Part 1:Echinacoside Isolated From Cistanche Tubulosa Putatively Stimulates Growth Hormone Secretion Via Activation Of The Ghrelin Receptor

Echinacoside Isolated from Cistanche tubulosa Putatively Stimulates Growth Hormone Secretion via Activation of the Ghrelin Receptor

Chieh-Ju Wu 1, Mei-Yin Chien 2, Nan-Hei Lin 1, Yi-Chiao Lin 1, Wen-Ying Chen 3, Chao-Hsiang Chen 2,4,* and Jason T. C. Tzen 1,*

1 Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan; baby159357520@gmail.com (C.-J.W.); CMNHEI@mohw.gov.tw (N.-H.L.); s9755702@gmail.com (Y.-C.L.)

2 Ko Da Pharmaceutical Co. Ltd., Taoyuan 324, Taiwan; rd1@koda.com.tw

3 Department of Veterinary Medicine, National Chung-Hsing University, Taichung 402, Taiwan; wychen@dragon.nchu.edu.tw

4 Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan

* Correspondence: gm@koda.com.tw (C.-H.C.); TCTZEN@dragon.nchu.edu.tw (J.T.C.T.); Tel.: +886-4-22840328 (ext. 776) (J.T.C.T.); Fax: +886-4-22853527 (J.T.C.T.)

Academic Editor: Pinarosa Avato

Received: 22 January 2019; Accepted: 14 February 2019; Published: 17 February 2019

Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791

Abstract: Cistanche species, the ginseng of the desert, has been recorded to possess many biological activities in traditional Chinese pharmacopeia and has been used as anti-aging medicine. Three phenylethanoid glycosides—echinacoside, tubuloside A, and acteoside—were detected in the water extract of Cistanche tubulosa (Schenk) R. Wight and the major constituent, echinacoside, was further purified. Echinacoside of a concentration higher than 10_6 M displayed significant activity to stimulate growth hormone secretion of rat pituitary cells. Similar to growth hormone-releasing hormone-6, a synthetic analog of ghrelin, the stimulation of growth hormone secretion by echinacoside was inhibited by [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P, an inverse agonist of the ghrelin receptor. Molecular modeling showed that all the three phenylethanoid glycosides adequately interacted with the binding pocket of the ghrelin receptor, and echinacoside displayed a slightly better interaction with the receptor than tubuloside A and acteoside. The results suggest that phenylethanoid glycosides, particularly echinacoside, are active constituents putatively responsible for the anti-aging effects of C. tubulosa and may be considered to develop as non-peptidyl analogs of ghrelin.

Keywords: cistanche tubulosa; echinacoside; ghrelin; growth hormone secretion; phenylethanoid glycosides

Pls click here to Part 2

Echinacoside in cistanche can improve the immune system

1. Introduction

Cistanche species (Orobanchaceae), the so-called ginseng of the desert, is a holoparasitic plant found in the desert region of northwestern China. This herbal medicine has been recorded to possess several biological activities such as constipation relief, longevity, and aphrodisiac properties in traditional Chinese pharmacopeia [1]. Different species of Cistanche are commonly named according to their hosts and growing environments, such as Cistanche deserticola Y.C. Ma and Cistanche tubulosa (Schenk) R. Wight. C. deserticola, hosted by Haloxylon ammodendron mainly found in Inner Mongolia, was originally regarded as the standard Cistanche source in Chinese Pharmacopoeia but has been listed as an endangered plant species in China since 1984. C. tubulosa, hosted by Tamarix ramosissima grown in Xinjiang Hetian, tends to possess a fast growth rate, and thus agricultural cultivation of this Molecules 2019, 24, 720; doi:10.3390/molecules24040720 species leads to a much better yield than the traditional C. deserticola. Nowadays, both of the above two species are collected in the Chinese Pharmacopoeia [2].

C. deserticola and C. tubulosa are found to possess similar chemical constituents and pharmacological activities, thus both of them are regarded to be potential herbal medicine for aging-related diseases [3–5]. Phenylethanol glycosides are common active ingredients found in these two species, and echinacoside is identified as the major phenylethanol glycoside in C. tubulosa [6]. In the past decade, echinacoside and the extract of C. tubulosa rich in echinacoside were demonstrated to possess many pharmacological activities, such as anti-aging, neuroprotection, enhancement of learning and memory, improvement of cardiac function, reduction of hyperlipidemia and hyperglycemia, and prevention of obesity-induced diabetes and metabolic syndrome [7–12].

Known as the “hunger hormone”, ghrelin is a peptide hormone composed of 28 amino acid residues in which the third serine residue is acylated with an n-octanoyl group essential for its biological functions [13]. Ghrelin has been demonstrated to be an endogenous ligand for a G-protein-coupled receptor named growth hormone secretagogue-1a receptor (GHS-R1a), which is mainly expressed in the hypothalamus and the pituitary gland [14]. Activation of GHS-R1a by ghrelin results in the stimulation of growth hormone secretion and the regulation of several physiological reactions [15]. Clinical studies show that exogenous supplementation with growth hormones could alleviate the symptoms of aging-related diseases in the elderly, such as increasing muscle strength and bone density and improving sleeping quality and cognitive ability [16,17]. However, concerns of serious side effects (e.g., prostate cancer, joint pain, insulin resistance, fluid retention, carpal tunnel syndrome, and gynecomastia) have been brought up for the treatment with exogenous growth hormones [18,19]. Therefore, the stimulation of growth hormone secretion by ghrelin via GHS-R1a activation has been considered as an alternative therapeutic approach for the aging-related diseases of the elderly, and its therapeutic effect is assumed to be superior to exogenous supplementation of growth hormone.

Recently, teaghrelins and ginkgoghrelins, unique acylated flavonoid glycosides found in Chin-shin oolong tea and Ginkgo biloba L. (Ginkgoaceae) leaves, were demonstrated to induce growth hormone secretion of rat primary anterior pituitary cells via binding to the ghrelin receptor [20–22]. Considering the therapeutic effects of Cistanche spp. on aging-related diseases were similar to the anti-aging effects of Chin-shin oolong tea and Ginkgo biloba leaves, we wondered whether the pharmacological activities of phenylethanol glycosides could be executed via the same pathway as those of teaghrelins and ginkgoghrelins by binding to the ghrelin receptor. Firstly, three phenylethanoid glycosides—echinacoside, tubuloside A, and acteoside—were identified in the water extract of C. tubulosa. The major constituent, echinacoside, was further purified and examined for its capability for stimulating growth hormone release of rat pituitary cells via activation of the ghrelin receptor. Molecular modeling of echinacoside, tubuloside A, and acteoside docking to the ghrelin receptor was performed and compared for the possible interactions within the binding pocket.

2. Results

2.1. Separation and Identification of Three Major Phenylethanol Glycosides in the Water Extract of Two Cistanche Species

In the HPLC analysis of two Cistanche species, C. tubulosa was found to possess much higher contents of phenylethanol glycosides than C. deserticola (Figure 1A,B). Three major phenylethanol glycosides were observed in C. tubulosa and identified as echinacoside, tubuloside A, and acteoside by mass spectrometric analysis (Figure 2) in comparison with the HPLC profiles and MS data of known compounds published previously [23,24]. According to the molecular fragmentation 785 m/z in MS1 and 623 m/z in MS2, the major peak in the water extract of C. tubulosa was identified as echinacoside (Figure 2A). Similarly, tubuloside A was assigned by its molecular fragmentation 827 m/z in MS1 and 665, 623, and 782 m/z in MS2 (Figure 2B); acteoside was confirmed by the molecular fragmentation 623 m/z in MS1 and 461 m/z in MS2 (Figure 2C). Echinacoside was further purified to near homogeneity (Figure 1C) and used in the following biological assays.

Figure 2. Cont.

Figure 2. Mass spectrometric analyses of echinacoside (A), tubuloside A (B), and acteoside (C) in MS1 (left panels) and MS2 (right panels) fragmentation.

2.2. Effect of Echinacoside on the Induction of Growth Hormone Secretion in Rat Pituitary Cells

To evaluate if echinacoside could be an agonist of growth hormone secretagogue receptor (GHSR), it was subjected to the examination of the induction of growth hormone secretion from primary rat anterior pituitary cells. Similar to GHRP-6, a synthetic GHSR agonist, echinacoside was able to stimulate the secretion of growth hormones from rat pituitary cells (Figure 3). Significant increases in growth hormone secretion were detected when rat pituitary cells were treated with echinacoside of a concentration higher than 10_6 M for 15 and 30 min. The treatment of echinacoside (from 10_8 to 10_5 M) for 15 and 30 min was found to stimulate growth hormone secretion of the rat pituitary cells in a dose-dependent manner. Moreover, a GHSR inverse agonist, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P, obviously inhibited the stimulatory effects of GHRP-6 and echinacoside on the induction of growth hormone secretion from rat pituitary cells (Figure 4). The results suggest that echinacoside is a potential agonist of GHSR.

Figure 3. Effect of echinacoside administration on growth hormone secretion of pituitary cells. Growth hormone (GH) secretion from rat primary pituitary cells was measured

after incubation with medium (control), GHRP-6 (GHSR agonist, 10_7 M), and various concentrations (from 10_8 to 10_5 M) of echinacoside (ECH) for 15 (A) and 30 min (B),

respectively. Data were presented as means o SD with n = 6. Significance levels seen by T-Test were * p < 0.05 and ** p < 0.005 versus the control.