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Article

Highly Oxygenated Triterpenoids and Diterpenoids from Fructus Rubi (Rubus chingii Hu) and Their NF-kappa B Inhibitory Effects

by
Jiang Wan
1,†,
Xiao-Juan Wang
1,†,
Nan Guo
1,
Xi-Ying Wu
1,
Juan Xiong
1,
Yi Zang
2,
Chun-Xiao Jiang
3,
Bing Han
1,*,
Jia Li
2,* and
Jin-Feng Hu
1,3,*
1
Minhang Hospital & School of Pharmacy, Fudan University, Shanghai 201199, China
2
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China
3
Institute of Natural Medicine and Health Products, School of Advance Study, Zhejiang Provincial Key Laboratory of Plant Ecology and Conservation, Taizhou University, Taizhou 318000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2021, 26(7), 1911; https://doi.org/10.3390/molecules26071911
Submission received: 21 February 2021 / Revised: 18 March 2021 / Accepted: 24 March 2021 / Published: 29 March 2021
(This article belongs to the Section Natural Products Chemistry)
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Abstract

:
During a phytochemical investigation of the unripe fruits of Rubus chingii Hu (i.e., Fructus Rubi, a traditional Chinese medicine named “Fu-Pen-Zi”), a number of highly oxygenated terpenoids were isolated and characterized. These included nine ursane-type (1, 2, and 4–10), five oleanane-type (3, 11–14), and six cucurbitane-type (15–20) triterpenoids, together with five ent-kaurane-type diterpenoids (21–25). Among them, (4R,5R,8R,9R,10R,14S,17S,18S,19R,20R)-2,19α,23-trihydroxy-3-oxo-urs-1,12-dien-28-oic acid (rubusacid A, 1), (2R*,4S*,5R*,8R*,9R*,10R*,14S*,17S*, 18S*,19R*,20R*)-2α,19α,24-trihydroxy-3-oxo-urs-12-en-28-oic acid (rubusacid B, 2), (5R,8R,9R,10R, 14S,17R,18S,19S)-2,19α-dihydroxy-olean-1,12-dien-28-oic acid (rubusacid C, 3), and (3S,5S,8S,9R, 10S,13R,16R)-3α,16α,17-trihydroxy-ent-kaur-2-one (rubusone, 21) were previously undescribed. Their chemical structures and absolute configurations were elucidated on the basis of spectroscopic data and electronic circular dichroism (ECD) analyses. Compounds 1 and 3 are rare naturally occurring pentacyclic triterpenoids featuring a special α,β-unsaturated keto-enol (diosphenol) unit in ring A. Cucurbitacin B (15), cucurbitacin D (16), and 3α,16α,20(R),25-tetrahydroxy-cucurbita-5,23- dien-2,11,22-trione (17) were found to have remarkable inhibitory effects against NF-κB, with IC50 values of 0.08, 0.61, and 1.60 μM, respectively.

Graphical Abstract

1. Introduction

The Rubus genus (family Rosaceae) is large and diverse (with about 700 species distributed worldwide), and Flora of China lists 139 species as endemic to China [1]. They are usually deciduous or semi-evergreen perennial herbs or shrubs, which are often spiny, with a characteristic fruit formed as a head of one-seeded drupelets. The unripe fruits of Rubus chingii Hu (Fructus Rubi, referred to as “Fu-Pen-Zi” in Chinese, bog-bun-ja in Korean, and gosho-ichigo in Japanese) have been widely used as a herb tonic for the treatment of various diseases, mainly associated with kidney deficiency, in East Asian countries [2,3]. As a top-grade traditional Chinese medicine, Fructus Rubi was recorded in one of the earliest collections in the Pharmacopeia of the People’s Republic of China [4]. The phytochemistry and pharmacology of R. chingii have recently been well-documented by two review articles [2,5], and both triterpenoids and diterpenoids are encountered in the fruits. Modern pharmacological studies have revealed that the chemical components from Fructus Rubi exhibit a broad spectrum of bioactivities, such as being anti-aging [6], anti-cancer [7], anti-oxidant [7,8], and anti-diabetic [9]. With the aim of obtaining more structurally interesting and bioactive naturally occurring triterpenoids and diterpenoids [10,11,12], a phytochemical investigation of a commercially available sample of Fructus Rubi was carried out, which resulted in the isolation of three new highly oxygenated pentacyclic triterpenoids (1–3) and one new ent-kaurane-type diterpenoid (21), along with 21 related known terpenoid compounds (4–20 and 22–25) (Figure 1). Herein, the isolation, structural elucidation, and NF-κB inhibitory activities of these compounds are reported.

2. Results and Discussion

A 70% ethanol extract of the unripe fruits of R. chingii (28.0 kg) was suspended in H2O and then partitioned successively with petroleum ether, EtOAc, and n-BuOH. The entire EtOAc-soluble fraction was repeatedly subjected to column chromatography (CC) over silica gel, MCI gel, Sephadex LH-20, and semi-preparative HPLC to afford 20 triterpenoids (1–20) and 5 diterpenoids (21–25) (Figure 1). By comparing the observed and reported physicochemical properties and spectroscopic data, the previously known ones were identified as fupenzic acid (4) [13], 2α,3α,19α-trihydroxy-urs-12-en-28-oic acid (5) [14], 2α,3α,23-trihydroxy-urs-12-en-28-oic acid (6) [15], 2α,19α-dihydroxy-3-oxo-urs-12-en-28-oic acid (7) [16], 2α,3β,19α,24-tetrahydroxyurs-12-en-28-oic acid (8) [17], 1β,3β,19α-trihydroxy-2-oxo-urs-12-en-28-oic acid (9) [18], 3β,19α-dihydroxy-2-oxo-urs-12-en-28-oic acid (10) [19], 2α,3α,19α,23-tetrahydroxy-olean-12-en-28-oic acid (11) [20], 2α,3α,23-trihydroxy-urs-12-en-28-oic acid (12) [21], arjunic acid (13) [22], 2α,3β,19α,24-tetrahydroxyolean-12-en-28-oic acid (14) [23], cucurbitacin B (15) [24], cucurbitacin D (16) [25], 3α,16α,20(R),25-tetrahydroxy-cucurbita-5,23-dien-2,11,22-trione (17) [26], 2,16α,20(R),25-tetrahydroxy-cucurbita-1,5,23-trien-3,11,22-trione (18) [27], 25-acetoxy-2α,16α,20(R)-trihydroxy-cucurbita-5,23-dien-3,11,22-trione (19) [28], 25-acetoxy-3β,16α,20(R)-trihydroxy-cucurbita-5,23-dien-2,11,22-trione (20) [26], 3β,16α,17-trihydroxy-ent-kaur-19-yl acetate (22) [29], 16α,17-dihydroxy-ent-kaur-3-one (23) [30], 3β,16α,17-trihydroxy-ent-kaurane (24) [31], and 16α,17,19-trihydroxy-ent-kaur-3-one (25) [32], respectively.
Rubusacid A (1) was obtained as a white powder. Its molecular formula was established as C30H44O6 from its HRESIMS (m/z 499.3067 [M − H]−, calcd. for C30H43O6, 499.3065) and 13C NMR data (Table 1). The IR spectrum exhibited characteristic absorptions for a cyclic enone (1703, 1644 cm−1) group, which was supported by its UV absorption band at 265 nm [33,34]. In the upfield region of the 1H NMR spectrum of 1, resonances of five tertiary methyl groups at δH 0.90 (3H, s, Me-26), 1.23 (3H, s, Me-29), 1.29 (3H, s, Me-24), 1.30 (3H, s, Me-25), and 1.36 (3H, s, Me-27), and one secondary methyl group at δH 0.95 (3H, d, J = 7.1 Hz, Me-30) were observed (Table 2). In addition, signals resonating at δH 3.69 and 3.71 (ABq, each 1H, d, J = 12.0 Hz, H2-23) for a hydroxymethylene group, and two olefinic protons at δH 5.37 (1H, dd, J = 3.7, 3.5 Hz, H-12) and 6.29 (1H, s, H-1) were also readily distinguished. A total of 30 carbon signals, including a ketone carbonyl at δC 200.5 (C-3), a carboxyl carbon at δC 182.2 (C-28), four olefinic carbons at δC 145.8 (C-2), 140.4 (C-13), 129.5 (C-1), and 129.0 (C-12), and two oxygenated carbons at δC 73.5 (C-19) and 65.9 (C-23), were displayed in its 13C NMR spectrum. The aforementioned NMR data of 1 highly resembled those of fupenzic acid (4) [13], a co-occurring ursane-type triterpenoid with an α,β-unsaturated keto-enol moiety (i.e., diosphenol chromophore [13,35,36]) in ring A. The only difference between them was that the Me-23 in 4 was hydroxylated in 1, which was confirmed by the H3-24/C-23 and H2-23/C-3 correlations (Figure 2) in its HMBC NMR spectrum. The relative configuration of 1 was determined by ROESY data analysis (Figure 3). The ROE correlation of H2-23 with H-5, along with the absence between H2-23 and H3-25, confirmed the α-orientation of the 23-CH2OH group. Moreover, the ROE correlations of H-18 with H-12/H-20/H3-29, and of H3-29 with H-12/H-20 demonstrated that H-18, H-20, and H3-29 were all β-oriented, thus requiring OH-19 in the α-orientation. The electronic circular dichroism (ECD) spectrum of 1 exhibited Cotton effects (CEs) at 217 (Δε −16.3), 274 (Δε +13.9), and 330 (Δε −3.0) nm (Figure S6, Supporting Information), corresponding to the n→π* and π→π* transitions of the unsaturated cyclohexanone chromophore. The ECD data of 1 were similar to those of (+)-cyrillin A [34] and sapiumic acid F [37], two pentacyclic triterpenoids possessing a common enone group in ring A, which then allowed the assignment of a 10R configuration in 1. Consequently, the structure of 1 was characterized as (4R,5R,8R,9R,10R,14S,17S,18S,19R,20R)-2,19α,23-trihydroxy-3-oxo- urs-1,12-dien-28-oic acid.
The HRESIMS data permitted the molecular formula of rubusacid B (2) to be assigned as C30H46O6 (m/z 525.3184 [M + Na]+, calcd. for C30H46O6Na, 525.3187), which was consistent with its 13C NMR data (Table 1). The close similarity of the 1H (Table 2) and 13C NMR spectroscopic data of 2 with those of 1, indicated that 2 is also an ursane-type triterpenoid. The difference of two mass units between 1 and 2, and the absence of the enol group (δH 6.29, s (H-1); δC 129.5 (C-1), 145.8 (C-2)) in ring A when compared with those of 1, suggested that the Δ1 double bond in 1 was hydrogenated in 2. This was confirmed by the HMBC correlation from H3-25 to C-1 (δC 51.2) (Figure 2). The large coupling constant of J1α,2 = 12.7 Hz suggested the axial position for H-2. Moreover, the correlations of H-2/H3-25, H-2/H2-24, and H2-24/H3-25 in the ROESY NMR experiment (Figure 3) confirmed the β-orientation for both H-2 and the CH2OH group at C-4. In addition, the α-orientation of OH-19 was also determined by ROESY NMR experiment, in a similar way to 1 (Figure 3). Accordingly, the structure of 2 was deduced as (2R*,4S*,5R*,8R*,9R*,10R*,14S*,17S*,18S*,19R*,20R*)-2α,19α,24-trihydroxy-3-oxo-urs-12-en-28-oic acid.
Rubusacid C (3) had a molecular formula of C30H44O5, as determined by HRESIMS ([M + H]+ ion at m/z 485.3254, calcd for C30H45O5, 485.3262) and 13C NMR data (Table 1). Like 1, the IR (1695, 1634 cm−1) and UV (266 nm) absorption bands of 3 also indicated a cyclic enonic group. The 1H (Table 2) and 13C NMR data of 3 showed similarities to those of 1, with the obvious differences being the presence of a pair of germinal methyl groups at C-20 (δH 0.97, s; 0.94, s) and an oxygenated methine at C-19 (δH 3.26, d, J = 3.9 Hz; δC 82.4) in 3, rather than the secondary methyl group and tertiary methyl group in 1. This implied that 3 is an oleanane-type triterpenoid, which agreed with the co-occurring terpenoid compounds 11–14 [20,21,22,23]. This was further supported by HMBC correlations (Figure 2) from H3-29 and H3-30 to C-19, C-20, and C-21, and from H-18 to C-19. In addition, the OH-23 in 1 was absent in 3, which was corroborated by the HMBC cross-peaks from H3-24 to C-23. The relative configuration of 3 was determined by analyzing the coupling constants and ROESY data. The small J value (3.9 Hz) between H-18 and H-19 was indicative of the equatorial orientation of H-19. Moreover, the ROE correlations from H-19 to H-12, H-18, and H3-29 confirmed that OH-19 was α-oriented (Figure 3). The chirality of C-10 was assigned to be R, as evidenced from a positive CE at 274 nm and negative CEs at 215 and 333 nm in its ECD spectrum, which were comparable with (+)-cyrillins A and relevant analogues [34,37]. Therefore, the structure of 3 was defined as (5R,8R,9R,10R,14S,17R,18S,19S)-2,19α-dihydroxy-olean-1,12-dien-28-oic acid.
Rubusone (21) was obtained as a white, amorphous powder. It had a molecular formula of C20H32O4 according to HRESIMS data analysis ([M + H]+ m/z 337.2389, calcd for C20H33O4, 337.2373) and the 13C NMR data (Table 1). The IR absorption bands at 3379 and 1701 cm−1 suggested the presence of hydroxyl and carbonyl groups, respectively. Inspection of the 1H NMR spectroscopic data (in C6D6, Table 3) indicated the presence of three tertiary methyl groups (δH 0.67 (3H, s, Me-19), 0.69 (3H, s, Me-20), and 1.10 (3H, s, Me-18)), an oxymethine resonance (δH 3.62 (1H, d, J = 3.8 Hz, H-3)), and a hydroxymethylene group at δH 3.37 and 3.44 (ABq, each 1H, d, J = 10.3 Hz, H2-17). A total of 20 carbon signals, including one ketone carbonyl at δC 210.2 (C-2) and three oxygenated at δC 82.8 (C-3), 81.2 (C-16), and 66.3 (C-17), were displayed in its 13C NMR spectrum. The aforementioned NMR data were similar to those of the co-occurring compounds 22–25 [29,30,31,32], suggesting 4 is an ent-kaurane derivative. Detailed comparisons suggested that the 1D NMR data were closely related to those of 3α,16β,17-trihydroxy-ent-kaurane-2-one, which was previously isolated from Homalanthus acuminatus [38]. The only noticeable difference was that the chemical shifts of CH2OH-17 (δH: 3.37 and 3.44, δC: 66.3) in 21 were significantly distinguished when compared with 3α,16β,17-trihydroxy-ent-kaurane-2-one (δH: 3.09 and 3.12; δC: 69.8), indicating that these two compounds are epimeric at C-16. The α-orientation of OH-16, as with 22–25 [29,30,31,32], was confirmed by comparing the chemical shifts of C-16 and C-17 with reported data [31,39]. The magnitude of JH-5/H-6α (11.6 Hz) indicated that H-5 was in axial position, and the ROE correlations of H-5 with H-3/H-9, and of H-3 with H3-18 revealed their cofacial relationship (arbitrarily assigned as β-oriented). In turn, the ROE correlation of H3-19 and H3-20 was indicative of their α-orientation. Additionally, the ECD spectrum of 21 showed a negative CE at 280 nm arising from the n→π* transition of the C-2 carbonyl group, indicative of an S configuration for C-3 by using the octant rule [40,41]. Thus, the structure of 21 was elucidated as (3S,5S,8S,9R,10S,13R,16R)-3α,16α,17-trihydroxy-ent-kaur-2-one.
Nuclear factor-κB (NF-κB) is an important transcription factor controlling different biological processes, such as immune differentiation and activation [42]. It has been regarded as a potential target for the regulation of dysfunction of immunity and inflammation. Recently, a few triterpenoids and diterpenoids from Stewartia sinensis [11] and Pseudotsuga sinensis [12] were found to have significant NF-κB inhibitory effects. In the present study, all the isolated terpenoids (1–25) were evaluated for their NF-κB inhibitory activities. Among them, only three cucurbitane-type compounds, 15–17, showed potent inhibitory effects, with IC50 values of 0.08, 0.61, and 1.60 μM (Table 4), respectively. The rest of the isolates were inactive (inhibition ration < 50% at 20 μM). Bortezomib (PS-341) was used as the positive control (IC50: 0.44 μM) [43]. Interestingly, some ent-labdane-type diterpenoid glycosides from Fructus Rubi have been reported to suppress the NF-κB signaling pathway [44], but the ent-kaurane-type diterpenoids isolated herein were inactive against NF-κB.
In addition, the 70% ethanol extract of Fructus Rubi combined with fluconazole (FLC) has been previously found to have an anti-fungal activity against twenty-two FLC-resistant Candida albicans strains [45]. Hence, all the isolated compounds were subjected to the same bioassay, but none of them were active. They were also evaluated for their anti-fungal effects against the C. albicans SC5314 sensitive strain, and only compound 12 showed a moderate inhibition (MIC80: 32 μg/mL), whereas the other twenty-four isolates were inactive (MIC80 > 64 μg/ mL). Fluconazole (MIC80: 0.125 μg/mL) was used as the positive control [46].

3. Materials and Methods

3.1. General Experimental Procedures and Agents

Optical rotations were obtained with a Rudolf Autopol IV at 21 °C. UV and IR spectra were recorded on a Hitachi U-2900E UV spectrophotometer (Hitachi High-Techologies Corporation, Tokyo, Japan) and a Thermo Scientific Nicolet Is5 FT-IR spectrometer (Thermo Fisher Scientific, San Jose, CA, USA), respectively. ECD spectra were collected on a JASCO-810 spectropolarimeter (Jasco Analytical Instruments, Easton, PA, USA). ESIMS and HRESIMS were acquired on an Agilent 1100 LC/MSD mass spectrometer (Agilent, Santa Clara, CA, USA) and an AB Sciex Triple TOF 5600 spectrometer (AB Sciex Pte. Ltd, Singapore), respectively. 1D and 2D NMR spectra were recorded on Bruker Avance III 400 or 600 MHz spectrometers (Bruker, Fallanden, Switzerland), using the residual solvent signals as the internal standard. All chemical shifts were expressed in ppm. Semi-preparative HPLC was performed on a Waters e2695 system coupled with a 2998 photodiode array (PDA) detector and a 2424 evaporative light-scattering detector (ELSD) (Waters, Milford, MA, USA). A Cosmosil C18 column (5 μM, 10 × 250 mm; flow rate: 3.0 mL/min) and an X-bridge C18 column (5 μM, 10 × 250 mm; flow rate: 3.0 mL/min) were utilized. Thin-layer chromatography (TLC) was performed on pre-coated plates (GF254, 0.25 mm, Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, China). TLC spots were visualized under UV light (254 or 365 nm) and by spraying with 5% H2SO4/vanillin, followed by heating to 120 °C. Candida albicans strains (the resistant strain 901, and the sensitive strain 5314) were provided by Dr William A. Fonzi from the Department of Microbiology and Immunology, Georgetown University, Washington DC, USA. RPMI-1640 medium was purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Fluconazole (FLC) was purchased from Pfizer-Roerig Pharmaceuticals (New York, NY, USA).

3.2. Plant Material

The unripe fruits of R. chingii were purchased from Shanghai Chinese Traditional Medicine Drinking Tablet Co., Ltd., Shanghai, China. They were taxonomically identified by one of the authors (Mr. B. Han). A voucher specimen (No. 20190319) was deposited at the herbarium of the School of Pharmacy at Fudan University.

3.3. Extraction and Isolation

The air-dried and powdered unripe fruits (28.0 kg) were extracted with 70% ethanol (5 × 20 L, each time for 24 h) at room temperature. After filtration, the solvent was removed under vacuum to give a dark green residue (5.7 kg, semi-dry), which was suspended in H2O (6 L) and then extracted successively with petroleum ether (PE, 3 × 6 L), EtOAc (3 × 6 L), and n-BuOH (3 × 6 L). The EtOAc-soluble extract (325.3 g) was subjected to a silica gel column with a stepwise gradient-elution of PE-EtOAc (30:1 → 20:1 → 10:1 → 5:1 → 1:1 → 1:5 → neat EtOAc), to afford nine fractions (Fr.1–9), according to TLC analysis. Fr.3 (10.4 g) was chromatographed over a silica gel column (CH2Cl2-MeOH, 10:1 → 5:1 → 1:1) to give six subfractions, Fr.3A−F. Compound 4 (3.0 mg) was obtained from Fr.3C (104 mg) by Sephadex LH-20 (MeOH), followed by HPLC purification (MeOH-H2O, 70:30, tR = 17.5 min). Fr.4 (6.4 g) was fractionated on an MCI column with a step gradient elution of MeOH-H2O (50:50 → 70:30 → 85:15 → 100:0), and six fractions (Fr.4A−F) were collected. Separation of Fr.4E (700 mg) over Sephadex LH-20 (MeOH) and semi-preparative HPLC (MeOH-H2O, 88:12) afforded compound 7 (28 mg, tR = 15.7 min). Fr.5 (23.0 g) was chromatographed over a silica gel column (CH2Cl2-MeOH, 20:1 → 10:1 → 5:1 → 1:1) to give eight fractions, Fr.5A–H. Compounds 1 (1.0 mg, tR = 12.9 min) and 3 (1.0 mg, tR = 18.5 min) were obtained from Fr.5A (1.2 g) by Sephadex LH-20 (MeOH), followed by semi-preparative HPLC (MeOH-H2O, 70:30). Purification of Fr.5B (400 mg) by semi-preparative HPLC (MeCN-H2O, 45:55) yielded compounds 16 (0.8 mg, tR = 18.7 min) and 17 (2.5 mg, tR = 17.3 min). Fr.5C (600 mg) was further purified by Sephadex LH-20 (MeOH) to give subfractions Fr.5C-1–4. Compounds 18 (1.5 mg, tR = 16.0 min) and 19 (2.0 mg, tR = 17.4 min) were isolated from Fr.5C-1 (130 mg) by semi-preparative MeCN-H2O, 43:57. By employing the same HPLC (MeOH-H2O, 67:33) system, Compound 15 (6.0 mg, tR = 14.2 min) was purified from Fr.5C-2 (264 mg), whereas compound 20 (2.2 mg, tR = 23.5 min) was obtained from Fr.5C-3 (80 mg). Fr.5D (1.2 g) was fractionated by Sephadex LH-20 (MeOH) followed by semi-preparative HPLC (MeCN-H2O, 50:50) to furnish compounds 2 (2.0 mg, tR = 16.5 min), 8 (5.0 mg, tR = 12.3 min), and 14 (6.0 mg, tR = 11.2 min). Fr.6 (6.7 g) was fractionated on an MCI column with a step gradient elution of MeOH-H2O (30:70 → 50:50 → 70:30 → 85:15 → 100:0), and eight fractions (Fr.6A–H) were collected. Fr.6B (840 mg) was further fractionated by Sephadex LH-20 (MeOH) to give the subfractions Fr.6B-1–3. Fr.6B-2 (260 mg) was purified by semi-preparative HPLC (MeOH-H2O, 80:20) to afford compounds 9 (2.0 mg, tR = 14.5 min) and 10 (3.3 mg, tR = 16.7 min). Fr.6F (0.9 g) was separated over silica gel (CH2Cl2-MeOH, 20:1 → 10:1 → 5:1 → 1:1) to give four subfractions (Fr.6F-1–4). Compound 5 (25.1 mg, tR = 11.3 min) was purified from Fr.6F-1 by Sephadex LH-20 (MeOH), followed by semi-preparative HPLC purification (MeOH-H2O, 77:23). Fr.6F-3 was purified by semi-preparative HPLC (MeOH-H2O, 74:26) to afford compounds 6 (2.2 mg, tR = 21.7 min) and 12 (44.3 mg, tR = 20.6 min). Fr.6E (0.9 g) was rechromatographed by silica gel with CH2Cl2-MeOH (9:1), and six fractions (Fr.6E-1–6) were obtained. Compound 13 (7.0 mg, tR = 16.3 min) was isolated from Fr.6E-4 (170 mg) by semi-preparative HPLC (MeOH-H2O, 71:29). Fr.7 (4.9 g) was fractionated on an MCI column with a step gradient elution of MeOH-H2O (30:70 → 50:50 → 70:30 → 85:15→ 100:0) and seven fractions (Fr.7A−G) were obtained. Separation of Fr.7C (200 mg) over Sephadex LH-20 (MeOH) and semi-preparative HPLC (MeCN-H2O, 30:70) afforded compounds 21 (0.7 mg, tR = 13.7 min) and 22 (49.1 mg, tR = 17.1 min). Fr.7D (550 mg) was purified by semi-preparative HPLC (MeOH-H2O, 73:27) to furnish compounds 23 (18.5 mg, tR = 9.2 min) and 24 (18.7 mg, tR = 11.7 min). Fr.8 (16 g) was fractionated on an MCI column with a step gradient elution of MeOH-H2O (30:70 → 50:50 → 70:30 → 85:15 → 100:0) and six fractions (Fr.8A−F) were obtained. Fr.8B (1.09 g) was further separated on a Sephadex LH-20 (MeOH) to give subfractions Fr.8B-1−6. Fr.8B-5 (0.7 g) was purified by semi-preparative HPLC (MeOH-H2O, 55:45) to afford compound 25 (5.0 mg, tR = 11.2 min). Purification of subfraction Fr.8B-6 (0.8 g) by semi-preparative HPLC (MeCN-H2O, 35:65) yielded compound 11 (3.0 mg, tR = 22.4 min).
(4R,5R,8R,9R,10R,14S,17S,18S,19R,20R)-2,19α,23-Trihydroxy-3-oxo-urs-1,12-dien-28-oic acid (rubusacid A, 1). White powder; [α]D21 14.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 265 (2.31) nm; ECD (c 2.67 × 10−3 M, MeOH) λmax (Δε): 217 (−16.3), 274 (+13.9), 330 (−3.0) nm; IR (KBr) vmax 3576, 3446, 2970, 2937, 2870, 1721, 1703, 1690, 1644, 1464, 1402, 1379, 1270, 1242, 1157, 1058, 931, 863, 754 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; ESIMS m/z 499 [M − H]−; HRESIMS m/z 499.3067 [M − H]− (calcd for C30H43O6, 499.3065, Δ = 0.3 ppm).
(2R*,4S*,5R*,8R*,9R*,10R*,14S*,17S*,18S*,19R*,20R*)-2α,19α,24-Trihydroxy-3-oxo-urs-12-en-28-oic acid (rubusacid B, 2). White powder; [α]D21 11.7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (2.14) nm; ECD (c 1.33 × 10−3 M, MeOH) λmax (Δε): 217 (−5.3) nm; IR (KBr) vmax 3446, 2975, 2927, 2848, 1703, 1691, 1459, 1387, 1210, 1185, 1140, 1053, 1028, 975, 868 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; ESIMS m/z 501 [M − H]−, 525 [M + Na]+; HRESIMS m/z 525.3184 [M + Na]+ (calcd for C30H46O6Na, 525.3187, Δ = −0.4 ppm).
(5R,8R,9R,10R,14S,17R,18S,19S)-2,19α-Dihydroxy-olean-1,12-dien-28-oic acid (rubusacid C, 3). White powder; [α]D21 16.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 266 (1.53) nm; ECD (c 2.75 × 10−3 M, MeOH) λmax (Δε): 215 (−20.5), 274 (+12.8), 333 (−4.0) nm; IR (KBr) vmax 3571, 3446, 2972, 2935, 2868, 1706, 1695, 1634, 1462, 1407, 1384, 1237, 1212, 1157, 1050, 936, 756, 649 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; ESIMS m/z 485 [M + H]+ and 507 [M + Na]+; HRESIMS m/z 485.3254 [M + H]+ (calcd for C30H45O5, 485.3262, Δ = −1.6 ppm).
(3S,5S,8S,9R,10S,13R,16R)-3α,16α,17-Trihydroxy-ent-kaur-2-one (rubusone, 21). White powder; [α]D21 −30.2 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 202 (1.53) nm; ECD (c 1.18 × 10−3 M, MeOH) λmax (Δε): 285 (−8.7) nm; IR (KBr) vmax 3379, 2935, 2865, 1701, 1619, 1514, 1449, 1379, 1317, 1290, 1207, 1182, 1142, 1043, 916, 871 cm−1; 1H and 13C NMR data, see Table 1 and Table 3; ESIMS m/z 337 [M + H]+, 359 [M + Na]+; HRESIMS m/z 337.2389 [M + H]+ (calcd for C20H33O4, 337.2373, Δ = 4.6 ppm).

3.4. NF-κB Inhibitory Assay

HEK293 with a stable NF-κB expression cell line was used for the luciferase assay [11,12,47]. Cells were maintained at 37 °C and 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium with 100 U/mL benzylpenicillin 100 μg/mL streptomycin, 2 mM glutamine, and 10% fetal bovine serum. Before seeding in 96-well plates, the cells were stained for 1 h in serum-free medium supplemented with 2 μ cell Tracker Green CMFDA. Cells were seeded into 96-well plates and incubated for 24 h, and then treated with different concentrations of the tested compounds followed by stimulation with 20 ng/mL TNF-α. The luciferase substrate was added to each well after incubation for 6 h, and then the released luciferin signal was detected using an EnVision microplate reader. The IC50 value was derived from a nonlinear regression model (curve-fit), based on a sigmoidal dose response curve (variable slope) and computed using Graphpad Prism 5 (Graphpad Software). Bortezomib (PS-341, CAS No. 179324-69-7) was used as the positive control [11,12,43].

3.5. Anti-Fungal Susceptibility Assay

An anti-fungal assay was carried out on the basis of the Clinical and Laboratory Standards Institute (CLSI) method M27-A3 [45,48]. A single colony of Candida albicans (the resistant strain 901 or the sensitive strain 5314) was chosen from Sabouraud’s agar and then inoculated in yeast extract peptone dextrose medium (YEPD) for activation by shake bottled cultivation (200 rpm, 37 °C). After 16 h, fungi in the late-exponential growth phase were obtained, followed by being adjusted to 1 × 103–5 × 103 CFU/mL with RPMI 1640 medium. The density of the fungi in 96-well plates was 103 CFU/mL, while the final concentrations of the test compounds ranged from 64 to 0.125 μg/mL in triplicate. The assay volume in each well was 100 μL with MIC80 determined following 48 h incubation at 37 °C. Optical density was measured with a microplate reader (Multiskan MK3; Labsystems, Nantaa, Finland) at 630 nm. MIC80 was determined as the lowest concentration of the drugs that inhibited growth by 80% compared with the positive control. Fluconazole was used as the positive control [45,46].

4. Conclusions

Previous phytochemical studies on the fruits and leaves of R. chingii were extensively reviewed [2,5]. Around 15 triterpenoids and 17 diterpenoids have so far been obtained from R. chingii [2,5,49]. In the present work, we focused on triterpenoids and diterpenoids from the unripe fruits of R. chingii. Three highly oxygenated triterpenoids (rubusacids A-C, 1–3) and one ent-kaurane-type diterpenoid (rubusone, 21) were reported that were hitherto unknown. Compounds 1 and 3 possess a special diosphenol unit in ring A. Diosphenols are α-diketones, in which one of the carbonyls is enolized; they are part of the structure of several products of natural and synthetic origin, but are quite rare in naturally occurring ursane- and oleanane-type pentacyclic triterpenoids. To our knowledge, only a few plant-originated oleanane-/ursane-type [13,50,51,52,53] and one biotransformed ursane-type [54] triterpenoids have such a moiety in ring A. These cucurbitane-type tetracyclic triterpenoids (15–20) were obtained from the unripe fruits of R. chingii for the first time. Regarding the bioactivity evaluations, three isolates (15–17) showed potent inhibitory effects against NF-κB. It is worth noting that some cucurbitane-type tetracyclic triterpenoid glycosides from Momordica charantia [55] have also been found to have this kind of effect. The major component, 2α,3α,23-trihydroxy-urs-12-en-28-oic acid (12), might be the principle in the anti-fungal activity of 70% ethanol Fructus Rubi [45]. In general, the above findings expanded the terpenoic structure diversity of R. chingii and could provide useful clues for the discovery and development of new therapeutic or preventive agents for the treatment of NF-κB related diseases.

Supplementary Materials

The following are available online, 1H NMR, 13C NMR, 1H-1H COSY, HMBC, ROESY, and HRESIMS spectra of compounds 1–3 and 21 are available as supporting information.

Author Contributions

J.W., J.X. and C.-X.J. performed the isolation, purification, and structure determination. X.-J.W., N.G., X.-Y.W., Y.Z. and B.H. worked in bioassays and biological data analyses. J.L. and J.-F.H. designed the whole experiments and revised the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by NSFC grants (Nos. 21937002, 81773599, 21772025), the National Mega-Project for Innovative Drugs from MOST (No. 2019ZX09735-002), the Youth Project Fund of Minhang Hospital Affiliated to Fudan University (2018MHJC07), Shanghai Natural Science Fund (17ZR1425100), and a special fund for the talent development in Minhang District.

Data Availability Statement

All data and figures generated or used during the study appear in the submitted article.

Acknowledgments

All authors are grateful to William A. Fonzi (the Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA) for providing the Candida albicans strains.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds 1–25 are available from the authors.

References

  1. Lu, L.-D.; Boufford, D.E. Flora of China; Wu, Z.-Y., Raven, P.H., Eds.; Science Press & Missouri Botanical Garden Press: Beijing, China; St. Louis, MO, USA, 2003; Volume 9, pp. 195–285. [Google Scholar]
  2. Sheng, J.-Y.; Wang, S.-Q.; Liu, K.-H.; Zhu, B.; Zhang, Q.-Y.; Qin, L.-P.; Wu, J.-J. Rubus chingii Hu: An overview of botany, traditional uses, phytochemistry, and pharmacology. Chin. J. Nat. Med. 2020, 18, 401–416. [Google Scholar] [CrossRef]
  3. Ohtani, K.; Yang, C.-R.; Miyajima, C.; Zhou, J.; Tanaka, O. Labdane-type diterpene glycosides from fruits of Rubus foliolosus. Chem. Pharm. Bull. 1991, 39, 2443–2445. [Google Scholar] [CrossRef] [Green Version]
  4. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China part I; Chinese Medical Science and Technology Press: Beijing, China, 2020; pp. 399–400. [Google Scholar]
  5. Yu, G.-H.; Luo, Z.-Q.; Wang, W.-B.; Li, Y.-H.; Zhou, Y.-T.; Shi, Y.-Y. Rubus chingii Hu: A review of the phytochemistry and pharmacology. Front. Pharmacol. 2019, 10, 799. [Google Scholar] [CrossRef]
  6. Zeng, H.-J.; Liu, Z.; Wang, Y.-P.; Yang, D.; Yang, R.; Qu, L.-B. Studies on the anti-aging activity of a glycoprotein isolated from Fupenzi (Rubus chingii Hu.) and its regulation on klotho gene expression in mice kidney. Int. J. Biol. Macromol. 2018, 119, 470–476. [Google Scholar] [CrossRef] [PubMed]
  7. Zhang, T.-T.; Yang, L.; Jiang, J.-G. Bioactive comparison of main components from unripe fruits of Rubus chingii Hu and identification of the effective component. Food Funct. 2015, 6, 2205–2214. [Google Scholar] [CrossRef]
  8. Chen, Y.; Wang, Y.-J.; Xu, L.-L.; Jia, Y.-N.; Xue, Z.-H.; Zhang, M.; Phisalaphong, M.; Chen, H.-X. Ultrasound-assisted modified pectin from unripe fruit pomace of raspberry (Rubus chingii Hu): Structural characterization and anti-oxidant activities. LWT-Food Sci. Technol. 2020, 134, 110007. [Google Scholar] [CrossRef]
  9. Chen, Y.; Chen, Z.-Q.; Guo, Q.-W.; Gao, X.-D.; Ma, Q.-Q.; Xue, Z.-H.; Ferri, N.; Zhang, M.; Chen, H.-X. Identification of ellagitannins in the unripe fruit of Rubus chingii Hu and evaluation of its potential anti-diabetic activity. J. Agric. Food Chem. 2019, 67, 7025–7039. [Google Scholar] [CrossRef]
  10. Li, H.; Liang, Y.-R.; Chen, S.-X.; Wang, W.-X.; Zou, Y.-K.; Nuryyeva, S.; Houk, K.N.; Xiong, J.; Hu, J.-F. Amentotaxins C-V, structurally diverse diterpenoids from the leaves and twigs of the vulnerable conifer Amentotaxus argotaenia and their cytotoxic effects. J. Nat. Prod. 2020, 83, 2129–2144. [Google Scholar] [CrossRef] [PubMed]
  11. Wan, J.; Zang, Y.; Xiao, D.-A.; Li, N.; Li, J.-M.; Jin, Z.-X.; Chen, D.-L.; Xiong, J.; Li, J.; Hu, J.-F. Stewartiacids A-N, C-23 carboxylated triterpenoids from Chinese stewartia and their inhibitory effects against ATP-citrate lyase and NF-κB. RSC Adv. 2020, 10, 3343–3356. [Google Scholar] [CrossRef]
  12. Huang, T.; Ying, S.-H.; Li, J.-Y.; Chen, H.-W.; Zhang, Y.; Wang, W.-X.; Li, J.; Xiong, J.; Hu, J.-F. Phytochemical and biological studies on rare and endangered plants endemic to China. Part XV. Structurally diverse diterpenoids and sesquiterpenoids from the vulnerable conifer Pseudotsuga sinensis. Phytochemistry 2020, 169, 112184. [Google Scholar] [CrossRef]
  13. Hattori, M.; Kuo, K.-P.; Shu, Y.-Z.; Tezuka, Y.; Kikuchi, T.; Namba, T. A triterpene from the fruits of Rubus chingii. Phytochemistry 1988, 27, 3975–3976. [Google Scholar] [CrossRef]
  14. Wiemann, J.; Deckelmann, A.M.; Csuk, R. A remarkably simple and convergent partial synthesis of pomolic acid. Tetrahedron Lett. 2016, 57, 3952–3953. [Google Scholar] [CrossRef]
  15. Sashida, Y.; Ogawa, K.; Mori, N.; Yamanouchi, T. Triterpenoids from the fruit galls of Actinidia polygama. Phytochemistry 1992, 31, 2801–2804. [Google Scholar] [CrossRef]
  16. Xu, H.-X.; Zeng, F.-Q.; Wan, M.; Sim, K.-Y. Anti-HIV triterpene acids from Geum japonicum. J. Nat. Prod. 1996, 59, 643–645. [Google Scholar] [CrossRef]
  17. Zhou, X.-H.; Kasai, R.; Ohtani, K.; Tanaka, O.; Nie, R.-L.; Yang, C.-R.; Zhou, J.; Yamasaki, K. Oleanane and ursane glucosides from Rubus species. Phytochemistry 1992, 31, 3642–3644. [Google Scholar]
  18. Kashiwada, Y.; Wang, H.-K.; Nagao, T.; Kitanaka, S.; Yasuda, I.; Fujioka, T.; Yamagishi, T.; Cosentino, L.M.; Kozuka, M.; Okabe, H.; et al. Anti-AIDS agents. 30. Anti-HIV activity of oleanolic acid, pomolic acid, and structurally related triterpenoids. J. Nat. Prod. 1998, 61, 1090–1095. [Google Scholar] [CrossRef]
  19. D’Abrosca, B.; Fiorentino, A.; Monaco, P.; Pacifico, S. Radical-scavenging activities of new hydroxylated ursane triterpenes from cv. Annurca apples. Chem. Biodivers. 2005, 2, 953–958. [Google Scholar] [CrossRef] [PubMed]
  20. Zeng, N.; Shen, Y.; Li, L.-Z.; Jiao, W.-H.; Gao, P.-Y.; Song, S.-J.; Chen, W.-S.; Lin, H.-W. Anti-inflammatory triterpenes from the leaves of Rosa laevigata. J. Nat. Prod. 2011, 74, 732–738. [Google Scholar] [CrossRef]
  21. Ullah, F.; Hussain, H.; Hussain, J.; Bukhari, I.A.; Khan, M.T.H.; Choudhary, M.I.; Gilani, A.H.; Ahmad, V.U. Tyrosinase inhibitory pentacyclic triterpenes and analgesic and spasmolytic activities of methanol extracts of Rhododendron collettianum. Phytother. Res. 2007, 21, 1076–1081. [Google Scholar] [CrossRef]
  22. Joo, H.; Lee, H.-J.; Shin, E.-A.; Kim, H.; Seo, K.-H.; Baek, N.-I.; Kim, B.; Kim, S.-H. c-Jun N-terminal kinase-dependent endoplasmic reticulum stress pathway is critically involved in arjunic acid induced apoptosis in non-small cell lung cancer cells. Phytother. Res. 2016, 30, 596–603. [Google Scholar] [CrossRef]
  23. Yeo, H.; Park, S.-Y.; Kim, J. A-ring contracted triterpenoid from Rosa multiflora. Phytochemistry 1998, 48, 1399–1401. [Google Scholar] [CrossRef]
  24. Wu, P.-L.; Lin, F.-W.; Wu, T.-S.; Kuoh, C.-S.; Lee, K.-H.; Lee, S.-J. Cytotoxic and anti-HIV principles from the rhizomes of Begonia nantoensis. Chem. Pharm. Bull. 2004, 52, 345–349. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Sasamori, H.; Reddy, K.S.; Kirkup, M.P.; Shabanowitz, J.; Lynn, D.G.; Hecht, S.M.; Woode, K.A.; Bryan, R.F.; Campbell, J.; Lynn, W.S.; et al. New cytotoxic principles from Datisca glomerata. J. Chem. Soc. Perkin Trans. 1983, 7, 1333–1347. [Google Scholar] [CrossRef]
  26. Monte, F.J.Q.; Papa, S.M.A.; Kintzinger, J.P.; Braz-Filho, R. Total assignment of 1H and 13C NMR spectra of two isomeric cucurbitane triterpenoids. Magn. Reson. Chem. 2000, 38, 809–812. [Google Scholar] [CrossRef]
  27. Seger, C.; Sturm, S.; Mair, M.-E.; Ellmerer, E.P.; Stuppner, H. Spectral assignments and reference data. 1H and 13C NMR signal assignment of cucurbitacin derivatives from Citrullus colocynthis (L.) Schrader and Ecballium elaterium L. (Cucurbitaceae). Magn. Reson. Chem. 2005, 43, 489–491. [Google Scholar] [CrossRef]
  28. Lavie, D.; Shvo, Y.; Gottlieb, O.R.; Glotter, E. The constituents of Ecballium elaterium L. XVI. Stereochemical problems in the cucurbitacins. J. Org. Chem. 1963, 28, 1790–1795. [Google Scholar] [CrossRef]
  29. Zhang, M.; Ou, Y.-W.; Chen, X.-X.; Cao, Y.; Kuang, Y.; Gong, Z.-Q.; Peng, S.; Chen, Y.-J. Two ent-kaurane diterpenoids from Rubus corchorifolius L.f. Helv. Chim. Acta 2011, 94, 1820–1824. [Google Scholar] [CrossRef]
  30. Tinto, W.F.; Blyden, G.; Reynolds, W.F.; McLean, S. Diterpene and anthraquinone constituents of Glycydendron amazonicum. J. Nat. Prod. 1991, 54, 1127–1130. [Google Scholar] [CrossRef]
  31. Arciniegas, A.; Pérez-Castorena, A.-L.; Nieto-Camacho, A.; Villaseñor, J.L.; de Vivar, A.R. Terpenoids from Melampodium perfoliatum. J. Nat. Prod. 2016, 79, 2780–2787. [Google Scholar] [CrossRef]
  32. Ding, G.; Fei, J.-D.; Wang, J.; Xie, Y.; Li, R.-T.; Gong, N.-B.; Lv, Y.; Yu, C.-Y.; Zou, Z.-M. Fimbriatols A-J, highly oxidized ent-kaurane diterpenoids from traditional Chinese plant Flickingeria fimbriata (B1.) Hawkes. Sci. Rep. 2016, 6, 30560. [Google Scholar] [CrossRef] [Green Version]
  33. Dellagereca, M.; Fiorentino, A.; Monaco, P.; Previtera, L. Polyoxygenated oleanane triterpenes from Hydrocotyle ranunculoides. Phytochemistry 1994, 35, 201–204. [Google Scholar]
  34. Chacón-Morales, P.A.; Santiago-Dugarte, C.; Amaro-Luis, J.M. Unexpected reduction-allylic of hemisynthetic diosphenols from longipinene derivatives. Phytochem. Lett. 2020, 39, 105–110. [Google Scholar] [CrossRef]
  35. Munkombwe, N.M.; Maswabi, T.; Hughes, N.A. Diosphenols from Spirostachys africana. Phytochemistry 1997, 45, 1217–1220. [Google Scholar] [CrossRef]
  36. Ren, Y.-L.; VanSchoiack, A.; Chai, H.-B.; Goetz, M.; Kinghorn, A.D. Cytotoxic barrigenol-like triterpenoids from an extract of Cyrilla racemiflora housed in a repository. J. Nat. Prod. 2015, 78, 2440–2446. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Zhang, G.-J.; Pan, Q.-M.; Zhang, Y.-L.; Liao, H.-B.; Yang, Y.-Q.; Hou, Y.; Liang, D. Coumarinolignoids and taraxerane triterpenoids from Sapium discolor and their inhibitory potential on microglial nitric oxide production. J. Nat. Prod. 2018, 81, 2251–2258. [Google Scholar] [CrossRef]
  38. Gustafson, K.R.; Munro, M.H.G.; Blunt, J.W.; Cardellina II, J.H.; McMahon, J.B.; Gulakowski, R.J.; Cragg, G.M.; Cox, P.A.; Brinen, L.S.; Clardy, J.; et al. HIV inhibitory natural products. 3. diterpenes from Homalanthus acuminatus and Chrysobalanus icaco. Tetrahedron 1991, 47, 4547–4554. [Google Scholar] [CrossRef]
  39. Li, X.; Zhang, D.-Z.; Onad, M.; Konda, Y.; Iguchi, M.; Harigaya, Y. ent-Kauranoid diterpenes from Artemisia sacrorum. J. Nat. Prod. 1990, 53, 657–661. [Google Scholar] [CrossRef]
  40. Chen, Q.; Mao, Q.Q.; Bao, M.; Mou, Y.X.; Fang, C.Y.; Zhao, M.; Jiang, W.; Yu, X.; Wang, C.J.; Dai, L.H.; et al. Spongian diterpenes including one with a rearranged skeleton from the marine sponge Spongai officinalis. J. Nat. Prod. 2019, 82, 1714–1718. [Google Scholar] [CrossRef]
  41. Moffitt, W.; Woodward, R.B.; Moscowitz, A.; Klyne, W.; Djerassi, C. Structure and the optical rotatory dispersion of saturated ketones. J. Am. Chem. Soc. 1961, 83, 4013–4018. [Google Scholar] [CrossRef]
  42. Zhang, Q.; Lenardo, M.J.; Baltimore, D. 30 years of NF-κB: A blossoming of relevance to human pathobiology. Cell 2017, 168, 37–57. [Google Scholar] [CrossRef] [Green Version]
  43. Sunwoo, J.B.; Chen, Z.; Dong, G.; Yeh, N.; Bancroft, C.C.; Sausville, E.; Adams, J.; Elliott, P.; van Waes, C. Novel proteasome inhibitor PS-341 inhibits activation of nuclear factor-κB, cell suivival, tumor growth, and angiogenesis in squamous cell carcinoma. Clin. Cancer Res. 2001, 7, 1419–1428. [Google Scholar]
  44. He, J.-M.; Chen, S.-C.; Li, R.-P.; Yuan, L.-X.; Bao, J.-M.; Guo, M.-L. Suppresion of nuclear factor-kappa B and mitogen-activated protein kinase signalling pathways by goshonoside-F5 extracted from Rubi Fructus. Int. Immunopharmacol. 2015, 24, 182–190. [Google Scholar] [CrossRef]
  45. Han, B.; Chen, J.; Yu, Y.-Q.; Cao, Y.-B.; Jiang, Y.-Y. Anti-fungal activity of Rubus chingii extract combined with fluconazole against fluconazole-resistant Candida albicans. Microbiol. Immunol. 2016, 60, 82–92. [Google Scholar] [CrossRef] [Green Version]
  46. Pu, D.B.; Li, X.N.; Lin, J.; Zhang, R.H.; Luo, T.; Wang, Y.; Gao, J.B.; Zeb, M.A.; Zhang, X.J.; Li, X.L.; et al. Triterpenoids from Ganoderma gibbosum: A class of sensitizers of FLC-resistant Candida albicans to fluconazole. J. Nat. Prod. 2019, 82, 2067–2077. [Google Scholar] [CrossRef]
  47. Peng, Y.-M.; Zheng, J.-B.; Zhou, Y.-B.; Li, J. Characterization of a novel curcumin analog P1 as potent inhibitor of the NF-κB signaling pathway with distinct mechanisms. Acta Pharmacol. Sin. 2013, 34, 939–950. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Clinical and Laboratory Standards Institute. M27-A3 Reference Method for Broth Dilution Anti-fungal Susceptibility Testing of Yeats. In Approved Standard, 3rd ed.; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2008. [Google Scholar]
  49. He, Y.-Q.; Jin, S.-S.; Ma, Z.-Y.; Zhao, J.; Yang, Q.; Zhang, Q.; Zhao, Y.-J.; Yao, B.-H. The anti-oxidant compounds isolated from the fruits of Chinese wild raspberry Rubus chingii Hu. Nat. Prod. Res. 2020, 34, 872–875. [Google Scholar] [CrossRef]
  50. Deng, Y.H.; Tianasoa-Ramamonjy, M.; Snyder, J.K. Remangilone D, a new bisnoroleanane triterpene, and a new flavone from Physena madagascariensis. Z. Naturforsch. B 2001, 56, 1079–1083. [Google Scholar] [CrossRef]
  51. Deng, Y.H.; Jiang, T.-Y.; Sheng, S.J.; Tianasoa-Ramamonjy, M.; Snyder, J.K. Remangilones A-C, new cytotoxic triterpenes from Physena madagascariensis. J. Nat. Prod. 1999, 62, 471–476. [Google Scholar] [CrossRef] [PubMed]
  52. Duan, H.Q.; Takaishi, Y.; Momota, H.; Ohmoto, Y.; Taki, T.; Tori, M.; Takaoka, S.; Jia, Y.F.; Li, D. Immunosuppressive terpenoids from extracts of Tripterygium wilfordii. Tetrahedron 2001, 57, 8413–8424. [Google Scholar] [CrossRef]
  53. Tian, Y.-N.; Li, B.-L.; Hu, J.-J.; Xie, J.-D.; Xiao, W.-J.; Nie, L.-H.; Wu, J.-W. Rosanortriterpenes A-B, two new nortriterpenes from the fruits of Rosa laevigata var. leiocapus. Nat. Prod. Res. 2019. [Google Scholar] [CrossRef] [PubMed]
  54. Huang, F.X.; Lin, X.H.; He, W.N.; Song, W.; Ye, M.; Yang, W.Z.; Guo, D.A. Two new oxidation products obtained from the biotransformation of asiatic acis by the fungus Fusarium avenaceum AS 3.4594. J. Asian Nat. Prod. Res. 2012, 14, 1039–1045. [Google Scholar] [CrossRef] [PubMed]
  55. Nhiem, N.X.; Yen, P.H.; Nguyen, T.T.N.; Quang, T.H.; Kiem, P.V.; Minh, C.V.; Tai, B.H.; Cuong, N.X.; Song, S.B.; Kim, Y.H. Inhibition of nuclear transcription factor-kappa B and activation of peroxisome proliferator-activated receptors in HepG2 cells by cucurbatane-type triterpene glycosides from Momordica charantia. J. Med. Food 2012, 15, 369–377. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Molecules 26 01911 g001 550
Figure 1. Chemical structure of triterpenoids (1–20) and diterpenoids (21–25).
Figure 1. Chemical structure of triterpenoids (1–20) and diterpenoids (21–25).
Molecules 26 01911 g001
Molecules 26 01911 g002 550
Figure 2. Observed key HMBC correlations of triterpenoids 1–3.
Figure 2. Observed key HMBC correlations of triterpenoids 1–3.
Molecules 26 01911 g002
Molecules 26 01911 g003 550
Figure 3. Observed key ROE correlations of compounds 1–3 and 21.
Figure 3. Observed key ROE correlations of compounds 1–3 and 21.
Molecules 26 01911 g003
Table
Table 1. 13C NMR data a (δ in ppm, 150 MHz) of compounds 1–3 (in CD3OD) and 21 (in C6D6).
Table 1. 13C NMR data a (δ in ppm, 150 MHz) of compounds 1–3 (in CD3OD) and 21 (in C6D6).
No.12321No.12321
1129.551.2130.053.01626.626.629.481.2
2145.871.2146.0210.21748.749.046.766.3
3200.5215.4202.482.81855.255.145.329.6
451.656.445.545.61973.573.682.416.6
555.860.255.254.02043.143.136.118.4
619.920.920.220.42127.327.328.6
734.634.433.741.52238.938.934.0
841.741.241.345.12365.920.122.2
944.648.544.755.62422.066.028.0
1039.439.039.745.22520.517.620.0
1124.924.824.318.82617.817.318.0
12129.0128.9124.526.12724.825.025.1
13140.4140.2145.044.628182.2182.2182.3
1442.942.743.036.82927.027.128.1
1529.529.629.552.93016.616.625.1
a Assignments were made by a combination of 1D and 2D NMR experiments.
Table
Table 2. 1H NMR data a (δ in ppm, J values in Hz, 600 MHz) of 1–3 in CD3OD.
Table 2. 1H NMR data a (δ in ppm, J values in Hz, 600 MHz) of 1–3 in CD3OD.
No.123
1α6.29 s1.17 dd (12.7, 12.5)6.27 s
1β 2.31 dd (12.5, 6.6)
2 4.62 dd (12.7, 6.6)
51.70 dd (overlapped)1.22 dd (overlapped)1.61 m
6a1.66 m1.63 m1.60 m
6b1.62 m1.58 m1.58 m
7a1.76 m1.55 m1.76 m
7b1.63 m1.35 m1.58 m
92.00 dd (11.3, 6.3)1.78 m2.05 dd (10.7, 6.6)
11a2.27 ddd (17.3, 11.3, 3.5)2.06 m2.33 m
11b2.14 ddd (17.3, 6.3, 3.7)1.35 m2.19 m
125.37 dd (3.7, 3.5)5.30 dd (3.8, 3.3)5.37 dd (3.8, 3.1)
15α1.03 ddd (14.1, 4.2, 2.5)0.99 m1.02 m
15β1.83 ddd (14.1, 13.2, 4.4)1.81 m1.63 m
16α2.60 ddd (13.3, 13.2, 4.4)2.57 ddd (13.4, 13.4, 4.0)1.75 m
16β1.55 ddd (13.3, 4.2, 2.5)1.55 m1.60 m
182.50 s2.51 s3.07 d (3.9)
19 3.26 d (3.9)
201.37 m1.34 m
21a1.25 m1.22 m2.29 m
21b1.20 m1.16 m1.62 m
22a1.75 m1.73 m1.61 m
22b1.62 m1.62 m1.37 m
23a3.71 d (12.0)1.21 s1.11 s
23b3.69 d (12.0)
24a1.29 s4.11 d (11.4)1.18 s
24b 3.50 d (11.4)
251.30 s1.31 s1.22 s
260.90 s0.84 s0.84 s
271.36 s1.32 s1.30 s
291.23 s1.19 s0.97 s
300.95 d (7.1)0.93 d (6.3)0.94 s
a Assignments were made by a combination of 1D and 2D NMR experiment.
Table
Table 3. 1H (δ in ppm, 600 MHz) and 13C (δ in ppm, 150 MHz) NMR data a of 21.
Table 3. 1H (δ in ppm, 600 MHz) and 13C (δ in ppm, 150 MHz) NMR data a of 21.
No.21No.21
δH (J in Hz) bδH (J in Hz) cδH (J in Hz) bδH (J in Hz) c
11.37 d (12.4)2.06 d (12.2)121.62 m; 1.65 m1.87 m; 1.83 m
2.41 d (12.4)2.67 d (12.2)131.88 m2.46 m
33.62 d (3.8)4.16 s141.57 m; 0.69 m1.70 m; 0.85 m
50.91 br d (11.6)1.43 br d (11.5)151.39 m; 1.25 m1.63 m; 1.35 m
61.32 m; 1.05 m1.69 m; 1.37 m173.44 d (10.3)4.12 d (11.0)
71.22 m; 1.21 m1.56 m; 1.53 m3.37 d (10.3)4.05 d (11.0)
90.99 br d (6.7)1.22 br d (8.6)181.10 s0.96 s
111.62 m; 1.18 m1.84 m; 1.51 m190.67 s1.28 s
OH-33.72 d (3.8) 200.69 s0.87 s
a Assignments were made by a combination of 1D and 2D NMR experiments; b Measured in C6D6;c Measured in C5D5N.
Table
Table 4. Inhibitory activities of indicated compounds against NF-κB.
Table 4. Inhibitory activities of indicated compounds against NF-κB.
CompoundNF-κB (IC50) a
150.08 ± 0.03 μM
160.61 ± 0.12 μM
171.60 ± 0.32 μM
PS-341 b0.44 ± 0.08 μM
a These data are expressed as the mean SEM of triplicated experiments. b Positive control.
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Wan, J.; Wang, X.-J.; Guo, N.; Wu, X.-Y.; Xiong, J.; Zang, Y.; Jiang, C.-X.; Han, B.; Li, J.; Hu, J.-F. Highly Oxygenated Triterpenoids and Diterpenoids from Fructus Rubi (Rubus chingii Hu) and Their NF-kappa B Inhibitory Effects. Molecules 2021, 26, 1911. https://doi.org/10.3390/molecules26071911

AMA Style

Wan J, Wang X-J, Guo N, Wu X-Y, Xiong J, Zang Y, Jiang C-X, Han B, Li J, Hu J-F. Highly Oxygenated Triterpenoids and Diterpenoids from Fructus Rubi (Rubus chingii Hu) and Their NF-kappa B Inhibitory Effects. Molecules. 2021; 26(7):1911. https://doi.org/10.3390/molecules26071911

Chicago/Turabian Style

Wan, Jiang, Xiao-Juan Wang, Nan Guo, Xi-Ying Wu, Juan Xiong, Yi Zang, Chun-Xiao Jiang, Bing Han, Jia Li, and Jin-Feng Hu. 2021. "Highly Oxygenated Triterpenoids and Diterpenoids from Fructus Rubi (Rubus chingii Hu) and Their NF-kappa B Inhibitory Effects" Molecules 26, no. 7: 1911. https://doi.org/10.3390/molecules26071911

APA Style

Wan, J., Wang, X. -J., Guo, N., Wu, X. -Y., Xiong, J., Zang, Y., Jiang, C. -X., Han, B., Li, J., & Hu, J. -F. (2021). Highly Oxygenated Triterpenoids and Diterpenoids from Fructus Rubi (Rubus chingii Hu) and Their NF-kappa B Inhibitory Effects. Molecules, 26(7), 1911. https://doi.org/10.3390/molecules26071911

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