Phenolic Compounds from the Roots of Rhodiola crenulata and Their Antioxidant and Inducing IFN-γ Production Activities.

In the present study, two new phenolic compounds 1 and 11, a pair of lignan isomers 12 and 13 with their absolute configurations established for the first time, were isolated from the ethanol extract of the roots of Rhodiola crenulata, together with 13 known phenolic compounds, and their structures were elucidated via NMR, HRESIMS, UV, IR and CD analyses. All the isolated compounds were evaluated for their in vitro antioxidant activities using the 2,2-diphenyl-1-picryhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays. Ten of them exhibited significant antioxidant activities compared to ascorbic acid. Furthermore, the inducibilities of the isolated compounds to IFN-γ production were also assessed. Compounds 1, 8, 9, 12, 13, 14 and 15 could moderately stimulate IFN-γ expression.

1. Introduction

Rhodiola crenulata (HK. f. et.Thoms) H. Ohba (Dhua Hong-Jing-Tian in Chinese) is an important species among the Rhodiola genus, mainly found distributed in western regions of China including the provinces of Yunnan, Sichuan and Tibet [1]. As a functional food, R. crenulata is used for prevention against high-altitude illness and regarded by the tourists going to plateau as a traditional phyto-adaptogen to environmental challenges [2]. Moreover, the extracts of R. crenulata have been made into pharmaceutical preparations and cosmetics with varied bioactivities [3,4]. Due to its extensive application in food, medicine and cosmetics, the chemical constituents and pharmacological activities of R. crenulata have been widely investigated. The main chemical constituents of R. crenulata are phenolic compounds, such as flavonoids, phenylpropanoids, phenolic acids and so on [5,6,7,8,9,10]. Modern pharmacological investigations have revealed that Rhodiola preparations exhibit antioxidant, immunomodulatory, anti-aging, anti-fatigue [11,12], neuroprotective [13], anti-inflammatory [14], antidepressive, anxiolytic, nootropic, life-span increasing and central nervous system (CNS) stimulating activities [15]. Due to the diverse biological activities of R. crenulata, the chemical constituents of R. crenulata and the bioactivities of the isolates were investigated by our research group.

2. Results and Discussion

2.1. Identification of Compounds 1–17

Compound 1 was isolated as a light yellow solid. Its molecular formula was determined as C17H22O6 by HRESIMS [M − H]− at m/z 321.1338, (calcd, for C17H21O6, 321.1338). The IR spectrum displayed OH (3411 cm−1), C=O (1708 and 1734 cm−1), and C=C (1633 cm−1) functions. The 1H-NMR spectrum revealed the existence of five methylenes at δH 4.12 (2H, t, J = 6.6 Hz), 2.33 (2H, t, J = 7.4 Hz), 1.64 (2H, m), 1.58 (2H, m) and 1.37 (2H, m), an ABX aromatic protons at δH 7.33 (1H, d, J = 2.0 Hz), 7.12 (1H, dd, J = 8.2, 2.0 Hz) and 6.80 (1H, d, J = 8.2 Hz), and two methoxyls at δH 3.83 and 3.59. The signals at δH 7.55 (1H, d, J = 16.0 Hz) and 6.47 (1H, d, J = 16.0 Hz) suggested trans double bond protons. The 13C-NMR spectrum revealed the presence of 17 C-atoms, which were identified with DEPT-135 spectrum as five methylenes (δC 63.5, 33.1, 27.9, 25.0 and 24.1), two methoxyls (δC 55.7 and 51.2), two carbonyl groups (δC 173.3 and 166.7), as well as a pair of olefinic carbons with trans double bond features (δC 145.0 and 114.4). The chemical shifts from δC 111.2 to 149.5 were in the aromatic region, which indicated a benzene ring and a double bond existed. From the above NMR data (Table 1), a 3,4-disubstituted cinnamoyl group linked with a fatty alcohol was deduced. Thus, the chemical structure illustrated in Figure 1 were established on the basis of these data. This was further confirmed by the key HMBC correlations (Figure 2) from δH 3.59 (OCH3) to 173.3 (C-1′), from δH 4.12 (H-6′) to δC 166.7 (C-9) and 27.9 (C-5). So, compound 1 was determined as methyl 6-O-(3-methoxy-4-hydroxy-cinnamoyl)-caproate and named rhodiolate.

Table 1

1H-NMR and 13C-NMR data of compounds 1, 2 and 11–13 (400 MHz and 100 MHz, DMSO-d6, δ in ppm, J in Hz).

No.	1		2		11		12		13
	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC
1		125.5				131.8		127.7		127.6
2	7.33 (d, 2.0)	111.2		147.6	6.79 (d, 1.2)	114.0	7.01 (d, 1.8)	112.2	7.07 (s)	112.2
3		149.5		135.9		147.4		148.1		148.3
4		148.0		176.9		145.4		147.6		147.9
5	6.80 (d, 8.2)	115.5		152.7	6.74 a	115.8	6.80 (d, 8.0)	115.8	6.84 a	115.5
6	7.12 (dd, 8.2, 2.0)	123.1	6.56 (s)	95.4	6.64 (dd, 8.2, 1.8)	117.3	6.85 (dd, 8.0, 1.8)	121.0	6.85 a	120.0
7	7.55 (d, 16.0)	145.0		154.0	5.07 (d, 3.6)	83.3	4.95 (d, 7.8)	76.6	4.90 (d, 7.8)	76.5
8	6.47 (d, 16.0)	114.4		126.4	3.70 (dd, 9.2, 3.8)	53.0	4.20 (m)	78.4	4.16 (m)	78.5
9		166.7		144.2		177.7	3.55 (d, 11.6) 3.35 a	60.6	3.53 (d, 10.2) 3.35 (dd, 12.0, 4.2)	60.6
10				103.8
1′		173.3		122.3		131.0		128.2		130.5
2′	2.33 (t, 7.4)	33.1	8.14 (d, 8.8)	130.2	6.99 (d, 1.2)	111.1	7.30 (d, 2.0)	116.9	7.05 (d, 2.0)	115.9
3′	1.58 (m)	24.1	6.94 (d, 8.8)	115.9		148.2		144.3		144.4
4′	1.37 (m)	25.0		159.8		145.7		146.1		143.8
5′	1.64 (m)	27.9	6.94 (d, 8.8)	115.9	6.81 a	115.9	6.94 (d, 8.0)	117.6	6.87 (d, 8.0)	117.3
6′	4.12 (t, 6.6)	63.5	8.14 (d, 8.8)	130.2	6.85 (dd, 8.2, 1.8)	119.5	7.21 (dd, 8.0, 2.0)	122.1	7.00 (dd, 8.0, 1.8)	121.6
7′					5.39 (d, 3.6)	85.4	7.52 (d, 16.0)	143.9	7.12 (d, 15.8)	136.6
8′					3.28 (m)	49.3	6.38 (d, 16.0)	117.7	6.29 (d, 15.8)	127.6
9′					4.14 (dd, 9.2, 7.2) 3.95 (dd, 9.2, 4.0)	72.5		168.3		171.2
3-OCH3	3.83 (s)	55.7					3.78 (s)	56.1	3.76 (s)	56.2
5-OCH3			3.91 (s)	56.8
1′-OCH3	3.59 (s)	51.2
3′-OCH3					3.79 (s)	56.2

a Overlapped signals are reported without designating multiplicity.

Figure 1

Chemical structures of compounds 1–17.

Figure 2

Key HMBC or NOESY correlations of compounds 1, 2 and 11–13.

Compound 2 showed an [M + H]+ ion peak at m/z 317.0667 (calcd. for C16H13O7, 317.0661) in the HRESIMS spectrum, consistent with the molecular formula of C16H12O7. The IR spectrum displayed OH (3418 and 3309 cm−1), C=C (1662 cm−1) and MeO (1252 cm−1) bands. The 1H-NMR spectrum revealed the existence of four active hydrogen protons at δH 12.01, 10.11, 9.40 and 8.75, and a pair of double peaks (δH 8.14 (2H, d, J = 8.8 Hz) and 6.94 (2H, d, J = 8.8 Hz)) ascribed to a 1,4-disubstituted benzene ring, as well as one single peak at δH 6.56. The 13C-NMR spectrum revealed the presence of 16 C-atoms, including 15 aromatic carbons at δC 176.9, 159.8, 154.0, 152.7, 147.6, 144.2, 135.9, 130.2, 130.2, 126.4, 122.3, 115.9, 115.9, 103.8 and 95.4, and a MeO at δC 56.8. Comparison the NMR data (Table 1) of compound 2 with those of herbacetin [16] revealed that the NMR data of compound 2 were similar to those of herbacetin, except for an extra MeO group. Based on the above NMR data, a MeO group substituting the OH at C-7 was proposed. This was further supported by HMBC correlation (Figure 2) between δH 3.91 (OCH3) and δC 154.0 (C-7). When comparing its 13C-NMR data to the previously published data of herbacetin 7-methyl ether [17], wrong assignments of C-5 (δC 153.72), C-7 (δC 159.39) and C-9 (δC 152.40) were noted in those published results. Consequently, compound 2 was identified as herbacetin 7-methyl ether, and its 13C-NMR data were corrected (see Table 1).

The molecular formula of compound 11 was determined as C19H18O7 by its HRESIMS [M + Na]+ signal at m/z 381.0953 (calcd. for C19H18O7Na, 381.0950). The IR absorptions suggested the existence of OH (3402 cm−1), C=O (1755 cm−1) and C=C (1650 cm−1) functionalities. The chemical shifts from δH 6.99 to 6.64 were in the aromatic region in the 1H-NMR spectrum, which indicated the existence of two 1,3,4-trisubstituted benzene systems (δH 6.79 (1H, d, J = 1.2 Hz), 6.74 (1H, overlapped) and 6.64 (1H, dd, J = 8.2, 1.8 Hz), and 6.99 (1H, d, J = 1.2 Hz), 6.85 (1H, dd, J = 8.2, 1.8 Hz) and 6.81 (1H, overlapped)), furthermore, a pair of oxymethylene signals (δH 4.14 (1H, dd, J = 9.2, 7.2 Hz) and 3.95 (1H, dd, J = 9.2, 4.0 Hz)), two oxymethine signals (δH 5.39 (1H, d, J = 3.6 Hz) and 5.07 (1H, d, J = 3.6 Hz)) were also visible in the 1H-NMR spectrum. The 13C-NMR spectrum displayed 19 C-atom signals, including two benzene rings (δC 147.4, 145.4, 131.8, 117.3, 115.8 and 114.0) and (δC 148.2, 145.7, 131.0, 119.5, 115.9 and 111.0), a methylene (δC 72.5), four methines (δC 85.4, 83.3, 53.0 and 49.3) and an ester carbonyl carbon (δC 177.7). The NMR data of 11 (Table 1) were closely similar to those of 4-ketopinoresinol [18], the chemical structure with the loss of MeO at C-3 was deduced on the basis of these data. This was further confirmed by the key HMBC correlation (Figure 2) from δH 3.79 (OCH3) to δC 148.2 (C-3′). The relative configuration was established by NOESY correlations (Figure 2) between δH 3.70 (H-8)/6.79 (H-2), δH 5.07 (H-7)/3.95 (H-9′b)/5.39 (H-7′), δH 3.28 (H-8′)/6.99 (H-2′). However, compound 11 gave a positive specific rotation +4.97 (c 0.2, MeOH), opposite that of (−)-3,4,3′,4′-tetrahydroxy-9,7′β-epoxylignano-7β,9′-lactone ( −5.8 (c 0.1, MeOH)) [19]. Thus, compound 11 was identified as (7β,7′β,8α,8′α)-3′-methoxy-9-oxo-7,9′,7,9′-diepoxylignan-3,4,4′-triol.

Compound 12 was isolated as a white amorphous powder, the HRESIMS of compound 12 showed an [M + Na]+ ion peak at m/z 381.0950 (calcd. for C19H18O7Na: 381.0950) for the molecular formula C19H18O7. The IR spectrum showed 3438 cm−1 (OH) and 1688 cm−1 (C=O) groups. The 1H-NMR spectrum of compound 12 displayed two pairs of ABX proton signals: δH 7.30 (1H, d, J = 2.0 Hz), 7.21 (1H, dd, J = 8.0, 2.0 Hz) and 6.94 (1H, d, J = 8.0 Hz), as well as δH 7.01 (1H, d, J = 1.8 Hz), 6.85 (1H, d, J = 8.0, 1.8 Hz) and 6.80 (1H, d, J = 8.0 Hz). A pair of proton signals (δH 7.52 (1H, d, J = 16.0 Hz) and 6.38 (1H, d, J = 16.0 Hz) of trans-double bonds were also seen in the 1H-NMR spectrum. Accordingly, the 13C-NMR and DEPT-135 spectra gave 19 C-atom signals. Except for a MeO group at δC 56.1, there remained 18 carbons, and a 3,4-disubstituted cinnamic acid (δC 168.3, 146.1, 144.3, 143.9, 128.2, 122.1, 117.7, 117.6 and 116.9) linked with a 7,8-disubstituted 3-methoxy-4-hydroxy-phenylpropanol (δC 148.1, 147.6, 127.7, 121.0, 115.8, 112.2, 78.4, 76.6 and 60.6) through a C-8-O-C-3′ and C-7-O-C-4′ linkage pattern was deduced. By comparison with the NMR data (Table 1) of compound 12 with those of arteminorin D [20], the data were seen to be the same as those of this compound. In the previous report, the absolute configuration of arteminorin D was not determined, so, the absolute configuration of compound 12 was elucidated by 2D NMR and CD exciton chirality methods in our work. The relative configuration (Figure 2) of compound 12 was identified by NOESY spectrum and the same as the literature reported (He et al., 2009). The UV spectrum (Figure 3) of compound 12 showed an absorption at 222 nm attributable to the benzene moiety (π→π*). Corresponding to this UV absorption, the CD spectrum of compound 12 showed a negative Cotton effect at 222 nm due to the transition interaction between two different benzene moieties in the structure. The above information demonstrated a negative chirality for compound 12, and the two aforementioned chromophores should be oriented counterclockwise in space (Figure 3). Thus, compound 12 was elucidated as (7R,8R)-3-methoxy-8′-carboxy-7′-en-3′,8-epoxy-7,4′-oxyneolignan-4,9-diol.

Figure 3

CD and UV spectra of compounds 12 (A) and 13 (B), where the arrow denotes the electronic transition dipole of the chromophores. (−) means the two chromophores should be oriented counterclockwise in space; (+) means the two chromophores should be oriented clockwise in space.

The HRESIMS of compound 13 showed an [M + Na]+ ion peak at m/z 381.0952 [M + Na]+ (calcd. for C19H18O7Na:381.0950) for the same molecular formula (C19H18O7) as that of compound 12. Comparison the NMR data (Table 1) of compound 13 with those of compound 12 revealed that the NMR data of compound 13 were similar. A 3,4-disubstituted cinnamic acid linked with a 7,8-disubstituted 3-methoxy-4-hydroxy-phenylpropanol with different linkage patterns from that of compound 12 was deduced. Although no HMBC correlations from H-7 to C-3′ or H-8 to C-4′ were observed, the diagnostic chemical shifts of C-7 (δC 76.5), C-8 (δC 78.5), C-3′ (δC 144.4) and C-4′ (δC 143.8), combined with the established molecular formula, C19H18O7, suggested a linkage pattern of C-7-O-C-3′ and C-8-O-C-4′. Furthermore, a large coupling constant between H-7 and H-8 (J = 7.8 Hz) indicated a trans relationship of the two protons. This chemical structure (Figure 1) was reported in a Chinese patent [21] without the absolute configuration, so the absolute configuration of compound 13 was also elucidated using the CD exciton chirality method (Figure 3), and the absolute configuration of compound 13 was determined as (7R,8R). Thus, compound 13 was elucidated as (7R,8R)-3-methoxy-8′-carboxy-7′-en-3′,7-epoxy-8,4′-oxyneolignan-4,9-diol.

In addition, the other 12 known phenolic compounds were identified as 5,7,3′,5′-tetrahydroxy-dihydroflavone (3) [22], kaempferol (4) [23], luteolin (5) [24], kaempferol-7-O-α-l-rhamnoside (6) [23], rhodionin (7) [6], rhodiosin (8) [6], ternatumoside II (9) [25], crenuloside (10) [6], (+)-isolarisiresinol (14) [26], (+)-dihydrodehydrodiconiferyl alcohol (15) [27], methyl gallate (16) [28] and 2-(4-hydroxyphenyl) ethyl 3,4,5-trihydroxybenzoate (17) [29] by comparing their physical and spectral data with literature values.

2.2. Antioxidant Activities

The DPPH and ABTS radical-scavenging assays are effective methods used to evaluate the antioxidant activity of natural products. As shown in Table 2, new compound 1 exhibited no radical-scavenging activity, while compounds 2–10 showed potent radical-scavenging activities comparable to ascorbic acid, compounds 16 and 17 exhibited more intense radical-scavenging activities. In all, the radical-scavenging activity of compounds is also attributed to the hydroxyl groups substituted on the aromatic ring.

Table 2

The IC50 values in μM of antioxidant activities of 1–17 (n = 3).

Compounds	DPPH IC50 (μM) a	ABTS IC50 (μM) a
1	>500 b	>500
2	114.3 ± 4.4	87.0 ± 7.9
3	73.8 ± 2.9	129.8 ± 12.3
4	91.6 ± 1.4	125.4 ± 9.8
5	94.3 ± 2.3	123.9 ± 10.4
6	83.0 ± 3.5	123.7 ± 12.8
7	104.7 ± 1.8	63.7 ± 8.5
8	96.5 ± 2.3	53.1 ± 4.8
9	260.5 ± 36.4	320.2 ± 22.6
10	64.1 ± 3.3	110.8 ± 10.7
11	>500	>500
12	>500	>500
13	>500	>500
14	227.1 ± 33.9	160.2 ± 8.8
15	>500	>500
16	52.8 ± 3.3	50.0 ± 4.9
17	79.5 ± 1.7	65.0 ± 3.9
Ascorbic acid	88.6 ± 1.9	89.8 ± 6.8

a Data were represented as mean ± SD; b The IC50 value of sample is higher than 500 μM.

2.3. Compounds Treatment Stimulates IFN-γ Production Activities

As reported in the literature [30,31], nitric oxide production by activated macrophages in vitro and in vivo is dependent on IFN-γ, which is corresponding to the immunomodulatory effect [32]. To further evaluate the biological of these isolated phenolic compounds, the inducibility to IFN-γ release was measured in cell culture supernatants. As shown in Figure 4, compounds 1, 8, 9, 12, 13, 14 and 15 could moderately stimulate IFN-γ expression. Finally, CCK-8 assays were used to determine if these isolates suppress the production of IFN-γ. As Figure 5 shows, there was no significant reduction in cell viability by these compounds (100 μM concentration). Therefore, we can conclude that the four lignans (compounds 12, 13, 14 and 15) and the two flavonoid glycosides (compounds 8 and 9) from R. crenulata have the ability of inducing IFN-γ production, moreover, the new compound 1 also possesses the inducing IFN-γ release activity. These compounds would be the active chemical constituents related to the immunomodulatory effect of R. crenulata.

Figure 4

IFN-γ production by spleen lymphocyte cells treated with compounds 1–17. Valus are means ± S.E.M., n = 3; * p < 0.05; ** p < 0.01; *** p < 0.005.

Figure 5

Cell viability of mouse spleen lymphocyte cells after 48 h treatment by 100 μM concentration of compounds 1–17 using CCK-8 assay (data points represent the mean ± S.D., n = 3).

3. Experimental Section

3.1. General Procedures

All reagents were of HPLC or analytical grade and were purchased from Tianjin Concord Chemical Company (Tianjin, China). Optical rotation values were measured by a P-1020 polarimeter (JASCO International Co., Ltd., Tokyo, Japan). IR spectra were measured on a Perkin Elmer spectrum 65 FT-IR spectrometer (PerkinElmer, Waltham, MA, USA). HRESIMS data were determined by an Agilent 6210 ESI/TOF mass spectrometer (Agilent Co., Santa Clara, CA, USA), the full scan mass spectra data were acquired in positive and negative ion modes. Acquisition parameters are as follows: capillary voltage was 3000 V for ESI (+) and 2600 V for ESI (−); cone voltage was 45 V; the ESI source temperature was 100 °C; the desolvation temperature was 350 °C; the nitrogen (N2) was used as desolvation gas at flow rates of 600 L/h for both ESI (+) and ESI (−); and the range of full scan was set at m/z 150–1000 Da.). NMR spectra were recorded on a Bruker-AVIII-400/600M spectrometer (Bruker Co., Geneva, Switzerland). CD spectra were recorded on a JASCO J-720W spectropolarimeter (JASCO). The ODs were recorded on a Flexstation 3 (Molecular Devices Co., Sunnyvale, CA, USA). Preparative HPLC: Agilent 1260 system equipped with a preparative Cosmosil C18 (5 μm, 20 mm × 250 mm) column (Agilent Co.). Column chromatography (CC): silica gel (SiO2; 200–300/400–500 mesh, Qingdao Marine Chemical Factory, Qingdao, China); Sephadex LH–20: (GE Healthcare UK Ltd, Buckinghamshire, UK); D101 macroporous adsorption resin (Tianjin Haiguang Chemical Company, Tianjin, China); ODS (ODS-A-HG 5–50 μm, YMC Co., Kyoto, Japan) TLC: silica gel GF254 (SiO2; 400–500 mesh, Qingdao Marine Chemical Factory, Qingdao, China). 2,2-di-phenyl-1-picrylhydrazyl (DPPH) (Sigma Corporation, Ronkonkoma, New York, NY, USA). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Shanghai Sangon Biological Coporation, Shanghai, China). K2S2O8 (Tianjin Guangfukeji Development Corporation, Tianjin, China).

3.2. Plant Materials

The roots of Rhodiola crenulata (HK. f. et.Thoms) H. Ohba were obtained from Shijiazhuang Yiling Pharmaceutical Co., Ltd. (Shijiazhuang, China), and were identified by Dr. Chun-Hua Wang of Tianjin University of Traditional Chinese Medicine. A voucher specimen (No. 20121011CH) was deposited in Tianjin Key Laboratory of Modern Chinese Medicine, Taida, Tianjin, China.

3.3. Extraction and Isolation

The roots of R. crenulata (25.0 kg) were extracted three times with 95% EtOH (50 L) with heating for 2 h. The solvent was concentrated to give a crude extract (2500 g), which was subjected to column chromatography (CC) (D101 macroporous adsorption resin, EtOH 95%, 50% and 30%). Then 95% EtOH extract (400 g) was subjected to silica gel CC (120 cm × 15 cm) eluted with a gradient of CH2Cl2/MeOH (100:0–100:20) to afford eight fractions (F1–F8). F1 (5 g) was further separated by silica gel CC with petroleum ether/ethyl acetate (100:0–100:40) to afford eight subfractions F1–1 to F1–8. Subfraction F1–7 (270 mg) was purified by preparative HPLC with MeOH/H2O (72:28) to afford compound 1 (42 mg). F2 (42 g) was subjected to silica gel column chromatography eluting with CH2Cl2/MeOH (100:0–100:10) to afford 42 subfractions. Compound 4 (330 mg) was obtained from F2–2 and compound 5 (120 mg) from F2–10, respectively. Subfractions F2–12 to F2–20 were merged and purified by preparative HPLC with MeOH/H2O (58:42) to afford compounds 14 (25 mg) and 15 (33 mg). Subfraction F2–22 (310 mg) was subjected to Sephadex LH–20 column chromatography with CH2Cl2/MeOH (50:50) and preparative HPLC with MeOH/H2O (55:45) to afford compound 12 (44 mg). Subfractions F2–23 to F2–30 were merged and subjected to Sephadex LH–20 column chromatography with CH2Cl2/MeOH (50:50) and preparative HPLC with MeOH/H2O (48:52) to afford compound 13 (24 mg). F2–35 to F2–42 were merged and separated by preparative HPLC with MeOH/H2O (44:56) to afford compound 11 (37 mg). F3 (36 g) was separated by silica gel CC with CH2Cl2/MeOH (100:0–100:20) to afford 34 subfractions. Compound 3 (47 mg) was obtained from F3–1 using a Sephadex LH–20 column with CH2Cl2/MeOH (50:50). F3–8 (85 mg) was washed with methanol to obtain feathery crystals of compound 16 (33 mg). Fractions F3–6 to F3–13 were merged and subjected to Sephadex LH–20 column chromatography with CH2Cl2/MeOH (50:50) to obtain compound 2 (58 mg) and a mixture of compounds 2 and 17 (89 mg), which were purified by preparative HPLC with MeOH/H2O (55:45) to yield compound 17 (22 mg). F4 (26 g) was separated by ODS CC with MeOH/H2O (20:80, 40:60, 60:40 and 80:20) to afford 10 subfractions. Compound 6 (105 mg) was obtained with a needle crystals from F4–1. F4–4 (3.7 g) was subjected to Sephadex LH–20 CC with CH2Cl2/MeOH (50:50) to give compound 7 (69 mg). F5 (26 g) was subjected to ODS CC with MeOH/H2O (20:80, 40:60, 60:40, 80:20) to get 11 subfractions. F5–2 (1.4 g) was separated by Sephadex LH–20 CC with MeOH to afford compound 9 (52 mg) and preparative HPLC with MeOH/H2O (33:67) to afford compound 10 (24 mg). F5–4 (1.1 g) was separated by Sephadex LH–20 CC with MeOH to obtain needle crystals to give compound 8 (420 mg).

3.4. Compound Characterization

Rhodiolate (1): Light yellow solid; IR (KBr) νmax: 3411, 2925, 2854, 1734, 1708, 1633, 1592, 1514, 1208 cm−l; UV (MeOH) λmax: 324, 197.5 nm; HRESIMS m/z 321.1338 [M − H]− (calcd for C17H21O6: 321.1338), 1H- (400 MHz) and 13C-NMR (100 MHz) data, see Table 1.

Herbacetin 7-methyl ether (2): Yellow needles; IR (KBr) νmax: 3418, 3309, 2922, 1662, 1252 cm−l; UV (MeOH) λmax: 199.5, 274.5, 331, 380 nm; HRESIMS m/z 317.0667 [M + H]+ (calcd for C16H13O7: 317.0661), 1H- (400 MHz) and 13C-NMR (100 MHz) data, see Table 1.

(+)-Syzygiresinol A (11): brown viscous substance; +4.97 (c 0.2, MeOH); IR (KBr) νmax: 3402, 3280, 2921, 1755, 1650, 1232 cm−l; UV (MeOH) λmax: 203, 282.5 nm; HRESIMS m/z 381.0953 [M + Na]+ (calcd for C19H18O7 Na: 381.0950), 1H- (400 MHz) and 13C-NMR (100 MHz) data, see Table 1.

(7R,8R)-3-Methoxy-8′-carboxy-7′-en-3′,8-epoxy-7,4′-oxyneolignan-4,9-diol (12): White amorphous powder; −4.67 (c 0.2, MeOH); IR (KBr) νmax: 3438, 2978, 1688, 1612, 1505, 1265, 1125, 1048 cm−1; UV (MeOH) λmax: 202, 222, 288.5, 319 nm; HRESIMS m/z 381.0950 [M + Na]+ (calcd for C19H18O7Na: 381.0950), 1H- (400 MHz) and 13C-NMR (100 MHz) data, see Table 1.

(7R,8R)-3-Methoxy-8′-carboxy-7′-en-3′,7-epoxy-8,4′-oxyneolignan-4,9-diol (13): Yellow viscous powder; −9.00 (c 0.2, MeOH); IR (KBr) νmax: 3427, 2956, 1537, 1242, 1098, 1024 cm−1; UV (MeOH) λmax: 202, 224, 287.5, 316 nm; HRESIMS m/z 381.0952 [M + Na]+, calcd for C19H18O7Na: 381.0950), 1H- (400 MHz) and 13C-NMR (100 MHz) data, see Table 1.

3.5. Antioxidant Assay

The DPPH method was widely used to evaluate the antioxidant activity [33,34]. We divided them into three groups, including sample group, control group and blank group. In a 96-well microplate, 150 μL of DPPH solution (250 μM) was added to 50 μL of the test sample in methanol at different concentrations. The OD values of the reaction mixtures was recorded at 517 nm using a Flexstation 3 for 30 min at 30 °C. The DPPH-scavenging activity was calculated by the following formula: % scavenging activity = 100 × 1 − (ODsample − ODblank)/ODcontrol, ODsample = absorbance of sample and DPPH, ODblank = absorbance of sample and methanol, ODcontrol = absorbance of DPPH and methanol. IC50, the concentration of sample needed to scavenge 50% of DPPH radical and was obtained by plotting the DPPH-scavenging percentage of each sample against the sample concentration. Ascorbic acid was used as the positive control in this experiment.

The ABTS assay was adopted to evaluate the antioxidant activity of phenolic compounds as well as the free radical-scavenging assay [35]. This assay was based on the oxidation of radical cation ABTS+, which was read at 734 nm. The working ABTS reagent was prepared by mixing the same volume of ABTS methanol solution (7 mM) and K2S2O8 solution (6.63 mg to 10 mL 50% methanol). Then diluted with methanol 1:3. The ABTS scavenging activity share the same method. Except that the OD values of the reaction mixtures were recorded at 734 nm using a Flexstation 3 for 10 min at 30 °C.

3.6. Cell Culture and Cytokine Assay

The spleen lymphocyte cells obtained from Balb-c mouse were maintained in RPMI 1640 medium containing 10% heat inactivated fetal bovine serum, 1% penicillin-streptomycin at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. For compounds treatment tests, cells were cultured in triplicate in Coster flat-bottom cell culture plates (Corning Inc., Corning, NY, USA). Cells were plated at a density of 2 × 106 cells/well in 24-well cell culture plates. Compounds to be tested were initially dissolved in 10 μL of DMSO, and then RPMI 1640 was added to make solutions in a series of concentration. The final concentration of test compounds was 10 μM. Cells were supplemented with the test compounds as stimulation with 1 μg/mL Con A. The activated cells were further incubated for 48 h. Control cells were grown under indentical conditions but were only exposed to Con A.

After an incubation period (48 h), supernatants of cells were analyzed for IFN-γ secretion. Concentrations of IFN-γ were determined by ELISA kits (eBioscience, San Diego, CA, USA), following the manufacturer instructions.

3.7. Cell Viability Test

Cell viability was determined using a CCK-8 cell proliferation and cytotoxicity assay kit (Beyotime Institute of Biotechnology, Haimen, China), according to the supplier’s manual. Dispense 100 μL of cell suspension (5000 cells/well) in a 96-well plate. Pre-incubate the plate for 24 h in a humidified incubator (at 37 °C, 5% CO2). Add 10 μL of 100 μM concentrations of compounds to be tested to the plate for 48 h in the incubator. Briefly, at the end of the drug treatment period, 10 μL CCK-8 solutions were fed to each well of the culture plate (containing 100 μL medium). After 4 h incubation, the optical density of the assay solution was measured at 450 nm with a spectrophotometer (BioTek, Winooski, Vermont, VT, USA).

4. Conclusions

In summary, the functional food, R. crenulata (HK. f. et.Thoms) H. Ohba, with its unique medicinal effects, has attracted increasing attention in the food and pharmaceutical fields. In our work, isolation and characterization resulted in two new and 15 known phenolic compounds, which structures were elucidated by chemical and spectroscopic analyses, and the absolute configurations of two lignan isomers were confirmed for the first time by CD analyses. In addition, the 13C-NMR data of herbacetin 7-methyl ether was corrected by our group. The biological assay disclosed flavonoids exhibited potent antioxidant activity, but other phenylpropanoids, including the new compounds, showed weak antioxidant activity. Based on the antioxidant evaluation results of the isolated phenolic compounds, we think the various flavonoids are the major chemical constituents responsible for the anti-aging, life-span increasing and anti-radiation activities. To further evaluate the biological activity of these isolated phenols, the induction of IFN-γ production was also evaluated because of its known immunoregulatory activity. As a result, four lignans (compounds 12, 13, 14 and 15) had the ability to induce IFN-γ production as well as two flavonoid glycosides (compounds 8 and 9), moreover, the new compound 1 also possessed the inducing IFN-γ production ability based on the cytokine assay. In all, the result of phytochemical investigation further reveals the chemical composition of R. crenulata and the biological evaluations of these compounds can provide insights into the various bioactivities of the chemical constituents of R. crenulata. As to the immunomodulatory effect of R. crenulata, polysaccharides of R. crenulata will be further investigated by our group to reveal the chemical basis of this activity.