Three new abietane-type diterpene glycosides from the roots of Tripterygium wilfordii

Fitoterapia 120 (2017) 126–130

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Three new abietane-type diterpene glycosides from the roots of Tripterygium wilfordii

MARK

Jianqun Liua,⁎, Qiushan Wua, Jicheng Shua, Rui Zhanga, Lifang Liub,⁎ a Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, No.818 Xingwan Road, Nanchang 330004, Jiangxi Province, PR China b State Key Laboratory of Natural Medicines, Department of Chinese Medicines Analysis, China Pharmaceutical University,No.24 Tongjiaxiang Road, Nanjing 210009, Jiangsu Province, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords: Tripterygium wilfordii Abietane diterpene glycoside Tripterycoside a Tripterycoside B Tripterycoside C

Three new abietane-type diterpene glycosides named as tripterycoside A (1), tripterycoside B (2), tripterycoside C (3), along with two known ones, 11-O-β-D-glucopyranosyl-neotritophenolide (4), wilfordoside A (5), and nine other known compounds, 5-hydroxymethylmellein (6),1,2-bis-(3-methoxy-4-hydroxyphenyl)-1,3-propanediol (7), leptolepisol C (8), icariol A2 (9), tripfordine A (10), 16α-hydroxy-ent-kauran-19-oic acid (11),wilforine (12), wilfordine (13), 3-acetyloleanolic acid (14),were isolated from the roots of Tripterygium wilfordii. Their structures have been elucidated on the basis of NMR and MS data. To the best of our knowledge, abietane-type diterpene glycosides were rarely reported natural products, especially abietane-type diterpene glycoside containing 7-oxo group (compound 3) was reported here for the first time. Furthermore, compounds 6, 7, 8 and 14 were isolated from this plant for the first time. Compounds 1–5 showed statistically significant inhibitory effects on IL-1β secretion in LPS-induced rat primary synovial fibroblasts at 10 μM.

1. Introduction Tripterygium wilfordii Hook. f. (Celastraceae) mainly distributed in the middle and lower reaches of Yangtze River in China [1]. It has been used as Traditional Chinese Medicine for the treatment of inflammation, rheumatoid arthritis, chronic nephritis, lupus erythematosus and tumor and so on [2]. Previous chemical investigation indicated that the main components of Tripterygium wilfordii were abietane-type diterpenes, alkaloids, triterpenes and lignans [3–5]. A lot of abietane-type diterpene aglycones but only three glycosides have been isolated from this plant [6,7]. Many abietane-type diterpenes such as triptolide have demonstrated anti-inflammatory, immunosuppression and antitumor activities [8,9]. This paper reports on the structural elucidation and anti-inflammatory in vitro activities of three novel abietane-type diterpene glycosides named as tripterycoside A (1), tripterycoside B (2) and tripterycoside C (3). 2. Experimental 2.1. General experimental procedures Melting points were determined on an XT4-100A (Shanghai Jicheng

⁎

Corresponding authors. E-mail addresses: [email protected] (J. Liu), [email protected] (L. Liu).

http://dx.doi.org/10.1016/j.fitote.2017.06.001 Received 17 January 2017; Received in revised form 2 June 2017; Accepted 4 June 2017 Available online 07 June 2017 0367-326X/ © 2017 Elsevier B.V. All rights reserved.

Analytical Instrument Co., Ltd. China) micro-melting point apparatus and are uncorrected. Alumina gel, silica gel, MCI gel, Sephadex LH20 and Rp-C18 were used for column chromatography (CC). All preparative reverse-phase HPLC separation was carried out on an YMC-Pack ODS-A column (250 ∗ 10 mm, 5 μm, Jpn) and the detection wavelength was set at 210 nm. UV spectra were recorded on a Shimadzu UV-2501PC UV–VIS spectrometer (Shimadzu Corporation, Japan). One- and twodimensional NMR spectra were recorded on a Bruker AV-600 instrument (Bruker BioSpin Group, Switzerland) operating at 600 and 150 MHz for 1H and 13C NMR, respectively. Chemical shifts are reported in δ value in ppm using the solvent as reference. HR-TOF-MS spectra were recorded on an LC 30A-AB 5600 Triple TOF mass spectrometer (AB Sciex, Inc., USA). 2.2. Plant material The root of Tripterygium Wilfordii was collected from Pingxiang city of Jiangxi province, PR China, in October 2014, and authenticated by Prof. Jianqun Liu. A voucher specimen (No.20141001) is deposited in the Herbarium of Jiangxi University of Traditional Chinese Medicine.

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2.3.2 Tripterycoside B (2) Light yellow amorphous powder (MeOH); mp 172–173 °C; UV (MeOH): λmax (log ε) 286 nm (3.44), λmax (log ε) 219 nm (4.23); HR-TOFMS: [M + H]+, found: m/z 491.2269, calc: m/z 491.2276; [M − H]−, found: m/z 489.2126, calc: m/z 489.2130, for molecular formula C26H34O9; 1H NMR and 13C NMR spectroscopic data (see Table 1). 2.3.3 Tripterycoside C (3) Yellow amorphous powder (MeOH); mp 175–176 °C; UV (MeOH): λmax (log ε) 366 nm (3.61), λmax (log ε) 272 nm (3.89), λmax (log ε) 217 nm (4.40); HR-TOF-MS: [M + H]+, found: m/z 505.2064, calc: m/ z 505.2068; [M − H]−, found: m/z 503.1902, calc: m/z 503.1923, for molecular formula C26H32O10; 1H NMR and 13C NMR spectroscopic data (see Table 1).

2.3. Extraction and isolation The air-dried roots of Tripterygium wilfordii (119 kg) were extracted three times with 95% ethanol under condition of reflux, yielding a residue (12 kg) after evaporation under reduced pressure. The residue was partitioned with EtOAc, yielding EtOAc solubles (1.87 kg) and EtOAc unsolubles (10.13 kg) after evaporation under reduced pressure. The EtOAc solubles (1.87 kg) were subjected to alumina gel column chromatography (CC) (200–300 mesh, 22.5 kg), eluted with gradients of petroleum ether-EtOAc (1:0, 4:1, 3:2, 2:3, v/v) and EtOAc-MeOH (1:0, 3:1, 1:1, 0:1), then eight combined fractions (Fr.1–8) were obtained. Fr.7 (15.6 g) was separated by MCI gel CC, eluted with MeOHH2O gradient system (0:1, 1:9, 3:7, 1:1, 7:3, 9:1, 1:0), to give seven fractions (Fr. 7.1–7.7). Fr.7.5 was chromatographed over Sephadex LH20 CC, eluted with MeOH to give nine combined fractions (Fr.7.5.1–7.5.9). Fr.7.5.6 (300 mg) was separated by preparative reverse-phase HPLC (The mobile phase was MeOH-H2O 39:61), then the compounds 1 (10.3 mg, retention time 37.2 min), 2 (20.2 mg, retention time 24.0 min), Fr.A (98 mg, retention time 48 min) and Fr.B (63 mg, retention time 45 min) were obtained, respectively. Fr.A (98 mg) was separated by preparative reverse-phase HPLC (The mobile phase was acetonitrile-H2O 33:67), then the compounds 3 (6 mg, retention time 13.5 min) and 4 (15.2 mg, retention time 14.9 min) were obtained, respectively. Fr.B (63 mg) was separated by preparative reverse-phase HPLC (The mobile phase was MeOH-H2O 38:62), then the compound 5 (12 mg, retention time 49.3 min) was obtained. Combined fractions of Fr.5 and Fr.6 (Named as Fr.56, 142.2 g) were subjected to MCI gel CC, eluting with gradient MeOH-H2O (0:1, 1:3, 1:1, 3:1, 1:0) to yield five fractions (Fr.56.1–56.5). Fr.56.3 (13.3 g) was subjected to silica gel CC (200–300 mesh, 270 g), eluted with petroleum ether-EtOAc (1:0, 7:2, 7:4, 1:1, 4:7, 2:7) and EtOAc-MeOH (1:0, 1:1, 0:1), then nine combined fractions (Fr.56.3.1–56.3.9) were obtained. Fr. 56.3.3 (20 mg) was separated by preparative reverse-phase HPLC (the mobile phase was MeOH-H2O 42:58), then to give compound 6 (4.5 mg, Retention time 19.5 min).Fr.56.3.4 (17 mg) was separated by preparative reversephase HPLC (the mobile phase was MeOH-H2O 15:85), then the compound 7 (4.5 mg, Retention time 23.0 min) was obtained. Fr. 56.3.6 (80 mg) was separated by preparative reverse-phase HPLC (The mobile phase was MeOH-H2O 26:74), then the compounds 8 (9.1 mg, Retention time 36.0 min) and 9 (3.6 mg, Retention time 20.2 min) was obtained. Fr. 56.4(52.9 g) was subjected to MCI gel CC, eluting with gradient MeOH-H2O (0:1, 1:9, 3:7, 1:1, 7:3, 9:1, 1:0) to yield seven fractions (Fr.56.4.1–56.4.7). Fr. 56.4.4 (8 g) was subjected to silica gel CC (200–300 mesh, 200 g), eluted with petroleum ether-EtOAc (1:0, 1:1, 1:3, 1:5, 1:10,0:1) and EtOAc-MeOH (8:1, 4:1, 1:1, 1:3,0:1), then eleven combined fractions (Fr.56.4.4.1–56.4.4.11) were obtained, successively. Compound 10 was obtained from Fr.56.4.4.3 and purified by recrystallization in MeOH. Fr.56.4.5 (27 g) was isolated by silica gel CC (200–300 mesh, 540 g), eluted with CH2Cl2-acetone (1:0, 8:1, 7:3, 1:1,3:7, 1:9, 0:1) to give seven fractions (Fr.56.4.5.1–56.4.5.7). Fr.56.4.5.2 (50 mg) was separated by preparative reverse-phase HPLC (The mobile phase was MeOH-H2O 47:53), then to give compound 11 (20 mg, retention time 16.0 min). Fr.3(4 g) was subjected to a silica gel CC (200–300 mesh, 120 g) and eluted with CH2Cl2-acetone(1:0, 15:1, 10:1, 5:1, 0:1), to give compounds 12 and 13 from Fr.3.2 and Fr.3.4, respectively. Fr.4(25.7 g) was subjected to a silica gel CC (200–300 mesh, 642 g) eluting with petroleum ether-EtOAc (35:1, 21:4, 13:8, 1:1, 9:16, 1:4, 0:1) to give seven fractions(Fr.4.1–4.6). Compound 14 was obtained from Fr.4.2 and purified by recrystallization in MeOH.

2.4. Bioassay procedure Rat synovial fibroblasts were obtained as previously described in ref. [10]. The isolated synovium was minced and digested with collagenase II (4 mg/ml) for 2 h at 37 °C and then cultured in RPMI 1640 Medium until the cells outgrowing from the explants. Passages 3–6 of the synovial fibroblasts were used for the next experiments. The synovial fibroblasts were seeded in 48-well microplates at a density of 2 × 106 per well and were cultured in RPMI 1640 medium for 48 h, then treated with the test compounds. After 0.5 h in culture, cells were incubated with LPS (10 ng/ml)at 37 °C in a humidified atmosphere of 5% CO2 24 h. Finally, the concentration of cytokine IL-1β was assayed using ELISA kits. 3. Results and discussion Compound 1 was obtained as a white amorphous powder and its molecular formula was established as C26H34O9 by HR-TOF-MS spectrum ([M + H]+, found: m/z 491.2268, calc: m/z 491.2276; [M − H]−, found: m/z 489.2127, calc: m/z 489.2130). The UV spectrum (λmax: 221 and 286 nm) and NMR spectrum were suggestive of a structure containing a benzene ring. The 1H NMR spectrum showed three methyl signals (δH 1.14, d, J = 6.8 Hz, H-16; δH 1.15, d, J = 6.8 Hz, H-17; δH 1.14, s, H-20), an oxygenated methylene signal (δH 4.82, d, J = 17.6 Hz, H-19; δH 4.95, d, J = 17.6 Hz, H-19) and an aromatic proton signal (δH 6.52, s, H-12). The 13C NMR spectrum displayed twenty-six resonances, of which six resonances (δC 107.1, 75.8, 78.2, 71.9, 78.2, 63.0) were assigned to a glucopyranose moiety, and the other twenty resonances were assigned by HSQC technique to three methyls, five methylenes, three methines and nine quaternary carbons including a benzene ring, an olefinic and an ester carbonyl (δC 177.3, C-18) (Table 1). The 13C NMR (δC 154.5, C-11; δC 146.1, C-14) indicated that benzene ring should be substituted by a hydroxyl and a glucosyl. The presence of the glucose moiety was also confirmed by checking TLC on the acid hydrolysis. Furthermore, the configuration of the glucose anomeric position (δH 4.52, d) was judged to be β from a large 3JH-1′, H-2′ coupling constant (J = 7.7 Hz). This evidence indicated the presence of triptophenolide abietane-diterpene skeleton [6]. In general, the 1H and 13C NMR spectra of 1 were very similar to those of 11-O-β-D-glucopyranosyl-neotritophenolide [6] except for the absence of an oxygenated methyl signal in 1. The HMBC correlations of H-1′(δH 4.52, d) /C-14 (δC 146.1) correspond to the linkage between the diterpene moiety and the β-glucopyranose. In addition, The HMBC correlations of H-12 (δH 6.52, s)/C-15 (δC 26.6), H-12(δH 6.52, s)/C-14 (δC 146.1), H-12(δH 6.52, s)/C-9 (δC 130.2) and H-15(δH 3.64, m)/C-12 (δC 112.4) supported the location of hydroxyl group at C-11 (Fig. 2). Therefore, compound 1 was confirmed and named as tripterycoside A (1) (Fig. 1). Compound 2 was obtained as a light yellow amorphous powder and its molecular formula was established as C26H34O9 by HR-TOF-MS spectrum ([M + H]+, found: m/z 491.2269, calc: m/z 491.2276; [M − H]−, found: m/z 489.2126, calc: m/z 489.2130). MS spectrum indicated that 2 was an isomer of 1. The UV spectrum (λmax: 219 and

2.3.1. Tripterycoside A (1) White amorphous powder (MeOH); mp 204–205 °C; UV (MeOH): λmax (log ε) 286 nm (3.36), λmax (log ε) 221 nm (4.22); HR-TOF-MS: [M + H]+, found: m/z 491.2268, calc: m/z 491.2276; [M − H]−, found: m/z 489.2127, calc: m/z 489.2130, for molecular formula C26H34O9; 1H NMR and 13C NMR spectroscopic data (see Table 1). 127

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Table 1 1 H NMR (600 MHz) and

13

C NMR (150 MHz) spectral data

No

1

1

δC 32.3

2 3 4 5 6

19.6 125.3 167.6 45.7 20.7

7

28.1

8 9 10 11 12 13 14 15 16 17 18 19

133.6 130.2 38.5 154.5 112.4 141.2 146.1 26.6 24.3 24.5 177.3 72.6

20 1’ 2’ 3’ 4’ 5’ 6’

17.5 107.1 75.8 78.2 71.9 78.2 63.0

a

a

of compounds 1–3 in CD3OD (δ in ppm, J in Hz). 2

δH 1.49(m); 3.70(m) 2.31(m) – – 2.80(d,12.5) 1.68(qd,12.7,5.8); 1.86(dd,12.7,7.1) 3.01(dd,18.2,4.9); 3.11(m) – – – – 6.52(s) – – 3.64(m) 1.14(d,6.8) 1.15(d,6.8) – 4.82(d,17.6); 4.95(d,17.6) 1.14(s) 4.52(d,7.7) 3.47(dd,8.7,7.7) 3.12(m) 3.40(t,8.9) 3.36(dd,9.2,8.7) 3.65(m); 3.79(dd,11.8,2.3)

3

δC 33.0

δH 1.44(m); 3.83(m) 2.31(m) – – 2.73(m) 1.76(qd,12.8,6.0); 1.91(dd,12.8,7.7) 2.75(m); 2.89(dd,17.5,5.5) – – – – 6.88(s) – – 3.26(hept,6.8) 1.18(d,6.8) 1.21(d,6.8) – 4.82(d,17.6); 4.94(dd,17.6,1.5) 1.18(s) 5.00(d,7.6) 3.52(dd,9.1,7.6) 3.48(t,8.2) 3.38(m) 3.40(m) 3.68(dd,11.9,5.4); 3.87(dd,11.9,1.9)

19.7 125.7 167.1 45.7 20.5 27.1 127.0 132.8 38.6 151.7 111.7 135.3 147.4 28.0 23.3 23.6 177.3 72.6 18.2 102.3 75.4 79.1 71.7 78.3 62.8

δC 32.5 19.1 126.5 163.8 42.7 49.7

δH 1.66(m); 3.92(dt,13.1,3.8) 2.37(m) – – 3.31(m) 3.33(t,6.9)

205.6

–

116.7 137.4 39.4 148.8 158.6 138.2 125.9 27.8 22.7 22.6 176.6 72.2

– – – – – – 7.44(s) 3.33(hept,6.9) 1.25(d,6.9) 1.21(d,6.9) – 4.87(m)

17.4 103.9 75.3 78.5 71.6 78.8 62.7

1.31(s) 4.94(d,7.6) 3.53(t,7.6) 3.38(m) 3.39(m) 3.48(t,9.1) 3.68(dt,11.9,2.7); 3.87(dd,11.9,1.3)

Assignments were confirmed by coupling constants, HSQC and HMBC analysis.

were assigned by HSQC technique to three methyls, five methylenes, three methines and nine quaternary carbons including a benzene ring, an olefinic and an ester carbonyl (δC 177.3, C-18) (Table 1). The 13C NMR (δC 151.7, C-11; δC 147.4, C-14) indicated that benzene ring should be substituted by a hydroxyl and a glucosyl. The presence of the glucose moiety was also confirmed by checking TLC on the acid hydrolysis. Furthermore, the configuration of the glucose anomeric position (δH 5.00, d) was judged to be β from a large 3JH-1′, H-2′ coupling

286 nm) and NMR spectrum were suggestive of a structure containing a benzene ring. The 1H NMR spectrum showed three methyl signals (δH 1.18, d, J = 6.8 Hz, H-16; δH 1.21, d, J = 6.8 Hz, H-17; δH 1.18, s, H20), an oxygenated methylene signal (δH 4.82, d, J = 17.6 Hz, H-19; δH 4.94, dd, J = 17.6, 1.5 Hz, H-19) and an aromatic proton signal (δH 6.88, s, H-12). The 13C NMR spectrum displayed twenty-six resonances, of which six resonances (δC 102.3, 75.4, 79.1, 71.7, 78.3, 62.8) were assigned to a glucopyranose moiety, and the other twenty resonances

Fig. 1. Structures of compounds 1–5.

6'

HO

O

5'

O HO

R1

4' HO

3'

R2

2'

O

1'

2

14

9 10

8

3 H

7

O

4

18

1 R1=H, R2=β-D-glucose 2 R1=β-D-glucose, R2=H 4 R1=β-D-glucose, R2=CH3

5 H

19

O

3

HO

OH

O OH O O H

HO O

HO OH

15

13

17

1

O

12

11

20

OH

O

O

16

OH

5 128

6

O

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J. Liu et al.

HO

HO

O HO

O

HO OH

O

OH O O

O HO O

O

2

HO HO HO

OH

OH

O

1

HO

O

HO OH

O

O O

H

3 C HMBC

Fig. 2. Key HMBC correlations of compounds 1–3.

constant (J = 7.6 Hz). In general, the 1H and 13C NMR spectra of 2 were also very similar to those of 11-O-β-D-glucopyranosyl-neotritophenolide [6] except for the absence of an oxygenated methyl signal in 2. The HMBC correlations of H-1′(δH 5.00, d)/C-11 (δC 151.7) correspond to the linkage between the diterpene moiety and the βglucopyranose. In addition, The HMBC correlations of H-12(δH 6.88, s)/ C-15 (δC 28.0), H-12(δH 6.88, s)/C-9 (δC 132.8), H-12(δH 6.88, s)/C-14 (δC 147.4), H-15 (δH 3.26, hept)/C-12 (δC 111.7) and H-7(δH 2.75, m; δH 2.89, m)/C-14 (δC 147.7) supported the location of hydroxyl group at C-14 (Fig. 2). Therefore, compound 2 was confirmed and named as tripterycoside B (2) (Fig. 1). Compound 3 was obtained as a yellow amorphous powder and its molecular formula was established as C26H32O10 by HR-TOF-MS spectrum ([M + H]+, found: m/z 505.2064, calc: m/z 505.2068; [M − H]−, found: m/z 503.1902, calc: m/z 503.1923). The UV spectrum (λmax: 217, 272 and 366 nm) and NMR spectrum indicated that 3 contained a benzene ring. The 1H NMR spectrum showed three methyl signals (δH 1.25, d, J = 6.9 Hz, H-16; δH 1.21, d, J = 6.9 Hz, H-17; δH 1.31, s, H-20), an oxygenated methylene signal (δH 4.87, m) and an aromatic proton signal (δH 7.44, s, H-14). The 13C NMR spectrum displayed twenty-six resonances, of which six resonances (δC 103.9, 75.3, 78.5, 71.6, 78.8, 62.7) were assigned to a glucopyranose moiety, and the other twenty resonances were assigned by HSQC technique to three methyls, four methylenes, three methines and ten quaternary carbons including a benzene ring, an olefinic, an ester carbonyl (δC 176.6, C-18) and ketone group (δC 205.6, C-7) (Table 1). The 13C NMR (δC 148.8, C11; δC 158.6, C-12) indicated that benzene ring should be substituted by a hydroxyl and a glucosyl. The presence of the glucose moiety was also confirmed by checking TLC on the acid hydrolysis. Furthermore, the configuration of the glucose anomeric position (δH 4.94, d) was judged to be β from a large 3JH-1′, H-2′ coupling constant (J = 7.6 Hz). The above evidence indicated 3 was a triptophenolide abietane-diterpene, too. The HMBC correlations of H-1′(δH 4.94, d) /C-11 (δC 148.8) correspond to the linkage between the diterpene moiety and the β-

glucopyranose. The HMBC correlations of H-14(δH 7.44, s)/C-15 (δC 27.8), H-14(δH 7.44, s)/C-8 (δC 116.7), H-14(δH 7.44, s)/C-9 (δC 137.4), H-14(δH 7.44, s)/C-12 (δC 158.6), H-15(δH 3.33, hept)/C-14 (δC 125.9) and H-15(δH 3.33, hept) /C-12 (δC 158.6) supported the location of hydroxyl group at C-12. In addition, The HMBC correlations of H-5(δH 3.31)/C-7 (δC 205.6) and H-6(δH 3.33, t) /C-7 (δC 205.6) supported the location of ketone group at C-7 (Fig. 2), which was further confirmed by UV spectrum. The UV absorption (λmax: 366 nm) showed an extended conjugated system in 3, so the ketone group should be adjacent to the benzene ring. Thus, compound 3 was confirmed and named as tripterycoside C (3) (Fig. 1). The known compounds were identified by comparing with the corresponding literature spectroscopic data as 11-O-β-D-glucopyranosyl neotritophenolide (4) [6], Wilfordoside A (5) [7], 5-Hydroxymethylmellein (6) [11],1,2-bis-(3-methoxy-4-hydroxyphenyl)-1,3-propanediol (7) [12], leptolepisol C (8) [13], icariol A2 (9) [14], tripfordine A (10) [15], 16αHydroxy-ent-kauran-19-oic acid (11) [16],wilforine (12) [17], wilfordine (13) [18], 3-acetyloleanolic acid (14) [19], respectively. Tripterygium genus includes three species, Tripterygium wilfordii Hook. f., Tripterygium hypoglaucum (Levl.) Hutch and Tripterygium regelii Sprague et Takeda. Previous phytochemistry studies indicated that a lot of abietane-type diterpene aglycones were isolated as characteristic secondary metabolites from this genus, but only three glycosides were isolated from Tripterygium wilfordii [6,7]. In this paper, five rare abietane-type diterpene glycosides were isolated from Tripterygium wilfordii. Therefore, these glycosides might be useful taxonomic markers of Tripterygium wilfordii. Compounds 1–5 were tested for their activities against inflammation. In an in vitro bioassay, their inhibitory effects on IL-1β secretion in LPS-induced Primary synovial fibroblasts were evaluated. They showed statistically significant inhibitory effects on IL-1β secretion at 10 μM (Table 2).

129

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Table 2 Inhibitory effect of the compounds 1–5 against LPS-induced IL-1β secretion in primary synovial fibroblasts cells (mean ± SD, n = 6). Groups

C (μM)

IL-1β (pg/mL)

Control Model 1 2 3 4 5 Prednisolone

– – 10 10 10 10 10 10

96.37 ± 4.96⁎⁎⁎ 123.98 ± 5.11 96.58 ± 4.16⁎⁎⁎ 89.35 ± 9.57⁎⁎⁎ 98.05 ± 11.39⁎⁎⁎ 91.35 ± 10.18⁎⁎⁎ 87.17 ± 9.25⁎⁎⁎ 92.46 ± 7.87⁎⁎⁎

⁎⁎⁎

p < 0.001.

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