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. 2017 Dec 12;26(6):1491–1500. doi: 10.1007/s10068-017-0234-8

Phenolics and eudesmanolide from aged common sage exudate with sugar

Tae Yeon Kim 1, Jeong-Yong Cho 1, Yu Geon Lee 1, Hang Yeon Jeong 1, Hyoung Jae Lee 1, Jae-Hak Moon 1,✉
PMCID: PMC6049721  PMID: 30263686

Abstract

Nineteen compounds including one new flavanone were isolated from the juice of aged common sage exudate with sugar (ACSE). The isolated compounds were identified by NMR and MS analyses as levodopa methyl ester (1), 3,4-dihydroxybenzoic acid (2), (S)-8-hydroxy-4-hydroxy-phenylpropanoic acid (3), 4-hydroxybenzoic acid ethyl ester (4), cis-caffeic acid (5), trans-caffeic acid (6), esculetin (7), (S)-8-hydroxy-3,4-dihydroxy-phenylpropanoic acid ethyl ester (8), cis-rosmarinic acid (9), trans-rosmarinic acid (10), trans-rosmarinic acid methyl ester (11), 6-methoxy-7,8,3′,5′-tetrahydroxyflavanone (12), nepetin (13), trans-caffeic acid ethyl ester (14), luteolin (15), cis-caffeic acid ethyl ester (16), 6-methoxynaringenin (17), 1α-acetoxy-2-oxo-eudesman-3,7(11)-dien-8β,12-olide (18), and hispidulin (19). Compound 12 was isolated for the first time from nature and seven compounds (1, 3, 4, 7, 8, 14, and 18) were newly identified from common sage. Of them, 15 isolated phenolic compounds (1–3, 5–8, 10–15, 17, and 19) were detected in ACSE juice, while only 10 was detected in the fresh common sage.

Keywords: Salvia plebeia, Common sage, Fermented juice, Flavanone, Eudesmanolide

Introduction

Aged products of plant exudates (APE) are manufactured by aging after food and medicinal materials (vegetables, fruits, herbs, etc.) are mixed and extracted with a high concentration of sugar [1–3]. These products are widely consumed in Korea due to the belief that they provide health benefits [1]. Several APE have been reported to exert beneficial biological properties, such as antioxidant, anti-obesity, and anticancer activities [3–5]. Nevertheless, information on the chemical constituents and biological effects of APE is much more limited than that of the original plant materials. In addition, scientists question whether APE are fermented by microorganisms at such high sugar concentrations. In fact, it has been reported that various microorganisms including yeasts are involved in the fermentation of plant exudates by sugar [6]. In particular, osmophilic yeasts, Candida sp., Zygosaccharomyces sp., and Saccharomyces sp. were dominant at high sugar concentrations during natural fermentation of plant materials, such as aloe, onion, Prunus mume, kale, and sea tangle [6]. Therefore, it is likely that microorganisms produce bioactive compounds and metabolize native compounds contained in raw materials by fermentation during the aging of APE.

Salvia plebeia R. Br. (Lamiaceae, common sage) is widely distributed in the Asian countries, of Korea, China, and Japan. This plant has been used in folk medicine for healing hepatitis, inflammation, and hemorrhoids [7]. Previous studies on the chemical constituents of common sage established the presence of flavonoids, terpenes, lignans, and caffeic acid derivatives [7–10]. Common sage is especially rich in flavonoids and phenolic acids. Several recent studies have reported that common sage exerts certain biological activities including liver and gastric protection, as well as antioxidant, antibacterial, anticancer, anti-inflammatory, anti-allergic, and anti-anaphylactic effects [11–14]. Therefore, the ingestion of common sage for the prevention of various diseases has recently received increased attention.

The aged common sage exudate by sugar (ACSE) is commonly consumed in Korea. However, the fermentation characteristics, as well as the chemical constituents and biological effects of ACSE have not yet been established. We hypothesized that fermentation during the aging process of ACSE would produce metabolites absent in fresh common sage. In our preliminary study [15], we confirmed that ACSE contained diverse compounds different from those found in fresh common sage through a chemical profiling study using high-pressure liquid chromatography (HPLC) analysis. The chemical analyses of the constituents of ACSE would provide important information concerning quality control and evaluation of the biological effects of ACSE. Therefore, we carried out the isolation and structural elucidation of the chemical constituents of ACSE.

In this study, 19 compounds [two p-hydroxybenzoic acid derivatives, eight phenylpropanoid derivatives, three rosmarinic acid derivatives, five flavonoids (one new compound), and one eudesmanolide] were isolated and structural-determined from ACSE. In addition, the presence of 15 isolated compounds in ACSE was confirmed by HPLC analysis.

Materials and methods

Materials and chemicals

Fermented common sage juice was provided from Saeselwon Company in Damyang County, South Korea. Briefly, fresh common sage was collected in June of 2013 in Damyang County, and the fresh leaf (8.5 kg) was mixed with sucrose (5.7 kg). The leaf was removed after 3 months and the ACSE juice was naturally aged in a sealed jar at room temperature for 9 months. Deuterated methanol (CD3OD) and acetone-d 6, with tetramethylsilane as an internal standard, were purchased from Merck Co. (Darmstadt, Germany). Methanol (MeOH), acetonitrile (MeCN), n-butanol (n-BuOH), and ethyl acetate (EtOAc) were obtained from Duksan (Ansan, Korea).

HPLC analysis of fresh common sage and its ACSE juice

ACSE juice (100 mL) was added to distilled water (250 mL) and successively partitioned with n-hexane (350 mL, 3 times), EtOAc (350 mL, 3 times), and water-saturated n-BuOH (BuOH, 350 mL, 3 times). Each layer was concentrated with rotary evaporation. Meanwhile, a mixture of fresh common sage and sucrose (6:4, w/w) was homogenized and extracted with MeOH (1.2 L). The MeOH extract obtained after rotary evaporation was partitioned using the same method as for the ACSE juice. The layers obtained after the solvent fractionation of both samples were analyzed by octadecylsilane (ODS)-HPLC using an ODS-80Ts (TSK-gel, 5 μm, 4.6 mm × 250 mm, Tosoh, Tokyo, Japan). The compounds were eluted with a gradient system of 5% MeOH containing 2% acetic acid in H2O → 100% MeOH for 50 min. The flow rate was 1.0 mL/min, and the detection wavelength was 254 nm.

Extraction and isolation

ACSE juice (4 L) was added to distilled water (6 L) and partitioned with n-hexane (10 L, 3 times). The aqueous layer was successively partitioned with EtOAc (10 L, 3 times) and BuOH (10 L, 3 times). After concentration, the EtOAc layer (11.135 g) was loaded onto a Diaion HP-20 column (5.0 cm × 83 cm; Mitsubishi Chemical Industrial, Tokyo, Japan) and eluted by a step-wise system of 10% MeOH in H2O → 100% MeOH (each step, 3.5 L). Twenty fractions (A-T) were obtained by the analysis of ODS-HPLC (column: ODS-80Ts, 4.6 mm × 250 mm Tosoh; gradient: 30% MeOH containing 2% acetic acid in H2O → 70% MeOH in H2O for 50 min; flow rate: 1.0 mL/min; wavelength: 254 nm). Fractions C + D, F, L, M, P, and S were purified by ODS-HPLC using a Shim-pack Prep-ODS (H) Kit (10 μm, 20 cm × 250 mm; Shimadzu, Kyoto, Japan; flow rate: 9.9 mL/min; wavelength: 280 nm). Fraction C + D (223.2 mg) was subjected to ODS-HPLC (gradient: 10% MeOH in H2O with pH adjusted to 2.65 by TFA → 25% MeOH in H2O for 40 min) to obtain 1 (t R 30.6 min, 4.0 mg). Compounds 2 (t R 19.0 min, 2.8 mg) and 3 (t R 23.0 min, 3.4 mg) were isolated from fraction F (108.1 mg) by ODS-HPLC (gradient: 15% MeOH in H2O with pH adjusted to 2.65 by TFA → 40% MeOH in H2O for 40 min). Compounds 4 (t R 3.2 min, 2.6 mg) and 5 (t R 6.0 min, 4.2 mg) were isolated from fraction L (207.9 mg) by ODS-HPLC (gradient: 20% MeOH in H2O with pH adjusted to 2.65 by TFA → 60% MeOH in H2O for 25 min). Compounds 6 (t R 16.2 min, 30.8 mg), 9 (t R 27.9 min, 3.67 mg), 10 (t R 29.5 min, 5.16 mg), and 11 (t R 29.8 min, 5.06 mg) with subfraction M2 (t R 18.6 min, 5.6 mg) were isolated from fraction M (2113.2 mg) by ODS-HPLC (gradient: 25% MeOH in H2O with pH adjusted to 2.65 by TFA → 65% MeOH in H2O for 40 min). Compounds 7 (t R 14.3 min, 1.1 mg) and 8 (t R 19.9 min, 1.2 mg) were isolated from subfraction M2 (5.6 mg) by ODS-HPLC using an ODS-80Ts column (TSK-gel, 4.6 mm × 250 mm, Tosoh; gradient: 25% MeOH in H2O with pH adjusted to 2.65 by TFA → 65% MeOH in H2O for 40 min; flow rate: 1.0 mL/min; wavelength: 254 nm). Compounds 12 (t R 12.6 min, 52.1 mg), 13 (t R 14.7 min, 1.0 mg), and 14 (t R 15.2 min, 50.4 mg), among with subfraction P4 (t R 18.1 min, 30.6 mg) from fraction P (359.8 mg) and 17 (t R 16.7 min, 3.0 mg) and 18 (t R 23.5 min, 3.0 mg) from fraction Q (156.0 mg) were purified by ODS-HPLC (gradient: 50% MeOH in H2O with pH adjusted to 2.65 by TFA → 80% MeOH for 40 min). Compounds 15 (t R 16.3 min, 3.0 mg) and 16 (t R 18.4 min, 0.9 mg) was isolated from subfraction P4 (30.6 mg) by ODS-HPLC (column: μBondapak C18, 10 μm, 7.8 mm × 300 mm, Waters, USA; gradient: 100% H2O with pH adjusted to 2.65 by TFA → 60% MeOH for 40 min; flow rate, 5.0 mL/min; wavelength: 254 nm). Compound 19 (t R 14.2 min, 2.6 mg) was isolated from fraction Q (97.7 mg) by ODS-HPLC (gradient: 30% MeOH in H2O with pH adjusted to 2.65 by TFA → 80% MeOH in H2O for 25 min).

Structural analysis

The nuclear magnetic resonance (NMR) spectra of 1–19 were recorded using unityINOVA 500 and 600 spectrometers (Varian, Walnut Creek, CA, USA). All mass spectra were recorded on a hybrid ion-trap time-of-flight mass spectrometer (LCMS-IT-TOF, Shimadzu) equipped with an electrospray ionization (ESI) source (ESIMS).

Compound 1

1H-NMR (500 MHz, CD3OD) δ 46.66 (1H, br. s, H-2), 6.67 (1H, d, J = 7.0 Hz, H-5), 6.52 (1H, dd, J = 7.0, 2.0 Hz, H-6), 2.77 (1H, dd, J = 11.5, 4.0 Hz, H-7a), 2.89 (1H, dd, J = 11.5, 6.0 Hz, H-7b), 4.29 (1H, dd, J = 6.0, 4.0 Hz, H-8), 3.65 (3H, s, −OCH3); ESIMS (negative) m/z 153.05 [M–H]−.

Compound 2

1H-NMR (500 MHz, CD3OD) δ 7.43 (1H, br. s, H-2), 6.79 (1H, d, J = 7.5 Hz, H-5), 7.41 (1H, dd, J = 7.5, 2.0 Hz, H-6); ESIMS (negative) m/z 153.05 [M–H]−.

Compound 3

1H-NMR (500 MHz, CD3OD) δ 7.07 (2H, d, J = 8.0 Hz, H-2 and H-6), 6.70 (2H, d, J = 8.0 Hz, H-3 and H-5), 2.81 (1H, dd, J = 14.0, 4.5 Hz, H-7a), 3.01 (1H, dd, J = 14.0, 7.5 Hz, H-7a), 4.27 (1H, dd, J = 7.5, 4.5 Hz, H-8); ESIMS (negative) m/z 181.0 [M–H]−.

Compound 4

1H-NMR (500 MHz, CD3OD) δ 7.87 (2H, d, J = 8.5 Hz, H-2 and H-6), 6.81 (2H, d, J = 8.5 Hz, H-3 and H-5), 4.13 (2H, m, H-1′), 1.25 (3H, J = 7.0 Hz, H-2′); ESIMS (negative) m/z 165.05 [M–H]−.

Compound 5

1H-NMR (500 MHz, CD3OD) δ 7.03 (1H, br. s, H-2), 6.76 (1H, d, J = 8.5 Hz, H-5), 6.93 (1H, br. d, J = 8.5 Hz, H-6), 7.75 (1H, d, J = 9.5 Hz, H-7), 6.16 (1H, d, J = 9.5 Hz, H-8); ESIMS (negative) m/z 179.0 [M–H]−.

Compound 6

1H-NMR (500 MHz, CD3OD) δ 7.03 (1H, d, J = 2.0 Hz, H-2), 6.78 (1H, d, J = 8.0 Hz, H-5), 6.94 (1H, dd, J = 8.0, 2.0 Hz, H-6), 7.53 (1H, d, J = 16.0 Hz, H-7), 6.22 (1H, d, J = 16.0 Hz, H-8); ESIMS (negative) m/z 179.0 [M–H]−.

Compound 7

1H-NMR (500 MHz, CD3OD) δ 6.17 (1H, d, J = 9.0 Hz, H-3), 7.78 (1H, d, J = 9.5 Hz, H-4), 6.94 (1H, s, H-5), 6.75 (1H, s, H-8); ESIMS (negative) m/z 176.85 [M–H]−.

Compound 8

1H-NMR (500 MHz, CD3OD) δ 6.67 (1H, d, J = 2.4 Hz, H-2), 6.66 (1H, dd, J = 7.8 Hz, H-5), 6.53 (1H, dd, J = 7.8, 2.4 Hz, H-6), 2.88 (1H, dd, J = 13.8, 5.4 Hz, H-7a), 2.77 (1H, dd, J = 13.8, 7.2 Hz, H-7b), 4.26 (1H, dd, J = 7.2, 5.4 Hz, H-8), 4.13 (2H, m, H-1′), 1.22 (3H, t, J = 7.2 Hz, H-2′); 13C-NMR (125 MHz, CD3OD) δ 130.0 (C-1), 117.8 (C-2), 146.3 (C-3), 145.2 (C-4), 116.3 (C-5), 122.0 (C-6), 41.3 (C-7), 73.5 (C-8), 175.7 (C-9), 62.1 (C-1′), 14.6 (C-2′); ESIMS (negative) m/z 225.05 [M–H]−.

Compound 9

1H-NMR (600 MHz, CD3OD) δ 6.70 (1H, d, J = 2.0 Hz, H-2), 6.68 (1H, d, J = 8.0 Hz, H-5), 6.53 (1H, dd, J = 8.0, 2.0 Hz, H-6), 2.96 (1H, d, J = 14.5, 7.0 Hz, H-7a), 3.05 (1H, d, J = 14.5, 4.3 Hz, H-7b), 5.12 (1H, dd, J = 8.5, 4.3 Hz, H-8), 7.38 (1H, d, J = 2.0 Hz, H-2′), 6.77 (1H, d, J = 8.0 Hz, H-5′), 7.04 (1H, dd, J = 8.0, 2.0 Hz, H-6′), 6.82 (1H, d. J = 13.0 Hz, H-7′), 5.78 (1H, d, J = 13.0 Hz, H-8′); ESIMS (negative) m/z 359.05 [M–H]−.

Compound 10

1H-NMR (600 MHz, CD3OD) δ 6.74 (1H, d, J = 2.0 Hz, H-2), 6.69 (1H, d, J = 8.0 Hz, H-5), 6.61 (1H, dd, J = 8.0, 2.0 Hz, H-6), 3.00 (1H, dd, J = 14.0, 8.5 Hz, H-7a), 3.10 (1H, dd, J = 14.0, 4.3 Hz, H-7b), 5.18 (1H, dd, J = 8.3, 4.3 Hz, H-8), 7.04 (1H, d, J = 2.0 Hz, H-2′), 6.77 (1H, d, J = 8.0 Hz, H-5′), 6.95 (1H, dd, J = 8.0, 2.0 Hz, H-6′), 7.54 (1H, d, J = 16.0 Hz, H-7′), 6.26 (1H, d, J = 16.0 Hz, H-8′); ESIMS (negative) m/z 359.05 [M–H]−.

Compound 11

1H-NMR (600 MHz, CD3OD) δ 6.76 (1H, d, J = 2.0 Hz, H-2), 6.72 (1H, d, J = 8.0 Hz, H-5), 6.56 (1H, dd, J = 8.0, 2.0 Hz, H-6), 2.96 (1H, d, J = 14.5, 7.0 Hz, H-7a), 2.73 (1H, d, J = 14.5, 4.3 Hz, H-7b), 5.42 (1H, dd, J = 8.5, 4.3 Hz, H-8), 7.53 (1H, d, J = 2.0 Hz, H-2′), 6.90 (1H, d, J = 8.0 Hz, H-5′), 7.21 (1H, br. d, J = 8.0, 2.0 Hz, H-6′), 7.83 (1H, d. J = 16.0 Hz, H-7′), 6.34 (1H, d, J = 16.0 Hz, H-8′), 3.67 (3H, s, -OCH3); ESIMS (negative) m/z 373.1 [M–H]−.

Compound 12

1H- and 13C-NMR data are shown in Table 1; HR-ESIMS (negative) m/z 317.0660 [M–H]− (calculated for C16H12O7, m/z 317.0661, +0.1 mDa).

Table 1.

1H- (500 MHz) and 13C- (125 MHz) NMR data of 12 in acetone-d 6

Position δH (int., mult., J in Hz) δC
2 5.38 (1H, dd, 13.0, 3.0) 80.4
3a
3b		 3.14 (1H, dd, 17.0, 13.0)
2.73 (1H, dd, 17.0, 3.0)		 43.7
4 – 198.2
5 6.00 (1H, s) 95.7
6 – 129.9
7 – 156.4
8 – 156.1
9 – 159.6
10 – 103.4
1′ – 131.6
2′ 6.86 (1H, br. s) 116.1
3′ – 146.1
4′ 7.03 (1H, s) 114.8
5′ – 146.4
6′ 6.86 (1H, br. s) 119.2
-OCH3 3.77 (3H, s) 60.8
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Compound 13

1H-NMR (500 MHz, CD3OD) δ 6.55 (2H, s, H-3 and H-8 overlapping signals), 7.38 (1H, br. s, H-2′), 6.90 (1H, d, J = 8.4 Hz, H-5′), 7.39 (1H, dd, J = 8.4, 1.8 Hz, H-6′), 3.88 (3H, s, -OCH3); 13C-NMR (125 MHz, CD3OD) δ 166.6 (C-2), 103.6 (C-3), 184.4 (C-4), 154.8 (C-5), 133.0 (C-6), 158.9 (C-7), 95.4 (C-8), 154.2 (C-9), 105.9 (C-10), 123.8 (C-1′), 114.2 (C-2′), 147.2 (C-3′), 151.2 (C-4′), 116.9 (C-5′), 120.4 (C-6′), 61.1 (-OCH3); ESIMS (negative) m/z 315.0 [M–H]−.

Compound 14

1H-NMR (500 MHz, CD3OD) δ 7.03 (1H, br. s, H-2), 6.77 (1H, d, J = 8.0 Hz, H-5), 6.91 (1H, br. d, J = 8.0 Hz, H-6), 7.51 (1H, d, J = 16.0 Hz, H-7), 6.22 (1H, d, J = 16.0 Hz, H-8), 4.17 (2H, m, H-1′),1.27 (3H, t, J = 7.0 Hz, H-2′); ESIMS (negative) m/z 206.9 [M–H]−.

Compound 15

1H-NMR (500 MHz, CD3OD) δ 6.54 (1H, s, H-3), 6.20 (1H, br. s, H-6), 6.44 (1H, br. s, H-8), 7.38 (1H, br. s, H-2′), 6.89 (1H, d, J = 8.4 Hz, H-5′), 7.39 (1H, dd, J = 8.4, 1.8 Hz, H-6′); 13C-NMR (125 MHz, CD3OD) δ 166.6 (C-2), 103.6 (C-3),184.4 (C-4), 154.8 (C-5), 100.2 (C-6), 166.1 (C-7), 95.1 (C-8), 163.3 (C-9), 104.0 (C-10), 123.8 (C-1′), 115.2 (C-2′), 146.9 (C-3′), 149.6 (C-4′), 115.4 (C-5′), 120.4 (C-6′); ESIMS (negative) m/z 286.9 [M–H]−.

Compound 16

1H-NMR (600 MHz, CD3OD) δ 7.03 (1H, d, J = 2.0 Hz, H-2), 6.73 (1H, d, J = 8.4 Hz, H-5), 7.01 (1H, dd, J = 8.4, 2.0 Hz, H-6), 7.53 (1H, d, J = 13.0 Hz, H-7), 6.25 (1H, d, J = 13.0 Hz, H-8), 4.16 (2H, dd, J = 14.4, 7.2 Hz, H-1′), 1.31 (3H, t, J = 7.2 Hz, H-2′); 13C-NMR (150 MHz, CD3OD) δ 127.8 (C-1), 115.2 (C-2), 145.8 (C-3), 148.3 (C-4), 116.9 (C-5), 123.0 (C-6), 144.8 (C-7), 115.9 (C-8), 168.6 (C-9), 61.3 (C-1′), 14.6 (C-2′); ESIMS (negative) m/z 206.9 [M–H]−.

Compound 17

1H-NMR (500 MHz, CD3OD) δ 5.32 (1H, br. d, J = 13.2 Hz, H-2), 2.70 (1H, dd, J = 17.3, 13.2 Hz, H-3a), 3.11 (1H, dd, J = 17.3, 2.8 Hz, H-3b), 5.97 (1H, s, H-8), 7.31 (2H, d, J = 8.5 Hz, H-2′ and H-6′), 6.82 (2H, d, J = 8.5 Hz, H-3′ and H-5′), 3.79 (3H. s, -OCH3); ESIMS (negative) m/z 301.05 [M–H]−.

Compound 18

1H- and 13C-NMR data are shown in Table 2; ESIMS (negative) m/z 302.95 [M–H]−.

Table 2.

1H- (600 MHz) and 13C- (150 MHz) NMR data of compound 18 in CD3OD

Position δH (int., mult., J in Hz) δC
1 6.00 (1H, s) 80.4
2 – 194.6
3 5.27 (1H, s) 126.5
4 – 164.0
5 3.33 (1H, m) 49.7
6a
6b		 3.24 (1H, dd, 12.9, 3.6)
2.50 (1H, dd, 12.9, 6.6)		 25.6
7 – 122.4
8 5.00 (1H, m) 79.0
9a
9b		 2.52 (1H, m)
0.90 (1H, t, 7.2)		 43.1
10 – 44.2
11 – 163.0
12 – 172.0
13 1.87 (3H, s) 8.4
14 1.09 (3H, s) 12.3
15 2.09 (3H, s) 22.0
1′ – 176.8
2′ 2.19 (3H, s) 20.6
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Compound 19

1H-NMR (600 MHz, CD3OD) δ 6.95 (1H, s, H-3), 6.93 (1H, s, H-8), 7.96 (2H, d, J = 8.4 Hz, H-2′ and H-6′), 7.26 (2H, d, J = 8.4 Hz, H-3′ and H-5′), 3.98 (3H, s, -OCH3); 13C-NMR (125 MHz, CD3OD) δ 164.9 (C-2), 103.8 (C-3), 183.5 (C-4), 154.5 (C-5), 132.9 (C-6), 159.2 (C-7), 95.5 (C-8), 154.0 (C-9), 105.7 (C-10), 122.6 (C-1′), 129.3 (C-2′ and C-6′), 117.2 (C-3′ and C-5′), 163.0 (C-4′), 60.6 (-OCH3); ESIMS (negative) m/z 299.05 [M–H]−.

HPLC analysis of the isolated compounds in MeOH extract of fresh common sage and ACSE juice

The fresh common sage (15.4 g fresh wt.) was homogenized with 100% MeOH (150 mL), extracted at room temperature for 24 h, and vacuum filtered through No. 2 filter paper (Whatman). The remaining residue was extracted again with 80% MeOH (150 mL) using the same extraction procedure described above. The 100 and 80% MeOH solutions were combined and concentrated with rotary evaporation. The concentrates (3.2 g) were suspended in distilled water (70 mL) and partitioned with n-hexane (70 mL, 3 times) and EtOAc (70 mL, 3 times), successively. After concentration, the EtOAc layer (2 mg/mL) was dissolved in MeOH. The ACSE juice (7 mL, 15.4 g fresh common sage eq. wt.) was added to distilled water (63 mL). The solvent fractionation of the ACSE juice was performed under the same conditions used for that of the fresh common sage MeOH extract. The EtOAc layers were injected to ODS-HPLC using Shiseido C18 (UG 120, 5 μm, 4.6 mm × 250 mm, Tokyo, Japan). The compounds were eluted using a gradient system of 10% MeOH containing 2% acetic acid in H2O to 70% MeOH in H2O over 40 min and holding at 70% MeOH for 45 min. The flow rate was 1.0 mL/min, and the detection wavelength was 254 nm. The 15 phenolic compounds isolated in this study were used as external standards.

Results and discussion

Isolation and identification of compounds from the EtOAc layer of ACSE juice

To compare the chemical constituents of fresh common sage and ACSE juice, the MeOH extract of fresh common sage + sucrose and ACSE juice were successively solvent fractionated with n-hexane, EtOAc, and BuOH. These layers were analyzed by analytical scale ODS-HPLC using an ODS-80Ts column and a gradient system of 5% MeOH containing 2% acetic acid in H2O → 100% MeOH. The HPLC results showed that the compounds contained in ACSE differed significantly from those of fresh common sage + sucrose (data not shown). In particular, new peaks were observed on the HPLC chromatograms of the n-hexane and EtOAc layers of ACSE. The HPLC chromatogram of the BuOH layer of ACSE was very similar to that of fresh common sage + sucrose. These results indicate that various compounds are likely being produced during the aging process of ACSE, and that diverse compounds produced during aging are mainly present in the EtOAc layer of the ACSE juice. Therefore, we purified and isolated the chemical constituents from the EtOAc layer of ACSE juice.

The ACSE juice (4 L) was successively solvent fractionated with n-hexane and EtOAc to afford the EtOAc layer. The concentrated EtOAc layer (5.17 g) was chromatographed on a Diaion HP-20 column (step-wise system: 10% MeOH → 100% MeOH). The resulting 20 fractions (A-T) were grouped by chemical profiles using ODS-HPLC. Nineteen compounds (1–19) were isolated from fractions C + D, F, L, M, P, Q, and S by ODS-HPLC purification under various HPLC conditions. The isolation and purification procedures for 1–19 are summarized in Fig. 1. Of them, 17 compounds were identified as 3,4-dihydroxybenzoic acid (2) [16], (S)-8-hydroxy-4-hydroxy-phenylpropanoic acid (3) [17], cis-caffeic acid (5) [18], trans-caffeic acid (6) [18], esculetin (7) [19], cis-rosmarinic acid (9) [19], trans-rosmarinic acid (10) [19], trans-rosmarinic acid methyl ester (11) [19], nepetin (13) [10], trans-caffeic acid ethyl ester (14) [20], luteolin (15) [21], cis-caffeic acid ethyl ester (16) [22], 6-methoxynaringenin (17) [10], and hispidulin (19) [10] (Fig. 2) by comparison of their NMR and MS data to those previously reported. Seven compounds (9–11, 13, 14, 16 and 17) were also confirmed by 2D-NMR experiments. Three compounds were determined by MS and 1D- and 2D-NMR experiments to be levodopa methyl ester (1), 4-hydroxybenzoic acid ethyl ester (4), and (S)-8-hydroxy-3,4-dihydroxy-phenylpropanoic acid ethyl ester (8).

Fig. 1.

Fig. 1

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Procedure used to isolate compounds 1–19 from the fermented common sage juice

Fig. 2.

Fig. 2

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Chemical structures of the isolated compounds 1–19

Structural-elucidation of isolated compounds 12 and 18

The ESI–MS (negative) spectrum of 12 revealed a deprotonated ion peak at m/z 317.06 [M–H]−, which was indicative of a molecular weight of 318. In addition, the molecular formula (C16H12O7) of 12 was determined by the deprotonated peak [M–H]− at m/z 317.0660 (calculated m/z 317.0661) in the HR–ESI–MS (negative) spectrum. The 1H- and 13C-NMR spectra of 12 indicated the presence of a typical flavanone. The 1H-NMR spectrum of 12 showed four singlet proton signals assigned to the benzene ring at δ 6.00 (1H, s, H-5), 6.86 (2H, br. s, H-2′ and H-6′) and 6.86 (1H, br. s, H-4′), one sp 3 methine proton signal at δ 5.38 (1H, dd, J = 13.0, 3.0 Hz, H-2), and methylene proton signals at δ 3.14 (1H, dd, J = 17.0, 13.0 Hz, H-3a) and 2.73 (1H, dd, J = 17.0, 3.0 Hz, H-3b). A methoxy proton signal at δ 3.77 (3H, s, −OCH3) was also observed (Table 1). The 1H-NMR structural assignment of 12 was supported by the presence of 16 carbon signals, including one carbonyl carbon at δ 198.2 (C-4) in the 13C-NMR spectrum (Table 1). The MS and 1D-NMR results suggested that 12 was a tetrahydroxyflavanone possessing a methoxy group. Furthermore, the complete structure of 12 was determined by 1H-1H COSY, HSQC, NOE, and HMBC experiments. The correlations of H-2′ to C-4′ and C-6′, H-4′ to C-2′ and C-6′, and H-6′ to C-2′ and C-4′ in the HMBC spectrum confirmed the presence of three B ring protons of a flavanone skeleton (Fig. 3). Therefore, the remaining methoxy and two hydroxyl groups are located on the A-ring, which was confirmed by HMBC and NOE experiments. The observation of the HMBC correlations of H-2 and H-5 to C-4 was indicative of the C-5 proton in a flavanone structure. The enhancement of H-5 by irradiation of the methoxy proton at δ 3.77 (3H, s, −OCH3) in the NOE experiment indicated that the methoxy group was connected to the C-6 of 7,8,3′,5′-tetrahydroxyflavanone (Fig. 2). Therefore, the planar structure of compound 12 was unambiguously determined to be 6-methoxy-7,8,3′,5′-tetrahydroxyflavanone (Fig. 2). The absolute stereochemistry for 3,5-dihydroxybenzene group in C-2 of 12 should be further determined by analysis of circular dichroism or X-ray crystallography.

Fig. 3.

Fig. 3

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HMBC (arrows) and NOE/NOESY (dotted lines) correlations of the isolated compounds 12 and 18

The ESI–MS (negative) spectrum of 18 showed a pseudomolecular ion peak at m/z 302.95 [M–H]−, indicating a molecular weight of 304. The 1H-NMR spectrum of 18 exhibited the presence of one double bond proton signal at δ 5.27 (H-3), and two methylene proton signals at δ 3.24 (H-6a), 2.50 (H-6b), 2.52 (H-9a), and 0.90 (H-9b) (Table 2). Additionally, three methine proton signals at δ 6.00 (H-1), 3.33 (H-5), and 5.00 (H-8) and four methyl proton signals at δ 1.87 (H-13), 1.09 (H-14), 2.09 (H-15), and 2.19 (H-1′) were observed. The 1H-NMR structural assignment of 18 was supported by the presence of 17 carbon signals, including three carbonyl carbons at δ 194.6 (C-2), 172.0 (C-12), and 176.8 (C-1′), in the 13C-NMR spectrum (Table 2). The presence of three quaternary carbons of double bond at δ 164.0 (C-4), 122.4 (C-7), and 163.0 (C-11), and one quaternary sp 3 carbon at δ 44.2 (C-10), was also confirmed by the HSQC experiment. Among with the molecular formula of 18, the chemical shifts of four sp 2 quaternary carbons indicated the presence of one ketone carbon at δ 194.6 (C-2) and two carboxylic groups with the signals at δ 172.0 (C-12) and 176.8 (C-1′). The MS and 1D-NMR results suggested that 18 was eudesmanolide with an acetyl group. The complete structure of 18 was determined by 1H-1H COSY, HSQC, NOESY, and HMBC experiments. The HMBC correlations were indicative of eudesma-3(4),7(11)-dien-8,12-olide and an acetyl group. In particular, a correlation of H-1 at δ 6.00 to C-1′ at δ 176.8 was observed in the HMBC spectrum, indicating that the acetyl group was esterified with C-1 of eudesma-3(4),7(11)-dien-8,12-olide (Fig. 3). The observation of NOESY correlations of H-1 at δ 6.00 and H-5 at δ 3.33 as well as H-8 at δ 5.00 and H-14 at δ 1.09 indicated that H-1 and H-5 had the same configuration, and those of H-8 and H-14 configuration were different (Fig. 3). Therefore, the structure of compound 18 was unambiguously determined to be 1α-acetoxy-2-oxo-eudesman-3,7(11)-dien-8β,12-olide (Fig. 2).

Occurrence of the isolated phenolic compounds in fresh common sage and ACSE juice by HPLC analysis

To confirm the occurrence of the isolated compounds in fresh common sage + sucrose and ACSE juice, the EtOAc layers were analyzed by an analytical scale ODS-HPLC using a Shiseido C18 column and a gradient system of 10% MeOH containing 2% acetic acid in H2O → 70% MeOH in H2O. For the caffeic acids (5 and 6), rosmarinic acids (9 and 10), and caffeic acid ethyl esters (14 and 16), both the trans and cis isomers were present. The cis forms (5, 9, and 16) might be respectively produced as artifact from the respective trans forms (6, 10, and 14) during the purification and isolation procedures. Therefore, 15 isolated phenolic compounds, namely 1–3, 5–8, 10–15, 17, and 19 (excluding 5, 9, 16, and 18) were used as external standards. Detection of these 15 compounds on the HPLC chromatogram of the ACSE juice indicated they were also present in ACSE juice [Fig. 4(B)]. Of the 15 isolated compounds, only compound 10 was detected on the HPLC chromatogram of the fresh common sage + sucrose [Fig. 4(A)]. Caffeic acid (6), esculetin (7), rosmarinic acid (10), nepetin (13), luteolin (15), 6-methoxynaringenin (17), and hispidulin (19) have been previously found in common sage [8–10]. Therefore, most of the isolated compounds might be present in small amounts in common sage, and/or produced during the aging of the common sage exudate with sugar.

Fig. 4.

Fig. 4

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HPLC chromatograms for the EtOAc layers of (A) fresh common sage + sucrose MeOH extract and (B) the ACSE juice and (C) external standards of phenolic compounds. 1: levodopa methyl ester; 2: 3,4-dihydroxybenzoic acid; 3: (S)-8-hydroxy-4-hydroxy-phenylpropanoic acid; 4: 4-hydroxybenzoic acid ethyl ester; 6: trans-caffeic acid; 7: esculetin; 8: (S)-8-hydroxy-3,4-dihydroxy-phenylpropanoic acid ethyl ester; 10: trans-rosmarinic acid; 11: trans-rosmarinic acid methyl ester; 12: 6-methoxy-7,8,3′,5′-tetrahydroxyflavanone; 13: nepetin; 14: trans-caffeic acid ethyl ester; 15: luteolin; 17: 6-methoxynaringenin; 19: hispidulin

Of the compounds isolated from the ACSE juice, five compounds (1, 4, 8, 11, and 14) were in the methyl- or ethyl-esterified forms. Such compounds are widely present in foods [23]. The carboxylic acids of these compounds could be esterified with MeOH, EtOH, or EtOAc under acidic conditions [24, 25]. Therefore, these compounds could likely be produced as artifacts during the extraction and purification steps. Methyl-esterified forms (11), as well as ethyl-esterified forms (1, 4, 8, and 14), were detected in the ACSE juice [Fig. 4(B)], whereas they were not detected in the fresh common sage [Fig. 4(A)]. Previous studies have reported that phenolic acids are esterified by microorganisms during fermentation [23, 26]. Therefore, the presence of 1, 4, 8, 11, and 15 in the ASCE juice was definitively verified and might be produced by fermentation during its aging.

Flavonoids are widely distributed as both free and glycoside forms in the plant kingdom, and their glycosides are hydrolyzed during fermentation [27, 28]. Flavonoids 13, 15, 17, and 19 identified in this study and their glycosides have already been found in common sage [8–10]. Here we confirmed the presence of the flavonoid aglycones 13, 15, 17, and 19 in the ACSE juice [Fig. 4(B)]. Their presence could not be confirmed in fresh common sage in this study, though their aglycone and glycoside forms are known to be in fresh common sage [8–10]. It is suggested that flavonoid aglycones 13, 15, 17, and 19 may be present in common sage at concentrations near the limit of detection of the HPLC method. In addition, these flavonoid aglycones might be produced from their glycosides by enzymatic hydrolysis from fermentation during ACSE manufacturing. A new flavonoid 12 isolated in this study was detected in the ACSE juice [Fig. 4(B)], but not in fresh common sage [Fig. 4(A)]. Therefore, compound 12 might exist as a glycoside in common sage.

In this study, 19 compounds, including two p-hydroxybenzoic acid derivatives (2 and 4), eight phenylpropanoid derivatives (1, 3, 5–8, 14, and 16), three rosmarinic acid derivatives (9–11), five flavonoids (12, 13, 15, 17, and 19), and one eudesmanolide (18), were isolated and identified from the ACSE (Fig. 2). Previous studies have reported the presence of phenylpropanoid derivatives, rosmarinic acid derivatives, flavonoids and their glycosides, and eudesmanolide in common sage [8–10]. However, to the best of our knowledge, compounds 1, 3, 4, 7, 8, 14, 18, and 19 were newly identified from common sage and compound 12 was isolated for the first time from nature. In addition, we confirmed the occurrence of 15 phenolic compounds (1–4, 6–8, 10–15, 17, and 19) in the ACSE juice. To understand what occurs in common sage exudate during the ACSE manufacturing, further chemical profiling will be conducted using the 15 phenolic compounds isolated in this study as external standards.

Diverse previous studies have reported that phenolic compounds, including flavonoids, exhibit various biological effects, such as antioxidant, anti-inflammatory, and anticancer activities [29–31]. Additionally, several eudesmanolides, including compounds 18 identified in this study, have anti-inflammatory, anti-tumor, and anticancer properties [32–34]. Therefore, phenolics and eudesmanolides could partially contribute to the beneficial health effects of ACSE juice. Therefore, this investigation may provide important information in understanding the biological effects of ACSE juice. However, high sugar intake can cause the health risks, such as obesity and diabetes [35]. Because the ACSE juice is comprised of a high sugar concentration, it is very important to find out optimum intake of ACSE juice to maintain and enhance our health. Therefore, further studies on the relationship between health and the optimum intake of ACSE juice are necessary.

Acknowledgements

This study was supported by the High Value-Added Food Technology Development Program through the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (315069-3), Republic of Korea. LC–ESI–MS and NMR spectral data were obtained from the Korea Basic Science Institute, Gwangju.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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