Induction of Growth Hormone Release by D. Y. Jung H. Ha Pueraria thunbergiana BENTH. C. Kim rgnlBasic Original

Abstract 10±30 min, while the Cmax value was increased by approximately 12-fold compared to the control group 198.2  25.0 pM) and the

Puerariae Radix PR), Puerariae Flos PF), and Puerariae Surculus AUC0±45 was increased to 10 times the level of the control group

PS) as well as their constituents were tested for induction of rat 10,840.9  845.5 min. pM). On the other hand, Tmax for the HPS

growth hormone rGH) release by both rat pituitary cell culture was 60 min, while Cmax was increased approximately to 5.8 fold

and in vivo experimentation in order to develop them to novel compared to control 244.1  36.4 pM). Cmax for was drugs. Through a calibration curve of the rGH released by addi- 1,028.6  502.7 pM, that is, approximately 5.2 times as high as tion of rat growth hormone-releasing hormone rGHRH) to rat the control level. However, 20 g/kg) was of no sta- pituitary cells, the 70% ethanol extracts of PR and PS increased tistical significance. Therefore, we suggest that the HPS and rGH release by about 1.6 and 1.7 times as high, respectively, as puerarin act either on GH secretagogue receptors or on GHRH re- the control group 264.6  13.6 pM). However, each puerarin ceptor of somatotrophin as possible agonists or an inhibitor on type as a representative constituent of PR in Korea Pharmacopeia somatostatin receptor to release rGH, respectively. KP) and tectorigenin and an important ingredient of PF were twice as effective as in the control group. The acid hydrolysate Key words

86 of Puerariae Surculus HPS) increased rGH release concentra- Growth hormone GH) ´ Growth hormone releasing hormone tion-dependently, and its EC50 was approximately 10.4 g/ml. GHRH) ´ Growth hormone secretagogue GHS) ´ Pituitary cells ´

The Tmax value for rGH after injection of 20 g/kg of rGHRH was Puerariae Radix ´ Puerariae Surculus ´ Tectorigenin

Introduction PS) is the young leaf of Pueraria thunbergiana BENTH., and is re- garded as a tonic and has similar usage to Astragali Radix [1] and Usage of traditional herbal medicine depends on their variable Ginseng Radix in complementary and alternative herbal medi-

efficacy. We investigated the effect of Pueraria thunbergiana cine. Pueraria thunbergiana BENTH. has been reported to contain Downloaded by: University of Washington at Seattle. Copyrighted material.

BENTH. Fabaceae), one of the most popular edible herbs in north- not only plenty of starches over 20%) but also , isofla- eastern Asia, on GH release. Puerariae Radix PR) is the dried root vone glycosides daidzin and puerarin), triterpenoidal sapogen- of Pueraria thunbergiana BENTH. Fabaceae), a perennial herb ols sophoradial as well as soyspagenol A and B), b-sitosterols, al- that has been used as an antipyretic, sedative, hypotensive herb lantoins, and other compounds [2]. Especially puerarin is consid- amongst other applications. Puerariae Flos PF) is the flower of ered as a reference and identifying ingredient for PR by Korea Pueraria thunbergiana BENTH., and used for curing hangover in Pharmacopeia KP). Tectorigenin, an shown in Fig. 2, complementary and alternative medicine. Puerariae Surculus was isolated and purified from PF. The storage period of PF was

Affiliation Drug Research and Development Team, Korea Institute of Oriental Medicine, Seoul, Korea

Correspondence C. Kim, Ph. D. ´ Drug Research and Development Team ´ Korea Institute of Oriental Medicine ´ 129-11 Chungdam-dong, Kangnam-ku ´ Seoul, 135-100 ´ Korea ´ Phone: + 82 2) 3442-1994-223, + 82 2) 3442-2120 ´ Fax: + 82 2) 3442-0220, + 82 2) 3442-1030 ´ E-Mail: [email protected]

Received 8 October 2002 ´ Accepted after revision 19 August 2003 Bibliography Horm Metab Res 2004; 36: 86±91  Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0018-5043 DOI 10.1055/s-2004-814216 above, this study tested the effects of Pueraria thunbergiana 2.0 BENTH. and its constituents on GH release.

1.5 Materials and Methods

Preparation of samples 1.0 Puerariae Radix Kim-cheon, Korea; specimen number in our

rGH (nM) herbarium: KIOM-99-3-0019) and Puerariae Flos China; speci- men number: KIOM-99-3-0021) were purchased from Korea Or- 0.5 iental Medicinal Herb Association Seoul, Korea); Puerariae Sur- y = 0.0013x + 0.1292 culus specimen number: KIOM-99-3-009) was collected from R2 = 0.91 Yangsu-ri area Yang-pyeong, Korea) in mid May, 1999. Samples

0.0 were dried, cut finely, and dipped in 70% ethanol at room tem- Basic Original 0 200 400 600 800 1000 perature for 7 days, and then filtered to give 70% ethanol ex- rGHRH (nM) tracts. The filtrate was concentrated using a rotary evaporator Fig. 1 Representative standard curve of rGHRH nM) on concentra- and then freeze-dried. The acid hydrolysate of PS HPS) was pre- tions of rGHs ng/ml) released in rat pituitary cells. Each point indicates pared by hydrolyzing the dried extract of PS with 1 N HCl 1 g/3 mean value of triplicate and each bar represents standard error of the ml) at 1008C for 2 hours, neutralizing with 10 N NaOH, then mean SEM). freeze-drying. Tectorigenin as an indicator of freshness of PF has already been isolated and purified from the ethanol extract of PF in our laboratory earlier [8]. The other constituents of Pueraria thunbergiana BENTH., , , ononine, and critical in activity of PF. The level of freshness of PF indicated a daidzin Fluka, Buchs, Switzerland), and puerarin as a reference high concentration of tectorigenin, which was converted to kak- ingredient of PR in KP, , , daidzein, kalide after over five years' storage [8]. and Sigma Chem. Co., St. Louis, U.S.A.) were pur- chased and dissolved in 0.1% DMSO Sigma Chem. Co., St. Louis,

GH is a peptide hormone consisting of 191 amino acids MW U.S.A.) prior to use. Plant hormones such as giberrelic acid GA3), 21,500) elaborated by specific somatotropes in the anterior pitui- indoleacetic acid IAA) and 2,4-dichlorophenoxyacetic acid 2,4- tary gland. Synthesis and release of GH by the specific somato- D: Gibco BRL, N.Y., U.S.A.) were also used with the same method tropes is modulated either by GHRH as a stimulator to the ability as described earlier. of somatotropes to release GH or by somatostatin as an inhibitor to GH release by the somatotropes. Somatostatin has inhibitory GHinduction test in pituitary cell culture 87 effects on the secretion of other hormones, including insulin A male Sprague-Dawley SD) rat 3±4 weeks old; Daehan Bio- and glucagon. The secretion of GH is controlled by the balance link, Eum-seong, Korea) was acclimatized for 1 week and then of the stimulatory and inhibitory hypothalamus peptides. GH is decapitated to isolate pituitary therefrom. The isolated pituitary mainly used for the treatment of dwarfism; however, recently, it was washed with cold HBSS Sigma Chem. Co., St. Louis, U.S.A.) is variously applicable to the prevention of chronic degenerative incubated with 0.2% hyaluronidase and 0.2% collagenase Sigma, diseases [3]. In addition, a newly discovered peptide, ghrelin, is Chem. Co., St. Louis, U.S.A.) at 378C for 15 min, which was repeat- synthesized in both the hypothalamus and the stomach, and is ed 3 times [9±11]. The isolated and separated pituitary cells known to be an endogenous GH secretagogue GHS) [4]. Other were incubated at 378C for 3 days in a 5% CO2 incubator with

synthetic peptide GHSs are GH-releasing peptides GHRPs), DMEM medium Gibco BRL, N.Y., U.S.A) containing 2.5% FBS and Downloaded by: University of Washington at Seattle. Copyrighted material. GHRP-1, GHRP-2, GHRP-6, ipamorelin, ep51 and Try-Ala-hexare- 10% horse serum. The pre-incubated pituitary cells were collect- lin [5], as well as non-peptide GHSs MK0677 and L-692585 [6]. ed, washed with cold HBSS, and diluted to 7.5 ” 104 cells/ml. Var- Neurotransmitters regulating the release of GHRH and somato- ious concentrations of rGHRH Bachem, Budendorf, Switzerland) statin also include serotonin, g-aminobutyric acid, dopamine and 0±1,000 nM) were added and the concentration of rGH released others, and stimulate GH secretion through interaction of a2- was analyzed to make a standard calibration curve. Each one of adrenergic receptors to release GHRH. Neuropeptides, including the ethanol extracts of PR, PF, and HPS corresponding to 1 mg pituitary adenylate cyclase activating peptide and neurotensin, of dried herb/ml) or each one of various single constituents in- increase in GH release, while calcitonin- and corticotropin-re- cluding tectorigenin isolated from PF 10 g/ml) were incubated leasing hormones reduce it [7]. GH release in humans decreases in 1 ml of the pituitary cell suspension in 24 well plate Becton with age, as does GH release through GHRH stimulation. GH con- Dickinson Labware, N.J., U.S.A.) at 378C for 15 min as described centration is at its maximum during adolescence, but after age above, respectively. The cultured medium of the cells was centri- 60, the somatopause starts with a decline in GH release to one fuged at 14,000 rpm for 10 min Centrifuge 5402, Eppendorf-Ne- quarter of the level in the twenties and causes the deficiency of theler-Hinz GmbH, Germany) and the supernatant was recov- GH. Further, obesity exceeding 15% of an ideal body weight may ered and stored at ± 208C until analysis of rGH. Dose-dependen- inhibit GH release. GH release is stimulated by hypoglycemia, ex- cy of rGH release was performed in puerarin, tectorigenin, and ercise, stress and the heavy protein intake, and is increased by the HPS at 2.5±20.0 g/ml each. , progesterone, , and thyroid hormones. Since GH release is regulated by various factors as described

Jung DY et al. Induction of Growth Hormone Release ´ Horm Metab Res 2004; 36: 86±91 OH OH OH O OH O OH OH O HO OMe OO O OH HO HO OH O OH O Tectorigenin Puerarin Genistein OH OH O OCH3 OH O O rgnlBasic Original

HO O HO HO

O O O Daidzein Coumesterol Biochanin A

OCH3 O OCH3 OH O O HO

OO O HO OH

O O OH OH HO OH Apigenin Formononetin Ononine

OH O O H OH OH O 88 HO HO O O O OO HO O OH OH OH O H3C CH OH 2 OH OOH OH OH Genistin Daidzin Gibberellic acid (GA3)

O O Downloaded by: University of Washington at Seattle. Copyrighted material.

O OH OH

O O

NH 2,4-dichlorophenoxyacetic acid (2,4-D) 3-indole acetic acid (IAA) Fig. 2 Structures of the components analyzed for GH release.

rGHrelease in vivo 50 g/kg), and tectorigenin 20 g/kg) were each administered A male SD rat 250±300 g; Daehan Biolink, Eum-seong, Korea) through the jugular vein, and 0.5 ml of blood was then collected was anesthetized with 50 mg/kg pentobarbital i.p. Hanlim every 10 min from the tail vein. The plasma was recovered after Pharm. Co., Seoul, Korea). rGHRH 10 or 20 g/kg), HPS 20 or centrifugation of the blood at 3,000 rpm for 15 min and then

Jung DY et al. Induction of Growth Hormone Release ´ Horm Metab Res 2004; 36: 86 ±91 Fig. 3 rGH concentrations % of controls) 600 ** induced by various herbal extracts and their Ctrl constituents on pituitary cells n = 3). PR, 500 Extracts Puerariae radix; PS, Puerariae surculus; HPS, Compounds acid hydrolysate of PS; T, tectorigenin isolat- Plant growth hormones 400 ed from Puerariae flos; P, puerarin; Ge, gen- istein; De, daidzein; C, coumestrol; B, bio- chanin A; A, apigenin; F, formononetin; O, 300 ononine; Gi, genistin; Di, daidzin; GA3, gib- % of Control ** ** berelic acd; 2,4-D, 2,4-dichlorophenoxy- 200 aceticacid; IAA, indoleacetic acid. **: p < 0.01 vs. control. 100

0 Ctrl PR PS HPS T P Ge De C B A F O G D GA3 2,4-D IAA rgnlBasic Original

3.0 Puerarin Tectorigenin ** 2.5 HPS

2.0

1.5 **

rGH (nM) 1.0 * ** ** 0.5 ## ## ## 0.0 0 10203040506070 Drug Concentration (µΜ)

0 5 10 15 20 25 Fig. 5 Time course of rGH levels in plasma after an i.v. dose of rGHRH 89 HPS Concentration (µg/ml) 10 g/kg or 20 g/kg), HPS-20 acid hydrolysate of Pueraiae Surculus: HPS, 20 g/kg), HPS-50 HPS, 50 g/kg), puerarin 20 g/kg), and tec- Fig. 4 Dose-response curves of puerarin as a reference ingredient of torigenin 20 g/kg), each. Data points indicate the mean values PR in KP, HPSacid hydrolysate of Pueraiae Surculus), and tectorigenin n = 4). isolated from Puerariae Flos on the concentration of rGH released in pi- tuitary cells. Data points represent the mean values of triplicate with bars indicate SEM. EC50 values for puerarin, tectorigenin and HPS were 7.6 M, 4.3 M, and 10.4 g/ml. **: p < 0.01; *: p < 0.05; #: 0.05 < p < 0.1 vs. control. also calculated using the calibration curve plotted by various concentrations of standard rGH. Downloaded by: University of Washington at Seattle. Copyrighted material. Statistical analysis stored at ± 20 8C for rGH analysis. All drugs were prepared with a Data are represented as the mean  SEM; p < 0.05 was taken as saline solution. Saline solution was administered in the equiva- statistically significant, determined by ANOVA or Bonferroni's lent amount to the control group. multiple-comparison method. rGHassay The rGH concentrations in both plasma and the pituitary cell cul- Results ture medium were analyzed with a radioimmunoassay kit RIA; Amersham Pharmacia Biotech, U.K.). The concentration of stand- A standard calibration curve of rGH ard rGH was 72.7±4,545.5 pM. GH-antibody complexes were ob- The concentration of rGH released by the addition of rGHRH 0± tained by a competitive reaction of anti-GH antibodies between 1,000 nM) was in proportion to the concentration of rGHRH and constant amount of [125 I]-labeled GH and various concentrations plotted as a calibration curve of Y = 0.028 X + 2.843 X: rGHRH of the standard GH or samples. The immunocomplexes formed concentration, Y: rGH concentration, r2 = 0.91, p < 0.01) n = 3) were precipitated by addition of second antibodies, and any ex- Fig.1). rGH release was increased by approximately 1.2 times cess labeled GH removed. The rest of radioactivity in the precipi- and 6 times the level in the control group by addition of rGHRH tates was counted with a g-counter Wallac, Turku, Finland) 0.1 M and 1 M, respectively Fig.1). [12,13]. The rGH concentration in the medium or plasma was

Jung DY et al. Induction of Growth Hormone Release ´ Horm Metab Res 2004; 36: 86±91 rGHinduction test in pituitary cell culture release is decreased and hGH plays an important role in the acti- The rGH concentrations released by the ethanol extracts of PR vation of immunocytes [14±16]. The reason why pituitaries of and PS corresponding to 1 mg of dried herb/ml) were increased 4±5 weeks old rats were isolated for the primary cell culture to approximately 1.6 times and 1.7 times the control group val- was that these young animals were very active on GH release. In ues without addition of rGHRH 264.6  13.6 pM), respectively. this study, our aim was to demonstrate that GH release was in- However, these differences were not statistically significant. The duced by activating GHRH, which plays an important role in the rGH concentration released by HPS was increased to approxi- induction of hGH release in vivo Fig. 5) or by addition of tradi- mately 5.4 times control group values p < 0.001) Fig. 3). Daid- tional herbal medicines [1]. Herbal medicines used in the study zein, genistein, apigenin, biochanin A, coumestrol, formononetin, were hardy herbaceous plants that can be relied on for food in ononine, daidzin, and genistin had no statistically significant ef- famine years [2]. They have less toxicity, contain various consti- fect on rGH concentration compared with the control group as tuents, and are thus broadly applicable. Even though rGH release shown in Fig. 2, while puerarin as a reference ingredient of PR in of PR and PS was about 1.5 times as high as controls, HPS induced KP and tectorigenin isolated from PF increased rGH release to ap- rGH release by approximately 5.4 times in vitro Fig. 3). There-

rgnlBasic Original proximately twice the level of the control group p < 0.01; see fore, we suggest that aglycones of active constituents or hydroly-

Fig. 3). Plant growth hormones, GA3, 2,4-D, and IAA, also had no sates of PS have an excellent effect on rGH release. However, pos- effect on rGH release Fig. 3). sible activities of other compounds formed through the weak acid hydrolysis described above cannot be ruled out. Another Puerarin as a reference ingredient of PR increased rGH release possibility is that increased ionic strength due to high concentra- concentration-dependently, but the increase in rGH release was tion of the salt formed through acid hydrolysis and neutraliza- saturated at 47.4 M n = 3; see Fig. 4). As shown in Fig. 4, puerar- tion might encourage the destruction of cell membranes, result- in induced the highest rGH release at 23.7 M p < 0.01). Tectori- ing in a dramatic increase in GH release. This possibility can be genin isolated from PF increased rGH release concentration-de- clarified by detection of cytotoxicity or measurement of concen- pendently and showed the highest rGH release at 16.7 M trations of other pituitary hormones such as TSH, ACTH, LH, FSH,

p < 0.05), and its EC50 was 4.3 M. HPS induced rGH release to and prolactin. Cytotoxicity was not likely since the correspond- approximately 2±9 times control levels depending on the con- ing levels in pituitary cells were not elevated by the addition of centration of herbal extracts, and the difference in rGH concen- HPS during incubation using the LDH cytotoxicity detection kit trations released at 10 g/ml and 20 g/ml compared to the con- Roche Diagnostics GmbH, Germany) in our preliminary experi- trol group were statistically significant p < 0.01). Especially HPS ment. Unfortunately, concentrations of other pituitary hormones induced rGH release corresponding to 1 M of rGHRH at 20 g/ were not measured, so whether rGH release induced by HPS is

ml, and its EC50 was 10.4 g/ml. specific is not clear.

rGHrelease in vivo GH release is regulated by GHRH as a positive regulator and by

90 After 10 or 20 g/kg of rGHRH was administered intravenously, somatostatin as a negative regulator. In the present study, the concentration of plasma rGH was increased by over 2.7 nM rGHRH increased rGH release concentration-dependently consistently within 10±30 min after administration, and then Fig.1), but the linearity of rGH release was no longer applicable

rapidly decreased again Fig. 5). Cmax of plasma rGH was 12 times above 2 M of rGHRH. There are many possible reasons, how-

as high as the control group 198.2  25.0 pM), and AUC0±45 for ever; one of them might be a complicated regulatory mechanism the rGHRH group increased to 10 times that of the control group of GH release and based on recent reports, whereby GH release 10,840.9  845.5 min.pM) p < 0.01, see Fig. 5). When 20 g/kg was induced by binding GHRH with a GHRH-receptor or binding

HPS was administered intravenously, Cmax for plasma rGH oc- GHS with a GHS-receptor [17,18] or through inhibition of so- curred approximately 60 min after the dose and around the matostatin binding with its receptor.

peak area, AUC30±90, it was 5.8 times as high as controls. Cmax of Downloaded by: University of Washington at Seattle. Copyrighted material. plasma rGH after administration of the HPS at high concentra- Several constituents of Pueraria thunbergiana BENTH. were test- tion 50 g/kg) was comparatively lower than that of 20 g/kg, ed for their stimulatory effects on GH release. Although studies

while AUC30±90 at peak concentration was increased to 1.7 times on constituents of PS are rare, both PR and PF contain daidzein, the control group value p < 0.05, see Fig. 5). In the case of a biochanin A, genistein, apigenin, formononetin, ononine, genis- 20 g/kg injection of puerarin, as a reference ingredient of PR in tin, and daidzin, whose effects on GH release were not signifi-

KP, its Tmax and Cmax of plasma rGH were approximately 30 min cantly different compared to the control group Fig. 3). However, and 5.2 times control values. When tectorigenin 20 g/kg) was only puerarin in PR and tectorigenin in PF were identified as ac-

administered intravenously, its Tmax was 45 min and Cmax level tive compounds affecting GH release, and their effect on GH re-

and AUC30±60 at peak concentration were increased to approxi- lease was saturated at 2.5±20 g/ml Figs. 3, 4). Puerarin as a re- mately twice and 1.2 times control group values, respectively. ference ingredient of PR in KP had the greatest effect on GH re- However, these were not statistically significant Fig. 5). lease among these constituents and induced GH release to twice the level of the control group Fig. 3). Even though some of herbal medicine constituents increased rGH release, plant growth hor- Discussion mones themselves did not affect the growth of animal cells but that of plant cells. Thus, a role for each different constituent was Both decrease in hGHRH release and increase in somatostatin demonstrated in the growth of animal and plant cells. As shown tone with aging result in a reduction of hGH release. When aged in Fig. 4, some of puerarin-treated groups showed statistical sig- humans are administered hGH for a period of 6 months, hGH nificance in the concentration of rGH released in pituitary cell

Jung DY et al. Induction of Growth Hormone Release ´ Horm Metab Res 2004; 36: 86 ±91 culture; their activity was low compared to HPS. Further, HPS 4 Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghre- showed higher rGH release activity than PS extract in vitro. lin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999; 402 6762): 656 ±660 Therefore, we suggest that HPS contains other active constitu- 5 Bowers CY, Momany F, Reynolds GA, Chang D, Hong A, Chang K. Struc- ents apart from puerarin and tectorigenin that induce rGH re- ture-activity relationships of a synthetic pentapeptide that specifical- lease; a the study on this topic is now in progress. ly releases growth hormone in vitro. Endocrinology 1980; 106 3): 663±667 6 Gertz BJ, Barrett JS, Eisenhandler R, Krupa DA, Wittreich JM, Seibold JR, Plasma rGH concentration was increased to 12 times control lev- Schneider SH. Growth hormone response in man to L-692,429, a novel els within 10±30 min after rGHRH administration, and then ra- nonpeptide mimic of growth hormone-releasing peptide-6. J Clin En- pidly returned to basal levels. This showed similar results to the docrinol Metab 1993; 77 5): 1393±1397 7 Argente J, Pozo J, Chowen JA. The growth hormone axis: control and other report that presented its results as Tmax was at 30 min and effects. Horm Res 1996; 45 Suppl 1): 9±11 C was about 20 g/l after an i.v. bolus dose [19]. However, T max max 8 Kim C, Shin S, Ha H, Kim JM. Study of substance changes in flowers of values of tectorigenin and HPS were 45 min and 60 min after ad- Pueraria thunbergiana BENTH. during storage. Arch Pharm Res 2003; ministration, respectively. These differences in Tmax may be ex- 26 2): 210 ±213 9 Bieglmayer C, Spona J, Schoeder R. Modulation of LH-RH stimulated plained by active compounds on GH release acting through the Basic Original gonadotropin release by progestagens and 17 beta- in pri- drug itself or its metabolites. Under in vivo conditions, tectori- mary pituitary cell culture. Endocrinol Exp 1980; 14 3): 171±182 genin did not show the same rGH release effect as that in vitro 10 Sliutz G, Speiser P, Schultz AM, Spona J, Zeillinger R. Agnus castus ex- and the HPS showed less induction of rGH release than rGHRH. tracts inhibit prolactin secretion of rat pituitary cells. Horm Metab Res We suggest that rGH release may be induced by constituents of 1993; 25 5): 253 ±255 11 Suter DE, Schwartz NB. Effects of glucocorticoids on secretion of lu- herbal extract such as tectorigenin and puerarin binding with teinizing hormone and follicle-stimulating hormone by female rat pi- various receptors, GHS-R or GHRH-R, in pituitary cells [20], or tuitary cells in vitro. Endocrinology 1985; 117 3): 849±854 stimulating directly cellular membrane [21] or inhibiting so- 12 Wehrenberg WB, Brazeau P, Luben R, Ling N, Guillemin R. A noninva- matostatin from binding with its receptor. It is known that pepti- sive functional lesion of the hypothalamo-pituitary axis for the study of growth hormone-releasing factor. Neuroendocrinology 1983; 36 domimetics and GHRP-6 activate phospholipase C to liberate the 6): 489±491 second-messenger IP3 and diacylglycerol, which use a different 13 Tannenbaum GS, Ling N. The interrelationship of growth hormone pathway from GHRH. GHS receptors have been identified in the GH)-releasing factor and somatostatin in generation of the ultradian pituitary and hypothalamus and have also been shown to be a rhythm of GH secretion. Endocrinology 1984; 115 5): 1952 ±1957 14 Lesniak MA, Gorden P, Roth J, Gavin JR 3rd. Binding of 125I-human unique G-protein-coupled receptor, and has little homology growth hormone to specific receptors in human cultured lympho- with the GHRH receptor. Thus, puerarin and tectorigenin are cytes. Characterization of the interaction and a sensitive radioreceptor hardly considered as true ligands acting on GHS-receptors in pi- assay. J Biol Chem 1974; 249 6): 1661±1667 tuitary cells. The affinity of puerarin and tectorigenin with GHS- 15 Kiess W, Butenandt O. Specific growth hormone receptors on human peripheral mononuclear cells: reexpression, identification, and char- receptors might be considered as weak ligands since rGH release acterization. J Clin Endocrinol Metab 1985; 60 4): 740±746 in vitro is saturated at about 20±30 nM Fig. 4). A true ligand of 16 Hattori N, Saito T, YagyuT, Jiang BH, Kitagawa K, Inagaki C. GH, GH re- endogenous GHS-receptor, ghrelin, induces plasma GH release ceptor, GH secretagogue receptor, and ghrelin expression in human T 91 similar to that by GHRH [4]. Thus, puerarin and tectorigenin cells, B cells, and neutrophils. J Clin Endocrinol Metab 2001; 86 9): 4284 ±4291 may be weak ligands to GHS-receptor. Further, it may be induced 17 Cheng K, Chan WW, Barreto A Jr, Convey EM, Smith RG. The synergistic by metabolites of administered ingredients such as HPS. Further effects of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 on growth hormone studies are warranted. GH)-releasing factor-stimulated GH release and intracellular adeno- sine 3¢,5¢-monophosphate accumulation in rat primary pituitary cell culture. Endocrinology 1989; 124 6): 2791±2798 18 Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum Acknowledgements CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, ChouM, LiuKK, McKee KK, Pong SS, ChaungLY, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJ, Dean DC, Melillo DG, Van

This research was partially supported by a grant PF 002 201-01) Downloaded by: University of Washington at Seattle. Copyrighted material. der Ploeg LHT. A receptor in pituitary and hypothalamus that func- from Plant Diversity Research Center of 21st Century Frontier Re- tions in growth hormone release. Science 1996; 273 5277): 974±977 search Program funded by Ministry of Science and Technology of 19 De Marinis L, Mancini A, Valle D, Izzi D, Bianchi A, Gentilella R, Giam- the Korean government. pietro A, Desenzani P, Giustina A. Role of food intake in the modula- tion of hexarelin-induced growth hormone release in normal human subjects. Horm Metab Res 2000; 32 4): 152±156 20 Bowers CY, Reynolds GA, Durham D, Barrera CM, Pezzoli SS, Thorner References MO. Growth hormone GH)-releasing peptide stimulates GH release in normal men and acts synergistically with GH-releasing hormone. J 1 Kim C, Ha H, Kim JS, Kim YT, Kwon S-C, Park SW. Induction of Growth Clin Endocrinol Metab 1990; 70 4): 975±982 Hormone by the roots of Astragalus membraceus in Pituitary cell Cul- 21 Arvat E, Maccario M, Di Vito L, Broglio F, Benso A, Gottero C, Papotti M, ture. Arch Pharm Res 2003; 26 1): 34±39 Muccioli G, Dieguez C, Casanueva FF, Deghenghi R, Camanni F, Ghigo E. 2 Endocrine activities of ghrelin, a natural growth hormone secreta- Kim C, Ha H, Kim H, Lee JH, Song KY. Pueraria lobata OHWL. as an Osteo- porosis therapeutics. Korean J Food Sci Technol 2002; 34 4): 710±718 gogue GHS), in humans: comparison and interactions with hexarelin, 3 Finch CE, Tanzi RE. Genetics of aging. Science 1997; 278 5337): 407± a nonnatural peptidyl GHS, and GH-releasing hormone. J Clin Endocri- 411 nol Metab 2001; 86 3): 1169±1174

Jung DY et al. Induction of Growth Hormone Release ´ Horm Metab Res 2004; 36: 86±91