Part 3: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

echinacoside in cistanche has many effects

Pls click here to Part 2

4. Materials and Methods

4.1. Chemicals and Plant Materials

Cistanche deserticola Y.C. Ma was obtained from a local market and authenticated by Dr. Nan-Hei Lin. Cistanche tubulosa (Schenk) R. Wight was purchased from Sinopharm Tian-Li Pharmaceutical Co., Ltd., (Hangzhou, China). HPLC-grade acetonitrile, formic acid, and methanol were purchased from ECHO Chemical Co., Ltd, (Miaoli, Taiwan). Dulbecco’s modified Eagle’s medium (DMEM), dialyzed fetal bovine serum (DFBS), and Trypsin-EDTA were bought from Invitrogen (Carlsbad, CA, USA). DNase I was bought from Worthington Biochemical (Lakewood, NJ, USA). Growth hormone-releasing hormone-6 (GHRP-6) was obtained from Gen Way Biotech, Inc. (San Diego, CA, USA). Collagenase type I and [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P were bought from Sigma-Aldrich Co. (St. Louis, MO, USA). Rat growth hormone ELISA kit was purchased from Sunred Biological Technology Corporation (Shanghai, China).

4.2. HPLC/UV and LC_MSn Analyses of the Water Extraction of Cistanche spp.

Dried stem (25 g) of Cistanche deserticola Y.C. Ma or Cistanche tubulosa (Schenk) R was extracted three times with 500 mL distilled water for 60 min at 50 ℃ in a water bath. The solution was filtered through a 13 mm Syrian Filter with 0.45 um PP membrane filter (Pall Corporation, Glen Cove, NY, USA), and subjected to the following analyses. The chemical constituents in the extracts were analyzed

using a Syncronis C18 column (4.6 × 250 mm inner diameter, 5 um, Thermo Scientific, Waltham, MA, USA) in the HPLC system coupled with a model 600E photodiode array detector (Waters Corporation,

Milford, MA, USA). The mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile. The eluting gradient was as follows: 0–60 min, linearly gradient from 14% B; 0–3 min, 14% to 17% B; 3–4 min, 17% B; 4–15 min, 17% to 20% B; 15–20 min, 20% B; 20–50 min, 20% to 14% B; 50–60

min, 14% B. The ultraviolet (UV) absorbance detection wavelength was set at 330 nm. A linear trap quadrupole (LTQ) tandem mass spectrometer (Thermo Electron, San Jose, CA, USA) equipped with electrospray ionization (ESI) interface were connected to a Surveyor LC system (Thermo Electron)

with a 5 uL sample loop. The eluting gradient was as follows: 0–90 min, linearly gradient from 14% B; 0–24 min, 14% to 17% B; 24–25 min, 17% B; 25–36 min, 17% to 20% B; 36–37 min, 20% B; 37–80 min,

20% to 14% B; 80–90 min, 14% B. The heated capillary temperature was set at 300℃ with a spray voltage of 4.5 kV. Negative ESI mode was firstly scanned ranging from m/z 400–1000. Data-dependent MSN was obtained using the high purity helium (>99.99%) as the collision gas.

4.3. Isolation of Echinacoside

The water extract from the dried stem (25 g) of C. tubulosa was concentrated under reduced pressure to give deep brown syrup. The crude extraction was suspended with distilled water and lyophilized by a freeze dryer. The powder of 100 mg was resolved in distilled water of 5 mL and subjected to purification using Sephadex LH-20 column (100 mL; GE Healthcare Bio-Sciences AB, Sweden) eluted with 10% aqueous methanol solution and monitored by HPLC. The fractions containing echinacoside were detected by reading the absorbance at 245 nm and harvested by using an autosampler.

4.4. Animals

The experiments were approved by the Institutional Animal Care and Use Committee of the National Chung_Hsing University with the approval number of IACUC 106-079. Male Sprague_Dawley rats weighing 250_300 g were purchased from BioLASCO, Taiwan Co., Ltd. (Taipei, Taiwan). Two animals per cage were maintained in a controlled environment of 23 o2, 60 o 10% humidity, and a 12 h light/dark cycle. The rats were fed with a standard chow diet (calories provided

by 28.7% protein, 13.4% fat, and 57.9% carbohydrate, 5001 Rodent LabDiet, St. Louis, MO, USA) and distilled water ad libitum.

4.5. Primary Pituitary Cell Culture

Pituitary cells were isolated according to a modified enzymatic dispersion method developed by Yamazaki et al. [29]. Briefly, male Sprague Dawley rats were anesthetized with Zoletil 50 (40 mg/kg, IP; Virbac Laboratories, Carros, France), and the anterior pituitary glands were removed and dispersed to culture pituitary cells in suspension, as described previously [30].

4.6. Growth Hormone Secretion Assay

The primary anterior pituitary cells of 4 × 104 cells/well were cultured at 37 ℃ under 5% CO2 for 2 days prior to the growth hormone secretion assay according to the protocol described previously [30].

After the removal of the culture medium, cells were starved in serum-free Dulbecco’s Modified Eagle’s medium (DMEM) for 90 min to stabilize basal hormone secretion. The starvation medium was replaced with fresh DMEM containing echinacoside (from 10_8 to 10_5 M) or GHRP-6 (an agonist of a human ghrelin receptor, GHSR, 10_7 M) as a positive control, and cells were incubated for 15 and 30 min at 37 ℃ under 5% CO2. For detection of antagonist effect, the cells were incubated with a GHSR

inverse agonist, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P (0.5 uM), and then treated with DMEM containing echinacoside (10_5 M) or GHRP-6 (10_7 M) for 30 min. The medium was collected for

determination of growth hormone secretion by a rat growth hormone ELISA kit (Shanghai Sunred Biological Technology Corporation).

4.7. Statistical Analysis

The data were presented as mean values o SD. The differences were analyzed by T-Test. Statistical calculations were performed by GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA). A level of p < 0.05 was considered to be statistically significant.

4.8. Homology Modeling and Docking

Homology modeling and docking to a human ghrelin receptor, growth hormone secretagogue receptor (GHSR, accession number AAI13548), was established by following our previous construction [21,24]. Briefly, crystal structures of β1 and β2 adrenergic receptors (PDB 2YCY and 3PDS) with bound ligands, cyanopindolol, and FAUC50 were used as templates to construct the GHSR structure [31,32]. The GHSR structure with the lowest PDF total energy was selected for further docking with GHRP-6, echinacoside, tubuloside A, and acteoside. All modeling processes were performed using the Discovery Studio 2.1 platform (http://accelrys.com/).

The 3D structure of GHRP-6 was downloaded from the Pub-Chem compound database on the NCBI website (http://www.ncbi.nlm.nih.gov/). The 3D structures of echinacoside, tubuloside A, and acteoside were built using the Chem3D program (http://www.cambridgesoft.com/). The ligand-binding site of GHSR was defined as the spherical space with a 14 Å radius from the center of the binding pocket in the docking simulation. Docking of GHRP-6, echinacoside, tubuloside A, or acteoside to the binding site of GHSR was performed in silico by employing the LibDock module in the Discover Studio 2.1 package and further minimized by smart minimize algorithm with CHARMm force field in the Discover Studio 2.1 package [33]. To compare the relative binding affinities of echinacoside, tubuloside A, and acteoside in GHSR, the active center area constructed with GHRP-6 in GHSR was used for the docking, and the binding energy was calculated by GEMDOCK (The Institute of Bioinformatics, National Chiao Tung University, Taiwan).

Author Contributions: Animal experiments, C.-J.W, Y.-C.L., and W.-Y.C.; Identification and purification: M-Y.C., N.-H.L., and C.-H.C.; Molecular modeling: C.-J.W., Y.-C.L., and J.T.C.T.; Project design and writing: C.-H.C. and J.T.C.T.

Funding: The work was partly supported by a grant to Jason TC Tzen of National Chung-Hsing University (NCHU-102D604).

Acknowledgments: The authors thank Tian-Shun Weng for sharing his professional experience in the usage of Cistanche species.

Conflicts of Interest: All authors declare no conflicts of interest.

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