionally used in various societies for their pharmacoloical properties. Some plants which are reputed to be lactoenic have the capacity to enhance milk secretion in lactatin women. ecent studies in this field have indicated that these extracts induced -casein synthesis in rat mammary land in vivo (Sawadoo & Houdebine, 988; Sawadoo et al., 988). It was observed that this effect is mediated throuh an effect on prolactin () secretion (Sawadoo & Houdebine, 988) (Sawadoo & Houdebine, 988; Sawadoo & Houdebine, 989). The extracts proved to stimulate secretion of rowth hormone and cortisol in addition to, in vivo (Sawadoo & Houdebine, 988). It is admitted that some plants in Malvaceae, inaceae, Euphorbiaceae and Umbelliferae families have

2 ÎÍÍ H. Sepehri et al., IIJS, (Biol.), the lactoenic capacity (Sawadoo & Houdebine, 988). Chemical analysis of these extracts revealed that active compounds are polysaccharides rich in pectins in most cases and rich in -lucan in other cases (Sawadoo & Houdebine, 988,989). Both pure pectic acid and -lucan showed a stron capacity to trier and GH secretion in experimental animals when injected intravenously (Sawadoo & Houdebine, 988). From these data, it was concluded that pectic substances and -lucan are the active lactoenic compounds existin in plants, and that their effects are mediated throuh the secretion of and possibly of GH and cortisol. In vitro experiments have shown that pectic acid is able to induce the secretion of, GH, H and -endorphin from rat hypophysis (Sawadoo, 988). Moreover, this compound was shown to stimulate -casein secretion from rabbit mammary land in (Sawadoo & Houdebine, 988). The mechanism of action of the plant extracts is not yet known. Whether the lactoenic substances stimulate secretion in vivo throuh a direct action on pituitary cells or throuh an indirect effect on hypothalamus hypophysis axis is not known. To et an answer for this question, the effect of various concentrations of lactoenic substances on secretion from incubated ewe hypophysis framents was investiated. esults confirm those previously obtained in a preliminary experiment (Sepehri et al.,99). In this study, the optimal concentrations of lactoenic substances which have maximal stimulatory effects were determined. Materials and Methods Chemical ectic acid (poly-d-alacturonic acid) was from Fluka and - lucan from Sima. These materials were dissolved in.7% NaCl (ph=7.8) after stirrin for hour at C. The ph of these solutions ( m which was. and 5.6 respectively, was neutralized by.m NaOH. Since above the mentioned compounds are only partially soluble in water, the solutions were centrifued at. for minutes and only the supernatant was added to the incubation Medium 99 at ph 8.. Animals: Mature ewes which have not been prenant were uses ( months old,.5-6.5k).

3 Effect of ectic Acid And?s-Glucan on ÎÍÎ ººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººº Incubation of hypophysis framents hypophysis were harvested immediately after the sacrifice and transported to the laboratory in culture medium. The land was cut into framents of mm with a razor blade. About m of tissue were spread on each stainless rids and incubated in Medium 99 ml/ rid in 5 mm culture dishes at 7 C and under 95% O, 5% CO atmosphere. In this experiment, the samples were first incubated in a medium with none of the compounds for minutes (preincubation) to allow the dischare of lactotropic cells, and to determine the basal level of secretion in each dish. The medium was collected and a fresh medium was then added without (contro or with the above-mentioned compounds at various concentrations and incubation was pursued for one additional hour (first incubation). After collectin the media, the samples were incubated for one additional hour in conditions similar to the first incubation (second incubation). The medium was then collected and kept frozen at C until prolactin measurements. Sterile condition was not necessary in this work since incubation time was short. Nevertheless, all the culture steps were carried out in a culture room under O flux to minimize contamination. rolactin measurement was measured in the culture medium usin a radioimmunoassay (IA) in duplicate samples. In all cases, the hormone concentration was measured in incubation media of each dish after the pre-, the first and the second incubation. The hormonal content of the preincubation medium reflects the basal secretion level of each hypophysis, and it depends on the amount and the quality of the tissue present in each dish. The content of the first and second incubation media reflects of the added compounds. In each experiment, ten dishes were used as control and ten dishes with added compounds for each assay. Each result is the mean ± SD of ten dishes. As expected, the concentration of in the first and second incubation media of the control dishes was lower than in the preincubation medium. This drop in the second incubation was slihtly more intensive. Therefor, the level in the first and second incubation media of control dishes was taken as a

4 ÎÍÏ H. Sepehri et al., IIJS, (Biol.), reference (% secretion) and levels in each roup of dishes were compared to it. esults Effect of pectic acid on secretion Framents of hypophysis were incubated for the sequential in culture media with various concentrations of pectic acid (5-4 µ. reliminary experiments have shown that some pectic derivatives such as pectic acid are capable of stimulatin release (Sepehri et al., 99). The present results confirm this fact. The maximum stimulation was in the rane of 5-5 µl concentrations. ectic acid was less stimulatory at concentrations hiher than 5 µl. These results were observed in the first and second incubations in a similar way (Fis. & ). Fiure shows that various concentrations of pectic acid stimulate secretion in a sinificant manners (p<.5) at the concentration of 5 and µl. In the dishes containin variable amounts of lactotropic cells, secretion level was different. It was therefore difficult to compare the levels, directly. For this reason, the results are expressed as the percentae, of stimulation with respect to the control (Fi. ). Fiure, shows that pectic acid stimulated secretion (from 5% to 78%). Effect of?ª-lucan on Secretion Various concentrations of -lucan (5-5 µ added to the incubation media of hypophysis framents showed a clear effect on secretion (Fis. & 4). The maximum stimulation was in the - 4 µl rane of concentration. At concentrations more than 4 µl, -lucan had less stimulatory effect. These results were observed in the first and second incubations in a similar way. esults in Fiure shows the stimulatory effect of -lucan on secretion, which is sinificant (<.5 or <.) at concentrations of to 5 µl. Fiure 4 which shows the percentae of secretion in various concentrations of -lucan, indicates that secretion was stronly stimulated (from % to %).

5 Effect of ectic Acid And?s-Glucan on ÎÍÐ ººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººº Control [5] [5] [] ectic acid (?? Control [5] [] [5] ectic acid (?W Control [] [5] [4] ectic acid (? Fiure Effect of various concentrations of petctic acid on secretion from ewe pituity framents in the first?œand second?œ incubations. Each result is the mean? SEM of ten dishes.? p<.5

6 ÎÍÑ H. Sepehri et al., IIJS, (Biol.), ) n tio la u tim 5 s (% 5 [] [5] [5] [] [5] [] [5] [] [5] [4] ectic acid (?ĝ Fiure Effect of pectic acid on secretion in the first?ó and second? incubations. esults are expressed in percentae in respect to controls. Control [5] [5] []? -lucan (? Control [5] [] [5]? -lucan (? Control [] [5] [4]? -lucan (?! Fiure Effect of various concentrations of?+-lucan on secretion from ewe pituitary framents in the first?k and second?k incubations. Each result is the mean? SEM of ten dishes.? p<.5;?? p<. Control [45] [5]? -lucan (?!

7 Effect of ectic Acid And?s-Glucan on ÎÍÒ ººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººº 5 ) n tio la u 5 tim s (% 5 [] [5] [5] [] [5] [] [5] [] [5] [4] [45] [5]? -lucan (?h Fiure 4 Effect of?ø-lucan on secretion in the first? and second?x incubations. esults are expressed in percentae in respect to controls. Conclusion The mechanism of action of lactoenic plant extracts on secretion is unknown. ectic substances and -lucan were shown to stimulate, in vivo,, GH and possibly cortisol secretion, to induce -casein synthesis in mammary land. The data reported here clearly show that these compounds stimulate secretion when added to ewe hypophysis framents. It was therefore concluded from these data that these compounds act as unspecific secretaoues throuh a direct effect on secretion on lactotropic cells of pituitary. revious work carried out in vivo has shown that GH secretion was also stimulated by these compounds, whereas it was never stimulated in vitro. One possible explanation is that GH releasin factor (GHH) rather than GH secretion is directly affected by the plant extracts in vivo (Sepehri,99). Alternatively, GH secretion may be too low in vitro to be sinificantly stimulated by the added compounds. ectic substances and -lucan exhibit rather limited homoloies of structure and it seems that they act throuh different mechanisms. However, one point remains strikin. Both polymeric compounds studied here are direct precursors of substances named elicitors which reconize specific

8 ÎÍÓ H. Sepehri et al., IIJS, (Biol.), receptors on plant cells, act as veetal hormones and induce the expression of certain enes and defensive responses (yan, 987). No receptor has been identified in the cells of hiher vertebrates for these compounds. A possible hypothesis is that pectic substances and - lucan have some structural homoloies with extracellular matrix of mammalian cells and the active compounds of plant extracts miht affect cellular secretion by bindin to these receptors. It remains of find which cellular receptors are involved in which part of cell and by which mechanism of action the stimulatory effects of active compounds of plant extracts on secretion are mediated Acknowledments The Tehran University which provided financial support of this study is fully acknowleded. The authors wish to thank Dr. K. Bahar who provided facilities to perform radioimmunoassays. eferences yan, C.A., (987) Oliosaccharide sinallin in plants, Ann. ev. Cell. Biol.,, Sawadoo,., (987) These de Doctorat d Etat, Universite de Tours, France. Contribution a l etude des plantes medicinales et de la pharmacopee traditionnelle africaine. Sawadoo,., and Houdebine,.M., (988) Indentification of the lactoenic compound present in beer, Ann. Biol. Clin., 46, 9-4. Sawadoo,., and Houdebine,.M., (988) Induction de la synthese de casin-?ódans la lande mammaire de rates traitees par des extraits de plants,c.. Acad. Sci. (III), 6, Sawadoo,., Houdebine,.M., Gueuen, J., and Berot, S. (988) Mise en evidence des propriet alactoenes de divers extraits de raine de coton, Bull. Med. Trad Sawadoo,., Houdebine,.M., Thibault J.F., and ouau X. (988) Mise en evidence d une activate lactoene dans des extraits veetaux,bull. Med. Trad.,, 9-. Sawadoo,., Houdebine,.M., Thibault J.F., ouau X., and Ollivier- Bousquet M. (988)Efect of pectic substances on prolactin and

9 Effect of ectic Acid And?s-Glucan on ÎÍÔ ººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººººº rowth hormone secretion in the ewe and on the induction of casein synthesis in the rat, eprod. Nutr ; Develop. 8 (A), 9-. Sawadoo,., Sepheri, H., and Houdebine,.M., (989) Mise en evidence d une facteur stimulant la secretion de prolactin et de l hormone do croissance dans les dreches de brasserie, eprod. Nutr. Develop., 9, Sepehri, H., (99) effet de l acide pectique,la fraction active obtenue des extraits de plantes lactoenes sur la secretion de la prolactine,?í-endorphine et luteinizin hormone (H) par l hypophyse isolee de rate, J.Sci. Univ. Tehran,, 5-4. Sepehri, H., (99) a synthese de?ã-caseine par l acide pectique obtenu des extraits des plantes lactoenes, J.Sci. Univ. Tehran,, 6-7. Sepehri, H., Houdebine,.M., and Kann, G., (99)ouvoir lactoene pontentiel de quelques extraits de plantes Iraniennes, Cahiers Aricultures., 5-9. Sepehri, H., enard, C., and Houdebine,.M., (99)?-lucan and pectine derivatives stimulate secretion from hypophysis In Vitro, roc. Soc. Exp. Biol. Med. 94, 9-97.