Introduction

Cnidium officinalis Makino (Umbelliferae) is a perennially grown herb either semi-shaded or not shaded in moist soil. It originates from China but is now extensively cultivated in Korea, Japan, and China [1]. The ground parts have been used as a medicinal, conspicuous aroma, and as condiment materials in beverages, baking, cosmetics, and the pharmaceutical industry [1]. The dried rhizomes (Cnidii Rhizoma) have been especially utilized in East-Asia countries for the treatment of female menstrual disorders and headaches through a decrease in inflammation and an improvement in blood circulation [2, 3]. Many studies have also reported the rhizomes as having anti-cancer [4], analgesic [5], antibacterial [6], anticonvulsive [7], anti-inflammatory [4, 5], febrifuge, hypotensive [8], sedative [9], and vasodilator [10] effects. The major constituents of the rhizomes were revealed to be phthalides, alkaloids, ceramides, polyphenols, and flavonoids [1]. Phthalides in particular are the most important constituents copiously contained in the essential oil [6]. Many reported biological activities of C. officinalis rhizomes are due to the presence of phthalides [6]. Phenols are also major constituents of rhizomes and are reported to have various pharmacological activities [11]. However, phenyl alkanoids of this plant are rarely studied. The authors isolated five phenyl alkanoids from C. officinalis rhizomes in this study. Among them, two compounds were revealed to be new and three others have never been reported for C. officinalis. This paper describes the isolation procedure for phenyl alkanoids and the structure determination including stereostructures.

Materials and methods

Plant materials

Cnidium officinalis rhizomes were provided and identified by Dr. J. T. Jeong, Department of Herbal Crop Research, RDA, Korea. A standard sample (NPCL-20200023) was put up at NPCL Laboratory, KyungHee University, Yongin, Korea.

General experimental procedures

The instruments and materials used for the isolation and identification of the phenyl alkanoids were the same as those in literatures [12]. The silica gel and the octadecyl silica gel (ODS) resins used for column chromatography (CC) were Kiesel gel 60 (Merck, Darmstadt, Germany) and the Lichroprep RP-18 (40–60 m\(\mu\), Merck) respectively. Sephadex LH-20 was purchased from Amersham Biosciences (Uppsala, Sweden). Thin layer chromatography (TLC) was carried out using Kiesel gel 60 F254 and RP-18 F254S (Merck) TLC plates, and the spots were detected using a UV lamp Spectroline Model ENF-240 C/F (Spectronics Corporation, Westbury, NY, USA) and a 10% H2SO4 solution. Deuterium solvents were purchased from Merck Co. Ltd and Sigma Aldrich Co. Ltd (St. Louis, MO, USA). Nuclear magnetic resonance (NMR) spectra were recorded on a 600 MHz FT-NMR spectrometer (Bruker AVANCE 600, Billerica, MA, USA). Infrared (IR) spectra were obtained using a Perkin Elmer Spectrum One FT-IR spectrometer (Buckinghamshire, England). The specific rotation value was measured with JASCO P-1010 digital polarimeter (Tokyo, Japan). ESIMS spectra were recorded on a AB SCIEX Q-TOF 5600 (Framingham, MA, USA). Solvents were supplied by Burdick & Jackson (Muskegon, MI, USA).

Isolation of phenyl alkanoids from Cnidium officinalis rhizomes

C. officinalis rhizomes were dried at room temperature and 10 kg of dried materials were powdered and soaked overnight in ethanol (70%, 54 L × 2) at room temperature. The obtained solution was evaporated using rotary vacuum evaporator at 40℃ affording a brownish extract (2.1 kg). The residue was divided using systemic solvent fractionation using polarity as ethyl acetate (COE, 280 g), n-butanol (COB, 125 g), and water (COW, 1.695 kg) Fr. The column chromatography (CC) for COE (270 g) and COB (120 g) was performed as seen in Figs. 1, 2. Finally, five phenyl alkanoids (1, COE-11–5; 2, COE-13–2-3; 3, COE-13–2-5; 4, COE-13–11-12–2; 5, COB-13–9-13–2) were isolated.

Fig. 1
figure 1

Isolation of phenyl alkanoids from the rhizomes of Cnidium officinalis

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Fig. 2
figure 2

Chemical structure of phenyl alkanoids from the rhizome of Cnidium officinalis Makino. 1, coniferyl alcohol methyl ether; 2, vanillin; 3, coniferyl aldehyde; 4, 7S-7-methoxyeugenol; 5, cnidiumoside

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Coniferyl alcohol methyl ether (1): Rf value on silica gel TLC, 0.72, n-hexane–ethyl acetate (1:1); Rf value on octadecyl silica gel TLC, 0.85, acetone–H2O (3:1); IR (LiF plates) νmax 3393, 2933, 1676, 1599, 1512, 1463 cm−1; 13C and 1H NMR (CMR and PMR): Tables 1 and 2. EI-MS: m/z 180 [M]+.

Table 1 13C-NMR data of phenylalkanoids from the rhizome of Cnidium officinalis Makino (125 MHz, δC)
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Table 2 1H-NMR data of phenyl alkanoids from the rhizome of Cnidium officinalis Makino (600 MHz, δH, coupling pattern, J in Hz)
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Vanillin (2): Rf value on silica gel TLC, 0.29, n-hexane– ethyl acetate (2:1); Rf value on octadecyl silica gel TLC, Rf 0.70, acetone–H2O (2:1); IR (LiF plates) νmax 3234, 2923, 1675, 1585, 1509 cm−1; CMR and PMR: Tables 1 and 2. EI-MS: m/z 152 [M]+.

Coniferyl aldehyde (3): Rf value on silica gel TLC, 0.20, n-hexane– ethyl acetate (2:1); Rf value on octadecyl silica gel TLC, Rf 0.72, acetone–H2O (2:1); IR (LiF plates) νmax 3183, 2919, 1748, 1660, 1584, 1512 cm−1; CMR and PMR: Tables 1 and 2. EI-MS: m/z 178 [M]+

7-Methoxyeugenol (4): Rf value on silica gel TLC, 0.76, n-hexane– ethyl acetate (1:1); Rf value on octadecyl silica gel TLC, 0.39, acetone–H2O (2:1); [α]D + 23.2 (c 0.12, CH3OH); IR (LiF plates) νmax 3153, 2916, 1673,1594, 1510, 1450 cm−1; CMR and PMR: Tables 1 and 2. EI-MS: m/z 194.0940 [M]+ (Calcd for C11H14O3 194.0937).

Cnidiumoside (5): Rf value on silica gel TLC, 0.54, chloroforem–methanol–H2O (6:4:1); Rf value on octadecyl silica gel TLC, 0.19, methanol–H2O (1:2); [α]D + 15.7 (c 0.10, CH3OH); IR (LiF plates) νmax 3425, 2996, 1675, 1465 cm−1; CMR and PMR: Tables 1 and 2. High resolution positive ESI–MS: m/z 845.3072 [M + H]+ (Calcd for C38H53O21 845.3074).

Acid hydrolysis of cnidiumoside (5)

The solution of 10 mg of 5 in 5 mL of 1N HCl was refluxed for 2 hrs. 15 mL of H2O was added and treated with ethyl acetate (20 mL × 2). The ethyl acetate phase was concentrated in vacuo and purified via open silica gel CC (2 × 8 cm) using n-hexane-ethyl acetate (2:1) as the eluting solution to give a dehydrodiconiferyl alcohol (5a, 4 mg).

Dehydrodiconiferyl alcohol (5a): [α]D -49 (c 0.18, CHCl3); PMR (600 MHz, CDCl3, δH, coupling pattern, J in Hz) 6.84 (1H, d, 1.9, H-2), 6.82 (1H, s, H-2′ or H-6′), 6.79 (1H, s, H-2′ or H-6′), 6.50 (1H, dd, 1.9, 8.3, H-6), 6.46 (1H, d, 8.3, H-5), 6.18 (1H, br. d, 16.0, H-7′), 6.14 (1H, dt, 16.0, 5.2, H-8′), 5.50 (1H, d, 6.8, H-7), 4.20 (2H, dd, 5.2, 1.3, H-9′), 3.89 (1H, dd, 11.9, 5.5, H-9a), 3.88 (3H, s, 3-OMe or 3′-OMe), 3.81 (1H, dd, 11.9, 6.3, H-9b), 3.78 (3H, s, 3-OMe or 3′-OMe), 3.56 (1H, ddd, 6.8, 6.3, 6.5, H-8).

Results and discussion

Five phenyl alkanoids 1–5 were isolated from the C. officinalis rhizome through repeated open column chromatography using octadecyl silica gel, silica gel, and Sephadex LH-20 as the stationary phase. 1–3 were identified as coniferyl alcohol methyl ether (1) [13], vaniline (2), and coniferyl aldehyde (3) [14], respectively, from the spectroscopic data and confirmed by previously reported data.

Compound 4 showed a molecular ion peak at m/z 194.0940 [M]+ in the high resolution EI-MS, establishing the molecular formula as C11H14O3 (Calcd. for C11H14O3 194.0937). The IR data exhibited the absorbing bands due to hydroxyl (3153 cm−1) and olefin (1673, 1594 cm−1) groups. CMR spectrum (150 MHz, CDCl3) showed 11 signals including two methoxy signals (δC 56.06, OMe-3; 56.40, OMe-7) suggesting 4 to be a phenylpropanoid. Two oxygenated olefin quaternaries (δC 145.36, C-4; 146.84, C-3), one olefin quaternary (δC 132.99, C-1), four olefin methines (δC 109.14, C-2; 114.24, C-5; 120.32, C-6; 139.00, C-8), one exomethylene (δC 116.21, C-9), and one oxygenated methine (δC 84.64, C-7) were detected. PMR spectrum (600 MHz, CDCl3) included the olefin signals due to a 1,2,4-trisubstituted benzene moiety [δH 6.81, dd, J = 1.8, 7.8 Hz, H-6 (δH, coupling pattern, J in Hz), H-5; 6.86, d, 1.8, H-2; 6.88, d, 7.8), the signals derived from a vinyl moiety (5.20, br. d, 10.2, H-9a; 5.26, br. d, 17.4, H-9b; 5.93, ddd, 6.6, 10.2, 17.4, H-8), an oxygenated methine (4.55, d, 6.6, H-7), and two methoxies (3.31, s, OMe-7; 3.90, s, OMe-3) signals. The NMR data described above were almost identical to eugenol [15] excluding an oxygenated methine and a methoxy moieties instead of a methylene moiety, suggesting that an additional methoxy moiety was linked to C-7. The HMBC spectrum affirmed it; a methoxy proton (δH 3.31, OMe-7) correlated with an oxygenated methine carbon (δC 84.64, C-7), which also correlated with three olefin methine protons (δH 5.93, H-8; 6.81, H-6; 6.86, H-2) and an exomethylene proton (δH 5.20, 5.26, H-9). As a result, the planar structure of compound 4 was decided as a 2-methoxy-4-(1-methoxy-2-propen-1-yl) phenol, 7-methoxyeugenol. The stereostructure of chiral center, C-7, could be determined as S by comparison of a specific rotation value, [α]D + 23.2, with a previously reported value for 7-hydroxyeugenol [16, 17]. Compound 4 was identified to be 7S-7-methoxyeugenol.

Compound 5 exhibited a molecular ion peak at m/z 845.3072 [M + H]+ in the high resolution ESI–MS, established the molecular formula as C38H52O21 (Calcd. for C38H53O21 845.3072). The IR data showed the absorption bands due to hydroxyl (3153 cm−1) and phenyl (1673, 1594 cm−1) moieties. Though CMR spectrum (150 MHz, CD3OD) showed about 50 carbon signals, some of them were revealed from those of impurities included in compound 5. Among them, 38 carbon signals were defined as those of the compound. The 20 signals including two methoxy signals as those of the aglycone moiety and 18 carbon signals due to three hexoses were detected, suggesting that 5 was a lignan triglyceride. CMR spectrum showed two hemiacetals (δC 102.89, C-1′′; 104.71, C-1′′′) and one hemiketal (δC 99.34, C-2′′′′) carbon signals as well as PMR spectrum showed only two hemiacetal proton signals (4.37, d, 7.8, H-1′′′; 4.89, d, 7.8, H-1′′), indicating that three sugars in compound 5 were composed of two aldohexoses and one ketohexose. CMR chemical shifts of three sugars confirmed the presence of one terminal β- d -galactopyranosyl (δC 62.95, C-6′′; 69.54, C-4′′; 71.99, C-2′′; 75.33, C-3′′; 77.99, C-5′′; 102.89, C-1′′) and one terminal β-d-allulofuranosyl (δC 62.65, C-6′′′′; 64.69, C-1′′′′; 71.42, C-3′′′′; 71.79, C-4′′′′; 83.46, C-5′′′′; 99.34, C-2′′′′) moieties as well as one β- d -glucopyranosyl (δC 71.49, C-4′′′; 75.05, C-2′′′; 78.23, C-5′′′; 78.35, C-3′′′; 104.71, C-1′′′) with a down-shifted oxygenated methylene signal (δC 66.06, C-6′′′) due to a glycosidation shift [18] indicating a terminal sugar to be connected to the hydroxyl group at C-6′′′ of a glucopyranosyl moiety. The coupling constants of two hemiacetal protons were 7.8 and 7.8 Hz confirming the anomer hydroxyl groups of both aldohexose moieties to have a β-configuration. The carbon signals of the aglycone moiety included four oxygenated olefin quaternaries (δC 145.71, C-3′; 147.74, C-4; 149.29, C-4′; 151.04, C-3), three olefin quaternaries (δC 129.89, C-5′; 132.91, C-1′; 138.22, C-1), seven olefin methines (δC 111.50, C-2; 112.37, C-2′; 116.92, C-6′; 118.13, C-5; 119.62, C-6; 127.86, C-8′; 132.05, C-7′), one oxygenated methine (δC 89.01, C-7), two oxygenated methylenes (δC 63.99, C-9′; 72.57, C-9), two methoxies (δC 56.92 × 2), and one methine (δC 53.39, C-8) signals. The oxygenated methylene carbon was shifted down-field compared to the usual one, confirming it to be linked to a sugar. PMR spectrum exhibited proton signals due to one 1,2,4-trisubstituted benzene ring (6.98, br. d, 8.4, H-6; 7.08, br. s, H-2; 7.14, d, 8.4, H-5), one 1,2,3,5-tetrasubstituted benzene ring (6.85, br. s, H-2′; 7.01, br. s, H-6′), a double bond with a trans configuration (6.23, dt, 15.6, 5.0, H-8′; 6.53, br. d, 15.6, H-7′), one oxygenated methine (5.70, d, 8.0, H-7), two oxygenated methylenes (4.20, d, 5.0, H-9′; 3.77, overlapped, H-9a; 4.24, dd, 4.0, 10.2, H-9b), two methoxies (3.84, s, OMe-3; 3.89, s, OMe-3′), and one methine (δH 3.64, overlapped, H-8) moieties. The aforementioned NMR data suggested the aglycone to be a neolignan with a coniferyl alcohol and a dehydroconiferyl alcohol moieties. In the HMBC spectrum, the oxygenated methine proton signal (δH 5.70, H-7) showed cross peaks with one oxygenated olefin quaternary (δC 149.29, C-4′), two olefin quaternaries (δC 129.89, C-5′; 138.22, C-1), two olefin methines (δC 111.50, C-2; 119.62, C-6), one oxygenated methylene (δC 72.57, C-9), and one methine (δC 53.39, C-8) carbon signals. The methine proton signal (δH 3.64, H-8) correlated with the carbon signals due to one oxygenated olefin quaternary (C-4′), two olefin quaternaries (C-1 and C-5′), one olefin methine (δC 116.92, C-6′), and one oxygenated methylene (C-9) moieties. Therefore, the two phenylpropanoid moieties of neolignan were linked at C-7 and C-4′ through an ether bond and at C-8 and C-5′. The anomer proton signals of one β-d-galactopyranosyl and one β-d-glucopyranosyl moieteies (δH 4.37, H-1′′′ and 4.89, H-1′′) showed correlations with the oxygenated olefin quaternary (δC 147.74, C-4) and the oxygenated methylene (C-9) carbon signals, respectively, as well as the anomer carbon signal of a β-d-glucopyranosyl moiety (δC 104.71, C-1′′′) correlated with the proton signals of an oxygenated methylene moiety (δH 3.77 and 4.24, H-9). Long-range coupling between the anomer carbon signal of the β-d-allulopyranosyl moiety (δC 99.34, C-2′′′′) and the oxygenated methylene proton signals (δH 3.47 and 3.65, H-6′′′) of the β-d-glucopyranosyl moiety in the HMBC spectrum were detected. The HMBC correlations described above concluded the linkage position of three sugars as seen in Fig. 1. The relative stereostructure between H-7 and H-8 was determined to be a trans configuration because the cross peak was not observed between H-7 (δH 5.70) and H-8 (δH 3.64) but was observed between H-7 and H-9 (δH 3.77 and 4.24) in the NOESY spectrum. The glycoside, compound 5, was treated with 1 N HCl and refluxed at 90℃ for two hours to finally yield the aglycone, dehydrodiconiferyl alcohol (5a). The dehydroconiferyl alcohol moiety with two chiral carbons, C-7 and C-8, has four stereoisomers, that showed typical specific rotation value [19]. 5a had an erythro-configuration between C-7 and C-8 and the specific rotation value was measured to be -49, confirming the absolute stereostructue of C-7 and C-8 to be R and S, respectively. Finally, the chemical structure of compound 5 was determined to be (2R,3S)-4-O-β-d-galactopyranosyl-9-[(6-O-β-d-allulofuranosyl-β-d-gucopyranosyl)oxy] dehydrodiconiferyl alcohol as seen in Fig. 1. It was revealed to be a new compound, named cnidiumoside.