Identification and quantification of potential anti-inflammatory hydroxycinnamic acid amides from wolfberry

Article abstract of DOI:10.1021/acs.jafc.6b05136

Wolfberry or Goji berry, the fruit of Lycium barbarum, exhibits health-promoting properties that leads to an extensive study of their active components. We synthesized a set of hydroxycinnamic acid amide (HCCA) compounds, including trans-caffeic acid, trans-ferulic acid, and 3,4-dihydroxyhydrocinnamic acid, with extended phenolic amine components as standards to identify and quantify the corresponding compounds from wolfberry and to investigate anti-inflammatory properties of these compounds using in vitro model. With optimized LC-MS/MS and NMR analysis, nine amide compounds were identified from the fruits. Seven of these compounds were identified in this plant for the first time. The amide compounds with a tyramine moiety were the most abundant. In vitro studies indicated that five HCCA compounds showed inhibitory effect on NO production inuded by lipopolysaccharides with IC50 less than 15.08 μM (trans-N-feruloyl dopamine). These findings suggested that wolfberries demonstrated anti-inflammatory properties.

Full text of DOI:10.1021/acs.jafc.6b05136

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Article
Identification and quantification of potential anti-inflammatory
hydroxycinnamic acid amides from Wolfberries, fruits of Lycium barbarum
Siyu Wang, Joonhyuk Suh, Xi Zheng, Yu Wang, and Chi-Tang Ho
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05136 • Publication Date (Web): 23 Dec 2016
Downloaded from http://pubs.acs.org on December 26, 2016
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Page 1 of 36
Journal of Agricultural and Food Chemistry
Identification and quantification of potential antiꢀinflammatory hydroxycinnamic acid
amides from Wolfberry
Siyu Wang,† Joon Hyuk Suh,§ Xi Zheng,& Yu Wang,§,* and ChiꢀTang Ho†,*
† Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ
08901, USA
§
Food Science and Human Nutrition, Citrus Research and Education Center, University
of Florida, 700 Experiment Station Rd, Lake Alfred, FL 33850 USA
& Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical
Biology, Ernest Mario School of Pharmacy, Rutgers University, 164 Frelinghuysen Rd.,
Piscataway, NJ 08854
* Corresponding authors:
Yu Wang, Tel: (863)ꢀ956ꢀ8673; Fax: (863)ꢀ956ꢀ4631; Email: yu.wang@ufl.edu;
ChiꢀTang Ho, Tel: (848)ꢀ932ꢀ5553; Fax: (732)ꢀ932ꢀ6776; Email:
ctho@sebs.rutgers.edu
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Abstract
Wolfberry or Goji berry, the fruit of Lycium barbarum, exhibits healthꢀpromoting
properties that leads to an extensive study of their active components. We synthesized a
set of hydroxycinnamic acid amide (HCCA) compounds, including transꢀcaffeic acid,
transꢀferulic acid and 3,4ꢀdihydroxyhydrocinnamic acid with extended phenolic amine
components as standards to identify and quantify the corresponding compounds from
wolfberry, and to investigate antiꢀinflammatory properties of these compounds using in
vitro model. With optimized LCꢀMS/MS and NMR analysis, nine amide compounds
were identified from the fruits. Seven of these compounds were identified in this plant for
10 the first time. The amide compounds with a tyramine moiety were the most abundant. In
11 vitro studies indicated that five HCCA compounds showed inhibitory effect on NO
12 production inuded by LPS with IC50 less than 15.08 ꢁM (trans-Nꢀferuloyl dopamine).
13 These findings suggested that wolfberries demonstrated antiꢀinflammatory properties.
14
15
16 Keywords: Lycium Barbarum, wolfberries, hydroxycinnamic acid amides, organic
17 synthesis, antiꢀinflammation
18
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19 Introduction
20 Hydroxycinnamic acid amides (HCAAs) are commonly found in flowering
21 plants,1 when cinnamate derivatives conjugate with either tyramine, tryptamine or
22 dopamine derivatives.2 . HCCAs play antibacterial and antiviral roles in plants as
23 numerous studies strongly indicate certain amides contribute to plant defense
24 mechanisms in response to microbial challenges and wound healing.1ꢀ3 HCCA
25 compounds, as secondary metabolites, are often considered as potential nutraceutical
26 ingredients due to reports of health benefits such as antiꢀfungal,4 antioxidant,5 antiꢀ
27 inflammatory6 as well as antiꢀcancer7 properties.
28
The genus Lycium encompasses approximately 80 species unevenly distributed
29 throughout South America, southern Africa, North America, Eurasia, Australia and
30 several islands in the Pacific Ocean.8 Wolfberry, the fruit of Lycium barbarum, is widely
31 used in Asian cuisine. Recently, they have been marketed as dietary supplements and
32 functional foods in multiple regions.9 Their increase in popularity is due to numerous
33 health benefits that improve kidney and liver function, immune system modulation, as
34 well as provide antiꢀaging and cytoprotective effects.10ꢀ11 The health benefits associated
35 with wolfberry have led to investigations into isolating and identifying several categories
36 of compounds, including polysaccharides,12 polyphenols,13ꢀ14 phenolic amides,15
37 carotenoids,16 flavonoids, organic acids and their derivatives.17 Among these species,
38 hydroxycinnamic acid derivatives are found to be abundant in this fruit17. By using
39 preparative high performance liquid chromatography, Zhou et al. identified a set of
40 dicaffeoylspermidine derivatives that provide effective antioxidant activities and
41 protection against Alzheimer’s disease.15 Other bioactive HCCA compounds have been
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42 identified from wolfberry. These include cis-Nꢀferuloyltyramine, trans-Nꢀ
43 feruloyltyramine and its dimer through the use of an activityꢀguided method and NMRꢀ
44 based identification.18ꢀ19
45
The antiꢀinflammatory properties of natural products have attracted more
46 attention due to the large body of scientific evidence that supports the close relationship
47 between chronic inflammation and many human diseases and conditions, as well as the
48 potential health beneficial properties exerted by these foodꢀsourced components.20ꢀ21
49 Consequently, in addition to the antioxidant activities investigated in previous studies,
50 some of these HCCA species identified from wolfberry have been shown to possess
51 putative antiꢀinflammatory properties. For example, by following a bioactivityꢀguided
52 method, trans-N-caffeoyltyramine was identified as an NFꢀκB inhibitor, which is known
53 as a major transcription factor activated in response to inflammation and contributes to
54 proꢀinflammatory mediator production.6 trans-Nꢀferuloyltyramine was found to have an
55 inhibitory effect on LPSꢀinduced NO and prostaglandin E2 production through
56 transcription factor APꢀ1 and the MAPK signaling pathway.22
57
The bioactive HCCA species identified from wolfberry lead to the hypothesis that
58 there are potentially more HCCA species present in the fruit with possible antiꢀ
59 inflammatory properties. This prompted us to synthesize a series of amide compounds
60 with similar extended amine components that potentially exist in wolfberry. In our
61 investigation, we designed and synthesized three sets of HCCA compounds according to
62 different hydroxycinnamic acid species, transꢀcaffeic acid, transꢀferulic acid and 3,4ꢀ
63 dihydroxyhydrocinnamic acid. The objective was to use these synthetic amide
64 compounds as references to identify and quantify compounds extracted from wolfberrry,
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65 and further investigate their antiꢀinflammatory activities in vitro.
66 Materials and Method
67 Chemicals and reagents
68
transꢀcaffeic acid, transꢀferulic acid, 3,4ꢀdihyroxyhydrocinnamic acid,
69 phenethylamine, tryptamine, tyramine, dopamine hydrochloride, 3ꢀphenylpropylamine,
70 Nꢀ(3ꢀdimethylaminopropyl)ꢀN′ꢀethylcarbodiimide hydrochloride, sulfanilamide,
71 naphthylethylenediamine dihydcrochloride, molecular biology grade dimethyl sulfoxide
72 (DMSO), lipopolysaccharides (LPS) (Escherichia coli O127: E8), NGꢀmethylꢀLꢀarginine
73 acetate (LꢀNMMA) were purchased from SigmaꢀAldrich (St. Louis, MO, USA).
74 Triethylamine, 3,4ꢀdimethoxyphenethylamine, LCꢀMS grade methanol, acetonitrile,
75 water, and formic acid were purchased from Fisher Scientific (Fair Lawn, NJ, USA).
76 Dimethylformamide (DMF), ethyl acetate, and hexane were purchased from Pharmcoꢀ
77 AAPER (Brookfield, CT, USA). Gibco Dulbecco's Modified Eagle Medium (DMEM),
78 fetal bovine serum (FBS), and streptomycin/penicillin solution were purchased from
79 ThermoFisher Scientific (Hagerstown, MD, USA). Dimethyl sulfoxideꢀd6 was purchased
80 from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA)
81 General synthetic procedure
82
5 mM of hydroxycinnamic acid (transꢀcaffeic acid, transꢀferulic acid, or 3,4ꢀ
83 dihydroxyhydrocinnamic acid) were mixed with 5 mM triethylamine in 10 mL DMF and
84 placed on ice for 15 minutes. 7.5 mM of phenolic amine (phenethylamine, tryptamine,
85 tyramine, 3ꢀphenylpropylamine, dopamine hydrochloride or 3,4ꢀ
86 dimethoxyphenethylamine) and 5 mM of Nꢀ(3ꢀdimethylaminopropyl)ꢀN′ꢀ
87 ethylcarbodiimide hydrochloride were added to DMF under nitrogen atmosphere at room
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88 temperature for 12 hours. The reaction solution was then mixed with 100 mL distilled
89 water and extracted three times using 100 mL ethyl acetate. The organic layer was next
90 washed with 0.2 M hydrochloric acid and brine, dried, evaporated and purified by using
91 silica gel (standard grade, pore size 60 Å, 230ꢀ400 mesh particle size, 40ꢀ63 ꢁm particle
92 size) column chromatography (ethyl acetate and hexane), then finally freeze dried
93 resulting in the target compounds. The purity of synthetic compounds was determined by
94 TLC and NMR. The chemical structures of HCCA compounds are shown in Figure 1.
95
96 NMR analysis
97
Proton nuclear magnetic resonance spectra (1HꢀNMR) were recorded on a Varian
98 VNMRSꢀ500 MHz, and Varian VNMRS 400 MHz instrument and reported in ppm using
99 solvent containing TMS as an internal standard (CDCl3 at 7.26 ppm, (CD3)2SO at 2.50
100 ppm, CD3OD at 3.31 ppm). Data are reported as s = singlet, d = doublet, t = triplet, dd =
101 doublet of doublets, m = multiplet; integration; coupling constant(s) in Hz.
102
103 N-trans-caffeoyl phenethylamine (1, Figure 1). Yellow powder; HESIMS m/z 284.1 [M +
104 H] (calcd for C17H17NO3, 283.33); 1H NMR (500 MHz, DMSOꢀd6) δ 9.22 (d, J = 80.8
105 Hz, 2H), 8.04 (t, J = 5.7 Hz, 1H), 7.31−7.14 (m, 6H), 6.93 (d, J = 2.1 Hz, 1H), 6.81 (dd, J
106 = 8.2, 2.1 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.31 (d, J = 15.7 Hz, 1H), 3.41−3.33 (m,
107 2H), 2.75 (t, J = 7.4 Hz, 2H). 13C NMR (125 MHz, DMSOꢀd6) δ 165.80, 147.70, 145.96,
108 139.97, 139.47, 129.06, 128.76, 126.82, 126.51, 120.82, 118.93, 116.18, 114.23, 40.76,
109 35.68.
110
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111 N-trans-caffeoyl 3,4-dimethoxyphenethylamine (2, Figure 1). Yellow powder; HESIMS
112 m/z 344.1 [M + H] (calcd for C19H21NO5, 343.38); 1H NMR (500 MHz, DMSOꢀd6) δ 9.20
113 (s, 2H), 7.99 (t, J = 5.7 Hz, 1H), 7.21 (d, J = 15.6 Hz, 1H), 6.91 (d, J = 2.1 Hz, 1H), 6.84
114 (d, J = 8.2 Hz, 1H), 6.81−6.79 (m, 2H), 6.73–6.69 (m, 2H), 6.31 (d, J = 15.7 Hz, 1H),
115 3.71 (s, 3H), 3.69 (s, 3H), 3.37–3.33 (m, 2H), 2.67 (t, J = 7.3 Hz, 2H). 13C NMR (125
116 MHz, DMSOꢀd6) δ 165.76, 149.03, 147.68, 147.65, 145.95, 139.40, 132.40, 126.83,
117 120.87, 120.79, 118.99, 116.17, 114.21, 112.97, 112.33, 55.95, 55.80, 40.90, 35.21.
118
119 N-trans-caffeoyl tryptamine (3, Figure 1). Dark Yellow powder; HESIMS m/z 321.1 [M ꢀ
120 H] (calcd for C19H18N2O3, 322.36); 1H NMR (500 MHz, DMSOꢀd6) δ 10.78 (s, 1H), 9.32
121 (s, 1H), 9.09 (s, 1H), 8.07 (t, J = 5.8 Hz, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.33 (s, 1H), 7.23
122 (d, J = 15.7 Hz, 1H), 7.14 (d, J = 2.3 Hz, 1H), 7.08−7.01 (m, 1H), 6.99−6.92 (m, 1H),
123 6.93 (d, J = 2.1 Hz, 1H), 6.82 (dd, J = 8.2, 2.0 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.32 (d,
124 J = 15.7 Hz, 1H), 3.48−3.39 (m, 2H), 2.86 (t, J = 7.4 Hz, 2H). 13C NMR (125 MHz,
125 DMSOꢀd6) δ 165.79, 147.66, 145.95, 139.37, 136.68, 127.67, 126.88, 123.06, 121.34,
126 120.77, 119.12, 118.71, 118.65, 116.17, 114.24, 112.30, 111.79, 40.02, 25.77.
127
128 N-trans-caffeoyl tyramine (4, Figure 1). Yellow powder; HESIMS m/z 300.1 [M + H]
129 (calcd for C17H17NO4, 299.33); 1H NMR (500 MHz, DMSOꢀd6) δ 9.31 (s, 1H), 9.14 (s,
130 1H), 9.08 (s, 1H), 7.98 (t, J = 5.7 Hz, 1H), 7.20 (d, J = 15.6 Hz, 1H), 6.99 (d, J = 8.4 Hz,
131 2H), 6.91 (d, J = 2.1 Hz, 1H), 6.80 (dd, J = 8.2, 2.1 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H),
132 6.66 (d, J = 8.3 Hz, 2H), 6.29 (d, J = 15.7 Hz, 1H), 3.32−3.25 (m, 2H), 2.62 (t, J = 7.4
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133 Hz, 2H). 13C NMR (125 MHz, DMSOꢀd6) δ 165.72, 156.06, 147.67, 145.95, 139.37,
134 129.98, 129.90, 126.84, 120.79, 119.01, 116.17, 115.54, 114.23, 41.12, 34.90.
135
136 N-trans-caffeoyl dopamine (5, Figure 1). Yellow powder; HESIMS m/z 316.1 [M + H]
137 (calcd for C17H17NO5, 315.33); 1H NMR (500 MHz, DMSOꢀd6) δ 8.92 (s, 4H), 7.98 (t, J
138 = 5.7 Hz, 1H), 7.21 (d, J = 15.7 Hz, 1H), 6.92 (d, J = 2.0 Hz, 1H), 6.81 (dd, J = 8.2, 2.1
139 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.62 (d, J = 7.9 Hz, 1H), 6.58 (d, J = 2.0 Hz, 1H), 6.43
140 (dd, J = 8.0, 2.1 Hz,1H), 6.30 (d, J = 15.6 Hz, 1H), 3.28 (dd, J = 14.0, 6.5 Hz, 2H), 2.59–
141 2.52 (m, 2H). 13C NMR (125 MHz, DMSO) δ 165.73, 147.67, 145.95, 145.48, 143.95,
142 139.37, 130.70, 126.85, 120.79, 119.65, 119.04, 116.41, 116.18, 115.93, 114.23, 41.16,
143 35.19.
144
145 N-trans-feruloyl phenethylamine (6, Figure 1). Colorless powder; HESIMS m/z 298.1 [M
146 + H] (calcd for C18H19NO3, 297.35); 1H NMR (500 MHz, DMSOꢀd6) δ 9.39 (s, 1H), 8.01
147 (t, J = 5.6 Hz, 1H), 7.34−7.24 (m, 3H), 7.24−7.16 (m, 3H), 7.12−7.07 (m, 1H), 6.96 (dd, J
148 = 8.0, 1.9 Hz, 1H), 6.77 (dd, J = 8.0, 0.8 Hz, 1H), 6.42 (dd, J = 15.6, 0.8 Hz, 1H), 3.78 (s,
3.28 (m, 2H), 2.75 (t, J = 7.3 Hz, 2H). 13C NMR (125 MHz, DMSOꢀd6) δ
149 3H), 3.44
−
150 165.78, 148.68, 148.24, 139.95, 139.35, 129.05, 128.75, 126.84, 126.50, 121.94, 119.39,
151 116.07, 111.19, 55.95, 40.73, 35.66.
152
153 N-trans-feruloyl 3,4-dimethoxyphenethylamine (7, Figure 1). Colorless powder; HESIMS
154 m/z 358.2 [M + H] (calcd for C20H23NO5, 357.41); 1H NMR (500 MHz, DMSOꢀd6) δ
155 9.39 (s, 1H), 7.96 (t, J = 5.7 Hz, 1H), 7.30 (d, J = 15.6 Hz, 1H), 7.09 (d, J = 1.9 Hz, 1H),
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156 6.96 (dd, J = 8.3, 1.9 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.77 (d,
157 J = 8.1 Hz, 1H), 6.71 (dd, J = 8.1, 2.0 Hz, 1H), 6.43 (d, J = 15.7 Hz, 1H), 3.78 (s, 3H),
158 3.72 (s, 3H), 3.69 (s, 3H), 3.43−
3.34 (m, 2H), 2.68 (t, J = 7.3 Hz, 2H). 13C NMR (125
159 MHz, DMSOꢀd6) δ 165.76, 149.04, 148.66, 148.24, 147.65, 139.31, 132.36, 126.86,
160 121.91, 120.86, 119.45, 116.08, 112.95, 112.32, 111.18, 55.95, 55.93, 55.80, 40.86,
161 35.19.
162
163 N-trans-feruloyl tryptamine (8, Figure 1). Pale yellow powder; HESIMS m/z 337.2 [M +
164 H] (calcd for C20H20N2O3, 336.39); 1H NMR (500 MHz, DMSOꢀd6) δ 10.80 (s, 1H), 9.39
165 (s, 1H), 8.04 (t, J = 5.7 Hz, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.35
−
7.32 (m, 2H), 7.16 (d, J =
6.94 (m,
166 2.0 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 7.05 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 6.99
−
167 2H), 6.78 (d, J = 8.1 Hz, 1H), 6.45 (d, J = 15.7 Hz, 1H), 3.79 (s, 3H), 3.46 (dd, J = 13.1,
168 7.3 Hz, 2H), 2.87 (t, J = 6.9 Hz, 2H). 13C NMR (125 MHz, DMSOꢀd6) δ 165.79, 148.65,
169 148.25, 139.28, 136.70, 127.68, 126.91, 123.07, 121.93, 121.36, 119.60, 118.72, 118.66,
170 116.09, 112.28, 111.80, 111.19, 55.96, 39.99, 25.75.
171
172 N-trans-feruloyl tyramine (9, Figure 1). Colorless powder; HESIMS m/z 314.1 [M + H]
173 (calcd for C18H19NO4, 313.35); 1H NMR (500 MHz, DMSOꢀd6) δ 9.38 (s, 1H), 9.14 (s,
174 1H), 7.95 (t, J = 5.7 Hz, 1H), 7.29 (d, J = 15.7 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 6.99 (d,
175 J = 8.4 Hz, 2H), 6.96 (dd, J = 8.2, 2.0 Hz, 1H), 6.77 (d, J = 8.1 Hz, 1H), 6.66 (d, J = 8.4
176 Hz, 2H), 6.41 (d, J = 15.8 Hz, 1H), 3.78 (s, 3H), 3.33−3.29 (m, 2H), 2.63 (t, J = 7.4 Hz,
177 2H). 13C NMR (125 MHz, DMSOꢀd6) δ 165.72, 156.06, 148.64, 148.23, 139.27, 129.95,
178 129.89, 126.86, 121.92, 119.47, 116.07, 115.54, 111.17, 55.95, 41.09, 34.87.
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179
180 N-trans-feruloyl 3-methoxytyramine (10, Figure 1). Colorless powder; HESIMS m/z
181 344.2 [M + H] (calcd for C19H21NO5, 343.38); 1H NMR (500 MHz, DMSOꢀd6) δ 9.38 (s,
182 1H), 8.68 (s, 1H), 7.95 (t, J = 5.7 Hz, 1H), 7.29 (d, J = 15.7 Hz, 1H), 7.09 (d, J = 1.9 Hz,
183 1H), 6.96 (dd, J = 8.3, 1.9 Hz, 1H), 6.80−6.73 (m, 2H), 6.66 (d, J = 8.0 Hz, 1H), 6.58 (dd,
184 J = 8.0, 1.9 Hz, 1H), 6.42 (d, J = 15.7 Hz, 1H), 3.78 (s, 3H), 3.72 (s, 3H), 3.38 ꢀ 3.29 (m,
185 2H), 2.64 (t, J = 7.4 Hz, 2H). 13C NMR (125 MHz, DMSOꢀd6) δ 165.73, 148.64, 148.24,
186 147.82, 145.22, 139.27, 130.65, 126.86, 121.90, 121.16, 119.49, 116.08, 115.77, 113.20,
187 111.17, 55.95, 55.94, 40.98, 35.24.
188
189 N-trans-feruloyl dopamine (11, Figure 1). Colorless powder; HESIMS m/z 330.1 [M + H]
190 (calcd for C18H19NO5, 329.35); 1H NMR (500 MHz, DMSOꢀd6) δ 9.38 (s, 1H), 8.72 (s,
191 1H), 8.62 (s, 1H), 7.94 (t, J = 5.6 Hz, 1H), 7.29 (d, J = 15.7 Hz, 1H), 7.09 (d, J = 2.0 Hz,
192 1H), 6.96 (dd, J = 8.3, 1.9 Hz, 1H), 6.77 (d, J = 8.1 Hz, 1H), 6.62 (d, J = 7.9 Hz, 1H),
193 6.58 (d, J = 2.1 Hz, 1H), 6.48 ꢀ 6.35 (m, 2H), 3.78 (s, 3H), 3.33−3.23 (m, 2H), 2.56 (t, J =
194 7.4 Hz, 2H). 13C NMR (125 MHz, DMSOꢀd6) δ 165.70, 148.64, 148.23, 145.48, 143.95,
195 139.26, 130.68, 126.87, 121.92, 119.64, 119.50, 116.41, 116.07, 115.93, 111.17, 55.95,
196 41.12, 35.15.
197
198 N-3,4-Dihydroxyhydrocinnamoyl phenethylamine (12, Figure 1). Colorless liquid;
199 HESIMS m/z 286.1 [M + H] (calcd for C17H19NO3, 285.34); 1H NMR (500 MHz, DMSOꢀ
200 d6) δ 8.63 (s, 1H), 7.84 (t, J = 5.5 Hz, 1H), 7.29
−7.21 (m, 2H), 7.17 (dt, J = 8.2, 1.8 Hz,
201 2H), 7.16 7.12 (m, 2H), 6.61 6.57 (m, 1H), 6.55 (d, J = 2.1 Hz, 1H), 6.40 (dd, J = 8.0,
−
−
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202 2.2 Hz, 1H), 3.27−3.18 (m, 2H), 2.67−2.63 (m, 2H), 2.59 (t, J = 7.7 Hz, 2H), 2.24 (dd, J =
203 8.7, 6.9 Hz, 2H). 13C NMR (125 MHz, DMSOꢀd6) δ 171.85, 145.40, 143.72, 139.98,
204 132.60, 129.06, 128.71, 126.43, 119.16, 116.10, 115.82, 40.63, 38.00, 35.66, 31.04.
205
206 N-3,4-Dihydroxyhydrocinnamoyl 3,4-dimethoxyphenthylamine (13, Figure 1). Yellow
207 amorphous powder; HESIMS m/z 346.2 [M + H] (calcd for C19H23NO5, 345.40); 1H
208 NMR (500 MHz, DMSOꢀd6) δ 8.69 (s, 1H), 8.59 (s, 1H), 7.81 (t, J = 5.6 Hz, 1H), 6.82
209 (d, J = 8.1 Hz, 1H), 6.75 (d, J = 2.0 Hz, 1H), 6.64 (dd, J = 8.2, 2.0 Hz, 1H), 6.60 (d, J =
210 8.0 Hz, 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.40 (dd, J = 8.0, 2.1 Hz, 1H), 3.71 (s, 3H), 3.69 (s,
211 3H), 3.21 (dt, J = 7.7, 6.2 Hz, 2H), 2.62–2.58 (m, 4H), 2.28−2.21 (m, 2H).13C NMR (125
212 MHz, DMSOꢀd6) δ 172.35, 149.53, 145.92, 144.24, 133.15, 132.97, 121.41, 121.39,
213 119.67, 116.61, 116.35, 113.44, 112.84, 56.46, 56.30, 41.32, 38.53, 35.74, 31.59.
214
215 N-3,4-Dihydroxyhydrocinnamoyl tryptamine (14, Figure 1). Yellow amorphous powder;
216 HESIMS m/z 325.2 [M + H] (calcd for C19H20N2O3, 324.38); 1H NMR (500 MHz,
217 DMSOꢀd6) δ 10.76 (s, 1H), 8.63 (s, 2H), 7.88 (t, J = 5.8 Hz, 1H), 7.51 (d, J = 7.9 Hz,
218 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.10 (s, 1H), 7.06−7.02 (m, 1H), 6.96 (t, J = 7.4 Hz, 1H),
219 6.60 (d, J = 7.9 Hz, 1H), 6.57 (d, J = 2.1 Hz, 1H), 6.41 (dd, J = 8.0, 2.1 Hz, 1H), 3.30
220 (dd, J = 13.8, 7.0 Hz, 2H), 2.77 (t, J = 7.5 Hz, 2H), 2.61 (dd, J = 8.9, 6.8 Hz, 2H),
221 2.30−
2.21 (m, 2H). 13C NMR (125 MHz, DMSOꢀd6) δ 172.30, 145.90, 144.20, 137.15,
222 133.18, 128.17, 123.50, 121.80, 119.65, 119.18, 119.12, 116.59, 116.33, 112.83, 112.26,
223 40.42, 38.61, 31.57, 26.21.
224
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225 N-3,4-Dihydroxyhydrocinnamoyl tyramine (15, Figure 1). Colorless amorphous powder;
226 HESIMS m/z 302.1 [M + H] (calcd for C17H19NO4, 301.34); 1H NMR (500 MHz, DMSOꢀ
227 d6) δ 8.88 (d, J = 153.8 Hz, 3H), 7.80 (t, J = 5.6 Hz, 1H), 6.95 ꢀ 6.91 (m, 2H), 6.67 ꢀ 6.63
228 (m, 2H), 6.60 (d, J = 8.0 Hz, 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.40 (dd, J = 8.0, 2.1 Hz, 1H),
229 3.20−3.12 (m, 2H), 2.63−2.56 (m, 2H), 2.57−2.50 (m, 2H), 2.27−
2.20 (m, 2H). 13C NMR
230 (125 MHz, DMSOꢀd6) δ 171.81, 156.01, 145.40, 143.71, 132.63, 130.01, 129.90, 119.16,
231 116.10, 115.83, 115.51, 41.01, 38.02, 34.89, 31.06.
232
233 N-3,4-Dihydroxyhydrocinnamoyl dopamine (16, Figure 1). Colorless liquid; HESIMS
234 m/z 318.2 [M + H] (calcd for C17H19NO5, 317.34); 1H NMR (500 MHz, DMSOꢀd6) δ 8.63
235 (s, 4H), 7.79 (t, J = 5.6 Hz, 1H), 6.63 ꢀ 6.56 (m, 2H), 6.55 (d, J = 2.1 Hz, 2H), 6.39 (ddd,
236 J = 8.1, 6.0, 2.1 Hz, 2H), 3.15–3.11 (m, 2H), 2.61−2.57 (m, 2H), 2.48−2.45 (m, 2H),
237 2.25−2.21 (m, 2H).13C NMR (125 MHz, DMSOꢀd6) δ 171.76, 145.46, 145.39, 143.91,
238 143.69, 132.65, 130.71, 119.64, 119.14, 116.37, 116.07, 115.89, 115.83, 41.02, 38.06,
239 35.19, 31.08.
240
241 N-trans-feruloyl 3-phenylpropylamine (17, Figure 1). Yellow liquid; HESIMS m/z 312.1
242 [M + H] (calcd for C19H21NO3, 311.38); 1H NMR (500 MHz, DMSOꢀd6) δ 9.39 (s, 1H),
243 7.99 (t, J = 5.7 Hz, 1H), 7.32 (d, J = 15.7 Hz, 1H), 7.28
244 7.17 7.13 (m, 1H), 7.11 (d, J = 2.0 Hz, 1H), 6.97 (dd, J = 8.2, 1.9 Hz, 1H), 6.78 (d, J =
245 8.1 Hz, 1H), 6.44 (d, J = 15.7 Hz, 1H), 3.79 (s, 3H), 3.20 3.09 (m, 2H), 2.63 ꢀ 2.54 (m,
−7.24 (m, 2H), 7.21−7.17 (m, 2H),
−
−
246 2H), 1.78 ꢀ 1.68 (m, 2H). 13C NMR (125 MHz, DMSOꢀd6) δ 166.26, 149.14, 148.74,
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247 142.63, 139.77, 129.23, 129.20, 129.17, 127.37, 126.65, 122.43, 119.97, 116.57, 111.63,
248 56.43, 39.21, 33.52, 31.95, 15.01.
249
250 LC-MS/MS analysis
251
LC separation was performed with an Ultimate 3000 system (Dionex, Sunnyvale,
252 CA, USA) including an RS pump, an XRS Open autosampler, and an RS column
253 compartment. Sixteen amide compounds were simultaneously chromatographed on a
254 Synergi FusionꢀRP column (2.0 mm × 100 mm, 2.5 ꢁm particle size, Phenomenex,
255 Torrance, CA, USA). The column temperature was set to 25 °C. Gradient elution of
256 mobile phase A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) was
257 applied with a flow rate of 0.4 mL/min with a linear gradient from 20% to 100% solvent
258 B for 0ꢀ5 min, holding at 100% solvent B for 5ꢀ7 min, and then returning to 20% solvent
259 B for column equilibration. The injection volume was 10 µL.
260
MS detection was conducted on a triple quadrupole mass spectrometer (TSQ
261 Quantiva, Thermo Fisher Scientific, San Jose, CA, USA) with selected reaction
262 monitoring (SRM). The instrument was operated with a heatedꢀelectrospray ionization
263 (HESI) in both polarity modes. All compounds except for compound 3 were analyzed in
264 positive mode, and only compound 3 was detected in negative mode. Nitrogen gas was
265 used as both sheath and auxiliary gas. Argon gas was employed as the collision gas. The
266 ion source conditions were as follows: positive spray voltage, 3,500 V; negative spray
267 voltage, 2,500 V; sheath gas, 45 Arb; aux gas, 15 Arb; sweep gas, 1 Arb; ion transfer tube
268 temperature, 350 °C; and vaporizer temperature, 350 °C. The MS/MS parameters were
269 optimized as follows: collision gas pressure, 2 mTorr; source fragmentation voltage, 0 V;
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270 chrom filter, 3 sec; and dwell time, 100 msec. RF lens voltage was 85 V for compound 3
271 and 10, and was 65 V for the others. Data analysis was performed using Xcaliber
272 software (Ver. 3.0).
273
274 Preparation of standard solutions
275
Stock solutions were prepared at a concentration of 1,000 µg/mL, filtered through
276 0.45 ꢁm nylon membrane filters, and stored at ꢀ20 °C until use. 1, 2, 3, 4, 6, 7, 8 and 9
277 were dissolved in dimethyl sulfoxide, 12, 13, 14 and 15 were dissolved in dimethyl
278 sulfoxide:methanol (1:1) mixture, and 5, 10, 11, and 16 were dissolved in dimethyl
279 sulfoxide:methanol (1:4) mixture. Standard working solutions were prepared by diluting
280 and mixing each stock solution with methanol to obtain proper concentrations. For
281 compound 17 (internal standard), a stock solution was prepared in dimethyl
282 sulfoxide:methanol (1:4) mixture at a concentration at 1,000 µg/mL, and a working
283 solution was prepared by diluting the stock solution with methanol. Each sample
284 contained 10 ng/mL of internal standard.
285
286 Preparation of samples
287
Dried wolberries were finely ground into powder, and then extracted using one of
288 two different methods. For compounds 9 and 10, which were found in relatively high
289 quantities in the sample, 10 mg of the powder was extracted using 4.5 mL of methanol
290 and 0.5 mL of internal standard solution in an ultrasonic bath at ambient temperature for
291 40 min. After vigorous agitation by using a multiꢀtube vortexer for 1 h, the resultant
292 solution was filtered through a 0.45 ꢁm membrane filter, and injected into the LCꢀMS.
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293 For all other compounds, an evaporation procedure was added during the extraction. 100
294 mg of the powder was extracted using 4.5 mL of methanol and 0.5 mL of internal
295 standard solution using the same procedure previously described. Then, 3 mL of the
296 filtrate was vaporized under nitrogen, and the residue was suspended in 0.3 mL of
297 methanol before injection into the LCꢀMS/MS system.
298
299 Cell culture
300
RAW264.7 murine macrophages were purchased from the American Type
301 Culture Collection (Manassas, VA, USA). Cells were cultured in highꢀglucose
302 Dulbecco’s Modified Eagle’s medium, supplemented with 100 IU/mL
303 penicillin/streptomycin, 1 mM sodium pyruvate and 10% fetal bovine serum. Cells were
304 incubated in 10 cm culture Petri dishes in 5% CO2 with 70% humidity at 37 °C.
305
306 Cell viability Assay
307
4×105 cells/mL were seeded into 96ꢀwell plates and incubated for 12 hours before
308 treatment. Compounds of interest were first dissolved in molecular biology grade DMSO
309 at a concentration of 100 ꢁM, and were further diluted with growth media to reach the
310 final assay concentration. Growth media with 0.01% v/v DMSO served as the control. 75
311 ꢁg/mL of LꢀNMMA was used as a positive control. Either the compound of interest or a
312 vehicle was added to the medium and then incubated for 24 hr. After treatment, cells
313 were washed twice with phosphate buffered saline. Phenol red free medium containing 5
314 mg/mL of 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide was added to
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315 cells and then incubated at 37 °C for 4 hours. After removing the supernatant, formazan
316 crystals were dissolved in 150 µL of DMSO. Optical densities were measured at 570 nm.
317
318 Nitrite Assay
319
100 ꢁM DMSO stock solutions of test compounds were diluted with growth
320 media to achieve assay concentrations.75 ꢁg/mL LꢀNMMA was used as positive control.
321 Cells (4×105 cells/mL) were treated with E. coli LPS (100 ng/mL) in either the presence
322 of the compound of interest or 0.01% dimethyl sulfoxide (DMSO) as a vehicle in phenol
323 red free medium for 24 hours. After a 12ꢀhour incubation period, 50 µL of conditional
324 supernatant was removed, mixed with an equal volume of Griess reagent (1%
325 sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride
326 in water), and incubated at room temperature for 10 minutes. Production of nitrite was
327 measured at an absorbance of 550 nm.
328
329 Statistical Analysis
330
All compound quantification results are shown as mean ± standard deviation.
331 Statistical analysis for IC50 was performed using Prism 7 by nonꢀlinear regression. IC50
332 values are shown as mean ± standard error. All experimental data were obtained
333 independently and replicated a total of three times. Significant differences were
334 determined as p < 0.05.
335
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336 Results and discussion
337 NMR of synthetic amide compounds
338
Amide structures were confirmed using the triplet with coupling constant of J
339 ~5.7 Hz. 1H NMR spectrum of caffeoyl and feruloyl amides had two vinyl doublets with
340 a coupling constant of J ~15.7 Hz, and feruloyl amides also had a methoxyl singlet at
341 3.80 ppm. 3,4ꢀdihydroxyhydrocinnamoyl amides lost the vinyl structure and alternately,
342 characteristic methylene chemical shifts around 2.58 ppm were found. 3,4ꢀ
343 dimethoxylphenethylamine moieties had two singlets with a chemical shift of ~3.70 ppm,
344 which were attributed to two methoxyl groups. An indole singlet above 10 ppm
345 confirmed the tryptamine moieties in compounds 3, 8, and 14.
346
347 Quantification of amide compounds
348
The LCꢀMS/MS analytical results showed that the fruits had amide compounds at
349 various concentrations. For example, the content of 9 was greater than 10,000ꢀfold more
350 than that of 7, whereas some compounds only presented in nanogram quantities.
351
The established LCꢀMS/MS method was applied to comprehensive analysis and
352 quantitative evaluation of the fruits samples. The analysis was performed in triplicate
353 (n=3). The chromatograms of 16 amide compounds are shown in Figure 2. The
354 compounds were fairly well separated from interferences. The retention times of
355 compounds 1ꢀ16 and 17 (internal standard) are 2.93 min, 2.66 min, 2.98 min, 2.32 min,
356 2.03 min, 3.25 min, 2.97 min, 3.25 min, 2.64 min, 2.36 min, 2.71 min, 2.46 min, 2.81
357 min, 2.04 min, 1.71 min, 2.68 min and 3.49 min, respectively. Quantification of each
358 analyte in the samples was calculated using the ratio of peak area (analyte peak area
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359 versus internal standard peak area) based on the calibration curve of each individual
360 standard. The correlation coefficient values of all models exhibited good linearity (R2 >
361 0.998) (Supplementary Figure 1). The results indicated successful application of the LCꢀ
362 MS/MS method for the quantification of amide compounds in different quantities. The
363 major constituents in the fruits were found to be 4, 5, 9, 10 and 11. Their contents were
364 between 107.2 and 11109.6 ng/g. The content of 3Tyra was 12.1 ng/g. All compounds
365 contained a tyramine or dopamine moiety, whereas compounds, 6ꢀ8, having a
366 phenethylamine, 3,4ꢀdimethoxyphenethylamine or tryptamine moiety, were found to be
367 minor ones (0.7 to 3.1 ng/g). Table 1 lists the mean concentrations of amide compounds
368 detected in the samples.
369
370 The methodology for identification of HCCA compounds from wolfberries
371
In our study, a novel identification method used synthetic standards as references
372 for LCꢀMS/MS analysis that compensated for the drawbacks of identification and
373 isolation of minor components from the plants. Our synthesis design was based on the
374 results from literatures and plant biosynthesis pathway of HCCA compounds. HCCA
375 compounds form a large class of secondary metabolites abundantly present in plants,
376 serving as growth and floral signaling compounds as well as metabolic intermediates.1, 23
377 In addition to the HCCA compounds isolated from the wolfberry fruits, several studies
378 reported that HCCA compounds with transꢀcaffeic, transꢀferulic and 3,4ꢀ
379 dihydroxyhydrocinnamic acid moieties were isolated from the root bark of the plant.24, 25
380 These hydroxycinnamoyl moieties derived from deaminated phenylalanine, and these
381 hydroxycinnamic acid precursors, including transꢀcaffeic acid and transꢀferulic acid were
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382 also identified from wolfberries17. Thus, we proposed that HCCA compounds were
383 synthesized from these three hydroxycinnamic acid families. Amine moieties could result
384 from decarboxylated amino acid; for instance, transꢀferuloyl tyramine is the most
385 common HCCA species conjugated from decarboxylated amino acid, tyrosine, which was
386 also found in wolfberry and other plant species.26ꢀ28 Plant aromatic Lꢀamino acid
387 decarboxylases generate phenethylamine, tryptamine and dopamine conjugates from Lꢀ
388 phenylalanine, Lꢀtryptophan and LꢀDopa, respectively.29 The amine conjugates can be
389 further modified through speciesꢀspecific hydroxylation or methylation reaction,30 which
390 gives possible methoxylated species, in our case, 3,4ꢀdimethoxyphenethylamine and 3ꢀ
391 methoxytyramine. Finally, the coupling of hydrocycinnamoyl and amine moieties is
392 catalyzed by a diverse set of specific hydroxycinnamoyl transferases.30 As a result, we
393 designed the synthesis from three hydroxycinnamic acid species with similar extended
394 amine moieties.
395
As shown in Table 1, our method did improve sensitivities to identify minor
396 components from the plants. We identified compounds 6, 7, 8 and 15 with concentrations
397 as low as 0.7 ng/g. Furthermore, by using reference standards, LCꢀMS/MS analysis
398 differentiated structural analogs with high sensitivities, which may remain a challenge
399 using traditional identification and isolation processes.
400
401 Nitric Oxide inhibition of HCCAs
402
Chronic inflammation is a complex process mediated by activation of
403 inflammatory or immune cells and has been shown to trigger chronic disorders. During
404 the inflammatory response, macrophages play a critical role in managing inflammatory
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405 phenomena such as the overproduction of proꢀinflammatory cytokines and inflammatory
406 mediators, including nitric oxide (NO). In order to select proper concentrations of the
407 compounds of interest for the antiꢀinflammatory studies, murine macrophage RAW264.7
408 cells were treated with the compounds of interest at various concentrations or 0.01%
409 DMSO vehicle for 24 hours, and cell viability was determined by MTT assay. Among the
410 IC50 values shown in Table 2, all of the tested compounds possessed IC50 values larger
411 than 100 ꢁM, indicating low cytotoxicity of these compounds, which was also shown in
412 Figure 3.
413
The NO production inhibitory effects of these HCCA compounds were
414 investigated by coꢀincubating RAW264.7 murine macrophages with the test HCCA
415 compounds and LPS (100 ng/mL) for 24 hours. The NO accumulation in cell medium
416 was measured by Griess reagent. After LPS stimulation, the NO production significantly
417 increased compared to the negative control groups, and could be observed in all
418 experimental groups, as seen in Figure 4. These results are shown in Table 2. A total of
419 five HCCA compounds exhibited NO inhibitory properties, including two HCCA
420 compounds with transꢀcaffeic acid moiety, compounds 4, and 5, two compounds with
421 transꢀferulic acid moiety, 6 and 11, and one compound with 3,4ꢀdihydrohydroxycinnamic
422 acid moiety, 15. Compound 5 was found to be the most potent compound that inhibited
423 NO production in LPSꢀinduced murine macrophage with the lowest IC50 value, followed
424 by 4, which also showed significant inhibitory effects on NO production. Hence, in
425 wolfberry, these two compounds are abundantly present (Table 1).
426
These results suggest excellent antiꢀinflammatory properties of the HCCA
427 compounds, especially the caffeic acid derivatives. Further efforts have been proposed to
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428 study the detailed structureꢀactivity relationship of HCCA compounds and their antiꢀ
429 inflammatory effects, as well as the underlying antiꢀinflammatory mechanism.
430
In our study, 16 HCCA compounds that potentially exist in wolfberry were
431 synthesized and used as reference standards for LC/MSꢀMS analysis. Among these
432 candidates, nine amide compounds were identified from the fruits. Furthermore, seven of
433 these compounds were identified for the first time in this plant (compounds 5ꢀ8, 11, 15),
434 and all the compounds found in the plant were directly quantified. By using HCCA
435 species from wolfberry as a model, we proposed a methodology for natural products
436 identification with improved sensitivities, which differentiate structural analogs as well as
437 identify minor chemical components.
438
The antiꢀinflammatory activities of these compounds were investigated in vitro
439 using an RAW264.7 cell model. We found that these compounds exhibited a promising
440 inhibitory effect on NO production. Compounds 4 and 11 showed significant potency in
441 NO inhibition. The major advantage of amide compounds is their stable characteristic at
442 physicological condition, leading to broader applications. Future plans include
443 elucidation of detailed molecular mechanisms of antiꢀinflammatory properties using both
444 in vitro and in vivo models, as well as their absorption and metabolism. The discovery of
445 novel HCCA compounds from worberry broadened the variety of this family in the plant,
446 enhancing potential use of wolfberry as functional ingredients in foods and dietary
447 supplements to prevent and treat of inflammation and inflammationꢀrelated diseases.
448
449 Funding
450 Researches fund by USDA NIFA Hatch Project NJ10136.
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451
452 Supporting Information
453 The supporting information contains SRM parameter setting for compound 1ꢀ17 and LCꢀ
454 MS/MS spectrum of compound 1ꢀ17
455
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456 References
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531 X.; Chen, X. H.; Bi, K. S.; Guo, D. A., Simultaneous determination of 24 constituents in
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Zhang, J.; Guan, S.; Sun, J.; Liu, T.; Chen, P.; Feng, R.; Chen, X.; Wu, W.; Yang,
535 M.; Guo, D.ꢀa., Characterization and profiling of phenolic amides from Cortex Lycii by
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Amaro, C.; GonzálezꢀCortazar, M.; HerreraꢀRuiz, M.; RománꢀRamos, R.;
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552
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553 Figure Captions
554 Figure 1. Chemical structure of HCCA synthetic compounds. Compounds 1ꢀ5 were
555 transꢀcaffeic acid species. Compounds 6ꢀ11 were transꢀferulic acid species, and
556 compounds 12ꢀ16 were 3,4ꢀdihydroxyhydrocinnamic acid species. Compound 17 was
557 synthesized by transꢀferulic acid and 3ꢀphenylpropylamine, and used as the internal
558 standard for quantification.
559
560 Figure 2. LCꢀMS/MS chromatograms of standards (A) and fruit extracts (B): 1 N-transꢀ
561 caffeoyl phenethylamine, 2 N-transꢀcaffeoyl 3,4ꢀdimethoxyphenethylamine, 3 Nꢀtransꢀ
562 caffeoyl tryptamine, 4 N-transꢀcaffeoyl tyramine, 5 N-trans-caffeoyl dopamine, 6 N-
563 transꢀferuloyl phenethylamine, 7 N-transꢀferuloyl 3,4ꢀdimethoxyphenethylamine, 8 Nꢀ
564 transꢀferuloyl tryptamine, 9 N-transꢀferuloyl tyramine, 10 N-transꢀferuloyl 3ꢀ
565 methoxytyramine, 11 N-transꢀferuloyl dopamine, 12 Nꢀ3,4ꢀ
566 Dihydroxyhydrocinnamoyl phenethylamine, 13 Nꢀ3,4ꢀDihydroxyhydrocinnamoyl 3,4ꢀ
567 dimethoxyphenethylamine, 14 N-3,4ꢀDihydroxyhydrocinnamoyl tryptamine, 15 N-3,4-
568 Dihydroxyhydrocinnamoyl tyramine, 16 N-3,4ꢀDihydroxyhydrocinnamoyl dopamine,
569 and 17 N-trans-feruloyl 3ꢀphenylpropylamine (internal standard)
570
571 Figure 3. Cytotoxicity of HCCA compounds on RAW264.7 cells. 75 ꢁg/mL LꢀNMMA
572 was used as a positive control. The cells were incubated with compounds of interest or
573 vehicle control (0.01% DMSO) for 24 hour. Asterisks indicate significant differences
574 from the control (0 ꢁM) determined using Dunnettʼs multiple comparison tꢀtest (*p <
575 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001).
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576
577 Figure 4. Effect of HCCA compounds on LPSꢀinduced NO release of RAW624.7 cell. 75
578 ꢁg/mL LꢀNMMA was used as the positive control. Compounds of interested were coꢀincu
579 bated with 100 ng/mL LPS for 24 hour. Negative control groups (ꢀ) were incubated with
580 growth media only without LPS or compounds of interest. The NO accumulation in cell
581 medium was measured by Griess reagent. Asterisks indicate significant differences from
582 the control (0 ꢁM) determined using Dunnettʼs multiple comparison tꢀtest (*p < 0.05, **
583 p < 0.005, *** p < 0.001, **** p < 0.0001).5
584
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