Teaghrelins, unique acylated flavonoid tetraglycosides in Chin-shin oolong tea, are putative oral agonists of the ghrelin receptor
Yuan-Hao LO1,#, Ying-Jie CHEN1,#, Chi-I CHANG2, Yi-Wen LIN3, Chung-Yu CHEN4 5 , Maw-Rong LEE4, Viola SY LEE1, Jason TC TZEN1,5,6,*
1Graduate Institute of Biotechnology and 4 8 Department of Chemistry, National Chung Hsing University, Taichung, 40227, Taiwan, China;
2 Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, China;
3 Graduate Institute of Acupuncture and Science School of Chinese Medicine, China Medical University, 40402, Taichung, Taiwan, China;
6 Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan, China
To whom correspondence should be addressed. E mail:
[email protected] # These two authors contributed equally to this work.
Abstract
22 Aim: Chin-shin oolong tea was empirically perceived to induce hunger and to accelerate 23 gastric emptying in a manner similar to the physiological effects of ghrelin, an endogenous 24 acylated peptide known as the hunger hormone. Here, we aimed to identify candidate 25 ingredients responsible for the empirical perception in this tea. 26 Methods: Two unique acylated flavonoid tetraglycosides, tentatively named teaghrelin-1 27 and teaghrelin-2, were identified and isolated from Chin-shin oolong tea, and functionally 28 examined via a food intake assay in rats and a
biochemical assay detecting the stimulation of 29 growth hormone secretion from rat pituitary cells. The metabolites of teaghrelins in rat bile 30 were detected after intravenous injection and structurally analyzed by mass spectroscopy. 31 Results:
Both teaghrelins were demonstrated to induce hunger of rats in the food intake 32 assay. Similar to GHRP-6, a synthetic analogue of ghrelin, teaghrelin-1 stimulated growth 33 hormone secretion of rat primary anterior pituitary cells in a dose
dependent manner, and the stimulation was inhibited by [D-Arg1, D-Phe5, D-Trp7,9, Leu11 34 ]-substance P, an antagonist of 35 the ghrelin receptor. While teaghrelin-2 remained unmodified, a meta-O-methylated 36 metabolite of teaghrelin-1was detected in bile of rats after intravenous injection . 37 Conclusion: Teaghrelins are responsible for the hunger induction of Chin-shin oolong tea 38 presumably via the same molecular mechanism activated by ghrelin. It seems that 39 teaghrelins are promising oral agonists of the ghrelin receptor, provided they undergo 40 necessary clinical trials. 41 42 Keywords: Chin-shin oolong tea; ghrelin; hunger induction; meta- O-methylation; teaghrelin.
Introduction
46 Tea, one of the most widely consumed beverages around the world, is generally assumed
to be originated from China as a medicinal herb[1] 47 , and its major ingredients, flavonols and
48 polyphenols, have been demonstrated by numerous studies to provide a variety of health
benefits[2-4] 49 . Oolong tea possessing a taste and color somewhere between green and black
50 teas is manufactured predominantly in Fujian and Guangdong of China as well as in Taiwan.
51 For the preparation of oolong tea, young leaves are freshly harvested and allowed to
52 undergoing a semi-fermentation process, where the term ‘fermentation’ refers to natural
browning reactions induced by oxidative enzymes in the cells of tea leaves[5] 53 . In the past
54 few decades, oolong tea has been the most favorite choice of Taiwanese due to its special
55 taste and flavor.
56 Various cultivars of Camellia sinensis are bred and cultivated in different mountain areas
57 of Taiwan. Tieguanyin cultivar is mainly planted in the Mushan area (altitude 250- 350 m)
58 of Northern Taiwan. Chin-shin cultivar is predominantly cultivated in the high- mountain
59 areas (altitude > 700 m) of Central Taiwan. Jin-xuan cultivar is popularly grown, 60 particularly in relatively low altitude areas, due to its higher growth rate and better disease
61 resistance than Chin-shin cultivar. Shy-jih-chuen cultivar is wildly farmed in low to medium
62 altitude (altitude 200-700 m) for its adequate growth in all four seasons. Among oolong teas
63 prepared from various tea cultivars in Taiwan, Chin-shin oolong tea is relatively expensive,
64 and empirically perceived to induce hunger and to accelerate gastric emptying in a manner
65 much stronger than oolong teas prepared from other cultivars. To rationalize this
empirical
66 perception, it is imaginarily speculated by local tea-consumers that some special ingredient(s)
67 in Chin-shin oolong tea may effectively absorb and remove oily components of food from the
68 gastrointestinal system, and thus strongly induce appetite for greasy food.
69 Ghrelin, generated from X/A-like cells in the oxyntic glands of the mucosa of the gastric Lo et al. 4
70 fundus, is a peptide hormone consisting of 28 amino acids, in which the third serine residue is
acylated with an n-octanoyl group essential for its biological functions[6-8] 71 . It is also known
72 as the "hunger hormone" and identified as the endogenous ligand for a G protein- coupled
receptor, named growth hormone secretagogue-1a receptor (GHS-R1a)[6, 9] 73 . The noteworthy
density of GHR-R1a is expressed in hypothalamus and pituitary gland[10] 74 , and also found in
75 vagal and spinal visceral afferents, thyroid, immune cells, spleen, myocardium and other
peripheral tissues[11-13] 76 . The remarkable physiological functions of ghrelin via activation of
77 GHS-R1a are promotion of appetite by stimulation of hypothalamic arcuate nucleus and
induction of growth hormone release from the anterior pituitary gland[14, 15] 78 . In addition,
79 several other biological effects of ghrelin have been reported, including influence on the
80 reproductive system, the gastrointestinal system, glucose metabolism, and cardiovascular
functions[16-18] 81 .
82 In light of the similarity between the empirical effects of Chin-shin oolong tea and the
83 physiological functions of ghrelin, we speculated if any ingredients in Chin-shin oolong tea
84 might mimic ghrelin to trigger physiological responses via the same molecular mechanism.
85 To inspect this speculation, we firstly identified two unique acylated flavonoid
tetraglycosides,
86 tentatively named teaghrelins, in Chin-shin oolong tea. Teaghrelins were purified and
87 examined for the speculative biological activities via a food intake assay in rats as well as a
88 biochemical assay detecting the stimulation of growth hormone secretion from rat primary
89 anterior pituitary cells. Moreover, metabolites of teaghrelins were examined in bile of rats
90 after intravenous injection.
91
92 Materials and methods 93 Chemicals and materials
94 HPLC grade acetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ, Lo et al. 5
95 USA). Acetic acid (99.7%) was obtained from J. T. Baker (Mallinckrodt Baker, Inc., 96 Phillipsburg, NJ, USA). Phosphoric acid (85%) was brought from Merck Millipore 97 (Gibbstown, NJ, USA). Purified water was afforded by a Millipore clear water purification
98 system (Direct-Q, Millipore, Billerica, MA, USA). Dulbecco’s Modified Eagle’s Medium
99 (DMEM) and dialyzed fetal bovine serum were bought from Invitrogen (Carlsbad, CA, USA).
100 DNase I was obtained from Worthington Biochemical (Lakewood, NJ, USA).
GHRP-6 was
purchased from Tocris Bioscience (Ellisville, MO, USA). Collagenase type I and [D-Arg1 101 ,
D-Phe5
, D-Trp7,9, Leu11 102 ]-substance P were obtained from Sigma-Aldrich Co. (St. Louis, MO,
103 USA). Different oolong teas prepared from various cultivars (Tieguanyin, Chin- shin,
104 Jin-xuan, Shy-jih-chuen, and so on) of tea plants (Camellia sinensis) were gifts or purchased
105 from local manufacturers.
106
107 Preparation and HPLC analysis of tea infusions
108 Tea infusions were prepared by adding 18 mL of boiling water to 1 g of various
oolong teas.
109 After 5 min, the brew was filtered through a 0.22 μm polyvinylidene difluoride (PVDF)
110 membrane filter (PALL Corporation, Glen Cove, NY, USA), and used for the following
111 analysis. Chemical constituents in the tea infusions were analyzed on a liquid 112 chromatography system coupled to a Model 600E photodiode array detector (Waters
113 Corporation, Milford, MA, USA) and performed using a 250 mm × 4.6 mm i.d., 5 μm, C18
reversed-phase column (Waters, USA) as described previously[19] 114 . The mobile phase
115 consisted of (A) water containing 0.026% phosphoric acid and (B) acetonitrile.
The gradient
116 was as follows: 0-60 min, linearly gradient from 10% to 30% B; 61-70 min, 30% B;
and
117 70-100 min, linear gradient from 30% to 10% B. The column was maintained at room
118 temperature and the injection volume was 5 μL at a flow rate of 1 mL/min. The UV
119 absorbance detection wavelength was set at 280 nm.
120 Lo et al. 6
121 Extraction and isolation of teaghrelins
122 Chin-shin oolong tea granules of 5 kg were powdered and extracted with 40 L of methanol for
123 three times (7 days for each time) at room temperature. Two unique acylated flavonoid
124 tetraglycosides (termed teaghrelin-1 and teaghrelin-2 in this study), quercetin (3-O-[2G-(E)-coumaroyl-3G-O-β-D-glucosyl-3R 125 -O-β-D-glucosylrutinoside]) and kaempferol
(3-O-[2G-(E)-coumaroyl-3G-O-β-D-glucosyl-3R 126 -O-β-D-glucosylrutinoside]), were firstly
127 isolated from the methanol extract as a mixture preparation, and then further separated to
obtain their individual compounds according to the protocol as described previously[20] 128 .
129
130 Animals
131 Male Sprague-Dawley rats of weighting 250-300 g were purchased from BioLasco, Taiwan
132 Co. Ltd (Taiwan, China), and adapted for 1 week before use. Two animals were housed per
133 cage and maintained in a controlled environment of 23 ± 2°C, 60 ± 10% humidity and 12-h
134 light/dark cycle. The rats were fed with hard rodent chow pellets (Fwusow Ind.
Corp.,
135 Taiwan, China) and purified water ad libitum. The animal experiments were approved by
136 the Institutional Animal Care and Use Committee of the National Chung-Hsing University
137 (IACUC Approval No: 102-92).
138
139 Food intake assay
140 The rats were fasted for 2 h with free access to drinking water, and then orally administrated
141 with aqueous solutions containing teaghrelins of 2.5 or 7.5 mg/kg. After oral administration
142 for 2 h, rodent chow was supplied to rats and its net consumption (food intake) was recorded
143 at 15 min and 2 h, respectively.
144
145 Primary pituitary cell culture
146 Pituitary cells were isolated according to a modified enzymatic dispersion method developed
by Yamazaki et al.
[21] 147 . Briefly, Sprague-Dawley male rats were anaesthetized with Zoletil Lo et al.
7
50® 148 (40 mg/kg, IP; Virbac Laboratories, Carros, France), and the anterior pituitary glands
149 were quickly removed after decapitation. The tissues were cut into small pieces and
150 dispersed by incubations in DMEM containing 0.25% (w/v) collagenase type I for 60 min,
151 0.25% (w/v) trypsin for 15 min, and then DMEM containing 16 U DNase I for 5 min. The
152 dispersed anterior pituitary glands were passed through a 100 μm nylon cell
strainer (BD
FalconTM 153 , Franklin Lakes, NJ, USA). Cells were finally suspended in DMEM containing
154 10% dialyzed fetal bovine serum. The yield of each anterior pituitary gland was approximately 1.5-2×106 155 cells.
156
157 Growth hormone secretion assay
The anterior pituitary cells were seeded on a 96-well plate at a density of 4×104 158 cell/well, and
159 cultured at 37°C under 5% CO2 for 2 days prior to assay. The culture medium was removed,
160 and cells were preincubated in serum-free DMEM for 90 min to stabilize basal hormone
secretion. The medium was transferred to fresh DMEM containing teaghrelin-1 (from 10-9 161
to 10-4
M) or GHRP-6 (GHS-R1a agonist, 10-7 162 M), and cells were incubated for 15, 30 and 60
163 min at 37°C under 5% CO2. To test the effect of antagonist, the cells were incubated with a
GHS-R1a antagonist, [D-Arg1 , D-Phe5
, D-Trp7,9, Leu11 164 ]-substance P (0.5 μM), and then treated with DMEM containing teaghrelin-1 (10-5 M) or GHRP-6 (10-7 165 M) for 30 min. The
166 medium was collected and growth hormone secretion was assayed by a rat growth hormone
167 enzyme-linked immunosorbent assay (ELISA) kit (EMD Millipore Corporation, Billerica,
168 MA, USA).
169
170 Bile collection and preparation
Male Sprague-Dawley rats (n = 3) were fasted for 18 h, and then anesthetized with Zoletil 50® 171
172 (40 mg/kg, IP). The rats were kept alive at the period of surgery. All groups were treated
173 with intravenous administration of normal saline containing teaghrelin-1 or teaghrelin-2 (30 Lo et al. 8
174 mg/kg). Bile fistulas of the rats were cannulated with PE-20 polyethylene tubing for
175 collection of bile. Bile was collected at 30 min intervals for 2.5 h after a single IV dosing.
176 Bile samples of 200 μL were vortex-mixed with two volumes of methanol containing 0.1%
177 phosphoric acid for 10 min, and centrifuged at 10,000 g for 20 min at 4°C. The supernatant
178 was filtered by a 0.22 μm PVDF membrane filter (PALL Corporation), and used for the
179 following analyses.
180
HPLC/UV and LC-MSn 181 analyses of bile extraction
Bile metabolites were analyzed by a HPLC system (Waters Corp.) with a Syncronis TM 182 C18
183 column (250×4.6 mm i.d., 5 μm) from Thermo Scientific (Waltham, MA, USA).
The HPLC
184 mobile phase comprised (B) acetonitrile and (C) water containing 0.5% acetic acid. The
185 gradient started at 5% solvent B and 95% solvent C, followed by a linearly increase of solvent
186 B to 25% for 10 min and raised to 30% for 30 min. Finally, the gradient decrease to 5% B
187 for 5 min. The sample injection volume was 20 μL with 1 mL/min flow rate at room
188 temperature. The detection wavelength was set at 280 nm. Mass spectrometric analysis
189 was performed on a LTQ linear ion trap tandem mass spectrometer (Thermo Electron, San
190 Jose, CA, USA) equipped with an electrospray ionization (ESI) interface and connected to a
191 Surveyor LC system (Thermo Electron, San Jose, CA, USA) with a 5 μL sample loop. The
192 analytes were separated on a Waters Xterra-RP18 column (250×4.6 mm i.d., 5 μm). The
193 tray temperature was set at 4°C. The mobile phase comprised (B) acetonitrile and (C) water
194 containing 0.5% acetic acid. The program for gradient elution started at 5% B,
increased to
195 25% B in 10 min, and then changed to 30% B in 30 min. Subsequently, the gradient
196 increased to 70% B in 1 min and held for 8 min. Finally, the gradient went back to 5% B in
197 1 min and held for 5 min. The flow rate was 1 mL/min. The mass spectra were obtained
198 with negative ESI mode. The spray voltage was 4.5 kV, and the heated capillary 199 temperature was at 300°C. Flow rates of sheath gas, auxiliary gas and sweep gas were 50, Lo et al. 9
200 13, and 3 arbitrary unit, respectively. Data-dependent acquisition (DDA) was used to
201 perform under automatic gain control conditions. The first scan was operated in full scan
mode ranging from m/z 150 to 1500. The other scans were set as the data- dependent MSn 202
203 scan by using the high purity helium (>99.99%) as the collision gas and the relative collision
204 energy of 33-35%. Isolation width of the precursor ion was set to 2 Da. The MS/MS
205 experiments of data-dependent acquisition were performed according to a previous scan.
206 The highest intensity ion of the previous scan was chosen as the precursor ion for the
207 successive MS/MS scans.
208
209 Statistical analysis
210 The data were presented as mean values ± S.E.M. The differences were analyzed by
211 one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc testing.
Statistical
212 calculations were performed by SigmaStat (Version 3.5). A level of p < 0.05 was considered
213 to be statistically significant.
214
215 Results
216 Identification of teaghrelins in Chin-shin oolong tea
217 To search for putative compounds responsible for the empirical effects
(induction of hunger
218 and acceleration of gastric emptying) of Chin-shin oolong tea, chemical compounds in the
219 infusion of Chin-shin oolong tea were analyzed and compared with those of other oolong teas
220 commonly found in Taiwan, as exemplified by Shy-jih-chuen oolong tea (Figure 1).
221 Comparable patterns of chemical compounds, such as caffeine and catechins, were observed
222 between Chin-shin oolong tea and Shy-jih-chuen oolong tea. Strikingly, two acylated
223 flavonoid tetraglycosides whose structures were chemically determined by NMR in our
previous study were found relatively abundant in Chin-shin oolong tea[22] 224 . Both compounds,
225 tentatively named teaghrelin-1 and teaghrelin-2, were purified as a mixture or as their Lo et al. 10
226 individual compounds, and used for the following assays.
227
228 Effect of teaghrelins on the food intake of rats
229 In comparison with the control group, food intake of rats was significantly enhanced after
230 ingestion with mixed teaghrelins of 2.5 mg/kg or 7.5 mg/kg (Figure 2A). The effect on the
231 enhancement of food intake was more drastic after ingestion for 15 min than that after
232 ingestion for 120 min. Apparently, ingestion of teaghrelins strongly induced appetite of rats
233 that ate relatively fast in the first 15 min while the total food consumption accumulated for
234 120 min was only mildly increased in the long run. The induction of appetite in rats after
235 ingestion with teaghrelins for 15 min was also dose dependent as higher effect on the
236 enhancement of food intake was observed in rats ingested with 7.5 mg/kg of teaghrelins
237 compared with those rats ingested with 2.5 mg/kg of teaghrelins. However, no statistical
238 difference was observed for rats ingested with 2.5 and 7.5 mg/kg of teaghrelins regarding
239 their total food consumption accumulated for 120 min. Similar outcomes and conclusion
240 were obtained when 7.5 mg/kg of teaghrelin-1 and teaghrelin-2 were used in the same food
241 intake assay, respectively (Figure 2B). Comparable effects on the enhancement of food
242 intake were observed for both teaghrelins, and it was obvious that the minor structural
243 difference in these two teaghrelins (one extra hydroxyl group at 3' position of teaghrelin-1)
244 did not cause appreciable variation for their effects on hunger induction of rats.
245
246 Effect of teaghrelin-1 on growth hormone secretion of rat pituitary cells 247 Stimulating growth hormone secretion of pituitary cells is a characteristic function of ghrelin.
248 To evaluate if teaghrelins are also capable of stimulating the secretion of growth hormone, the
249 relatively abundant teaghrelin-1 is used to examine for this characteristic function by
250 incubating with primary rat anterior pituitary cells. Similar to GHRP-6, a synthetic analogue
251 of ghrelin, teaghrelin-1 was able to stimulate the secretion of growth hormone from rat Lo et al. 11
252 anterior pituitary cells (Figure 3A). In our assay conditions, the stimulatory effects of
253 GHRP-6 and teaghrelin-1 on growth hormone secretion of rat pituitary cells were relatively
254 high after treatment for 15 min and 30 min, respectively. Treatment of teaghrelin-1 (from
10-9
to 10-4 255 M) for 30 min stimulated growth hormone secretion of the rat pituitary cells in a
256 dose dependent manner (Figure 3B). Moreover, both stimulatory effects of GHRP-6 and
teaghrelin-1 on growth hormone secretion of the rat pituitary cells were inhibited by [D-Arg1 257 ,
D-Phe5
, D-Trp7,9, Leu11 258 ]-substance P, an antagonist of the ghrelin receptor (Figure 4).
Taken
259 together, teaghrelin-1 seems to be an agonist of the ghrelin receptor.
260
261 Identification of a teaghrelin metabolite in rat bile
262 To examine biliary metabolites, bile samples of three rats were collected every 30 min for 2.5
263 h after IV injected with 30 mg/kg of teaghrelin-1 and teaghrelin-2, respectively.
Putative
metabolites of teaghrelins were analyzed by the LC-MSn 264 with data-dependent acquisition scan
265 function. A major metabolite, M1 was detected after IV injection of teaghrelin-1 (Figure
266 5A). In contrast, teaghrelin-2 seemed to be unmodified in rat bile after IV injection (Figure
267 5B). The unmodified teaghrelin-2 in rat bile was also confirmed by mass spectroscopic
268 analysis (data not shown). The structure of M1 was determined by multiple stage mass
spectra in the negative mode (Figure 5C). The deprotonated molecule 269 of M1 at m/z 1093
was indicated in the full mass spectrum. The m/z 947 ion in MS2 270 spectrum was owing to
neutral loss of 146 Da identified as p-coumaroyl moiety[23]
. In MS3 271 spectrum, the fragment
272 ion at m/z 315, produced from m/z 947 with the neutral loss of tetrasaccharide residue with
632 Da, was assigned as the monomethylated quercetin moiety of M1[23] 273 . The fragment of
m/z 300 was also found in the MS3 spectrum and assigned as [M–H–CH3
● ]
274 fragment of the 3'-methyl quercetin[24]
. The ion transition from m/z 315 to 300 observed in the MS4 275
276 spectrum confirmed that the monomethylation occurred in the metabolite of
teaghrelin-1.
277 Altogether, M1 was identified as 3'-methyl teaghrelin-1 (Figure 5D). Lo et al. 12 278
279 Discussion
280 In this study, we identified two unique acylated flavonoid tetraglycosides in Chin- shin
281 oolong tea, and successfully demonstrated that these two compounds, named teaghrelins,
282 were probably responsible for the empirical effects, such as hunger induction, of Chin-shin
283 oolong tea presumably via the same molecular mechanism of the endogenous hunger
284 hormone, ghrelin. Similar to ghrelin, teaghrelins are able to induce hunger sensation of rats
285 as well as to stimulate growth hormone secretion of rat primary anterior pituitary cells. The
286 identification of teaghrelins in Chin-shin oolong tea seems to provide a scientific clue to
287 resolve the mystery for the hunger experience strongly induced by Chin-shin oolong tea.
288 In a rough screening of Chin-shin oolong teas prepared from tea plants cultivated in
289 diverse locations with different fertilizations in the mountain areas of Central Taiwan, the
290 contents of teaghrelins varied substantially; in general, cultivation at a relatively high altitude
291 (> 700 m) and supplement with sufficient natural fertilizers seemed to be positively correlated
292 with the accumulation of teaghrelins in leaves of Chin-shin oolong (data not shown).
293 Moreover, several acylated flavonoid tetraglycosides with the same structural backbone but
294 different hexosyl (at least, glucosyl, rhamnosyl and arabinosyl) glycosides were detected in
295 substantially low contents in various oolong teas including Tieguanyin, Wuyi, Fenghuang,
Gaoshan Shibi, Laocong Shuixian, and Baihao oolong teas in our previous study[23]
296 . These
297 varied acylated flavonoid tetraglycosides are putatively regarded as different types of
298 teaghrelins, and the presence of teaghrelins in variable oolong teas in substantially low
299 contents presumably explains why many oolong teas are also able to induce hunger sensation
300 though not as strong as Chin-shin oolong tea.
301 Hepatic metabolism and biliary excretion mechanism are important for the pharmacological effects of drugs[25] 302 , and thus identification of drug metabolites in bile is
helpful to realize the drug disposition for the preclinical study[26] 303 . It has been shown that the Lo et al. 13
304 most abundant compounds in tea, flavonoids, tend to be methylated in their meta (3'-O-)
305 position of catecholic moiety by catechol-O-methyltransferase, and the 3'-O- methyl
derivatives are generally found as the major metabolites in bile[27, 28] 306 . Moreover, methylated
307 flavonoids have been reported to possess better intestinal absorption and oral bioavailability
than their unmethylated ones
[29, 30] 308 . Accordingly, a meta-O-methylated metabolite at 3'
309 position of teaghrelin-1 was detected in rat bile after intravenous injection in this study. As
310 expected, lacking meta (3'-O-) position of catecholic moiety, teaghrelin-2 was not methylated
311 and remained unmodified in bile.
312 Because of the multiple biological activities of ghrelin, synthetic analogues (both 313 peptidyl and nonpeptidyl GHS-R1a agonists), such as GHRP-6, L-692,429 and MK- 677, have
314 been developed for the potential therapeutic applications of several diseases, e.g.,
gastrointestinal deficiency and anorexia[31-34] 315 . In animal and human studies, ghrelin as well
316 as its peptidyl analogues has a short biological half-life that limits its utility as a pharmacological agent[35-37] 317 . In contrast, nonpeptide oral ghrelin analogues have been found
to possess better bioavailability[31, 34] 318 . However, such oral ghrelin analogues
like MK-677 in
319 clinical studies, lacked the efficacy for treatment of gastrointestinal deficiency in elderly
patients[38] 320 , and caused side effects in mild lower-extremity edema and muscle pain while the
raise of appetite was maintained for a short period in healthy older people[39] 321 . Moreover,
322 TZP-101 and TZP-102, the macrocylic, peptidomimetic ghrelin receptor agonists were
323 reported to show no significant effect on diabetic gastroparesis treatment in phase 2b program,
and caused minor adverse events, such as hyperglycemia, nausea and diarrhea[40- 42] 324 . In a
325 word, no synthetic oral agonists of the ghrelin receptor were satisfactory and approved for the
326 clinical application so far. According to this study, teaghrelins extracted from oolong teas
327 seem to be promising oral agonists of the ghrelin receptor, provided they undergo necessary
328 clinical trials.
329 Lo et al. 14 330
331 Acknowledgements
332 We thank Mr. Jim-Fang Huang, Mr. Chao-Jie Lee, Mr. Maw-Song Lee, Mr. Kang- Sheng
333 Fang, and Mr. Chien-Hong Chen for providing various oolong tea samples. The work was
334 partly supported by grants to Jason TC TZEN of National Chung-Hsing University 335 (NCHU-101D073, NCHU-102D604, NCHU-102S0503, and NSC 100-3114-B-005- 001).
336
337 Author contribution
338 Jason TC TZEN designed research; Yuan-Hao LO performed the growth hormone release
339 assay; Ying-Jie CHEN performed the food intake assay; Chi-I CHANG prepared and
340 identified teaghrelins; Yi-Wen LIN guided the animal experiments; Chung-Yu CHEN and
341 Maw-Rong LEE performed the LS/MS/MS analysis; Viola SY LEE screened oolong teas;
342 Yuan-Hao LO, Ying-Jie CHEN and Jason TC TZEN wrote the paper.
343
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457 Lo et al. 20 458 Figure legends
459 Figure 1. Comparison of the HPLC profiles of Chin-shin and Shy-jih-chuen oolong tea
460 infusions. Two unique peaks, teaghrelin-1 and teaghrelin-2, in Chin-shin oolong tea were
461 indicated by arrows, and their structures were shown on top of the peaks.
Caffeine and the
462 major catechin, EGCG (epigallocatechin-3-gallate) in both teas were labeled.
463
464 Figure 2. Effects of mixed teaghrelins (A) and individual teaghrelin (B) on food intake of rats.
465 Mean cumulative values of food intake of rats 15 and 120 min after orally administration with
466 mixed teaghrelins of 2.5 and 7.5 mg/kg as well as those with teaghrelin-1 or teaghrelin-2 of
467 7.5 mg/kg were recorded. Data were presented as means ± SEM with n = 8.
Significance
levels seen by one-way ANOVA were **p < 0.001 vs. control, ## 468 p < 0.001 significant
469 differences between columns.
470
471 Figure 3. Effect of teaghrelin-1 administration on growth hormone (GH) secretion of pituitary
472 cells. (A) GH secretion from rat primary pituitary cells was measured after incubation with
medium (control), GHRP-6 (GHS-R1a agonist, 10-7
M) and teaghrelins-1 (10-5 473 M) for 15, 30
474 and 60 min. (B) GH secretion from rat primary anterior pituitary cells was observed by
475 incubating with teaghrelin-1 of various concentrations for 30 min. Data were presented as
476 means ± SEM with n = 6. Significance levels seen by one-way ANOVA were *p <
0.05 vs.
477 control, **p < 0.001 vs. control.
478
Figure 4. Effects of [D-Arg1 , D-Phe5
, D-Trp7,9, Leu11 479 ]-substance P (GHS-R1a antagonist) on
480 pituitary growth hormone (GH) secretion induced by GHRP-6 and teaghrelin-1.
Rat primary
anterior pituitary cells were incubated with GHRP-6 (10-7 M) and teaghrelin-1 (10-5 481 M) in the
presence and absence of [D-Arg1 , D-Phe5
, D-Trp7,9, Leu11 482 ]-substance P (0.5 μM) for 30 min. Lo et al. 21
483 Data were presented as means ± SEM with n = 6. Significance levels seen by one- way
ANOVA were *p < 0.05 vs. control, **p < 0.001 vs. control, # 484 p < 0.05 significant differences
between columns, ## 485 p < 0.001 significant differences between columns.
486
487 Figure 5. HPLC chromatograms of teaghrelins and bile metabolites in rats. Bile samples
488 were collected from rats administrated with 30 mg/kg of teaghrelin-1 (A) and teaghrelin-2 (B)
489 at different time intervals, basal, 0-30 min, 31-60 min, 61-90 min, 91-120 min, and 121-150
490 min. The samples were analyzed in HPLC chromatogram by comparing with isolated
491 teaghrelins as standards (teaghrelin-1 and teaghrelin-2 in the top panel). (C) Data-dependent
MSn 492 spectra of teaghrelin-1 metabolite (M1). (D) Proposed chemical structure of M1.
493 Figure 1
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
Relative Absorbance 0.00 0.20
0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 Minutes
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Teaghrelin-1
Teaghrelin-2 EGCG Caffeine EGCG Caffeine O
O O OH HO O
O O O O O
HO O OH HO HO HOHO OH OHOH OH OH OH R 2 4 5 8 9 10 1' 3' 4' Glc-Ⅰ Rha Glc-Ⅲ Glc-Ⅱ O OH 1"
4"
7"
9"
Teaghrelin-1: R-OH Teaghrelin-2: R-H Chin-shin oolong tea
Shy-jih-chuen oolong tea Figure 2 B
0.0 0.5 1.0 1.5 2.0
** **
**
120
Food intake (g) Time (min) Control
Teaghrelins (2.5 mg/kg) Teaghrelins (7.5 mg/kg) 15
**
##
0.0 0.5 1.0 1.5 2.0 2.5
Food intake (g) Control
Teaghrelin-1 Teaghrelin-2 120
Time (min) 15
** **
**
**
A Figure 3 A
B 80 100 120
140 160
180 Control Teaghrelin-1 10-9
GH secretion (% of control) Concentration (M)
10-7 10-6 10-5 10-4 C 10-8
*
*
* 80 120 160 200 240 280
GH secretion (% of control) Control
GHRP-6 Teaghrelin-1 30 60 Time (min) 15
**
**
**Figure 4 80
120 160 200
#
*
**
GHRP-6 Teaghrelin-1
GH secretion (% of control)
None Antagonist Control
##Absolute Absorbance 0.00 0.40
0.80 0.00 0.40 0.80 0.00 0.40 0.80 0.00 0.40 0.80 0.00 0.40 0.80 0.00 0.40 0.80 0.00 0.40 0.80
Minutes 0.00 20.00 40.00 A
B MS MS/MS 1093 MS3 1093 947 MS4
1093 947 315 O
O O OH
HO O O O O O O
HO O OH HO HO HOHO OH OH OH OH
OH OH O O OH H3C C D Figure 5
Teaghrelin-1 Teaghrelin-2 Basal
0-30 min 31-60 min 61-90 min 91-120 min 121-150 min M1 Teaghrelin-1
Teaghrelin-1 Teaghrelin-2 Teaghrelin-2
Basal 0-30 min 31-60 min 61-90 min
91-120 min 121-150 min
Absolute Absorbance 0.00 0.40
0.80 0.00 0.40 0.80 0.00 0.40 0.80 0.00 0.40 0.80 0.00 0.40 0.80 0.00 0.40 0.80 0.00 0.40 0.80
Minutes 0.00 20.00 40.00
200 400 600 800 1000 1200 1400 m/z
0 50 100 0 50 100 0 50 100
Relative Abundance 0
50
100 575.46 1093.31 604.86 947.10 777.07 315.08 300.19 300.07
