Cinnamaldehyde

In this study, AB (wild-type), Nrf2-mutant (nfe2l2a^fh318) [13 (link)], and Nrf2-knockout (nfe2l2a^it321) zebrafish larvae were used. Both mutant and knockout lines were maintained by PCR-based genotyping. The former was maintained as described previously [14 (link)]. For the latter, the primer sets 5′-TATTGTGCAGCCCTAGTGTG and 5′-TAGCTGAAGTCGAACACCTC were used. Larvae used in these experiments were obtained from parents of AB, homozygous nfe2l2a^fh318 or homozygous nfe2l2a^it321 by natural mating. The nfe2l2a^fh318 and nfe2l2a^it321 lines can be obtained from the Zebrafish International Resource Center (http://zebrafish.org (accessed on 2022 January 5)) and the National BioResource Project Zebrafish (https://shigen.nig.ac.jp/zebra (accessed on 2022 January 5)), respectively.
Genistein, glycitin, glycitein, daidzin, daidzein, equol, and cinnamaldehyde were purchased from FUJIFILM Wako (Osaka, Japan). Genistin and sulforaphane were purchased from NAGARA Science (Gifu, Japan) and LKT Laboratories (St. Paul, MN, USA), respectively. For stock solutions, hydrogen peroxide and sodium arsenite were dissolved in MilliQ water (Merck-Millipore Billerica, MA, USA), sulforaphane in ethanol, and isoflavone compounds in dimethyl sulfoxide. They were diluted to final concentrations with E3+ medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄ and 0.1 µg/mL methylene blue).

Reference standards of amygdalin, coumarin, and cinnamic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Albiflorin, cinnamaldehyde, paeoniflorin, and paeonol were obtained from Wako (Osaka, Japan). The purity of the seven reference standards was ≥98.0%. The chemical structures of the seven marker components are shown in Fig. 1a. High-performance liquid chromatography (HPLC)-grade reagents methanol, acetonitrile, and water to obtain the aqueous extract of GJBRHE were obtained from J. T. Baker (Phillipsburg, NJ, USA). Acetic acid was purchased from Merck (Darmstadt, Germany).Fig. 1

HPLC chromatograms of (a) the reference standard mixture and (b) GJBRHE: 230 nm (I), 254 nm (II), and 280 nm (III)

In this study, wild-type (AB strain) and Nrf2-mutant (nfe2l2a^fh318) [15 (link)] zebrafish larvae were used. The Nrf2-mutant line was maintained by polymerase chain reaction based genotyping, as described previously [28 (link)]. Embryos were obtained by natural mating. Capsaicin, carnosic acid, cinnamaldehyde, eugenol, 6-gingerol, isoeugenol, quercetin, H₂O₂ and NaAsO₂ were purchased from FUJIFILM Wako (Osaka, Japan). Diallyl trisulfide and sulforaphane were bought from LKT Laboratories (St. Paul, MN, USA). Curcumin and 6-MSITC were purchased from Sigma-Aldrich Japan (Tokyo, Japan) and Abcam (Cambridge, UK), respectively. H₂O₂ and NaAsO₂ were dissolved in MiliQ water (Merck-Millipore, Billerica, MA), sulforaphane in ethanol and other phytochemicals were dissolved in dimethyl sulfoxide for the stock solution, and were diluted to the final concentration with E3+ medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄ and 0.1 μg/mL methylene blue).

CA (trans-Cinnamic acid, Wako Pure Chemical Industries, Osaka, Japan) and CD (Cinnamaldehyde, Wako Pure Chemical Industries, Osaka, Japan) were dissolved in 10% dimethyl sulfoxide (DMSO, Sigma) (adjusted pH 7 by using 2M HCl and 5M NaOH) and 1% Tween 20 (Sigma) respectively. The final volume of 10% DMSO and 1% Tween 20 used in the experiments is 2 µL/g rat. Different doses of CA and CD (10, 20, and 40 mg/kg) were administered (i.p.) in rats with cold and mechanical allodynia.

Cinnamaldehyde (≥98.0%), cinnamic acid (≥98.0%), liquiritin (≥99.0%), 6-gingerol (≥98.0%), and glycyrrhizin (≥99.0%) were purchased from Wako (Osaka, Japan). Amygdalin and coumarin (both ≥99.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). liquiritin apioside (≥98.0%) was purchased from Shanghai Sunny Biotech (Shanghai, China) and ephedrine HCl (≥95.0%) was provided from Ministry of Food and Drug Safety. High-performance liquid chromatography (HPLC) grade, methanol, acetonitrile, and water were purchased from J. T. Baker (Phillipsburg, NJ, USA). Reagent grade, trifluoroacetic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The reference standards, cinnamaldehyde and coumarin, were purchased from Wako (Osaka, Japan) and Sigma-Aldrich (St. Louis, CA, USA). The purity of the two reference standards was ≥98.0% by HPLC analysis. The HPLC-grade solvents, acetonitrile and water, methanol, were obtained from J.T. Baker (Phillipsburg, NJ, USA). The Oryeong-san samples used in this study consisted of five herbal medicines (Table 1) and were purchased from Kwangmyungdang Medicinal herbs (Ulsan, Korea). All herbal medicines were taxonomically confirmed by Professor Je-Hyun Lee, Dongguk University, Korea. Voucher specimens (2013-KE17-1 through KE17-5) have been deposited at the Herbal Medicine Formulation Research Group, Korea Institute of Oriental Medicine.Table 1

The combination of crude components of ORSE

Latin name	Amount (g)	Herbal name	Source
Alisma orientale Juzepzuk	9.375	Alismatis Rhizoma	Yeongcheom, Korea
Poria cocos	5.625	Poria Sclerotium	Pyeongchang, Korea
Atractylodes japonica Koidzumi	5.625	Atractylodis Rhizoma Alba	China
Polyporus umbellatus Fries	5.625	Polyporus	China
Cinnamomum cassia Presl	1.875	Cinnamomi Cortex	Vietnam
Total	28.1