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. Author manuscript; available in PMC: 2024 Jul 1.
Published in final edited form as: J Nat Prod. 2021 Feb 19;84(3):738–749. doi: 10.1021/acs.jnatprod.0c01117

Hepatoprotective Glucosyloxybenzyl 2‑Hydroxy-2-isobutylsuccinates from Pleione yunnanensis

Shao-wei Han 1, Xiao-juan Wang 2, Bao-song Cui 3, Hua Sun 4, Hui Chen 5, Daneel Ferreira 6, Shuai Li 7, Mark T Hamann 8
PMCID: PMC11215813  NIHMSID: NIHMS1993489  PMID: 33606538

Abstract

Nine new glucosyloxybenzyl 2-hydroxy-2-isobutylsuccinates, pleionosides M−U (1−9), and 12 known compounds (10−21) were isolated from the pseudobulbs of Pleione yunnanensis. Their structures and absolute configurations were established through a combination of HRESIMS and NMR data and supported by physical and chemical methods. Compounds 5, 6, 10, and 15 showed significant in vitro hepatoprotective activity against D-galactosamine (D-GalN)-induced toxicity in HL-7702 cells with increasing cell viability by 27%, 22%, 19%, and 31% compared to the model group (cf. bicyclol, 14%) at 10 μM, respectively. Compounds 4, 9, and 11 exhibited moderate hepatoprotective activity against N-acetyl-p-aminophenol (APAP)-induced toxicity in HepG2 cells with increasing cell viability by 9%, 16%, and 12% compared to the model group (cf. bicyclol, 9%) at 10 μM, respectively.

Graphical Abstract

graphic file with name nihms-1993489-f0007.jpg

Liver diseases, including viral hepatitis (predominantly hepatitis B virus [HBV] and hepatitis C virus [HCV]), alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), autoimmune liver disease, and drug-induced liver injury (DILI), and associated cirrhosis and hepatocellular carcinoma (HCC), have caused serious public health problems because of their high prevalence worldwide and poor long-term clinical outcome.1,2 Among the various types of liver disease, viral hepatitis and NAFLD have received significant attention due to their rapid growth in the past few decades.3,4 In order to reduce the burden of liver disease, pharmacologists have been looking for more effcient drugs, and research experience has shown that lead compounds derived from natural products are consistently highly promising as potential new drugs or supplements for many hard to treat chronic diseases.

The dry pseudobulbs of Pleione yunnanensis (Rolfe) Rolfe (Orchidaceae), together with P. bulbocodioides (Franch.) Rolfe, and Cremastra appendiculata (D. Don) Makino, are the main sources of Pseudobulbus Cremastrae seu Pleiones (Chinese name: “Shan-Ci-Gu”), a traditional Chinese medicine in clinical treatment for various cancers, peptic ulcer, or uroschesis coupled with other herbs.5,6 A large number of phytochemical studies showed that the main constituents of “Shan-Ci-Gu” were phenanthrenes,7–27 bibenzyl derivatives,10,11,14,27–32 glycosides,20,27,33–35 flavonoids,6,36–39 lignans,27,40 alkaloids,27,41 terpenoids,42 steroids,27 and simple aromatic compounds.36,37,39,43,44 Modern pharmacological studies showed that the chemical components of “Shan-Ci-Gu” exhibited cytotoxic,15–20,42 antiangiogenic,38 antioxidant,45 anticholinesterase,45 β-amyloid aggregation,45 and butyrylcholinesterase inhibitory activities35 and inhibited the binding of tritium-labeled N-methylscopolamine to the muscarinic M3 receptor.41

Our previous phytochemical research on P. bulbocodioides led to the isolation of a series of glucosyloxybenzyl succinate derivatives with hepatoprotective activity,46 and it was noted that P. yunnanensis contained similar constituents based on HPLC analysis.47 Therefore, a detailed phytochemical study to explore the glycosidic constituents with new structures and bioactivities from P. yunnanensis was performed and led to the isolation and identification of 17 glucosyloxybenzyl 2-hydroxy-2-isobutylsuccinates (1−17), including nine new compounds (1−9), as well as four simple aromatic compounds. It is reported that lesions of D-galactosamine (D-GalN)-induced hepatotoxicity can be considered to resemble those of the human virus hepatitis, because the D-GalN model of liver injury can mimic the severe state of intense inflammation and the activation of intracellular signaling pathways during human viral hepatitis.48,49 Thus, compounds having a hepatoprotective effect against D-GalN-induced toxicity may have potential therapeutic effects on viral hepatitis. Besides, there is growing evidence that compounds having a hepatoprotective effect against N-acetyl-p-aminophenol (APAP)-induced toxicity may have a positive effect on preventing NAFLD from causing more serious liver damage.50,51 Therefore, the isolated compounds were assayed for their biological activities on the two different hepatocyte injury models, respectively, in order to fully explore their hepatoprotective activities. Herein, we detail the isolation and structure elucidation of the new compounds 1−9 and the hepatoprotective activities of all the isolated compounds.

RESULTS AND DISCUSSION

The 50% EtOH extract of the dried pseudobulbs of P. yunnanensis was suspended in H2O and chromatographed successively over macroporous resin, ODS, Sephadex LH-20, MCI GEL CHP20, and preparative RP-HPLC to afford the new pleionosides M−U (1−9) and 12 known compounds.

Compounds 1−9 were obtained as white amorphous powders. The monosaccharide, glucose, obtained by acid hydrolysis of each compound was identified by TLC comparison with an authentic glucose sample. The (D)-absolute configuration of the glucose was defined via HPLC comparison with an authentic glucose sample after derivatization.52 The 3J1,2 value in the 1H NMR spectrum of the glucopyranose (7.0−8.0 Hz) indicated its β anomeric configuration. Assignment of all NMR signals was based on DEPT, HSQC, COSY, NOESY, and HMBC data.

Compound 1 was obtained as a white amorphous powder and assigned the molecular formula C49H62O24 based on the HRESIMS ion at m/z 1057.3555 [M + Na]+. The 1H NMR data (Table 1) showed the methylene protons at δH 3.07 (d, J = 18.0 Hz, H-3a) and 2.92 (d, J = 18.0 Hz, H-3b), and their HMBC correlations (Figure 1) to the oxygenated carbon at δC 78.9 (C-2) and two ester carbonyl carbons at δC 171.4 (C-1) and 170.1 (C-4) established the 2-hydroxysuccinic acid moiety. The two methyl doublets at δH 0.68 and 0.59 (d, J = 6.5 Hz, H3-8 and H3-7) and a methylene at δH 1.59 (dd, J = 14.0, 4.5 Hz, H-5a) and 1.51 (dd, J = 14.0, 7.0 Hz, H-5b), which all showed COSY correlations to a methine at δH 1.67 (dq, J = 13.0, 6.5 Hz, H-6), suggested the presence of a C-2 isobutyl moiety. The HMBC correlations from H2-5 to C-1, C-3 (δC 43.0), C-6 (δC 22.8), C-7 (δC 22.7), and C-8 (δC24.4) established the 2-hydroxy-2-isobutylsuccinic acid moiety.

Table 1.

1H and 13C NMR Data of Compounds 1−3 in DMSO-d6 (δ in ppm)

position 1a
 2a
 3a
δC, type δH (J in Hz) δC, type δH (J in Hz) δC, type δH (J in Hz)
1 171.4, C 171.4, C 171.2, C
2 78.9, C 78.9, C 78.8, C
3a 43.0, CH2 3.07, d (18.0) 43.1, CH2 3.08, d (18.0) 43.2, CH2 3.06, d (18.0)
3b 2.92, d (18.0) 2.93, d (18.0) 2.91, d (18.0)
4 170.1, C 170.1, C 170.1, C
5a 48.1, CH2 1.59, dd (4.5, 14.0) 48.2, CH2 1.60, dd (4.0, 14.0) 48.0, CH2 1.59, dd (4.5, 14.0)
5b 1.51, dd (7.0, 14.0) 1.51, dd (7.0, 14.0) 1.49, dd (7.0, 14.0)
6 22.8, CH 1.67, dq (6.5, 13.0) 22.9, CH 1.66, m 22.8, CH 1.66, dq (6.5, 13.0)
7 22.7, CH3 0.59, d (6.5) 22.8, CH3 0.60, d (6.5) 22.8, CH3 0.57, d (6.5)
8 24.4, CH3 0.68, d (6.5) 24.4, CH3 0.69, d (6.5) 24.4, CH3 0.72, d (6.5)
1’ 128.84, C 128.85, C 128.83, C
2’ 129.9, CHb 7.26, d (8.5) 129.9, CH 7.27, d (8.5) 129.9, CH 7.26, d (8.5)
3′ 116.1, CHc 7.01, d (8.5) 116.2, CH 7.02, d (8.5) 116.2, CH 7.01, d (8.5)
4’ 157.33, C 157.35, C 157.3, C
5′ 116.1, CHc 7.01, d (8.5) 116.2, CH 7.02, d (8.5) 116.2, CH 7.01, d (8.5)
6’ 129.9, CHb 7.26, d (8.5) 129.9, CH 7.27, d (8.5) 129.9, CH 7.26, d (8.5)
7′a 66.0, CH2 5.03, d (12.0) 66.0, CH2 5.03, d (12.0) 65.9, CH2 5.04, d (12.0)
7′b 4.95, d (12.0) 4.96, d (12.0) 4.91, d (12.0)
1″ 100.3, CH 4.861, d (7.5) 100.3, CH 4.86, d (7.5) 100.3, CH 4.86, d (7.0)
2″ 73.2, CH 3.24d 73.3, CH 3.22d 73.2, CH 3.24d
3″ 76.6, CH 3.26d 76.6, CH 3.25d 76.6, CH 3.25d
4″ 69.7, CH 3.16d 69.7, CH 3.14d 69.6, CH 3.17d
5″ 77.0, CH 3.32d 77.0, CH 3.31d 76.99, CH 3.31d
6″ 60.7, CH2 3.68, br d (11.5)
3.46d 		60.7, CH2 3.68d
3.45d 		60.6, CH2 3.68d
3.46d
1‴ 128.79, C 128.78, C 128.77, C
2‴ 129.8, CHb 7.26, d (8.5) 129.8, CH 7.26, d (8.5) 129.70, CH 7.24, d (8.5)
3‴ 116.0, CHc 7.01, d (8.5) 116.1, CH 7.01, d (8.5) 116.0, CH 6.98, d (8.5)
4‴ 157.29, C 157.33, C 157.2, C
5‴ 116.0, CHc 7.01, d (8.5) 116.1, CH 7.01, d (8.5) 116.0, CH 6.98, d (8.5)
6‴ 129.8, CHb 7.26, d (8.5) 129.8, CH 7.26, d (8.5) 129.70, CH 7.24, d (8.5)
7‴a 65.7, CH2 4.97, d (12.0) 65.7, CH2 4.98, d (12.0) 65.7, CH2 4.97, d (12.0)
7‴b 4.86, d (12.0) 4.86, d (12.0) 4.86, d (12.0)
1⁗ 100.3, CH 4.857, d (7.5) 100.3, CH 4.85, d (7.5) 100.2, CH 4.85, d (7.0)
2⁗ 73.2, CH 3.24d 73.3, CH 3.22d 73.2, CH 3.24d
3⁗ 76.6, CH 3.26d 76.6, CH 3.25d 76.6, CH 3.25d
4⁗ 69.7, CH 3.16d 69.7, CH 3.14d 69.6, CH 3.17d
5⁗ 77.0, CH 3.32d 77.0, CH 3.31d 76.97, CH 3.31d
6⁗ 60.7, CH2 3.68, br d (11.5) 60.7, CH2 3.68d 60.6, CH2 3.68d
3.46d 3.45d 3.46d
1⁗′ 96.9, CH 5.05, d (7.5) 96.9, CH 5.00, d (8.5) 96.8, CH 5.06, d (8.5)
2⁗′ 73.1, CH 4.59, dd (8.5, 9.0) 73.21, CH 4.59, t (9.0) 73.5, CH 4.65, dd (8.0, 9.5)
3′⁗ 74.1, CH 3.34d 74.1, CH 3.34d 74.2, CH 3.39d
4⁗′ 69.6, CH 3.24d 69.6, CH 3.23d 69.7, CH 3.26d
5′⁗ 76.3, CH 2.90, m 76.3, CH 2.90, m 76.4, CH 2.92, m
6⁗′ 60.3, CH2 3.48d 60.3, CH2 3.48d 60.3, CH2 3.49d
3.46d 3.43d 3.41d
1⁗″ 132.8, CH 128.1, C 128.0, C
2⁗″ 132.8, CH 7.74, d (8.5) 132.3, CH 7.78, d (8.5) 129.66, CH 7.62, d (8.5)
3′⁗′ 114.7, CH 6.73, d (8.5) 115.4, CH 6.99, d (8.5) 116.5, CH 7.07, d (8.5)
4⁗″ 158.7, C 158.1, C 159.0, C
5′⁗′ 114.7, CH 6.73, d (8.5) 115.4, CH 6.99, d (8.5) 116.5, CH 7.07, d (8.5)
6⁗″ 132.8, CH 7.74, d (8.5) 132.3, CH 7.78, d (8.5) 129.66, CH 7.62, d (8.5)
7⁗″ 142.9, CH 6.78, d (13.0) 142.3, CH 6.86, d (13.0) 143.5, CH 7.64, d (16.0)
8⁗″ 115.9, CH 5.62, d (13.0) 117.6, CH 5.70, d (13.0) 116.6, CH 6.38, d (16.0)
9⁗″ 164.8, C 164.7, C 165.7, C
1⁗‴ 100.0, CH 4.92, d (7.5) 100.0, CH 4.95, d (8.5)
2⁗‴ 73.18, CH 3.22d 73.2, CH 3.24d
3′⁗″ 76.6, CH 3.25d 76.5, CH 3.25d
4⁗‴ 69.7, CH 3.14d 69.6, CH 3.17d
5′⁗″ 77.0, CH 3.31d 77.1, CH 3.37, m
6⁗‴ 60.6, CH2 3.68d 60.6, CH2 3.68d
3.45d 3.46d
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a

1H and 13C NMR data (δ) were recorded at 500 and 125 MHz.

b,c

Interchangeable.

d

Overlapped and assigned by HSQC.

Figure 1.

Figure 1.

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Structure and key HMBC (H→C, blue) and NOESY (↔, magenta) correlations of 1.

The 1H NMR spectrum of 1 clearly showed two pairs of overlapped AA′BB′ signals at δH 7.26 (d, J = 8.5 Hz, H-2′, 6′, H-2‴,6″′) and 7.01 (d, J = 8.5 Hz, H-3′, 5′, H-3‴, 5″′) and four benzyloxy hydrogens at δH 5.03 (d, J = 12.0 Hz, H-7′a), 4.95 (d, J = 12.0 Hz, H-7′b) and 4.97 (d, J = 12.0 Hz, H-7‴a), 4.86 (d, J = 12.0 Hz, H-7‴b). The HMBC correlations from H2-7′ to C-1′ (δC 128.84) and C-2′/6′ (δC 129.9) and from H2-7‴ to C-1″′ (δC 128.79) and C-2‴/6″′ (δC 129.8) indicated the presence of two 1,4-disubstituted benzylic units in 1. There were three anomeric protons at δH 5.05 (d, J = 7.5 Hz, H-1⁗′), 4.861 (d, J = 7.5 Hz, H-1″), and 4.857 (d, J = 7.5 Hz, H-1⁗), together with partially overlapped signals attributed to oxymethylene and oxymethine hydrogens of the glucosyl moieties between δH 3.16 and 3.68, suggesting the presence of three β-configured glucosyl units. HMBC correlations from H-1″ to C-4′ (δC 157.33) and from H-1⁗ to C-4‴ (δC 157.29), in combination with NOESY correlations (Figure 1) between H-1″ and H-5′ and between H-1⁗ and H-5‴, showed that there were two 4-(β-D-glucopyranosyloxy)-benzyl units in 1. Furthermore, those two 4-(β-D-glucopyranosyloxy)benzyl units were esterified at C-1 and C-4 of the 2-hydroxy-2-isobutylsuccinic acid moiety by the HMBC correlations from H2-7′ to C-1 and from H2-7‴ to C-4, respectively

Another set of AA′BB′-type aromatic proton resonances was observed at δH 7.74 (d, J = 8.5 Hz, H-2⁗″/6⁗″) and 6.73 (d, J = 8.5 Hz, H-3⁗″/5⁗″), as well as the resonances due to the (Z)-double bond at δH 6.78 (d, J = 13.0 Hz, H-7⁗″) and 5.62 (d, J = 13.0 Hz, H-8⁗″). In addition, the connection of the aromatic ring to the double bond was deduced from the HMBC correlations from H-7⁗″ to C-2⁗″/6⁗″ (δC 132.8), from H-8⁗″ to C-1⁗″ (δC 125.6), and from H-2⁗″/6⁗″ to C-7⁗″ (δC 142.9). This information along with the presence of the carbonyl carbon at C-9⁗″ (δC 164.8) demonstrated the presence of a (Z)-4-hydroxycinnamoyl group in compound 1.53 The HMBC correlation from H-1⁗′ to C-2 revealed that the glucopyranosyl moiety with the anomeric proton at δH 5.05 was located at C-2 of the 2-hydroxy-2-isobutylsuccinate unit. Moreover, H-2⁗′ (δH 4.59) showed HMBC correlations to C-9⁗″ and C-8⁗″ (δC 115.9), thus defining the location of the (Z)-4-hydroxycinnamoyloxy group at C-2⁗′.

To define the absolute configuration, compound 1 was subjected to alkaline hydrolysis and afforded (R)-2-hydroxy-2-isobutylsuccinic acid as a white powder, identified by spectroscopic data and an [α]20 D value of −9 (c 0.2, MeOH), lit. [α]26 D −7.8 (c 0.05, MeOH).54 Based on these data, the structure of pleionoside M (1) was established as 1,4-bis(4-β-D-glucopyranosyloxybenzyl)-(R)-2-{2-O-[(Z)-4-hydroxycinnamoyl]-β-D-glucopyranosyloxy} 2-isobutylsuccinate (Figure 1).

Compound 2 was obtained as a white amorphous powder. The HRESIMS sodium adduct ion at m/z 1219.4086 [M + Na]+ indicated a molecular formula of C55H72O29, suggesting that 2 had one more glucopyranosyl unit (162 Da) compared with 1. The NMR spectra were closely related to those of 1, except for the presence of four instead of three β-configured anomeric protons at δH 5.00 (d, J = 8.5 Hz, H-1⁗′), 4.86 (d, J = 7.5 Hz, H-1″), 4.85 (d, J = 7.5 Hz, H-1⁗), and 4.92 (d, J = 7.5 Hz, H-1⁗′′′). The β-D-glucopyranosyl moiety with an anomeric proton at δH 4.92 (H-1⁗′′′) was located at C-4⁗″ (δC 158.1) based on the HMBC correlation from H-1⁗′′′ to C-4⁗″ (Figure S24). The other β-D-glucopyranosyl units as well as all other signals shown in Table 1 were assigned based on the DEPT, HSQC, COSY, NOESY, and HMBC spectra of 2. The absolute configuration of 2 was determined using the same method as for 1. Hence, the structure of pleionoside N (2) was defined as 1,4-bis(4-β-D-glucopyranosyloxybenzyl)-(R)-2-{2-O-[(Z)-4-β-D-glucopyranosyloxycinnamoyl]-β-D-glucopyranosyloxy} 2-isobutylsuccinate.

Compound 3 was obtained as a white amorphous powder, and a molecular formula, C38H46O19, assigned by the HRESIMS sodium adduct ion at m/s 1219.4068 [M + Na]+, indicating that 3 was an isomer of 2. The difference between 2 and 3 involved the signals of the cinnamoyl ester moiety of the C-2 glucosyloxy unit by comparison of their NMR data. The two definite hydrogen signals at δH 7.64 (d, J = 16.0 Hz, H-7⁗″) and 6.38 (d, J = 16.0 Hz, H-8⁗″) in 3 replaced the signals at δH 6.86 (d, J = 13.0 Hz, H-7⁗″) and 5.70 (d, J = 13.0 Hz, H-8⁗″) in 2. The coupling constant of 16.0 Hz of 3 indicated the presence of an (E)-cinnamate moiety in 3. Assignment of the position of the key units of 3 was based on the HMBC and NOESY data (Figure 2). Hence, the structure of pleionoside O (3) was defined as 1,4-bis(4-β-D-glucopyranosyloxybenzyl)-(R)-2-{2-O-[(E)-4-β-D-glucopyranosyl-oxycinnamoyl]-β-D-glucopyranosyloxy} 2-isobutylsuccinate.

Figure 2.

Figure 2.

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Structure and key HMBC (H→C, blue) and NOESY (↔, magenta) correlations of 3.

Compound 4 was obtained as a white amorphous powder and assigned a molecular formula of C47H62O23 based on the HRESIMS sodium adduct ion at m/z 1017.3584 [M + Na]+. The 1H NMR data of 4 (Table 2) showed two methylenes at δH 3.10 (d, J = 17.5 Hz, H-3a), 2.98 (d, J = 17.5 Hz, H-3b) and 1.71 (m, H-5a), 1.68 (m, H-5b), a methine at δH 1.72 (m, H-6), and two methyl doublets at δH 0.83 (d, J = 6.5 Hz, H-8) and 0.76 (d, J = 6.5 Hz, H-7). The HMBC correlations (Figure 3) from H2-3 to the ester carbonyl carbons at δC 172.1 (C-1) and 169.9 (C-4) and the oxygenated carbon at δC79.5 (C-2) established the 2-hydroxysuccinic acid moiety. The COSY correlations between H2-5 and H-6 and H-6 and H3-7/8 suggested the presence of an isobutyl unit. Moreover, the HMBC correlations from H2-5 to C-1 and C-2 confirmed that the isobutyl unit was located at C-2 and indicated the presence of a 2-hydroxy-2-isobutylsuccinic acid moiety in 4.

Table 2.

1H NMR Spectroscopic Data of Compounds 4−9 in DMSO-d6 (δ in ppm)

position 4a 5a 6a 7a 8a 9b
3a 3.10, d (17.5) 2.85, d (15.0) 2.85, d (15.0) 2.86, d (15.0) 2.85, d (15.0) 2.85, d (15.0)
3b 2.98, d (17.5) 2.59, d (15.0) 2.58, d (15.0) 2.59, d (15.0) 2.59, d (15.0) 2.59, d (15.0)
5a 1.71, m 1.58, dd (6.5, 14.0) 1.57, dd (6.5, 14.0) 1.58, dd (6.5, 14.0) 1.58, dd (6.5, 14.0) 1.58, dd (6.6, 13.8)
5b 1.68, m 1.53, dd (6.0, 14.0) 1.52, dd (6.0, 14.0) 1.53, dd (5.5, 14.0) 1.53, dd (6.0, 14.0) 1.53, dd (6.0, 13.8)
6 1.72, m 1.66, dq (6.5, 13.0) 1.66, dq (6.5, 13.0) 1.66, dq (6.5, 13.0) 1.67, dq (6.5, 13.0) 1.66, dq (6.6, 13.2)
7 0.76, d (6.5) 0.74, d (6.5) 0.73, d (6.5) 0.74, d (6.5) 0.74, d (6.5) 0.74, d (6.6)
8 0.83, d (6.5) 0.84, d (6.5) 0.83, d (6.5) 0.85, d (6.5) 0.84, d (6.5) 0.84, d (6.6)
2′ 7.27, d (8.5) 7.26, d (8.5) 7.27, d (8.5) 7.30, d (8.5) 7.28, d (9.0) 7.27, d (9.0)
3′ 7.01, d (8.5) 7.02, d (8.5) 7.02, d (8.5) 7.03, d (8.5) 7.06, d (9.0) 7.06, d (9.0)
5′ 7.01, d (8.5) 7.02, d (8.5) 7.02, d (8.5) 7.03, d (8.5) 7.06, d (9.0) 7.06, d (9.0)
6′ 7.27, d (8.5) 7.26, d (8.5) 7.27, d (8.5) 7.30, d (8.5) 7.28, d (9.0) 7.27, d (9.0)
7′a 5.07, d (12.0) 5.01, d (12.5) 5.02, d (12.5) 5.03, d (12.5) 5.01, d (12.5) 5.01, d (12.6)
7′b 4.97, d (12.0) 4.95, d (12.5) 4.97, d (12.5) 4.98, d (12.5) 4.97, d (12.5) 4.96, d (12.6)
1″ 4.86, d (7.5) 4.85, d (7.5) 4.89, d (7.5) 4.99, d (7.5) 4.84, d (7.5) 4.82, d (7.2)
2″ 3.22c 3.22c 3.24c 3.26c 3.22c 3.25c
3″ 3.25c 3.25c 3.28c 3.40c 3.25c 3.57c
4″ 3.17c 3.16, dd (4.0, 8.5) 3.12c 3.19c 3.16c 3.03, m
5″ 3.33c 3.31c 3.53, m 3.35c 3.32c 3.26c
6″ 3.67c 3.67c 3.69, br d (10.0) 3.69, br d (10.0) 3.68, br d (12.0) 3.98, d (10.2)
3.45c 3.46c 3.44c 3.47c 3.45, dd (5.5, 12.0) 3.58c
2‴ 7.26, d (8.5) 7.26, d (8.5) 7.26, d (8.5) 7.27, d (8.5) 7.27, d (9.0) 7.27, d (9.0)
3‴ 7.02, d (8.5) 7.01, d (8.5) 7.00, d (8.5) 7.01, d (8.5) 7.01, d (9.0) 7.01, d (9.0)
5‴ 7.02, d (8.5) 7.01, d (8.5) 7.00, d (8.5) 7.01, d (8.5) 7.01, d (9.0) 7.01, d (9.0)
6‴ 7.26, d (8.5) 7.26, d (8.5) 7.26, d (8.5) 7.27, d (8.5) 7.27, d (9.0) 7.27, d (9.0)
7‴a 4.99, d (12.0) 4.99, d (12.5) 4.98, d (12.5) 5.00, d (12.5) 4.99, d (12.5) 4.99, d (12.6)
7‴b 4.89, d (12.0) 4.94, d (12.5) 4.93, d (12.5) 4.94, d (12.5) 4.94, d (12.5) 4.93, d (12.6)
1⁗ 4.89, d (7.5) 4.89, d (7.5) 4.85, d (7.5) 4.85, d (7.5) 4.85, d (7.5) 4.86, d (7.8)
2⁗ 3.24c 3.24c 3.25c 3.23c 3.24c 3.23c
3⁗ 3.28c 3.27c 3.22c 3.27c 3.57c 3.27c
4⁗ 3.14c 3.13c 3.16c 3.16c 3.03c 3.16c
5⁗ 3.53c 3.53, ddd (2.0, 6.5, 10.0) 3.32c 3.33c 3.18c 3.32c
6⁗ 3.69, br d (10.0) 3.69, br d (10.0) 3.67c 3.67, br d (10.0) 3.98, br d (10.0) 3.68, ddd (1.8, 5.4, 12.0)
3.44c 3.44c 3.47c 3.44c 3.59c 3.45, dt (5.4, 12.0)
1⁗′ 4.66, d (7.5) 4.20, d (7.5) 4.20, d (7.8)
2⁗′ 2.96c 7.07, d (8.5) 7.07, d (8.5) 7.14, d (8.5) 2.97, t (8.5) 2.97, m
3′⁗ 3.12, dd (5.0, 9.0) 6.69, d (8.5) 6.69, d (8.5) 6.68, d (8.5) 3.10, br d (9.0) 3.10, td (4.8, 9.0)
4⁗′ 3.08c 3.16c 3.16c
5′⁗ 2.90, ddd (2.0, 5.0, 9.0) 6.69, d (8.5) 6.69, d (8.5) 6.68, d (8.5) 3.02c 3.01, m
6⁗′ 3.50c 7.07, d (8.5) 7.07, d (8.5) 7.14, d (8.5) 3.66, br d (12.0) 3.65, ddd (12.0, 5.4, 1.8)
3.42c 3.41, dd (5.5, 12.0) 3.41, dt (12.0, 5.4)
7⁗′ 4.33, s 4.33, s 4.71, d (11.0)
4.64, d (11.0)
2⁗″ 7.07, d (8.5)
3′⁗′ 6.69, d (8.5)
4⁗″
5′⁗′ 6.69, d (8.5)
6⁗″ 7.07, d (8.5)
7⁗″ 4.33, s
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a

1H NMR data (δ) were recorded for compounds 4–8 at 500 MHz.

b

1H NMR data (δ) were recorded for compound 9 at 600 MHz.

c

Overlapped and assigned by HSQC.

Figure 3.

Figure 3.

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Structure and key HMBC (H→C, blue) and NOESY (↔, magenta) correlations of 4.

In addition, the 1H NMR spectra of 4 showed three pairs of AA′BB′ signals at δH 7.27 (d, J = 8.5 Hz, H-2′, 6′), 7.01 (d, J = 8.5 Hz, H-3′, 5′), 7.26 (d, J = 8.5 Hz, H-2‴, 6″′), 7.02 (d, J = 8.5 Hz, H-3‴, 5″′), and 7.07 (d, J = 8.5 Hz, H-2⁗″, 6⁗″), 6.69 (d, J = 8.5 Hz, H-3⁗″, 5⁗″) and three benzyloxy signals at δH 5.07 (d, J = 12.0 Hz, H-7′a), 4.97 (d, J = 12.0 Hz, H-7′b), 4.99 (d, J = 12.0 Hz, H-7‴b), 4.89 (d, J = 12.0 Hz, H-7‴b), and 4.33 (s, H-7⁗″). The HMBC correlations from H2-7′ to C-2′/ 6′ (δC 129.9), from H2-7‴ to C-2″′/6′′′ (δC 129.8), and from H2-7″⁗ to C-2″⁗/6⁗″ (δC 129.2) indicated the presence of three 4-substituted benzylic units in 4, respectively. The 1H NMR spectrum showed three anomeric protons at δH 4.89 (d, J = 7.8 Hz, H-1⁗), 4.86 (d, J = 7.2 Hz, H-1″), and 4.66 (d, J = 7.5 Hz, H-1⁗′) together with partially overlapped proton signals between δH 2.96 and 3.69 indicating three β-glucopyranosyl moieties in 4. In the HMBC experiment, the long-range correlation between H-1⁗′ and C-2 suggested that the glucopyranosyl moiety with the anomeric proton at δH 4.66 (H-1⁗′) was located at C-2. The HMBC correlations from H-1″ to C-4′ (δC 157.3) and from H-1⁗ to C-4‴ (δC 157.2), in combination with NOESY correlations (Figure 3) between H- ″ and H-5′ and between H-1⁗ and H-5‴, showed that there were two 4-(β-D-glucopyranosyloxy)benzyl units in 4. Furthermore, the two 4-(β-D-glucopyranosyloxy) benzyl units were esterified at C-1 and C-4 of the 2-hydroxy-2-isobutylsuccinic acid moiety based on the HMBC correlations from H2-7′ to C-1 and from H2-7‴ to C-4, respectively. The HMBC correlations from H-7⁗″ to C-2⁗″/6⁗″ (δC 129.2) and C-6⁗ (δC69.1) and the NOESY correlations between H-7⁗″ and H-6‴′b (δH 3.44) indicated that this 4-substituted benzylic unit was located at C-6⁗ of the glucopyranosyl unit with the anomeric proton at δH 4.89 (H-1⁗). Thus, the structure of pleionoside P (4) was elucidated as 1-[4-(β-D-glucopyranosyloxy)benzyl]-4-{4-[6-O-(4-hydroxybenzyl)-β-D-glucopyranosyl]oxybenzyl}-(R)-2-isobutyl-2-(β-D-glucopyranosyloxy)succinate.

Compound 5 had a molecular formula of C41H52O18, as determined by HRESIMS data analysis, one glucopyranosyl unit less than in 4. A detailed comparison of the NMR spectroscopic data (Tables 2 and 3) of 4 and 5 indicated that the data assigned to the β-D-glucopyranosyl moiety at C-2 in 4 were absent in 5, and the C-2 signal of 5 was shielded (4.6 ppm) compared to 4, while C-1 and C-3 of 5 were deshielded (2.0 and 3.1 ppm, respectively). Assignment of the positions of the key units of 5 was based on the results of HMBC and NOESY data (Figure S57). Thus, the structure of pleionoside Q (5) was elucidated as 1-[4-(β-D-glucopyranosyloxy)benzyl]-4-{4-[6-O-(4-hydroxybenzyl)-β-D-glucopyranosyl]oxybenzyl}-(R)-2-hydroxy-2-isobutylsuccinate.

Table 3.

13C NMR Spectroscopic Data of Compounds 4–9 in DMSO-d6 (δ in ppm)

position 4, typea 5, typea 6, typea 7, typea 8, typea 9, typeb
1 172.1, C 174.1, C 174.1, C 174.1, C 174.1, C 174.1, C
2 79.5, C 74.9, C 74.9, C 74.9, C 74.9, C 75.0, C
3 41.6, CH2 44.7, CH2 44.7, CH2 44.7, CH2 44.8, CH2 44.7, CH2
4 169.9, C 169.5, C 169.5, C 169.5, C 169.6, C 169.6, C
5 46.0, CH2 47.5, CH2 47.6, CH2 47.6, CH2 47.6, CH2 47.6, CH2
6 23.1, CH 23.3, CH 23.3, CH 23.3, CH 23.4, CH 23.4, CH
7 23.8, CH3 23.3, CH3 23.3, CH3 23.3, CH3 23.4, CH3 23.4, CH3
8 24.2, CH3 24.3, CH3 24.2, CH3 24.3, CH3 24.3, CH3 24.3, CH3
1’ 129.0, C 129.2, C 129.0, C 129.2, C 128.9, C 129.1, C
2’ 129.9, CH 129.6, CH 129.62, CH 129.8, CH 129.7, CH 129.8, CH
3′ 116.1, CHc 116.11, CH 116.11, CH 116.1, CH 116.1, CH 116.3, CH
4′ 157.3, C 157.3, C 157.16, C 157.0, C 157.3, C 157.30, Cc
5′ 116.1, CHc 116.11, CH 116.11, CH 116.1, CH 116.1, CH 116.3, CH
6′ 129.9, CH 129.6, CH 129.62, CH 129.8, CH 129.7, CH 129.8, CH
7′ 66.3, CH2 65.9, CH2 65.9, CH2 65.9, CH2 66.0, CH2 65.9, CH2
1″ 100.3, CH 100.3, CH 100.1, CH 100.1, CH 100.28, CH 100.3, CH
2″ 73.17, CH 73.1, CH 73.21, CH 81.0, CH 73.22, CH 73.23, CHe
3″ 76.55, CH 76.6, CH 76.5, CH 76.0, CH 76.7, CH 75.9, CH
4″ 69.7, CH 69.7, CH 69.9, CH 69.9, CH 69.7, CH 70.1, CH
5″ 77.0, CH 77.0, CH 75.4, CH 76.9, CH 77.0, CH 76.5, CH
6″ 60.7, CH2 60.7, CH2 69.2, CH2 60.65, CH2 60.7, CH2 68.4, CH2
1‴ 128.8, C 129.0, C 129.2, C 129.1, C 129.2, C 129.0, C
2‴ 129.8, CH 129.6, CH 129.64, CH 129.6, CH 129.8, CH 129.7, CH
3‴ 116.2, CHc 116.07, CH 116.08, CH 116.0, CH 116.2, CH 116.1, CH
4‴ 157.2, C 157.1, C 157.22, C 157.2, C 157.2, C 157.27, Cc
5‴ 116.2, CHc 116.07, CH 116.08, CH 116.0, CH 116.2, CH 116.1, CH
6‴ 129.8, CH 129.6, CH 129.64, CH 129.6, CH 129.8, CH 129.7, CH
7‴ 65.7, CH2 65.3, CH2 65.3, CH2 65.3, CH2 65.4, CH2 65.4, CH2
1⁗ 100.1, CH 100.1, CH 100.3, CH 100.3, CH 100.31, CH 100.4, CH
2⁗ 73.21, CH 73.2, CH 73.16, CH 73.2, CH 73.20, CH 73.21, CHe
3⁗ 76.6, CH 76.5, CH 76.6, CH 76.6, CH 75.8, CH 76.7, CH
4⁗ 69.9, CH 69.9, CH 69.7, CH 69.7, CH 70.1, CH 69.71, CHf
5⁗ 75.4, CH 75.4, CH 77.0, CH 77.0, CH 76.5, CH 77.0, CH
6⁗ 69.1, CH2 69.1, CH2 60.7, CH2 60.61, CH2 68.4, CH2 60.7, CH2
1⁗′ 98.3, CH 128.7, C 128.6, C 129.0, C 103.4, CH 103.3, CH
2⁗′ 73.8, CH 129.1, CH 129.1, CH 129.4, CH 73.6, CH 73.6, CH
3′⁗ 76.53, CH 114.8, CH 114.9, CH 114.7, CH 76.6, CH 76.6, CH
4⁗′ 69.5, CH 156.7, C 156.7, C 156.7, C 69.6, CH 69.69, CHf
5′⁗ 76.9, CH 114.8, CH 114.9, CH 114.7, CH 76.9, CH 76.9, CH
6⁗′ 60.7, CH2 129.1, CH 129.1, CH 129.4, CH 61.1, CH2 61.1, CH2
7⁗′ 72.1, CH2 72.1, CH2 73.6, CH2
1⁗″ 128.7, C
2⁗″ 129.2, CH
3′⁗′ 114.9, CH
4⁗″ 156.7, C
5′⁗′ 114.9, CH
6⁗″ 129.2, CH
7⁗″ 72.1, CH2
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a

13C NMR data (δ) were recorded for compounds 4–8 at 125 MHz.

b

13C NMR data (δ) were recorded for compound 9 at 150 MHz.

c,d,e,f

Interchangeable.

Compounds 6 had the same molecular formula C41H52O18 as 5, as determined by HRESIMS data analysis, indicating that it was a regioisomer of 5. Although the 1H and 13C NMR data (Tables 2 and 3) of 5 and 6 were similar, the compounds had different retention times (16.1 and 18.0 min, respectively) on semipreparative HPLC (55% MeOH−H2O). In the HMBC data (Figure 4) of 6, correlations from H-7⁗′ (δH 4.33, s, 2H) to C-2⁗′/6⁗′ (δC 129.1) and C-6″ (δC 69.2) suggested that the p-hydroxybenzyl unit was located at C-6″ in 6. HMBC correlations from H2-7′ (δH 5.02 and 4.97, d, J = 12.5 Hz) to C-1 (δC 174.1) and C-2′/6′ (δC 129.62) and from H-1″ (δH 4.89, d, J = 7.5 Hz) to C-4′ (δC 157.16) indicated that the (6-p-hydroxybenzyl-4-O–β-D-glucopyranosyloxy)benzyl unit was located at C-1 in 6 instead of C-4 in 5. Thus, the structure of pleionoside R (6) was elucidated as 1-{4-[6-O-(4-hydroxybenzyl)-β-D-glucopyranosyl]oxybenzyl}−4-[4-(β-D-glucopyranosyloxy)benzyl]-(R)-2-hydroxy-2-isobutylsuccinate.

Figure 4.

Figure 4.

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Structure and key HMBC (H→C, blue) and NOESY (↔, magenta) correlations of 6.

Compound 7 was obtained as an amorphous powder. HRESIMS data analysis indicated that it was an isomer of 6. A detailed comparison of the NMR spectroscopic data (Tables 2 and 3) of 6 and 7 suggested that the p-hydroxybenzyl moiety was located at C-2″ in 7 instead of C-6″ in 6. This conclusion was supported by HMBC correlations (Figure S79) from H-7⁗′ (δH 4.71 and 4.64, d, J = 11.0 Hz) to C-2″ (δC 81.0), from H-7′ (δH 5.03 and 4.98, d, J = 12.5 Hz) to C-1 (δC 174.1) and from H-1″ (δH 4.99, d, J = 7.5 Hz) to C-4′ (δC 157.0), as well as the changes in chemical shifts of C-2″ and C-6″ [deshielded (7.79 ppm) and shielded (8.55 ppm) shifts compared to 6, respectively]. Thus, the structure of pleionoside S (7) was elucidated as 1-{4-[2-O-(4-hydroxybenzyl)-β-D-glucopyranosyl]oxybenzyl}−4-[4-(β-D-glucopyranosyloxy)-benzyl]-(R)-2-hydroxy-2-isobutylsuccinate.

Compound 8 was assigned the molecular formula C40H56O22 from the HRESIMS ion at m/z 911.3166 [M + Na]+. The 1H NMR data of 8 (Table 2) showed methylene proton resonances at δH 2.85 (d, J = 15.0 Hz, H-3a) and 2.59 (d, J = 15.0 Hz, H-3b), and their HMBC correlations (Figure 5) to the oxygenated carbon at δC 74.9 (C-2) and two carbonyl carbons at δC 174.1 (C-1) and 169.6 (C-4) established the presence of the 2-hydroxysuccinic acid moiety. The two methyl doublets at δH 0.84 and 0.74 (d, J = 6.5 Hz, H3-8 and H3-7) and methylene hydrogens at 1.58 (dd, J = 14.0, 6.5 Hz, H-5a) and 1.53 (dd, J = 14.0, 6.0 Hz, H-5b) all showed COSY correlations to a methine hydrogen at δH 1.67 (dq, J = 13.0, 6.5 Hz, H-6), suggesting the presence of a 2-isobutyl moiety. The HMBC correlations from H2-5 to C-1 (δC174.1) and C-3 (δC44.8) established the presence of the 2-hydroxy-2-isobutylsuccinic acid moiety.

Figure 5.

Figure 5.

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Structure and key HMBC (H→C, blue) and NOESY (↔, magenta) correlations of 8.

The 1H NMR spectrum of 8 clearly showed two pairs of partially overlapped AA′BB′ signals at δH 7.28 (d, J = 9.0 Hz, H-2′, 6′), 7.06 (d, J = 9.0 Hz, H-3′, 5′), and 7.27 (d, J = 9.0 Hz, H-2‴, 6″′), 7.01 (dd, J = 9.0 Hz, H-3‴, 5″′), two benzyloxy hydrogen signals at δH 5.01 (d, J = 12.5 Hz, H-7′a), 4.97 (d, J = 12.5 Hz, H-7′b), 4.99 (d, J = 12.5 Hz, H-7″′a), and 4.94 (d, J = 12.5 Hz, H-7″′b), and three anomeric protons at δH 4.85 (d, J = 7.5 Hz, H-1⁗), 4.84 (d, J = 7.5 Hz, H-1″), and 4.20 (d, J = 7.5 Hz, H-1⁗′), together with partially overlapped signals attributable to the oxymethylene and oxymethine hydrogens of three glucosyl moieties (Table 2). In addition, HMBC correlations from H-7′a and H-7′b to C-1, from H-3′/5′ to C-1′ (δC 128.9) and C-4′ (δC 157.3), and from H-1″ to C-4′, as well as the NOESY correlations (Figure 5) between H-1″ and H-3′/5′, confirmed that the 4-(β-D-glucopyranosyloxy)-benzyl unit was esterified at C-1 of the 2-hydroxy-2-isobutylsuccinic acid moiety. The HMBC correlations from H-7‴a and H-7″′b to C-4, from H-1⁗ to C-4′′′ (δC 157.2), and from H-1⁗′ to C-6‴′ (δC 68.4) as well as the NOESY correlations between H-1⁗ and H-3‴/5″′ revealed that the 4-[6-O-(β-D-glucopyranosyl)-β-D-glucopyranosyl]oxybenzyl unit was esterified at C-4 of the 2-hydroxy-2-isobutylsuccinic acid moiety in 8. Thus, the structure of pleionoside T (8) was defined as 1-[4-(β-D-glucopyranosyloxy)benzyl]-4-{4-[6-O-(β-D-glucopyranosyl)-β-D-glucopyranosyl]oxybenzyl}-(R)-2-hydroxy-2-isobutylsuccinate.

Compound 9 was obtained as an amorphous powder. HRESIMS data analysis indicated that 9 was an isomer of 8, and the NMR data (Tables 1 and 2) suggested a close structural similarity. The HMBC correlations (Figure S103) from H-1⁗′ (δH 4.20, d, J = 7.8 Hz) to C-6″ (δC 68.4), from H-1″ (δH 4.82, d, J = 7.2 Hz) to C-4′ (δC 157.30), and from H-7′ (δH 5.01 and 4.96, d, J =12.6 Hz) to C-1 (δC 174.1) and C-2/6 (δC 129.8) in 9 indicated that the 4-[6-O-(β-D-glucopyranosyl)-β-D-glucopyranosyl]oxybenzyl moiety was located at C-1 of the 2-hydroxy-2-isobutylsuccinic acid moiety instead of C-4 in 8. The assignment of all other signals (Tables 2 and 3) was based on HSQC, COSY, NOESY, and HMBC data. Therefore, the structure of pleionoside U (9) was defined as 1-{4-[6-O-(β-D-glucopyranosyl)-β-D-glucopyranosyl]-oxybenzyl}−4-[4-(β-D-glucopyranosyloxy)benzyl]-(R)-2-hydroxy-2-isobutylsuccinate.

Besides these new compounds, 12 known compounds were identified as shancigusin H (10),27 dactylochin A (11),55 gymnoside III (12),12 dactylorhin E (13),56 militarine (14),20 1-[4-(β-D-glucopyranosyloxy)benzyl]-4-methyl-(R)-2-hydroxy-2-isobutylsuccinate (15),57 gymnoside I (16),27 loroglossin (17),56 bis(4-hydroxybenzyl) ether,58 4-hydroxybenzyl alcohol,59 4-hydroxybenzoic acid,36 and 4-hydroxybenzyl methyl ether.60

The isolated compounds were assayed for their hepatoprotective effect by the D-GalN-induced normal human HL-7702 hepatocyte injury model and the APAP-induced HepG2 cell injury model, respectively, using the hepatoprotective drug bicyclol as the positive control (Table 4). Compared to the 23% viability for the D-GalN-treated cells, compounds 5, 6, 10, and 15 showed significant in vitro hepatoprotective activity with increasing cell viability by 27%, 22%, 19%, and 31% (cf. bicyclol, 14%) at 10 μM, respectively, which may imply a potential therapeutic effect on viral hepatitis.48 Besides, compounds 4, 9, and 11 exhibited moderate hepatoprotective activity with increasing cell viability by 9%, 16%, and 12% compared to the model group (cf. bicyclol, 9%) at 10 μM when added into resuscitated HepG2 cells incubated with APAP for 48 h, respectively, which may have a positive effect on preventing NAFLD from causing more serious liver damage.50,51 However, the pathophysiologies of liver injury after D-GalN and APAP overdose are complex and different. The characteristics of the D-GalN model of liver injury are multifocal degeneration and coagulative necrosis with infiltration of inflammatory cells, as well as reaction of M1-/M2-macrophages.48,61 The toxicity of APAP is initiated by the formation of a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which can deplete glutathione and binds to cellular proteins, resulting in mitochondrial oxidant stress, profound mitochondrial dysfunction, and overproduction of peroxynitrite, ultimately leading to cell death.51,62 Therefore, further study is needed to obtain reliable mechanistic information by which these compounds exert hepatoprotective effects in these two different liver injury models and their potential therapeutic effects on viral hepatitis and NAFLD in the future.

Table 4.

Hepatoprotective Effect of Compounds 1−17 (10 μM)

D-GalN model of liver injury in HL-7702 cells
 APAP model of liver injury in HepG2 cells
no. OD value cell survival rate increase of cell viability OD value cell survival rate increase of cell viability
control 1.14 ± 0.027 100% 1.29 ± 0.051 100%
D-GalNa 0.40 ± 0.090 ### 33% / / /
APAPb / / / 0.30 ± 0.062### 23%
bicyclolc 0.56 ± 0.010* 47% 14% 0.41 ± 0.024* 32% 9%
1 0.48 ± 0.039 39% 6% 0.27 ± 0.012 21% −2%
2 0.46 ± 0.011 38% 5% 0.22 ± 0.009 17% −6%
3 0.52 ± 0.050 43% 10% 0.33 ± 0.023 25% 2%
4 0.47 ± 0.036 39% 6% 0.41 ± 0.011* 32% 9%
5 0.70 ± 0.032* 60% 27% 0.35 ± 0.009 27% 4%
6 0.64 ± 0.022* 55% 22% 0.36 ± 0.014 28% 5%
7 0.58 ± 0.018 49% 16% 0.24 ± 0.008 19% −4%
8 0.47 ± 0.013 38% 5% 0.36 ± 0.016 28% 5%
9 0.44 ± 0.011 36% 3% 0.50 ± 0.012** 39% 16%
10 0.61 ± 0.025* 52% 19% 0.30 ± 0.021 23% 0
11 / / / 0.45 ± 0.015** 35% 12%
12 0.47 ± 0.008 39% 6% 0.18 ± 0.013 14% −9%
13 / / / 0.35 ± 0.027 27% 4%
14 / / / 0.30 ± 0.002 23% 0
15 0.74 ± 0.017* 64% 31% 0.36 ± 0.009 28% 5%
16 / / / 0.36 ± 0.005 28% 5%
17 / / / 0.30 ± 0.004 23% 0
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a

At 60 mM.

b

At 8 mM.

c

At 10 μM.

###

P < 0.001, compared with control group;

*

P < 0.05,

**

P < 0.01, compared with model group; / not evaluated

EXPERIMENTAL SECTION

General Experimental Procedures.

Optical rotations were measured on a JASCO P-2000 polarimeter and UV spectra with a JASCO J-1810 spectrophotometer. IR spectra were recorded on a Nicolet 5700 spectrometer by an FT-IR microscope transmission method. The 1H and 13C NMR spectra were obtained at 500/600 MHz for 1H and 125/150 for 13C, respectively, on an INOVA 500 MHz spectrometer, a Bruker AV-III-500 MHz spectrometer, and a VNS-600 MHz spectrometer. Chemical shifts are given in δ (ppm) values relative to those of the solvent signal [DMSO (δH 2.50; δC 39.51), D2O (δH 3.30)] on the TMS scale. The standard pulse sequences programmed into the instrument were used for each 2D measurement. ESIMS was performed using an Agilent 1100 series LC/MSD ion trap mass spectrometer. HRESIMS was performed using an Agilent 6520 Accurate-Mass Q-Tof LC/MS mass spectrometer. Reversed-phase preparative HPLC was carried out on a Shimadzu instrument (pump LC-6AD, UV detector SPD-20A, 224 nm) equipped with a Shimadzu shim-pack PRC-ODS column (20.0 i.d. × 250 mm, S-5 μm) or Thermo BDS HYPERSIL C18 column (10.0 i.d. × 250 mm, S-5 μm) eluted with MeOH–H2O or MeCN–H2O at room temperature. Analytical HPLC was performed on a Shimadzu instrument (pump LC-20AT, UV detector SPD-M20A, 224 nm) equipped with an Alltech Previal C18 column (4.6 i.d. × 250 mm, S-5 μm) eluted with water–MeOH (flow rate, 1 mL/min) at room temperature. Column chromatography (CC) was carried out on a CombiFlash Rf 200 chromatograph (Teledyine Isco) or ordinary pressure chromatograph, using RP-18 (50 μm, YMC) and Sephadex LH-20 (GE Healthcare Bio-Science AB). Fractions were monitored by TLC, and spots were visualized under UV light or by spraying with 10% H2SO4 in 95% EtOH followed by heating.

Plant Material.

The pseudobulbs of Pleione yunnanensis were collected in Guizhou Province, People’s Republic of China, in March 2011, and identified by Prof. Ma Lin (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, People’s Republic of China). A voucher specimen (No. ID-S-2430) is deposited at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College.

Extraction and Isolation.

The air-dried pseudobulbs of P. yunnanensis (20 kg) were powdered and extracted with 480 L of 95% EtOH at room temperature. The EtOH extract was concentrated in vacuo to yield a homogenate (2.7 kg). The extract was suspended in H2O (4 L, 1.025 g/mL) and was applied to an HP-20 macroporous adsorbent resin (14 × 100 cm) column eluting with H2O (200 L) and 15% (80 L), 40% (160 L), 75% (20 L), and 95% EtOH (100 L) successively. The 40% EtOH eluent (30 g) was suspended in H2O (200 mL) and subjected to RP-MPCC (3.7 i.d. × 46 cm), eluting with a H2O−MeOH gradient system (1:0 → 9:1 → 8:2 → 7:3 → 6:4 → 5:5 → 4:6 → 2:8 → 0:1, v/v, 1.5 L each) successively, to afford seven fractions. Fraction 3 (3.3 g) was chromatographed on a Sephadex LH-20 column (3.0 i.d. × 80 cm) and eluted with H2O to afford five subfractions (3A−3E) based on TLC. Subfraction 3C (460 mg) was chromatographed by semipreparative HPLC (20% MeCN−H2O) to give 13 (72 mg) and 17 (21 mg). Subfraction 3D (420 mg) was further purified by semipreparative HPLC (40% MeOH−H2O) to give 15 (25 mg) and 16 (34 mg). Fraction 4 (4.0 g) was chromatographed on a Sephadex LH-20 column (3.0 i.d. × 72 cm) and eluted with MeOH to afford four subfractions (4A−4D). Subfraction 4B (2.1 g) was chromatographed on an MCI CHP20 column (2.0 i.d. × 70 cm) and eluted with MeOH−H2O (30:70, v/v) to afford three subfractions, 4B-30A−4B-30C. Subfraction 4B-30B (620 mg) was further purified by semipreparative HPLC (32% MeCN−H2O) to give 8 (32 mg) and 9 (45 mg). Fraction 5 (6.2 g) was chromatographed on a Sephadex LH-20 column (3.0 i.d. × 72 cm) and eluted with MeOH to afford four subfractions (5A−5D) based on TLC. Subfraction 5C (2.0 g) was further chromatographed on a Sephadex LH-20 column (3.0 i.d. × 72 cm) and eluted with MeOH to afford five subfractions (5C1−5C5) based on TLC. Subfraction 5C1 (2.0 g) was purified by semipreparative HPLC (55% MeOH−H2O) to give 2 (10 mg), 3 (23 mg), 11 (146 mg), and 14 (21 mg). Subfraction 5C3 (1.2 g) was further purified by semipreparative HPLC (42% MeOH−H2O) to give bis(4-hydroxybenzyl) ether (54 mg), 4-hydroxybenzyl alcohol (17 mg), 4-hydroxybenzoic acid (25 mg), and 4-hydroxybenzyl methyl ether (76 mg). Fraction 6 (4.3 g) was chromatographed on a Sephadex LH-20 column (3.0 i.d. × 72 cm) and eluted with MeOH to afford six subfractions (6A−6F) based on TLC. Subfraction 6E (2.7 g) was purified by semipreparative HPLC (33% MeCN−H2O) to give 4 (27 mg) and 12 (30 mg). Fraction 7 (1.9 g) was chromatographed on a Sephadex LH-20 column (3.0 i.d. × 72 cm) and eluted with MeOH to afford seven subfractions (7A−7G) based on TLC. Subfraction 7B (1.5 g) was subjected to a Sephadex LH-20 column (1.5 i.d. × 170 cm) and eluted with MeOH to afford five subfractions (7B1−7B5). Subfraction7B3 (1.35 g) was subjected to an ODS column (2.0 i.d.× 40 cm) and eluted with MeOH−H2O (48:52 → 52:48 → 56:44 → 60:40, v/v, 240 mL each) to afford eight subfractions, 7B3A−7B3H. Subfraction7B3E (222 mg) was purified by semipreparative HPLC (55% MeOH−H2O) to give 1 (28 mg), 5 (32 mg), 6 (54 mg), and 10 (52 mg). Subfraction7B3F (668 mg) was purified by RP-HPLC with MeOH−H2O (55:45, v/v) to give eight subfractions, 7B3F-1−7B3F-8. Subfraction 7B3F-5 (109 mg) was purified by semipreparative HPLC (40% MeCN−H2O) to give 7 (49 mg).

Pleionoside M (1): white amorphous powder; [α]20D −16 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.41), 223 (4.48), 301 (4.15), 310 (4.17) nm; IR (KBr) νmax 3392, 2957, 2877, 1728, 1608, 1514, 1450, 1394, 1348, 1232, 1171, 1075, 1045, 896, 833 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, Table 1; ESIMS m/z 1057.4 [M + Na]+, and 1033.5 [M − H]–; HRESIMS m/z 1057.3555 [M + Na]+ (calcd for C49H62O24Na, 1057.3523).

PleionosideN (2): white amorphous powder; [α]20D −44 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.57), 223 (4.63), 311 (4.26) nm; IR (KBr) νmax 3408, 2956, 2877, 1728, 1608, 1514, 1451, 1393, 1232, 1171, 1075, 1040, 896, 853, 832 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, Table 1; ESIMS m/z 1219.5 [M + Na]+, and 1231.5 [M + Cl]–; HRESIMS m/z 1219.4086 [M + Na]+ (calcd for C55H72O29Na, 1219.4051).

PleionosideO (3): white amorphous powder; [α]20D −70 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 201 (4.52), 223 (4.62), 299 (4.32), 313 (4.37) nm; IR (KBr) νmax 3397, 2956, 2925, 1728, 1606, 1514, 1449, 1393, 1232, 1171, 1075, 1039, 897, 835 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, Table 1; ESIMS m/z 1219.5 [M + Na]+, 1195.4 [M − H]–, and 1231.4 [M + Cl]–; HRESIMS m/z 1219.4068 [M + Na]+ (calcd for C55H72O29Na, 1219.4051).

Pleionoside P (4): white amorphous powder; [α]20D −62 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.34), 224 (4.42), 271 (3.49), 276 (3.49) nm; IR (KBr) νmax 3404, 2920, 2875, 1734, 1614, 1514, 1452, 1394, 1232, 1173, 1074, 1016, 897, 829 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, Tables 2 and 3; ESIMS m/z 1017.4 [M + Na]+, 993.4 [M − H]–, and 1029.4 [M + Cl]–; HRESIMS m/z 1017.3584 [M + Na]+ (calcd for C47H62O23Na, 1017.3574).

Pleionoside Q (5): white amorphous powder; [α]20D −49 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.65), 224 (4.78), 271 (3.77), 276 (3.76) nm; IR (KBr) νmax 3410, 2876, 1733, 1614, 1514, 1452, 1232, 1074, 1016, 892, 829 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, Tables 2 and 3; ESIMS m/z 855.5 [M + Na]+, 871.3 [M + K]+, 831.3 [M − H]–, and 867.3 [M + Cl]–; HRESIMS m/z 855.3055 [M + Na]+ (calcd for C41H52O18Na, 855.3046).

Pleionoside R (6): white amorphous powder; [α]20D −42 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 201 (4.34), 224 (4.43), 271 (3.40), 276 (3.39) nm; IR (KBr) νmax 3398, 2953, 2876, 1733, 1614, 1514, 1452, 1392, 1311, 1232, 1073, 829 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, Tables 2 and 3; ESIMS m/z 855.3 [M + Na]+, 871.2 [M + K]+, 831.4 [M − H]–, and 867.4 [M + Cl]–; HRESIMS m/z 855.3054 [M + Na]+ (calcd for C41H52O18Na, 855.3046).

Pleionoside S (7): white amorphous powder; [α]20D −48 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (4.28), 224 (4.38), 271 (3.45), 276 (3.39) nm; IR (KBr) νmax 3395, 2955, 2875, 1733, 1614, 1514, 1452, 1393, 1309, 1232, 1074, 1016, 894, 828 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, Tables 2 and 3; ESIMS m/z 855.2 [M + Na]+, 831.2 [M − H]–, and 867.1 [M + Cl]–; HRESIMS m/z 855.3064 [M + Na]+ (calcd for C41H52O18Na, 855.3046).

Pleionoside T (8): white amorphous powder; [α]20D −46 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.22), 223 (4.36), 270 (3.23), 277 (3.15) nm; IR (KBr) νmax 3397, 2955, 2927, 1733, 1613, 1513, 1386, 1233, 1174, 1073, 927, 831 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) data, Tables 2 and 3; ESIMS m/z 911.3 [M + Na]+, 927.3 [M + K]+, 887.2 [M − H]–, and 923.4 [M + Cl]–; HRESIMS m/z 911.3166 [M + Na]+ (calcd for C40H56O22Na, 911.3155).

Pleionoside U (9): white amorphous powder; [α]20D −50 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (4.22), 223 (4.28), 270 (3.22), 277 (3.16) nm; IR (KBr) νmax 3390, 2958, 2928, 1735, 1613, 1513, 1384, 1233, 1175, 1233, 1075, 831 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz) data, Tables 2 and 3; ESIMS m/z 911.4 [M + Na]+, 887.3 [M − H]–, and 923.3 [M + Cl]–; HRESIMS m/z 911.3154 [M + Na]+ (calcd for C40H56O22Na, 911.3155).

Acidic Hydrolysis of 1−9 and the Discrimination of Glucose Enantiomers.

Authentic sugar samples D- and L-glucose (two reactions, 5 mg respectively) and L-cysteine methyl ester (5 mg) were dissolved in pyridine (1 mL) and heated at 60 °C for 1 h. o-Tolyl isothiocyanate (5 mg) was added, and the mixture was further heated for 1 h. The reaction mixture (2 μL) was analyzed by HPLC and detected at 250 nm. Analytical HPLC was performed on a Cosmosil 5C18-AR II column (250 × 4.6 mm i.d., Waters, America) at 25 °C with isocratic elution of 25% MeCN in 50 mM H3PO4 for 25 min at a flow rate of 0.8 mL/min. Peaks were detected with an SPD-M20A photodiode array detector (Shimadzu).

A solution of each compound (4−6 mg) in 1 N HCl (2.0 mL) was individually refluxed at 80 °C for 6 h. The reaction mixture was extracted with EtOAc (3 × 5 mL), and the aqueous phase was evaporated under reduced pressure. Each residue was subjected to the derivatization reaction and analyzed in the same way as the standard glucose sample. The absolute configuration of the glucose was defined via HPLC analysis at 250 nm by comparison with an authentic glucose sample after derivatization, whose product is methyl 2-(polyhydroxyalkyl)-3-(o-tolylthiocarbamoyl)-thiazolidine-4(R)-carboxylates.52 (Figure S1, Supporting Information).

Alkaline Hydrolysis of 1–9.

Compounds 1–9 (3–7 mg) were individually added to 3% aqueous NaOH solution (5 mL), and the mixture was stirred at room temperature for 2 h. The reaction mixture was acidified to pH 4 with 2 N HCl and partitioned with EtOAc. The organic layer was subjected to RP-HPLC using 30% MeOH as mobile phase to give (R)-2-hydroxy-2-isobutylsuccinic acid in each instance (0.52−1.46 mg, respectively).

(R)-2-Hydroxy-2-isobutylsuccinic acid: amorphous powder; [α]20D −9 (c 0.2, MeOH), lit. [α]26D −7.8 (c 0.05, MeOH);52 ESIMS m/z 190.9 [M + H]+, 212.9 [M + Na]+, 228.8 [M + K]+, and 188.6 [M − H]–; 1H NMR (CD3OD, 500 MHz): 2.90 (1H, d, J = 16.0 Hz, H-3a), 2.59 (1H, d, J = 16.0 Hz, H-3b), 1.82 (1H, dq, J = 6.5, 13.0 Hz, H-6), 1.68 (1H, dd, J = 6.0, 14.0 Hz, H-5a), 1.59 (1H, dd, J = 6.0, 14.0 Hz, H-5b), 0.97 (3H, d, J = 6.5 Hz, H-8), 0.91 (3H, d, J = 6.5 Hz, H-7).

Hepatoprotective Activity Assay.

All the compounds were tested for hepatoprotective activity against APAP-induced toxicity in HepG2 cells by means of a published MTT method,63 and compounds 1−10, 12, and 15 were also evaluated for their hepatoprotective activity against D-GalN-induced toxicity in HL-7702 cells by means of a published MTT method.64 Bicyclol was used as a positive control for both of them. The bioactivity data are collated in Tables 4 and S1.

Supplementary Material

SI_Hepatoprotective Glucosyloxybenzyl 2‑Hydroxy-2-isobutylsuccinates fromPleione yunnanensis

Chart 1.

Chart 1.

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ACKNOWLEDGMENTS

This study was financially supported by the National Natural Science Foundation of China (Nos. 81730093, 81173529).

Footnotes

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01117.

UV, IR, ESIMS, HRESIMS, 1H NMR, 13C NMR, DEPT, HMBC, HSQC, COSY, and NOESY data for compounds 1–9 and ESIMS and 1H NMR data for (R)-2-hydroxy-2-isobutylsuccinic acid (PDF)

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jnatprod.0c01117

The authors declare no competing financial interest.

DEDICATION

Dedicated to Dr. A. Douglas Kinghorn, The Ohio State University, for his pioneering work on bioactive natural products.

Contributor Information

Shao-wei Han, State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China.

Xiao-juan Wang, State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China; Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, Medical University of South Carolina, Charleston, South Carolina 29425, United States.

Bao-song Cui, State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China.

Hua Sun, State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China.

Hui Chen, State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China.

Daneel Ferreira, Department of BioMolecular Sciences, Division of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States.

Shuai Li, State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China.

Mark T. Hamann, Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, Medical University of South Carolina, Charleston, South Carolina 29425, United States

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Supplementary Materials

SI_Hepatoprotective Glucosyloxybenzyl 2‑Hydroxy-2-isobutylsuccinates fromPleione yunnanensis
NIHMS1993489-supplement-SI_Hepatoprotective_Glucosyloxybenzyl_2_Hydroxy-2-isobutylsuccinates_fromPleione_yunnanensis.pdf (11.5MB, pdf)

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