Identification of Characteristic Phenolic Constituents in Mousouchiku Extract Used as Food Additives

Abstract

Mousouchiku extract is prepared from the bamboo-sheath of Phyllostachys heterocycla MITF. (Poaceae), and is registered as a food manufacturing agent in the List of Existing Food Additives in Japan. This study describes the chromatographic evaluation of characteristic components of this extract to obtain the chemical data needed for standardized specifications. We isolated 12 known compounds from this extract: 5-hydroxymethyl-2-furfural, 4-hydroxybenzoic acid, trans-p-coumaric acid, trans-ferulic acid, N,N′-diferuloylputrescine, 4′-hydroxypropiophenone, β-arbutin, tachioside, isotachioside, 3,4′-dihydroxypropiophenone 3-O-glucoside, koaburaside, and (+)-lyoniresinol 9′-O-glucoside. Moreover, a new propiophenone glycoside, propiophenone 4′-O-(6-β-D-xylosyl)-β-D-glucoside (propiophenone 4′-O-primeveroside), was isolated. The structure of each isolated compound was elucidated based on NMR and MS data or direct HPLC comparisons with authentic samples. Among the isolates, (+)-lyoniresinol 9′-O-glucoside was found to be the major ingredients of the extract as observed using HPLC analysis. However, 2,6-dimethoxy-1,4-benzoquinone, which is considered the main constituent of mousouchiku extract, was only detected as a trace constituent and not isolated in this study.

The genus Phyllostachys (Poaceae) consists of approximately 50 species, with most of them being traditionally used as a material for preparation of basket, sieve, and other crafts. Bamboo is one of the most popular plants and has been used for a long time in preparation of food-related items in Japan. Thus, bamboo is one of the familiar plants to Japanese people. P. heterocycla MITF. (Japanese name: mousouchiku) is widely distributed in warm-humid East Asia. In the List of Existing Food Additives in Japan, mousouchiku extract is defined as “an additive obtained from the epidermis of stem of P. heterocycla MITF. with 2,6-dimethoxy-1,4-benzoquinone being its main constituent.” Concerning the origin, manufacturing method, and essential qualities of this additive, the official list states: “This additive is prepared from the epidermis of stem of P. heterocycla MITF. using extraction with ethanol.” However, previous phytochemical studies on secondary metabolites of this plant are limited. Nishina et al. reported an antibacterial component 2,6-dimethoxy-p-benzoquinone in the bark of mousouchiku.¹⁾ In addition, ethoxyquin, sesquiterpenes, and cyclohexanone derivatives were identified using GC/MS as antioxidants and antimicrobials.

Most existing food additives, which are officially registered under the Food Sanitation Act, are natural extracts containing various ingredients. However, the characteristic and effective components of the existing food additives are not always properly defined due to poor characterization of the ingredients in the respective raw material. Thus, we evaluated the characteristic constituents of the existing food additives from natural extracts such as gentian root extract²⁾ and grape skin extract.³⁾ There is little information available on the chemical constituents of mousouchiku extract. To ensure food safety, analysis and characterization of individual constituent and establishment of analytical standards for developing national specifications are required.

In this study, we investigated the constituents of mousouchiku extract to collect the chemical data requisite for the development of standardized specifications. Additionally, to evaluate the antimicrobial activity of commercial products and isolates, an antimicrobial susceptibility test utilizing Escherichia coli and Staphylococcus aureus was performed.

Results and Discussion

The commercial mousouchiku extract (commercial product A, ethanol extract of P. heterocycla) was suspended in water, and then partitioned into n-hexane-, ethyl acetate (EtOAc)-, n-butanol (n-BuOH)-, and water-soluble portions. The EtOAc extract was separated by column chromatography to extract six known compounds, 5-hydroxymethyl-2-furfural (2), 4-hydroxybenzoic acid (3), trans-p-coumaric acid (4), trans-ferulic acid (5), N,N′-diferuloylputrescine (6),⁴⁾ and 4′-hydroxypropiophenone (7) (Fig. 1). The n-BuOH extract was separated by combination of chromatography to furnish compound 1 and six known compounds, β-arbutin (8), tachioside (9),⁵⁾ isotachioside (10),⁵⁾ 3,4′-dihydroxypropiophenone 3-O-glucoside (11),⁶⁾ koaburaside (12),⁷⁾ and (+)-lyoniresinol 9′-O-glucoside (13)⁸⁾ (Fig. 1).

Fig. 1. Structure of Compounds (1–13)

Compound 1 was obtained as an off-white amorphous powder and assigned the molecular formula C₂₀H₂₈O₁₁ based on high resolution-electrospray ionization (HR-ESI)-MS data [m/z 467.1553 (M+Na)⁺, Calcd for 467.1524]. The ¹H-NMR spectrum showed two ortho-coupled 2H-doublets (δ 7.98, 7.19) in the aromatic proton region, and vicinal-coupled methylene [δ 3.02 (2H, dq, J=1.5, 7.5, 7.5, 7.5 Hz)] and methyl [δ 1.17 (3H, t, J=7.5 Hz)] protons were observed in the aliphatic proton region along with two pairs of sugar protons, which were assigned based on ¹H–¹H correlation spectroscopy (COSY) spectrum (Fig. 2). The ¹³C-NMR and heteronuclear single quantum coherence (HSQC) measurements indicated AA′BB′ signals [δ 162.8, 132.5, 131.4 (2C), 117.4 (2C)], methylene carbon (δ 32.4), methyl carbon (δ 8.8), two pairs of sugar carbons (five and six membered sugar), and carbonyl carbon (δ 202.1). These spectral data indicated the compound 1 to be a di-glycoside of propiophenone. The location of sugar moiety and side chain was confirmed based on heteronuclear multiple bond connectivity (HMBC) correlations (Fig. 2) from H-1′ (δ 4.99) to C-4 (162.8), H-1″ (δ 4.31) to C-6′ (δ 69.8), and H-2 (δ 7.98) and H-9 (δ 1.17) to C-7 (δ 202.1), respectively. Additionally, to confirm the absolute configuration of sugar moiety, compound 1 was hydrolyzed and subsequently derivatized according to the method reported by Tanaka et al.⁹⁾ and D-glucose and D-xylose were confirmed by direct HPLC comparisons with authentic samples treated under the same condition. Based on this data, the structure of compound 1 was assigned as propiophenone 4′-O-primeveroside.

Fig. 2. Key ¹H–¹H COSY () and HMBC (→) Correlations of Compound 1

To estimate the phenolic distribution in the mousouchiku extract, the commercial product (A) was analyzed by reversed-phase HPLC (Fig. 3) with authentic original samples, and (+)-lyoniresinol 9′-O-glucoside (13) and trans-p-coumaric acid (4) were found to be the major ingredients. This additive is characterized as containing 2,6-dimethoxy-1,4-benzoquinone as the major ingredient. The minor peak at 18.4 min was not identical to that of 2,6-dimethoxy-1,4-benzoquinone (t_R 17.9 min) from the difference of the retention time and the UV λ_max pattern from authentic compound at the photodiode array detector-equipped HPLC analysis. Additionally, other commercial food manufacturing agents (B–F) were analyzed under the same conditions, and 2,6-dimethoxy-1,4-benzoquinone was not detected as one of the major ingredient (Fig. 3). In contrast, compound 13 was detected in samples E and F; therefore, it is suggested that this compound is characteristic phenolic in this extract.

Fig. 3. RP-HPLC Profile (UV 280 nm) of Mousouchiku Extracts (A–F) and 2,6-Dimethoxy-1,4-benzoquinone (a)

The number on the chromatogram corresponds to the compound number. The HPLC condition is described in Experimental.

We also evaluated the antimicrobial activities of isolates, extracts, and commercial products of P. heterocycla against E. coli JM109 and S. aureus FDA209P by the standard two-fold dilution method. None of the isolates showed any antimicrobial activity, whereas 2,6-dimethoxy-1,4-benzoquinone exerted potent antimicrobial activity against S. aureus FDA209P [minimum inhibitory concentration (MIC): 4 µg/mL] (Table 1). Among the commercial products, sample D showed weak antimicrobial activity (MICs for E. coli JM109: 400 µg/mL, S. aureus FDA209P: 200 µg/mL, Table 2).

Table 1. Antimicrobial Activities of Isolated Compounds (1–13) from the Commercial Ethanol Extract (A) and 2,6-Dimethoxy-1,4-benzoquinone (a) against Escherichia coli JM109 and Staphylococcus aureus FDA209P

Compounds	MIC (µg/mL)
	E. coli JM109	S. aureus FDA209P
1	512	>512
2	>512	512
3	>512	512
4	>512	>512
5	>512	>512
6	>512	>512
7	>512	>512
8	>512	>512
9	>512	>512
10	>512	512
11	>512	>512
12	>512	>512
13	512	>512
a	128	4

Table 2. Antimicrobial Activities of Commercial Ethanol Extracts of Phyllostachys heterocycla (A–F)

Products	MIC (µg/mL)
	E. coli JM109	S. aureus FDA209P
A	3200	1600
B	3200	3200
C	3200	3200
D	400	200
E	3200	800
F	1600	800

In the present study, (+)-lyoniresinol 9′-O-glucoside (13) was found to be the major ingredient of the mousouchiku extract from HPLC analysis. On the other hand, 2,6-dimethoxy-1,4-benzoquinone, defined in the official list as main constituent, was not detected. Therefore, the standardized markers in mousouchiku extract should be defined. These results would contribute to establishing the quality standard of this food additive.

Experimental

General

NMR spectra were recorded on Bruker AVANCE 500 (500 MHz for ¹H and 126 MHz for ¹³C) with chemical shifts given in δ (ppm) values relative to those of the solvent (methanol-d₄ [δ_H 3.30, δ_C 49.0] and acetone-d₆ [δ_H 2.05, δ_C 29.8]) on a tetramethylsilane (TMS) scale. HR-ESI-MS spectra were obtained on a micrOTOF-Q (Bruker, Karlsruhe, Germany) mass spectrometer. Reversed-phase HPLC was performed on a Shimadzu LC-10Avp system (Kyoto, Japan), using YMC-pack ODS-AQ (ϕ2.0×150 mm) column at 40°C with flow rate of 0.25 mL/min.

The analyses were conducted using solvent A (100 µM phosphate buffer) and solvent B (methanol) at the following gradient: 0–50% B (linear gradient) from 0–30 min and 50–60% B (linear gradient) from 30–50 min, monitored at 210–400 nm.

Column chromatography was carried out on Diaion HP-20 (Mitsubishi Chemicals, Tokyo, Japan), Toyopearl HW-40 (coarse grade: Tosoh Co., Tokyo, Japan), YMC-gel ODSAQ12S50 (YMC Co., Ltd., Kyoto, Japan), Chromatorex ODS (Fuji Silysia Chemical Ltd., Aichi, Japan), and Sephadex LH-20 (GE Healthcare Japan, Tokyo, Japan). Each authentic sample (β-arbutin [Nacalai Tesque, Kyoto, Japan, 03314-94], 4′-hydroxypropiophenone [Tokyo Chemical Industry, Tokyo, Japan, H0299] was purchased from the manufactures. Each commercial product of food manufacturing agent was obtained from Japan Food Additives Association (JAFA) (Tokyo, Japan).

Isolation Procedure

The commercial ethanol extract of P. heterocycla was obtained from JAFA (Tokyo, Japan). The commercial product A (23.4 g) was suspended in water (700 mL) and then successively extracted with n-hexane (700 mL×3), EtOAc (700 mL×3), and n-BuOH (700 mL×3) to yield n-hexane (52.8 mg), EtOAc (682.5 mg), n-BuOH (2.7 g), and H₂O (20.9 g) extracts. The EtOAc extract was fractionated by column chromatography using a YMC-gel ODS-AQ (ϕ1.1×40 cm) with aqueous methanol (50→60%)→methanol to fractions (S1–S11, 50%; S1–S8, 60%; S9–S11, methanol), monitored by reversed-phase HPLC. Fraction S1 (121.9 mg) was further separated by Chromatorex ODS (ϕ1.1×20 cm) column chromatography to give 5-hydroxymethyl-2-furfural (2, 1.5 mg) and 4-hydroxybenzoic acid (3, 2.4 mg). Fraction S2 (112.6 mg) was further purified by Sephadex LH-20 and YMC-gel ODS-AQ column chromatography (ϕ1.1×20 cm) to give trans-p-coumaric acid (4, 5.4 mg) and trans-ferulic acid (5, 1.5 mg). Fraction S3 (40.0 mg) was separated by Chromatorex ODS column chromatography (ϕ1.1×20 cm) to yield 4′-hydroxypropiophenone (7, 1.1 mg). Fractions S4–S6 (109.7 mg) were combined and fractionated by Sephadex LH-20 subsequent YMC-gel ODS-AQ and Chromatorex ODS column chromatography (ϕ1.1×20 cm) to give N,N′-diferuloylputrescine (6, 1.3 mg). The n-BuOH extract (2.50 g) was separated by Diaion HP-20 column chromatography (ϕ2.0×40 cm) with H₂O→aqueous methanol (10→20→30→50%)→methanol, and yielded each eluate. The 10% methanol eluate (80.5 mg) was further separated by YMC-gel ODS-AQ column chromatography (ϕ1.1×20 cm) to give β-arbutin (8, 4.8 mg), tachioside (9, 3.8 mg), and isotachioside (10, 4.9 mg). The 20% methanol eluate (64.5 mg) was further purified by YMC-gel ODS-AQ column chromatography (ϕ1.1×20 cm) to yield tachioside (9, 4.6 mg), 3,4′-dihydroxypropiophenone 3-O-glucoside (11, 3.4 mg), and koaburaside (12, 17.2 mg). The 30% methanol eluate (95.5 mg) was further separated by YMC-gel ODS-AQ column chromatography (ϕ1.1×20 cm) to give 3,4′-dihydroxypropiophenone 3-O-glucoside (11, 6.2 mg). The 50% methanol eluate (363.5 mg) was further purified by YMC-gel ODS-AQ, Sephadex LH-20, Chromatorex ODS and Toyopearl HW-40 column chromatography (ϕ1.1×20 cm) to give (+)-lyoniresinol 9′-O-glucoside (13, 60.8 mg) and propiophenone 4′-O-primeveroside (1, 1.1 mg). Each structure of known compounds was elucidated based on NMR and MS data, or direct HPLC comparison with authentic sample.

Propiophenone 4′-O-Primeveroside (1)

A pale brown amorphous powder. ¹H-NMR (methanol-d₄) δ: 7.98 (2H, d, J=8.5 Hz, H-2, 6), 7.19 (2H, d, J=8.5 Hz, H-3, 5), 4.99 (1H, d, J=7.5 Hz, Glc H-1), 4.31 (1H, d, J=7.5 Hz, Xyl H-1), 4.11 (1H, dd, J=2.0, 12.0 Hz, Glc H-6), 3.82 (1H, m, Xyl H-5), 3.75 (1H, m, Glc H-6), 3.71 (1H, m, Glc H-5), 3.45–3.48 (3H, m, Glc H-2, 3, Xyl H-4), 3.37 (1H, m, Glc H-4), 3.26 (1H, dd, J=9.0, 9.5 Hz, Xyl H-3), 3.19 (1H, dd, J=7.5, 9.0 Hz, Xyl H-2), 3.08 (1H, dd, J=10.0, 11.0 Hz, Xyl H-5), 3.02 (2H, dq, J=1.5, 7.5, 7.5, 7.5 Hz, H-8), 1.17 (3H, t, J=7.5 Hz, H-9). ¹³C-NMR (methanol-d₄) δ: 202.1 (C-7), 162.8 (C-4), 132.5 (C-1), 131.4 (C-2, 6), 117.4 (C-3, 5), 105.4 (Xyl C-1), 101.5 (Glc C-1), 77.6, 77.7, 77.8 (each 1C, Glc C-3, 5, Xyl C-3), 75.0 (Xyl C-2), 74.8 (Glc C-2), 71.4 (Glc C-4), 71.2 (Xyl C-4), 69.8 (Glc C-6), 66.9 (Xyl C-5), 32.4 (C-8), 8.8 (C-9). HR-ESI-MS: m/z 467.1553 (M+Na)⁺ (Calcd for C₂₀H₂₈O₁₁+Na⁺, 467.1524).

D-,L-Confirmation of Sugar Moiety of Propiophenone 4′-O-Primeveroside (1)

A solution of compound 1 (0.4 mg) in 0.5 M HCl (0.1 mL) was heated at 105°C for 1 h. After neutralization with Amberlite IRA-400 (Organo Co.), the mixture was evaporated under N₂ stream and the residue was heated with L-cystein methyl ester hydrochloride (0.5 mg) in pyridine (0.5 mL) at 60°C for 1 h. o-Tolylisothiocyanate (0.5 mg/0.5 mL pyridine) was added to the mixture and further heated at 60°C for 1 h. The reactant was analyzed by HPLC equipped with C₁₈ column eluted with 25% acetonitrile in 50 mM H₃PO₄. The retention times of the product (t_R 10.6, 12.2 min) was identical to that of an authentic o-tolylthiocarbamate derivative prepared from D-glucose (t_R 10.4 min) and D-xylose (t_R 12.0 min) under the same conditions, and different from L-glucose (t_R 9.6 min) and L-xylose (t_R 11.2 min) derivatives.

Antimicrobial Assay

The MICs of the P. heterocycla extract, isolates, and commercial products for E. coli JM109 and S. aureus FDA209P were determined by the standard two-fold dilution method.¹⁰⁾ Briefly, bacterial cells (10⁵ colony forming unit (CFU)/mL) were added into 100 µL of the extracts dilutions in Muller–Hinton broth, and then incubated at 37°C for 24 h. The microbial growth was examined by visual inspection. In this assay, extracts and compounds were dissolved in dimethyl sulfoxide (DMSO). Liquid sample of commercial products were evaporated under N₂ stream and dried. The final concentration (µg/mL) of DMSO in broth was lower than 6.4% and did not affect the microbial growth.

Acknowledgment

This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare of Japan.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials (spectroscopic data for compounds including 1D-, 2D-NMR spectra).

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