CC BY-NC-ND 4.0 · PMIO 2017; 4(02): e43-e51
DOI: 10.1055/s-0043-106742
Original Article
Georg Thieme Verlag KG Stuttgart · New York

Tetra-cis/trans-Coumaroyl Polyamines as NK1 Receptor Antagonists from Matricaria chamomilla

Sait Byul Park1, Kwangho Song1, Yeong Shik Kim1
  • 1Natural Products Research Institute, College of Pharmacy, Seoul National University, Gwanaggu, Seou, South Korea
Further Information

Correspondence

Prof. Yeong Shik Kim
Natural Products Research Institute
College of Pharmacy
Seoul National University
1 Gwanangro
Gwanaggu
Seoul 08826
South Korea   
Phone: +82/2/880 2479   
Phone: Fax: +82/2/765 4768   

Publication History

received 11 January 2017
revised 07 March 2017

accepted 09 March 2017

Publication Date:
13 June 2017 (online)

 

Abstract

Cis-trans isomers of N 1,N 5,N 9,N 14-tetra-p-coumaroyl thermospermines and N 1,N 5,N 10,N 14-tetra-p-coumaroyl spermines were found in Matricaria chamomilla, German chamomile using countercurrent chromatography with methylene chloride-methanol-water (1:1:1, v/v/v). Their structures were elucidated based on spectroscopic and spectrometric data (1D and 2D NMR, and HRESIMS). The antagonistic activity against the neurokinin-1 receptor was examined by a calcium assay, measuring the cytosolic fluorescence triggered by substance P binding to the receptor. The compounds 1a/1b, 2a/2b, and 3a/3b potently suppressed the calcium flux compared to the known antagonist L-703,606 oxalate, indicating that the compounds competitively inhibited the binding of substance P. They also suppressed substance P-induced proliferation in MDA-MB-453, the HER2-amplified breast cancer cell line. It is suggested that tetracoumaroyl thermospermines and tetracoumaroyl spermines are promising antagonists, exerting positive effects on substance P/neurokinin-1 receptor-related diseases.


#

Introduction

Chamomile belongs to the Asteraceae family, and the species most commonly used as tea is Matricaria chamomilla L., known as German chamomile. It has been traditionally consumed to treat sleep disorders and to ameliorate anxiety and depression [1]. A broad range of biological activities attributed to chamomile has been studied in modern research, which has demonstrated that chamomile possesses antiallergic, anticancer, anti-inflammatory, and wound healing properties [2]. More than 120 chemical compounds involved in the bioactivities of chamomile have been identified, including flavonoids, terpenoids, and coumarins [3]. Tetracoumaroyl spermine (N 1,N 5,N 10,N 14-tetrakis[3-(4-hydroxyphenyl)-2-propenoyl]-1,5,10,14-tetraazatetradecane) was first isolated from Asteraceae plants by preparative HPLC, and its potency as a neurokinin-1 (NK1) receptor antagonist was demonstrated by the inhibition of substance P-induced contractions in the guinea pig ileum [4]. The cis-trans isomers of tricoumaroyl spermidine were separated from safflower using high-speed countercurrent chromatography (HSCCC) [5], and the antioxidant activities of tetracoumaroyl spermine (TCS) and tricoumaroyl spermidine have also been reported [6]. Thermospermine is the isomer of spermine converted from spermidine by thermospermine synthase [7], and it is abundant in the plant kingdom [8] [9]. Despite occurring in the majority of plants, hydroxycinnamic acid-conjugated thermospermine has not yet been reported. The antagonism of tetracoumaroyl thermospermine (TCTS) is deducible by the structural analogy with TCS; hence, the isolation and characterization of novel TCSs/TCTSs would be worthwhile for NK1 receptor-targeted drug development.

The NK1 receptor is one of the G protein-coupled receptors (GPCR), and the activation or deactivation of the receptor through the binding of an agonist/antagonist can be quantitatively assessed by intracellular calcium tagged with fluorescence. The activated GPCR triggers phospholipase C to hydrolyze the membrane phospholipid PIP2 to form IP3, and the IP3 binds to its receptor, releasing calcium from the endoplasmic reticulum [10]. The cytosolic calcium level is an indicator for the transient changes induced by the binding of substance P to the NK1 receptor. NK1 receptors and substance P are found in the brain regions that regulate emotion, such as the hypothalamus, amygdala, and the periaqueductal gray [11]. The binding of substance P to NK1 receptors is also related to the transmission of stress signals, inducing mood disorders and anxiety [12] [13]. NK1 receptor antagonists selectively suppress the substance P-mediated actions, showing antidepressant, anxiolytic, and antiemetic properties [14]. The NK1 receptor/substance P complex is also widely distributed in tumor cells, and the binding stimulates mitogenesis, inducing proliferation and inhibiting apoptosis through the mitogen-activated protein kinase (MAPK) pathway. The NK1 receptor antagonists L-732,138, L-703,033, and aprepitant have shown antitumor activity in human cancer cell lines [15] [16]. In breast cancer, substance P and NK1 receptors are involved in the acquisition of oncogenicity [17] [18], and substance P enhances the aggressiveness of breast cancer cells by promoting the activity of the receptor tyrosine-protein kinase ERBB family, including epidermal growth factor receptor 1 (EGFR) and 2 (HER2) [19]. It was also found that substance P and NK1 receptors are highly expressed in HER2+ primary breast tumors [20]. Breast cancer can be classified based on the immunohistochemical expression of estrogen receptors (ER), progesterone receptors (PR), and HER2 [21]. In this study, 2 subtypes of breast cancer cell lines (HER2-positive MDA-MB-453 and MDA-MB-231, characterized by the lack of expression of ER, PR, and HER2) were used to demonstrate that the antagonism against NK1 receptors inhibits substance P-induced proliferation in HER2-positive breast cancer.


#

Results and Discussion

3 peaks that represented identical ions were detected on liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), and the m/z values were estimated at [M−H] 785.43 and [M+Na]+ 809.53 ([Fig. 1]). Peaks 1, 2, and 3 (P1, P2, and P3) were revealed to be the isomers of TCS by comparing them to the molecular weights of reported compounds from chamomile [4]. Countercurrent chromatography (CCC) was used to separate the isomers due to the highly similar polarities among the compounds, which impede the separation by reversed-phase C18 chromatography. The partition coefficients of P1, P2, and P3 were first examined with chloroform-methanol-water (1:1:1, v/v/v) [5] that was used to separate the cis-trans isomers of tricoumaroyl spermidine. The solvent composition was modified ([Table 1]), and methylene chloride-methanol-water (1:1:1, v/v/v) was finally selected. P1, P2, and P3 were collected at 440, 750, and 920 min of the retention time on the CCC chromatogram ([Fig. 2]). The isomeric compounds presented unique UV spectra ([Fig. 3]), showing that the UV λ max values moved to longer wavelengths, implying that their structural differences were derived from cis-trans hydroxycinnamic acid groups conjugated on the tetra-amines. Meanwhile, P1, P2, and P3 were found to possess the unique mass spectral fragment exhibiting the [M−H] ion at m/z 144.4. P1, P2, and P3 were originally regarded as the isomers of spermine-based tetracoumaroyl moiety-conjugated compounds; however, the ions were estimated to be relationally generated from thermospermine due to the asymmetric carbon arrangement ([Fig. 4]).

Zoom Image
Fig. 1 HPLC chromatogram and mass spectra of the tetra-cis/trans-coumaroyl polyamines. 3 peaks possessing isomers of hydroxycinnamates-conjugated polyamines were detected by LC-ESI/MS a and the compounds exhibited the identical molecular weight, where the m/z values at [M−H] 785 and [M+Na]+ 809 estimated the formula as C46H50N4O8 b.
Zoom Image
Fig. 2 CCC chromatogram of the tetra-cis/trans-coumaroyl polyamines. Methylene chloride/MeOH/water (1:1:1, v/v/v) was selected for the CCC operation to separate hydroxycinnamates-conjugated polyamines.
Zoom Image
Fig. 3 HPLC chromatograms and UV spectra of P1, P2, and P3 separated by CCC. The hydroxycinnamates-conjugated polyamines exhibited different UV spectra that the λmax moved to a longer wavelength from P1 to P3. The result implied their structural differences were possibly derived from the variation of cis/trans hydroxycinnamate groups conjugated on aliphatic tetraamine.
Zoom Image
Fig. 4 Structural differences in the 2 polyamines and the unique mass fragment of thermospermine. The asymmetric aliphatic carbon arrangement leads thermospermine to have a unique MS2 fragment of m/z 144, which can be a marker to distinguish conjugated thermospermines from spermine. 

Table 1 Partition coefficients of P1, P2, and P3.

MC

Chl

EA

IPA

ACN

EtOH

MeOH

DW

P1a

P2a

P3a

1.250

1.000

1.000

1.206

2.065

2.666

1.125

1.000

1.000

1.465

2.173

2.685

1.000

1.000

1.000

1.199

2.293

2.782

1.000

0.250

0.750

1.000

0.617

0.795

0.984

1.000

0.250

0.750

1.000

0.387

0.361

0.353

1.000

0.250

0.750

1.000

0.214

0.165

0.174

0.875

0.125

1.000

1.000

1.034

2.239

1.881

0.500

0.500

1.000

1.000

0.608

1.382

1.099

0.125

0.875

1.000

1.000

0.653

0.802

0.933

1.000

1.000

1.000

0.546

0.925

0.964

0.875

0.125

1.000

1.000

0.479

0.484

0.481

a Partition coefficients (KD values) of P1-3 were evaluated using HPLC by the area integration of the upper phase divided by that of the lower phase on the chromatogram

The integration in the 1H NMR data of P1, P2, and P3 showed the double numbers of hydrogen atoms, 8 olefins and 20 non-overlapping aliphatic protons, suggesting the co-presence of TCS and TCTS in P1, P2, and P3 ([Table 2], [3]). The 1H and 13C NMR spectra were compared with previous data [4] [6]. In the 1H NMR spectrum of P1 (Fig. S1, Supporting Information), 16 protons, at δ H 6.49 (d, J=12.7 Hz), 6.48 (d, J=12.8 Hz), 6.45 (d, J=12.8 Hz), 6.38 (d, J=12.7 Hz), 5.95 (d, J=12.8 Hz), 5.83 (d, J=12.8 Hz), 5.79 (d, J=12.8 Hz), 5.75 (d, J=12.8 Hz), 6.51 (d, J=12.7 Hz), 6.50 (d, J=12.8 Hz), 6.40 (d, J=12.8 Hz), 6.38 (d, J=12.7 Hz), 5.92 (d, J=12.8 Hz), 5.87 (d, J=12.8 Hz), 5.78 (d, J=12.8 Hz), and 5.74 (d, J=12.8 Hz), indicated 8 cis-coumaroyl moieties. Based on the assignments with the heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) experiments, the 2 polyamines in P1 were identified to be N 1(Z)-N 5(Z)-N 10(Z)-N 14(Z)-tetra-p-coumaroyl spermine and N 1(Z)-N 5(Z)-N 9 (Z)-N 14(Z)-tetra-p-coumaroyl thermospermine, which were named 1a and 1b, respectively. In the 1H NMR spectrum of P2 (Fig. S7, Supporting Information), 16 protons, at δ H 7.33 (2H, d, J=15.7 Hz), 6.45 (d, J=12.6 Hz), 6.41 (d, J=15.7 Hz), 6.38 (d, J=12.6 Hz), 6.40 (d, J=15.7 Hz), 5.95 (d, J=12.6 Hz), 5.84 (d, J=12.6 Hz), 7.30 (2H, d, J=15.7 Hz), 6.41 (d, J=12.6 Hz), 6.39 (d, J=12.6 Hz), 6.36 (2H, d, J=15.7 Hz), 5.93 (d, J=12.6 Hz), and 5.89 (d, J=12.6 Hz), indicated 4 cis- and 4 trans-coumaroyl moieties. Based on the assignments with the HSQC and HMBC experiments, the 2 polyamines in P2 were identified to be N 1(E)-N 5(Z)-N 10(Z)-N 14(E)-tetra-p-coumaroyl spermine and N 1(E)-N 5(Z)-N 9(Z)-N 14(E)-tetra-p-coumaroyl thermospermine, which were named 2a and 2b, respectively. In the 1H NMR spectrum of P3 (Fig. S13, Supporting Information), 16 protons, at δ H 7.41 (2H, d, J=15.7 Hz), 7.34 (2H, d, J=15.7 Hz), 6.92 (d, J=15.7 Hz), 6.90 (d, J=15.7 Hz), 6.42 (d, J=15.7 Hz), 6.41 (d, J=15.7 Hz), 7.40 (2H, d, J=15.7 Hz), 7.31 (2H, d, J=15.7 Hz), 6.83 (d, J=15.7 Hz), 6.82 (d, J=15.7 Hz), and 6.40 (2H, d, J=15.7 Hz), indicated 8 trans-coumaroyl moieties. Based on the assignments with the HSQC and HMBC experiments, the 2 polyamines in P3 were identified to be N 1(E)-N 5(E)-N 10(E)-N 14(E)-tetra-p-coumaroyl spermine and N 1(E)-N 5(E)-N 9(E)-N 14(E)-tetra-p-coumaroyl thermospermine, which were named 3a and 3b, respectively. All chemical structures are shown in [Fig. 5].

Zoom Image
Fig. 5 Structures of tetra-cis/trans-coumaroyl polyamines 1a-3b isolated from M. chamomilla.

Table 2 1H NMR data of compounds 1a-3b (δ in ppm, J in Hz, 600 MHz).

Position

1a

1b

2a

2b

3a

3b

OH'

9.66, br s

9.66, br s

9.91, br s

9.91, br s

9.82, br s

9.82, br s

OH''

9.66, br s

9.66, br s

9.75, br s

9.75, br s

9.83, br s

9.83, br s

OH'''

9.66, br s

9.66, br s

9.75, br s

9.75, br s

9.84, br s

9.84, br s

OH''''

9.66, br s

9.66, br s

9.91, br s

9.91, br s

9.85, br s

9.85, br s

1-NH

7.98, m

7.96, m

7.97, m

7.95, m

7.97, m

7.97, m

14-NH

8.05, m

8.03, m

8.04, m

8.01, m

8.09, m

8.09, m

3'

6.49, d (12.7)

6.51, d (12.7)

7.33, d (15.7)

7.30, d (15.7)

7.34, d (15.7)

7.31, d (15.7)

3''

6.38, d (12.7)

6.38, d (12.7)

6.38, d (12.6)

6.39, d (12.6)

7.41, d (15.7)

7.40, d (15.7)

3'''

6.45, d (12.8)

6.40, d (12.8)

6.45, d (12.6)

6.41, d (12.6)

7.41, d (15.7)

7.40, d (15.7)

3''''

6.48, d (12.8)

6.50, d (12.8)

7.33, d (15.7)

7.30, d (15.7)

7.34, d (15.7)

7.31, d (15.7)

5', 9'

7.60, m

7.61, m

7.38, d (8.7)

7.38, d (8.7)

7.56, d (8.5)

7.39, d (8.4)

5'', 9''

7.18, d (8.5)

7.19, d (8.5)

7.18, d (8.7)

7.18, d (8.7)

7.49, d (8.5)

7.38, d (8.4)

5''', 9'''

7.23, d (8.5)

7.21, d (8.5)

7.22, d (8.7)

7.20, d (8.7)

7.46, d (8.5)

7.38, d (8.4)

5'''', 9''''

7.60, m

7.61, m

7.38, d (8.7)

7.38, d (8.7)

7.52, d (8.5)

7.39, d (8.4)

2'

5.79, d (12.8)

5.78, d (12.8)

6.41, d (15.7)

6.36, d (15.7)

6.42, d (15.7)

6.40, d (15.7)

2''

5.83, d (12.8)

5.87, d (12.8)

5.84, d (12.6)

5.89, d (12.6)

6.92, d (15.7)

6.83, d (15.7)

2'''

5.95, d (12.8)

5.92, d (12.8)

5.95, d (12.6)

5.93, d (12.6)

6.90, d (15.7)

6.82, d (15.7)

2''''

5.75, d (12.8)

5.74, d (12.8)

6.40, d (15.7)

6.36, d (15.7)

6.41, d (15.7)

6.40, d (15.7)

6', 8'

6.68, m

6.68, m

6.78, m

6.78, m

6.77, d (8.6)

6.79, d (8.6)

6'', 8''

6.70, m

6.70, m

6.69, m

6.69, m

6.71, d (8.4)

6.79, d (8.6)

6''', 8'''

6.70, m

6.70, m

6.69, m

6.69, m

6.7, d (8.4)

6.79, d (8.6)

6'''', 8''''

6.68, m

6.68, m

6.78, m

6.78, m

6.76, d (8.6)

6.79, d (8.6)

2

2.99, m

3.11, m

3.04, m

3.16, m

3.16, m

3.16, m

3

1.53, m

1.64, m

1.54, m

1.65, m

1.68, m

1.68, m

4

3.15, m

3.23, m

3.16, m

3.25, m

3.46, m

3.49, m

6

3.20, m

3.23, m

3.22, m

3.25, m

3.35, m

3.36, m

7

1.35, m

1.35, m

1.37, m

1.37, m

1.48, m

1.60, m

8

3.33, m

1.16, m

3.35, m

1.18, m

3.39, m

1.51, m

9

3.06, m

3.07, m

3.36, m

10

3.24, m

3.25, m

3.46, m

11

1.50, m

3.32, m

1.52, m

3.35, m

1.56, m

3.49, m

12

1.58, m

1.70, m

1.58, m

1.71, m

1.75, m

1.75, m

13

3.02, m

3.14, m

3.07, m

3.19, m

3.21, m

3.21, m

a Assignments were based on HSQC and HMBC experiments. Compounds 1a-3b were measured in DMSO-d6

Table 3 13C NMR data of compounds 1a-3b (δ in ppm, J in Hz, 150 MHz).

Position

1a a

1b a

2a a

2b a

3a a

3b a

1'

166.2, C

166.2, C

165.4, C

165.4, C

165.5, C

165.3, C

1''

168.1, C

168.0, C

168.5, C

168.1, C

165.6, C

165.3, C

1'''

168.2, C

168.1, C

168.5, C

168.2, C

165.6, C

165.3, C

1''''

166.2, C

166.2, C

165.4, C

165.4, C

165.5, C

165.3, C

7'

157.8, C

157.8, C

158.9, C

158.9, C

158.8, C

158.8, C

7''

157.6, C

157.6, C

157.7, C

157.7, C

158.8, C

158.8, C

7'''

157.6, C

157.6, C

157.7, C

157.7, C

158.8, C

158.8, C

7''''

157.8, C

157.8, C

158.9, C

158.9, C

158.8, C

158.8, C

3'

136.5, CH

136.4, CH

138.7, CH

138.7, CH

138.8, CH

138.6, CH

3''

131.9, CH

131.9, CH

131.9, CH

131.9, CH

141.5, CH

141.5, CH

3'''

131.9, CH

132.0, CH

131.9, CH

131.9, CH

141.5, CH

141.5, CH

3''''

136.5, CH

136.4, CH

138.7, CH

138.7, CH

138.8, CH

138.6, CH

5', 9'

131.9, CH2

131.9, CH2

129.3, CH2

129.3, CH2

129.8, CH2

129.2, CH2

5'', 9''

129.9, CH2

129.9, CH2

129.9, CH2

129.9, CH2

129.7, CH2

129.2, CH2

5''', 9'''

123.0, CH2

123.0, CH2

123.0, CH2

123.0, CH2

129.7, CH2

129.2, CH2

5'''', 9''''

131.9, CH2

131.9, CH2

129.3, CH2

129.3, CH2

129.8, CH2

129.2, CH2

4'

126.3, C

126.3, C

125.8, C

125.9, C

125.8, C

125.9, C

4''

126.4, C

126.4, C

126.4, C

126.5, C

126.2, C

126.1, C

4'''

126.5, C

126.4, C

126.4, C

126.5, C

126.2, C

126.1, C

4''''

126.3, C

126.3, C

125.8, C

125.9, C

125.8, C

125.9, C

2'

120.9, CH

120.9, CH

118.7, CH

118.5, CH

118.4, CH

118.7, CH

2''

121.0, CH

120.8, CH

121.1, CH

121.1, CH

114.7, CH

114.6, CH

2'''

120.0, CH

120.9, CH

121.1, CH

121.1, CH

114.7, CH

114.6, CH

2''''

120.7, CH

120.7, CH

118.7, CH

118.5, CH

118.4, CH

118.7, CH

6', 8'

115.2, CH2

115.2, CH2

115.8, CH2

115.8, CH2

115.7, CH2

115.7, CH2

6'', 8''

114.7, CH2

114.7, CH2

115.2, CH2

115.2, CH2

115.6, CH2

115.7, CH2

6''', 8'''

114.7, CH2

114.7, CH2

115.7, CH2

115.7, CH2

115.6, CH2

115.7, CH2

6'''', 8''''

115.2, CH2

115.2, CH2

115.8, CH2

115.8, CH2

115.7 CH2

115.7, CH2

2

36.0, CH2

36.5, CH2

36.2, CH2

36.7, CH2

36.6, CH2

36.6, CH2

3

28.3, CH2

27.0, CH2

28.6, CH2

27.3, CH2

27.9, CH2

27.9, CH2

4

45.5, CH2

42.1, CH2

45.4, CH2

42.1, CH2

44.9, CH2

46.9, CH2

6

43.6, CH2

47.5, CH2

43.6, CH2

47.6, CH2

45.5, CH2

43.7, CH2

7

24.0, CH2

25.8, CH2

24.1, CH2

25.8, CH2

25.0, CH2

26.7, CH2

8

43.9, CH2

25.5, CH2

43.9, CH2

25.5, CH2

45.1, CH2

26.7, CH2

9

47.4, CH2

47.4, CH2

43.7, CH2

10

45.6, CH2

45.8, CH2

44.8, CH2

11

24.5, CH2

42.2, CH2

24.5, CH2

42.3, CH2

24.8, CH2

46.9, CH2

12

28.3, CH2

27.0, CH2

28.6, CH2

27.3, CH2

29.7, CH2

29.7, CH2

13

36.0, CH2

36.5, CH2

36.2, CH2

36.7, CH2

36.2, CH2

36.2, CH2

a  Assignments were based on HSQC and HMBC experiments. Compounds 1a-3b were measured in DMSO-d6

The antagonism of P1-3 against the NK1 receptor was examined with the calcium assay to assess the potency suppressing cytosolic calcium fluorescence. The maximum fluorescent unit was induced by the minimum concentration of substance P at 3.29 nM, and it dose-dependently decreased with pretreated P1, P2, and P3, demonstrating that they were favorably potent compared to the known antagonist L-703,606 oxalate ([Fig. 6]). The antagonistic activities tended to decrease from P1 to P3; the IC50 values of P1-3 and L-703,606 oxalate were shown at 0.5, 1.3, 1.7, and 2.8 μM, respectively. It could be suggested that cis-coumaroyl was more advantageous than trans-coumaroyl for preoccupying the binding site on the NK1 receptor. The antagonistic activity of P1, 4 cis-coumaroyl moiety-conjugated thermospermine, and spermine, showing the strongest potency among the isomers, was visually represented by the real-time monitoring system (Supporting Information, video file). P1 suppressed substance P-induced calcium release compared to the selective NK1 receptor antagonist L-703,606. The emission indicating the calcium release induced by substance P was dramatically inhibited by the treatment of 0.49 and 6.19 μM P1. In addition, it was reported that substance P and NK1 receptors were relatively overexpressed, which activated EGFR and HER2-related signal transduction, modulating further proliferation of the breast cancer cells [20]. To examine whether P1, P2, and P3 possibly inhibited substance P-induced proliferation as the antagonists, HER2-positive (MDA-MB-453; ER-, PR-, HER2+) and HER2-negative (MDA-MB-231; ER-, PR-, HER2-) breast cancer cell lines were compared. The 5 nM of substance P selectively activated the proliferation up to 120% in MDA-MB-453, but the substance P-dependent proliferation was not observed in MDA-MB-231 ([Fig. 7]). The antagonistic activities of P1-3 that downregulated substance P/NK1 receptor-induced proliferation were consequentially shown in MDA-MB-453 only. The increased proliferation rate by substance P was recovered to the blank state with the treatment of P1-3, exhibiting potent proliferative inhibition compared to L-703,606 oxalate (P1, IC50=2.6 μM; P2, IC50=5.4 μM; P3, IC50=6.7 μM; L-703,606 oxalate, IC50=3.2 μM). Taken together, it was demonstrated that the isomers of TCS were potent antagonists of the NK1 receptor and were applicable for the treatment of substance P/NK1 receptor-related disease, including HER2-positive cancers, as well as pain, mood disorders, and insomnia.

Georg Thieme Verlag. Please enable Java Script to watch the video.

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Fig. 6 Antagonistic activity of P1-3 assessed by the calcium assay. The statistical analyses were performed by 2-way ANOVA with replication, followed by Dunnett’s test. *p<0.05, ** p<0.01, and ***p<0.001 indicated significant differences from the antagonistic activity of P1-3, suppressing the calcium fluorescence induced by substance P (EC90 3.29 nM).
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Fig. 7 Suppressing activity of P1-3 on the substance P-induced proliferation in MDA-MB-453 and MDA-MB-231. Substance P selectively induced the proliferation of HER2-positive (MDA-MB-453; ER-, PR-, HER2+) breast cancer cells up to 120 percent, while the proliferation rate in HER2-negative (MDA-MB-231; ER-, PR-, HER2-) breast cancer cells was not relatively affected by substance P treatment. The significant differences between the substance P non-treated and treated group were calculated by Dunnett’s test (*p<0.05, ** p<0.01, ***p<0.001).

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Materials and Methods

General experimental procedures

Mitsubishi Chemical Corporation Diaion HP-20 and GE Healthcare Life Sciences Sephadex LH-20 were used for open-column chromatography, and TLC was performed using 60 F254 silica gel-coated EMD Millipore plates. HPLC/MS analysis was performed with an Agilent Technologies HPLC 1100 series with a G1322A degasser, a G1311A quaternary pump, a G1313A autosampler, a G1316A column oven, and a G1315A diode array detector with a Zorbax SB-Aq C18 column (3.5 μm, i.d. 4.6×150 mm and i.d. 4.6×75 mm) and a Thermo Fisher Scientific Finnigan LCQ Deca MS system. The mobile phases were composed of water with 0.1% formic acid (A) and J.T. Baker Chemical Co. HPLC grade methanol with 0.1% formic acid (B). The flow rate was set to 0.3 mL/min. The running program was initiated with 25% B for 2 min, then 25–45% B for 5 min, and the gradient was further increased from 45–85% for 33 min. Mass spectra were obtained by electrospray ionization in both the negative and positive ionization modes at the range of m/z 100–1 000, and the analysis was conducted using the following conditions: spray voltage, 5.0 kV; sheath gas flow rate, 60 arb; auxiliary gas flow rate, 9.3 arb; capillary voltage, 45.0 V; capillary temperature, 290°C; and tube lens, 20 V. The NMR spectra were recorded on a Bruker AVANCE 600 spectrometer operated at 600 MHz for 1H and at 150 MHz for 13C.

The preparative CCC separation was performed using a Tauto Biotechnique Company TBE-300A CCC equipped with a 280-mL coil column composed of polytetrafluoroethylene tubing, including a 20-mL sample loop with the following components: a Hitachi L-6200A Intelligent Pump, a Sedere SEDEX 60 LT ELSD, a Sungchang Electrics automatic voltage regulator, an Amersham Biosciences circulator, an Advantec MFS SF-2120 super fraction collector, and a Younglin Instrument Autochro data module with Autochro-2000 1.0 software.

EMD Millipore Ready-to-Assay NK1 tachykinin receptor frozen cells, Sigma-Aldrich substance P acetate salt hydrate, Santa Cruz Biotechnology L-703,606 oxalate, Abcam Fluo-8 AM, TCI America probenecid, and Corning Inc. black 96-well clear bottom plates were used for the calcium assay. The purity of the L-703,606 oxalate was greater than 96%. The assay was conducted using a Molecular Devices SpectraMax M5 multiplate reader, and the real-time images were obtained with a PerkinElmer Operetta with Harmony® high-content imaging software.

MDA-MB-231 and MDA-MB-453 human breast cancer cells were obtained from the Korea Cell Bank. Dulbecco’s phosphate-buffered saline (DPBS), DMSO, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and substance P acetate salt hydrate were purchased from Sigma-Aldrich. DMEM (high-glucose) and fetal bovine serum (FBS) were obtained from GenDepot. Cell viability assessed by the MTT assay was measured using a Molecular Devices Emax Microplate Reader.


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Plant material

Dried German chamomile flowers were purchased from Agricultural Corporation Namwonherb Co., Ltd. A voucher specimen (SNU-14-517) is deposited in the herbarium of the Natural Products Research Institute (NPRI), College of Pharmacy, Seoul National University. The specimen was identified as M. chamomilla by Professor Young Bae Suh at the College of Pharmacy, Seoul National University.


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Extraction and isolation

5 kg of dried chamomile flower were soaked in 15 L of methanol for 24 h at room temperature. The methanolic extract was concentrated using a rotary vacuum evaporator at 45°C, and ethyl acetate was obtained by solvent partitioning. Next, 3 L of Diaion HP-20 resin were activated with the same volume of methanol in a 5-L bucket, and the resin was equilibrated in 50% aqueous methanol. The constituents in the dried ethyl acetate sample were adsorbed into the resin for 2 h, and then the resin was washed with 70% methanol. The 90% methanol fraction was collected, then further subfractionated through Sephadex LH-20 column chromatography with 80% MeOH. The enriched fractions were selected by silica gel thin-layer chromatography with the solvent composition of ethyl acetate-MeOH-water (10:1:0.5, v/v/v); the R f was 0.33 under UV 254 nm. Methylene chloride-MeOH-water (1:1:1, v/v/v) was selected as the solvent system, and the aqueous upper phase was used as the stationary phase. The tubing column within the CCC was entirely filled with the stationary phase at a flow rate of 9 mL/min. The operation was then performed at a rotation speed of 850 rpm, with a circulator temperature of 25°C, connecting the outlet through a split valve to the ELSD system at 35°C, a gain of 4, and nitrogen gas pressure at 2.5 bar. A 100-mg sample was dissolved in 6 mL of the lower and upper phases (1:1, v/v), and was injected when the mobile phase began to be pumped out of the column at a flow rate of 0.8 mL/min.


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Calcium assay

The antagonistic activities of TCTS and TCS for suppressing substance P binding to NK1 receptors were evaluated by a calcium assay using Ready-to-Assay™ NK1 tachykinin receptor frozen cells, following the assay steps as suggested in the manufacturer’s protocol. The excitation and emission wavelengths were set at 488 and 515 nm, respectively, and the known NK1 receptor antagonist L-703,606 oxalate was used as the positive control. The relative increase in the intracellular calcium fluorescence was calculated by the following formula (abbreviations: Max F, maximum fluorescence; B, buffer solution-treated (blank); C, compound treated with substance P; S, substance P-treated only):

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Antiproliferation assay

Cultures were maintained in DMEM supplemented with 10% FBS and antibiotics (penicillin 100 U/mL and streptomycin 100 μg/mL) in a humidified atmosphere incubator at 37°C with 5% CO2. The toxicity and antiproliferative activity of the compounds were evaluated by cell viability with the MTT assay. MDA-MB-453 and MDA-MB-231 breast cancer cells were seeded into 96-well plates at densities of 3×104 and 1×104 cells per well, respectively. The plates were maintained at 37°C for 24 h and the media in the plate was exchanged with FBS-excluded DMEM with the target compounds or the positive control L-703,606 oxalate. MDA-MB-453 was further incubated for 50 h and MDA-MB-231 for 24 h, considering their doubling times. The MTT solution (0.5 mg/mL) was added to each well, and the cells were incubated for another 2 h. The cell viability was evaluated by measuring the absorbance at a 540 nm wavelength using a microplate reader. The relative proliferation factors were calculated by the following formula:

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Statistical analysis

All data are presented as the mean ± standard deviation (SD) of n=3 independent experiments, and each concentration was performed in triplicate. Statistical analysis was performed using Microsoft Excel 2013, and the significant differences between the control and experimental groups were calculated by 2-way ANOVA with replication, followed by Dunnett’s test (*p<0.05, ** p<0.01,  *** p<0.001).


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Conflicts of Interest

The authors declare no competing financial interest.

Acknowledgements

This work was financially supported by the National Research Foundation of Korea (MRC2009-0083533). The research involves neither human participants nor animals.

Supporting Information


Correspondence

Prof. Yeong Shik Kim
Natural Products Research Institute
College of Pharmacy
Seoul National University
1 Gwanangro
Gwanaggu
Seoul 08826
South Korea   
Phone: +82/2/880 2479   
Phone: Fax: +82/2/765 4768   


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Fig. 1 HPLC chromatogram and mass spectra of the tetra-cis/trans-coumaroyl polyamines. 3 peaks possessing isomers of hydroxycinnamates-conjugated polyamines were detected by LC-ESI/MS a and the compounds exhibited the identical molecular weight, where the m/z values at [M−H] 785 and [M+Na]+ 809 estimated the formula as C46H50N4O8 b.
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Fig. 2 CCC chromatogram of the tetra-cis/trans-coumaroyl polyamines. Methylene chloride/MeOH/water (1:1:1, v/v/v) was selected for the CCC operation to separate hydroxycinnamates-conjugated polyamines.
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Fig. 3 HPLC chromatograms and UV spectra of P1, P2, and P3 separated by CCC. The hydroxycinnamates-conjugated polyamines exhibited different UV spectra that the λmax moved to a longer wavelength from P1 to P3. The result implied their structural differences were possibly derived from the variation of cis/trans hydroxycinnamate groups conjugated on aliphatic tetraamine.
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Fig. 4 Structural differences in the 2 polyamines and the unique mass fragment of thermospermine. The asymmetric aliphatic carbon arrangement leads thermospermine to have a unique MS2 fragment of m/z 144, which can be a marker to distinguish conjugated thermospermines from spermine. 
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Fig. 5 Structures of tetra-cis/trans-coumaroyl polyamines 1a-3b isolated from M. chamomilla.
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Fig. 6 Antagonistic activity of P1-3 assessed by the calcium assay. The statistical analyses were performed by 2-way ANOVA with replication, followed by Dunnett’s test. *p<0.05, ** p<0.01, and ***p<0.001 indicated significant differences from the antagonistic activity of P1-3, suppressing the calcium fluorescence induced by substance P (EC90 3.29 nM).
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Fig. 7 Suppressing activity of P1-3 on the substance P-induced proliferation in MDA-MB-453 and MDA-MB-231. Substance P selectively induced the proliferation of HER2-positive (MDA-MB-453; ER-, PR-, HER2+) breast cancer cells up to 120 percent, while the proliferation rate in HER2-negative (MDA-MB-231; ER-, PR-, HER2-) breast cancer cells was not relatively affected by substance P treatment. The significant differences between the substance P non-treated and treated group were calculated by Dunnett’s test (*p<0.05, ** p<0.01, ***p<0.001).
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