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Induction of Growth Hormone Release by Pueraria Thunbergiana BENTH

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Induction of Growth Hormone Release by Pueraria Thunbergiana BENTH Induction of Growth Hormone Release by D. Y. Jung H. Ha Pueraria thunbergiana BENTH. C. Kim Original Basic 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 puerarin 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, tectorigenin 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 daidzein, 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 isoflavone 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- Original Basic 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., coumestrol, formononetin, 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, genistein, genistin, daidzein, apigenin kalide after over five years' storage [8]. and biochanin A 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.
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