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Molecules 2015, 20(12), 21946-21959; doi:10.3390/molecules201219814

Article
Bioactive Constituents from the Aerial Parts of Lippia triphylla
Yi Zhang 1, Yue Chen 1, Shiyu Wang 2, Yongzhe Dong 1, Tingting Wang 2, Lu Qu 1, Nan Li 2 and Tao Wang 2,*
1
Tianjin State Key Laboratory of Modern Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China
2
Tianjin Key Laboratory of TCM Chemistry and Analysis, Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshan Road, Nankai District, Tianjin 300193, China
*
Correspondence: Tel./Fax: +86-22-5959-6163
Academic Editor: Christopher W. K. Lam
Received: 27 September 2015 / Accepted: 4 December 2015 / Published: 8 December 2015

Abstract

: Five new compounds, lippianosides A (1), B (2), C (3), D (4), and E (5), along with 26 (631) known ones were obtained from the 95% EtOH extract of Lippia triphylla (L. triphylla) aerial parts collected from Rwanda, Africa. Among the known compounds, 11 and 1730 were isolated from the Lippia genus for the first time. In addition, 12, 13, and 16 were firstly obtained from this species. The structures of them were elucidated by chemical and spectroscopic methods. The antioxidant and triglyceride accumulation inhibition effects of the 31 compounds were examined in L6 cells and HepG2 cells, respectively.
Keywords:
Lippia triphylla; lippianoside; structure elucidation; antioxidant; triglyceride accumulation inhibition; L6 cells; HepG2 cells

1. Introduction

The genus Lippia (L.) has a great economic value as a condiment and traditional medicine. Lippia leaves, flowers, and aerial parts were used in folk medicine for the treatment of respiratory and digestive system diseases [1]. Polyphenols from L. citriodora decreased triglyceride (TG) accumulation, the generation of reactive oxygen species (ROS) and restored mitochondrial membrane potential in adipocytes via ROS-mediated down-regulation of nuclear factor κB transcription factor, peroxisome proliferator-activated receptor γ-dependent transcription, upregulation of adiponectin and activation of AMP-activated protein kinase (AMPK) [2]. Essential oils from L. thymoides leaves had antimicrobial selectivity to Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus [3]. Methanolic extract of L. nodiflora leaves showed reduced effect on ROS production against LPS induced toxicity in HepG2 cells [4]. L. sidoides displayed immunomodulatory effects through the inhibition of cyclic nucleotide-dependent phosphodiesterase activity and activation of p38 MAPK pathway [5]. L. graveolens extract and its constituents, cirsimaritin, hispidulin, and naringenin, could inhibit dipeptidyl peptidase IV and protein tyrosine phosphatase, which indicated that L. graveolens was useful for type 2 diabetes management [6]. Oral administration of γ-sitosterol isolated from L. nodiflora once daily for 21 days in STZ-induced diabetic rats resulted in a significant decrease in blood glucose and glycosylated hemoglobin with a significant increase in plasma insulin level, and, subsequently, increased insulin secretion in response to glucose [7].

L. triphylla (L′Her.) O. Kuntze (syn. L. citrodora (Ort.) HBK) is a perennial, bushy plant of Verbenaceae family, commonly named lemon verbena. It grows spontaneously in many countries in South America, such as Brazil, Chile, Argentina, and Peru, had been introduced into Europe by the end of the 17th century, and has since been cultivated in North Africa and Southern Europe [8]. It contains special lemon-like fragrance, and is used against vertigo, nausea, and headaches in Greece [9].

During the course of our studies, we identified five new compounds, lippianosides A–E (15), along with 26 known ones (631) from the 95% EtOH extract of L. triphylla aerial parts collected from Rwanda. Their structures were elucidated by chemical and spectroscopic methods. Based on previous Lippia genus activity reports evidence, the antioxidant and TG accumulation inhibitory effects of the isolates were examined.

2. Results and Discussion

The 95% EtOH extract of L. triphylla was subjected to solvent partition, chromatographic isolation, and chemical and spectral analyses. As a result, five new compounds, lippianosides A–E (15) (Figure 1), together with 26 known ones (Figure 2), jionoside C(6) [10], trans-acteoside (7) [11], isoverbascoside (8) [12], cis-acteoside (9) [13], martynoside (10) [14], isomartynoside (11) [14], β-hydroxyacteoside (12) [15], campneoside I (13) [16], cistanoside F (14) [17], jaceosidin (15) [18], nepetin (16) [19], nepitrin (17) [20], dehydrodiconiferyl glucoside D (18) [21], dehydrodiconiferyl glucoside E (19) [21], (+)-lariciresinol-9-O-β-d-glucopyranoside (20) [22], (+)-pinoresinol 4-O-β-d-glucoside (21) [23,24], dihydrovomifoliol-O-β-d-glucopyranoside (22) [25,26], turpinionoside D (23) [27], 9-hydroxymegastigm-5-en-4-one (24) [28], (−)-loliolide (25) [29,30], eudesm-4(15)-ene-1β,6α-diol (26) [31], (6S)-3,7-dimethyl-7-hydroxy-2(Z)-octen-6-olide (27) [32], ursolic acid (28) [33], avicennone A (29) [34], benzyl alcohol O-β-d-glucopyranoside (30) [35], and icariside H1 (31) [36] were yielded and identified. Among the known ones, 11 and 1730 were isolated from the Lippia genus for the first time, and 12, 13, and 16 were obtained from this species for the first time.

Lippianoside A (1), [ α ] D 25 +7.9° (in MeOH), white powder. Its molecular formula, C27H36O12, was determined from the molecular ion peak at m/z 575.2113 [M + Na]+ by HR-Q-TOF-ESI-MS measurement. Acid hydrolysis of 1 with 1 M HCl yielded d-glucose, which was identified on the basis of retention time (HPLC) and optical rotation [37,38]. 1H- and 13C-NMR (DMSO-d6, Table 1) spectra suggested the following moieties presented in 1: a β-d-glucopyranosyl (δ 3.98 (1H, d, J = 8.0 Hz, H-1′′)), two ABX-type aromatic rings (δ 6.64 (1H, dd, J = 2.0, 8.0 Hz, H-6′), 6.71 (2H, J = 8.0 Hz, H-5 and 5′), 6.76 (1H, d, J = 2.0 Hz, H-2′), 6.77 (1H, dd, J = 2.0, 8.0 Hz, H-6), 6.88 (1H, d, J = 2.0 Hz, H-2)), three methoxy groups (δ 3.07, 3.73, 3.76 (3H each, all s, 7′, 3′, 3-OCH3)). In addition, the 1H and 13C-NMR spectra exhibited signals attributable to two methylenes bearing oxygen (δ 3.11 (1H, dd, J = 9.0, 9.0 Hz, H-9′a), 3.46 (1H, dd, J = 4.0, 9.0 Hz, H-9′b), 3.75 (1H, m, overlapped, H-9a), 4.05 (1H, dd, J = 4.5, 9.0 Hz, H-9b), two methines bearing oxygen (δ 3.97 (1H, d, J = 7.5 Hz, H-7′), 4.56 (1H, d, J = 7.5 Hz, H-7)), along with two aliphatic methines at δ 1.75 (1H, m, H-8) and 2.40 (1H, m, H-8′), respectively. According to the long-range correlations (Figure 3) observed from HMBC spectrum, the planar structure of 1 was determined. The coupling constant of H-7′ (J = 7.5 Hz) in 1 suggested an antiperiplanar orientation of H-7′ and H-8′. On the other hand, the CD spectrum of 1 (Δε: −121.7 (201 nm), −5.9 (228 nm)) was very similar to that of (7R,8S,7′S,8′R)-4,9,4′,7′-tetrahydroxy-3,3′-dimethoxy-7,9′-epoxylignan 9-O-β-d-glucopyranoside (Δε: −12.9 (205 nm), −2.5 (234 nm)) [39], which indicated the absolute configuration of 1 was 7R,8S,7′S,8′R. Furthermore, the NOE correlations between δH 4.56 (H-7) and δH 2.40 (H-8′) and 3.75 (H-9a); δH 1.75 (H-8) and δH 3.97 (H-7′); and δH 2.40 (H-8′) and δH 3.75 (H-9a), 6.64 (H-6′) and 6.76 (H-2′) observed in the NOESY spectrum confirmed the accuracy of configuration analysis. Finally, the structure of 1 was determined as (7R,8S,7′S,8′R)-4,9,4′-trihydroxy-3,3′,7′-trimethoxy-7,9′-epoxylignan 9-O-β-d-glucopyranoside.

Figure 1. The new compounds (15) obtained from L. triphylla.
Figure 1. The new compounds (15) obtained from L. triphylla.
Molecules 20 19814 g001 1024
Figure 2. The known compounds (631) obtained from L. triphylla.
Figure 2. The known compounds (631) obtained from L. triphylla.
Molecules 20 19814 g002 1024
Figure 3. The main 1H-1H COSY, HMBC, and NOE correlations of 1.
Figure 3. The main 1H-1H COSY, HMBC, and NOE correlations of 1.
Molecules 20 19814 g003 1024
Table 1. The 1H- and 13C-NMR data of 1.
Table 1. The 1H- and 13C-NMR data of 1.
No.1 a1 b
δCδH (J in Hz)δCδH (J in Hz)
1133.2-134.8-
2110.46.88 (d, 2.0)111.66.97 (d, 1.5)
3147.2-149.0-
4145.5-147.2-
5115.06.71 (d, 8.0)116.16.77 (d, 8.0)
6118.56.77 (dd, 2.0, 8.0)120.36.86 (dd, 1.5, 8.0)
782.74.56 (d, 7.5)85.34.76 (d, 7.0)
849.71.75 (m)51.61.96 (m)
969.53.75 (m, overlapped)70.43.17 (dd, 4.0, 10.0)
4.05 (dd, 4.5, 9.0) 3.55 (dd, 6.5, 10.0)
3-OCH355.53.76 (s)56.73.86 (s)
1′130.7-132.7-
2′110.96.76 (d, 2.0)112.16.79 (d, 1.5)
3′147.5-149.3-
4′145.9-147.6-
5′115.06.71 (d, 8.0)116.26.76 (d, 8.5)
6′120.26.64 (dd, 2.0, 8.0)121.86.70 (dd, 1.5, 8.0)
7′84.53.97 (d, 7.5)87.14.01 (d, 9.0)
8′48.52.40 (m)50.32.52 (m)
9′68.73.11 (dd, 9.0, 9.0)71.83.97 (dd, 9.0, 9.0)
3.46 (dd, 4.0, 9.0) 4.18 (dd, 4.0, 9.0)
3′-OCH355.73.73 (s)56.63.82 (s)
7′-OCH355.43.07 (s)56.73.16 (s)
1′′102.83.98 (d, 7.5)104.54.02 (d, 8.0)
2′′73.42.91 (dd, 7.5, 9.0)75.23.11 (dd, 8.0, 9.0)
3′′76.63.11 (dd, 9.0, 9.0)78.23.29 (dd, 9.0, 9.0)
4′′69.83.03 (dd, 9.0, 9.0)71.73.28 (dd, 9.0, 9.0)
5′′76.73.01 (m)77.93.16 (m)
6′′60.93.45 (dd, 5.5, 12.5)62.83.66 (dd, 5.5, 12.0)
3.61 (dd, 2.5, 12.5) 3.82 (dd, 2.5, 12.0)

a: determined in DMSO-d6; b: determined in CD3OD.

Lippianoside B (2) was obtained with negative optical rotation ( [ α ] D 25 −8.9° in MeOH). The molecular formula of 2 was revealed as C26H30O13 by HR-Q-TOF-ESI-MS (m/z 549.1604 [M − H], calcd for C26H29O13, 549.1614). Treatment of 2 with 1 M HCl gave d-glucose, which was identified by HPLC analysis [37,38]. 1H-, 13C-NMR (CD3OD, Table 2) and various 2D NMR spectra (Figure 4), indicated the presence of an iridoid moiety, a trans-feruloyl, and a β-d-glucopyranosyl in 2. In the HMBC experiment (Figure 4), the long-range correlations between the following proton and carbon pairs were observed: δH 4.98 (H-1) and δC 145.0 (C-8), 152.9 (C-3); δH 3.13 (H-5) and δC 145.0 (C-8), 171.0 (C-11); δH 2.69 (H-9) and δC 113.3 (C-4), 128.9 (C-7); δH 4.19, 4.25 (H2-10) and δC 46.7 (C-9), 128.9 (C-7), 145.0 (C-8); δH 4.73 (H-1′) and δC 98.9 (C-1); δH 4.40, 4.44 (H2-6′) and δC 169.1 (C-9′′). Finally, in the NOESY spectrum, the NOE correlations between δH 4.98 (H-1) and δH 1.96 (Hα-6); δH 3.13 (H-5) and δH 2.69 (H-9) and 2.75 (Hβ-6); and δH 2.75 (Hβ-6) and δH 2.69 (H-9) suggested the relative configuration of 2 was 1α,5β,9β. On the other hand, the 1H and 13C-NMR spectra of 2 were superimposable on those of 6′-O-trans-p-coumaroyl geniposidic acid [40], except for the signals due to trans-feruloyl group at 6′-position. Consequently, the structure of lippianoside B was elucidated to be 6′-O-trans-feruloyl geniposidic acid (2).

Figure 4. The main 1H-1H COSY, HMBC and NOE correlations of 2.
Figure 4. The main 1H-1H COSY, HMBC and NOE correlations of 2.
Molecules 20 19814 g004 1024
Table 2. The 1H- and 13C-NMR data of 2 in CD3OD.
Table 2. The 1H- and 13C-NMR data of 2 in CD3OD.
No.δCδH (J in Hz)No.δCδH (J in Hz)
198.94.98 (d, 8.0)3′77.83.43 (dd, 8.5, 9.0)
3152.97.49 (s)4′71.93.37 (dd, 9.0, 9.0)
4113.3-5′75.73.56 (m)
537.13.13 (q like, 8.0, 8.0)6′64.44.40 (dd, 6.0, 12.0)
639.91.96 (br. dd, ca. 8, 16) 4.44 (dd, 2.5, 12.0)
2.75 (br. dd, ca. 8, 16)1′′127.7-
7128.95.76 (br. s)2′′111.67.17 (br. s)
8145.0-3′′149.4-
946.72.69 (dd, 8.0, 8.0)4′′150.7-
1061.64.19 (d, 14.0)5′′116.56.81 (d, 8.0)
4.25 (d, 14.0)6′′124.37.05 (br. d, ca. 8)
11171.0-7′′147.17.59 (d, 16.0)
1′100.74.73 (d, 7.5)8′′115.36.35 (d, 16.0)
2′74.83.27 (dd, 7.5, 8.5)9′′169.1-
3′′-OCH356.53.88 (s)

Lippianoside C (3), [ α ] D 25 −61.4° (in MeOH), was isolated as a white powder. Its molecular formula, C27H38O12, was established by HR-Q-TOF-ESI-MS with m/z 557.2267 [M + Na]+ (calcd for C27H38O12Na, 557.2255). Its IR spectrum showed absorption bands due to hydroxyl (3384 cm−1), α,β-unsaturated carbonyl (1701 cm−1), aromatic ring (1603, 1514 cm−1), and ether function (1064 cm−1). Acid hydrolysis of 3 with 1 M HCl gave d-glucose and L-rhamnose [37,38]. The 1H-, 13C-NMR (CD3OD, Table 3) and various kinds of 2D NMR (Figure 5) including 1H-1H COSY, HMQC, and HMBC showed signals assignable to 2-hexene-1-alcohol, trans-p-coumaroyl, β-d-glucopyranosyl, and α-l-rhamnopyranosyl. In the HMBC experiment, the long-range correlations were observed between δH 4.36 (H-1′) and δC 70.7 (C-1); δH 5.19 (H-1′′) and δC 81.7 (C-3′); and δH 4.92 (H-4′) and δC 168.4 (C-9′′′), and the connectivities of the above-mentioned moieties were elucidated. Finally, in the NOESY experiment, the NOE correlations observed between δH 2.39 (H2-4) and δH 3.56, 3.87 (H2-1) as well as δH 5.47 (H-2) and δH 5.38 (H-3) indicated the double bond configuration of 2-position in 3 was Z.

Lippianoside D (4) was a white powder with negative optical rotation ( [ α ] D 25 −62.1° in MeOH). Its elemental composition was determined to be C19H34O8 by HR-Q-TOF-ESI-MS observed at m/z 413.2158 [M + Na]+. The 1H and 13C-NMR (CD3OD, Table 4) showed signals due to four methyl (δ 1.05, 1.07, 1.78 (3H each, all s, H3-11, 12, 13), 1.20 (3H, d, J = 6.0 Hz, H3-10)), three methylene (δ (1.38 (1H, dd, J = 5.0, 13.0 Hz), 1.74 (1H, ddd, J = 13.0, 13.0 Hz), H2-2), 1.57, 1.66 (1H each, both m, H2-8), (1.99 (1H, ddd, J = 5.0, 13.0, 13.0 Hz), 2.27 (1H, ddd, J = 5.0, 13.0, 13.0 Hz), H2-7)), three methine bearing an oxygen function (δ 3.71 (1H, ddd, J = 4.0, 5.0, 13.0 Hz, H-3), 3.73 (1H, br. d, ca. J = 4 Hz, H-4), 3.90 (1H, m, H-9)), together with a β-d-glucopyranosyl (δ 4.35 (1H, d, J = 7.5 Hz, H-1′)). The 1H-1H COSY experiment indicated the presence of partial structure written in bold lines (Figure 6). According to the long-range correlations (Figure 6) observed from HMBC spectrum, the planar structure of 4 was determined. Finally, treatment of 4 with 1 M HCl liberated d-glucose [37,38]. Enzymatic hydrolysis of it with β-glucosidase gave (3S,4R,9R)-3,4,9-trihydroxymegastigman-5-ene [27] as aglycon. Then, the structure lippianoside D was determined to be (3S,4R,9R)-3,4,6-trihydroxymegastigman-5-ene 9-O-β-d-glucopyranoside (4).

Figure 5. The main 1H-1H COSY, HMBC and NOE correlations of 3.
Figure 5. The main 1H-1H COSY, HMBC and NOE correlations of 3.
Molecules 20 19814 g005 1024
Figure 6. The main 1H-1H COSY and HMBC correlations of 4.
Figure 6. The main 1H-1H COSY and HMBC correlations of 4.
Molecules 20 19814 g006 1024
Table 3. The 1H- and 13C-NMR data of 3 in CD3OD.
Table 3. The 1H- and 13C-NMR data of 3 in CD3OD.
No.δCδH (J in Hz)No.δCδH (J in Hz)
170.73.56 (m) 3.87 (m)2′′72.43.91 (dd, 1.0, 3.0)
2134.65.47 (m)3′′72.13.57 (dd, 3.0, 9.5)
3125.95.38 (m)4′′73.83.27 (dd, 9.5, 9.5)
428.82.39 (q like, ca. 7)5′′70.43.58 (m)
521.62.08 (m)6′′18.51.08 (d, 6.5)
614.70.97 (t, 7.5)1′′′127.1-
1′104.24.36 (d, 8.0)2′′′131.47.46 (d, 8.5)
2′76.23.38 (dd, 8.0, 8.5)3′′′117.06.80 (d, 8.5)
3′81.73.82 (dd, 8.5, 9.0)4′′′161.6-
4′70.74.92 (dd, 9.0, 9.0)5′′′117.06.80 (d, 8.5)
5′76.13.54 (m)6′′′131.47.46 (d, 8.5)
6′62.43.52 (dd, 6.0, 12.0)7′′′147.77.66 (d, 16.0)
3.62 (br. d, ca. 12)8′′′114.86.34 (d, 16.0)
1′′103.15.19 (d, 1.0)9′′′168.4-
Table 4. The 1H- and 13C-NMR data of 4.
Table 4. The 1H- and 13C-NMR data of 4.
No.4 a4 b
δCδH (J in Hz)δCδH (J in Hz)
138.8-37.9-
242.01.38 (dd, 5.0, 13.0)42.41.72 (dd, 3.0, 12.5)
1.74 (dd, 13.0, 13.0) 2.20 (dd, 12.5, 12.5)
368.13.71 (ddd, 4.0, 5.0, 13.0)67.14.12 (ddd, 4.0, 5.5, 12.5)
473.13.73 (br. d, ca. 4)72.34.19 (br. d, ca. 4)
5128.1-128.4-
6143.7-141.6-
725.61.99 (ddd, 5.0, 13.0, 13.0)25.02.11 (ddd, 4.5, 13.0, 13.0)
2.27 (ddd, 5.0, 13.0, 13.0) 2.48 (ddd, 4.5, 13.0, 13.0)
838.31.57 (m), 1.66 (m)37.91.65 (m), 1.85 (m)
976.13.90 (m)74.84.14 (m)
1019.81.20 (d, 6.0)19.91.31 (d, 6.5)
1127.71.05 (s)27.41.03 (s)
1230.01.07 (s)29.61.04 (s)
1318.51.78 (s)18.51.97 (s)
1′102.34.35 (d, 7.5)102.44.93 (d, 7.5)
2′75.23.16 (dd, 7.5, 8.5)75.24.04 (dd, 7.5, 8.5)
3′78.23.36 (dd, 8.5, 9.0)78.64.29 (dd, 8.5, 9.0)
4′71.93.30 (dd, 9.0, 9.0)71.94.23 (dd, 9.0, 9.0)
5′77.93.26 (m)78.33.97 (m)
6′63.03.67 (dd, 5.5, 12.0)63.04.36 (dd, 5.0, 11.5)
3.85 (dd, 2.0, 12.0) 4.55 (dd, 2.0, 11.5)

a measured in CD3OD; b measured in C5D5N.

Lippianoside E (5) was obtained as a white powder. Its molecular formula, C19H30O9, was determined from the positive HR-Q-TOF-ESI-MS. The 1H- and 13C-NMR (CD3OD, Table 5) spectra showed signals assignable to four methyls (δ 1.15, 1.37, 1.47, 2.19 (3H each, all s, H3-12, 11, 13, 10)), two methylenes (δ (1.33 (1H, dd, J = 12.0, 12.0 Hz), 1.92 (1H, ddd, J = 2.5, 5.0, 12.0 Hz), H2-2), (1.37 (1H, dd, J = 12.0, 12.0 Hz), 2.48 (1H, ddd, J = 2.5, 4.5, 12.0 Hz), H2-4)), a methine bearing an oxygen function (δ 4.32 (1H, m, H-3)), a three-substituted double bond (δ 5.89 (1H, s, H-7)), two carboxyl groups (δC 200.7 (C-9), 213.0 (C-8)), and a β-d-glucopyranosyl (δ 4.52 (1H, d, J = 7.5 Hz, H-1′)). Selected long-range correlations observed in the HMBC experiment were shown in Figure 7. Treatment of 5 with 1 M HCl yielded d-glucose [37,38]. In the NOESY experiment, NOE correlations were observed between the following proton pairs: δH 1.92 (Heq-2) and δH 1.37 (H3-11) and 4.32 (H-3); δH 4.32 (H-3) and δH 1.37 (H3-11) and 1.47 (H3-13); and δH 5.89 (H-7) and δH 1.37 (H3-11), 1.47 (H3-13), and 2.19 (H3-10), which suggested the configuration of 5 was 3S*5S*6E.

Figure 7. The main 1H-1H COSY, HMBC, and NOE correlations of 5.
Figure 7. The main 1H-1H COSY, HMBC, and NOE correlations of 5.
Molecules 20 19814 g007 1024
Table 5. The 1H- and 13C-NMR data of 5.
Table 5. The 1H- and 13C-NMR data of 5.
No.5 a5 b
δCδH (J in Hz)δCδH (J in Hz)
137.1-35.4-
249.91.33 (dd, 12.0, 12.0)49.11.21 (dd, 12.5, 12.5)
1.92 (ddd, 2.5, 5.0, 12.0) 1.79 (ddd, 3.0, 4.5, 12.0)
363.84.32 (m)61.04.15 (m)
448.11.37 (dd, 12.0, 12.0)45.91.19 (dd, 12.0, 12.0)
2.48 (ddd, 2.5, 4.5, 12.0) 2.38 (ddd, 3.0, 5.0, 12.0)
578.7-76.9-
6141.6-117.4-
6119.1-117.4-
7101.45.89 (s)99.85.86 (s)
8213.0-210.6-
9200.7-197.5-
1026.72.19 (s)26.22.12 (s)
1130.11.37 (s)29.01.29 (s)
1232.51.15 (s)31.71.05 (s)
1326.61.47 (s)26.31.33 (s)
1′98.74.52 (d, 7.5)96.84.36 (d, 7.0)
2′75.33.14 (dd, 7.5, 9.0)73.62.91 (dd, 7.0, 8.0)
3′78.63.35 (dd, 9.0, 9.0)77.33.16 (dd, 8.0, 9.0)
4′71.73.25 (dd, 9.0, 9.0)70.13.01 (dd, 9.0, 9.0)
5′77.83.22 (m)76.63.06 (m)
6′62.93.61 (dd, 5.5, 12.0)61.13.37 (dd, 6.0, 12.0)
3.81 (dd, 2.0, 12.0) 3.62 (dd, 2.0, 12.0)

a measured in CD3OD; b measured in DMSO-d6.

Antimycin A, an electron transport chain inhibitor in mitochondria between cytochromes b and c, can produce ROS in cells, causing the leakage of superoxide radicals from cell mitochondria by inhibiting mitochondrial electron transport [41]. Compared with normal group, 20 µg/mL antimycin A induced significant L6 cell injury at a rate of 50%, while 10 µM resveratrol showed increased cell survival rate effects compared with the antimycin treated group. Except for 1, 2 and 20, all the compounds isolated from L. triphylla displayed significant protective effects against antimycin A-induced L6 cell injury at 30 µM, and 21 showed strongest protective activity (Figure 8).

Figure 8. Cell survival rate of 131 on L6 cells treated with antimycin A. Values represent the mean ± SD of determinations (n = 8). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control group. Final administrated concentration of revestrol was 10 μM, and 131 was 30 μM.
Figure 8. Cell survival rate of 131 on L6 cells treated with antimycin A. Values represent the mean ± SD of determinations (n = 8). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control group. Final administrated concentration of revestrol was 10 μM, and 131 was 30 μM.
Molecules 20 19814 g008 1024

Intracellular excess lipid accumulation (especially in liver and muscle) is a mediator of metabolic syndrome, which is comprised of a cluster of risk factors such as diabetes, hyperlipidemia, and hypertension. Free fatty acid (FFA) induced TG accumulation in HepG2 cells is commonly used for research on lipid metabolism regulation effects [42]. As shown in Figure 9, intracellular lipid contents were significantly increased after 0.2 mM oleic acid treatment. This accumulation effects were inhibited by orlistat at 0.5 µM. The TG accumulation inhibitory effects of the isolates were tested. Except for compounds 12, 13 and 2022, all isolates displayed inhibitory effects on TG accumulation in FFA induced HepG2 cells.

Figure 9. TG accumulation inhibitory effects of 131 in HepG2 cells. TG relative concentration: percentage of control group, which set as 100%. Values represent the mean ± SD of determinations (n = 8). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control group. Final administrated concentration of 131 was 10 μM, and oristat was 0.5 μM.
Figure 9. TG accumulation inhibitory effects of 131 in HepG2 cells. TG relative concentration: percentage of control group, which set as 100%. Values represent the mean ± SD of determinations (n = 8). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control group. Final administrated concentration of 131 was 10 μM, and oristat was 0.5 μM.
Molecules 20 19814 g009 1024

3. Experimental Section

3.1. General

The following instruments were used to obtain physical data: Optical rotations were determined on a Rudolph Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA, l = 50 mm), IR and UV spectra were recorded on Varian 640-IR FT-IR (Varian Australia Pty Ltd., Mulgrave, Australia)and Varian Cary 50 UV-VIS spectrophotometer (Varian, Inc., Hubbardsdon, MA, USA), respectively. 1H- and 13C-NMR spectra were measured on a Bruker 500 MHz NMR spectrometer (Bruker BioSpin AG Industriestrasse 26 CH-8117, Fällanden, Switzerland) at 500 MHz for 1H- and 125 MHz for 13C-NMR, with tetramethylsilane (TMS) as an internal standard. Positive- and negative-ion HR-ESI-Q-TOF-MS were recorded on an Aglient 6520 Q-TOF mass spectrometer (Agilent Corp., Santa Clara, CA, USA).

The following experimental conditions were used for chromatography: A macroporous synthetic resin (D101) (Haiguang Chemical Co., Ltd., Tianjin, China), Silica gel (74–149 μm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and ODS (50 μm, YMC Co., Ltd., Tokyo, Japan). HPLC was performed on ODS (Cosmosil 5C18-MS-II, Tokyo, Japan; Φ = 20 mm, L = 250 mm, flow rate 9.0 mL/min), and the eluate was monitored with a UV detector (Shimadzu RID-10ª UV-vis, Shimadzu Co. Ltd., Kyoto, Japan).

3.2. Plant Material

The aerial parts of Lippia triphylla were collected in Rwanda, Africa, and identified by Dr. Tianxiang Li at Tianjin University of TCM as M. indica L. A voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM.

3.3. Extraction and Isolation

The dried aerial parts of L. triphylla (2.85 kg) were extracted with 95% EtOH under reflux. Evaporation of the solvent under reduced pressure to yield a 95% ethanol–water extract (403 g). The extract partitioned with CHCl3–H2O (1:1, v/v) to give CHCl3 (105 g) and H2O (258 g) layers. The H2O layer (215 g) was subjected to D101 macroporous resin CC and eluted with H2O and 95% EtOH, successively, to yield H2O (131 g) and 95% EtOH (63 g) eluates, respectively.

The 95% EtOH eluate (50.0 g) was separated by SiO2 gel CC (CHCl3 → CHCl3-MeOH (100:2 → 100:4 → 100:6, v/v) → CHCl3–MeOH–H2O (10:3:1 → 7:3:1 → 6:4:1, v/v/v, lower layer) → MeOH) to afford 16 fractions (Fr. 1–16). Fraction 3 (1.0 g) was isolated by SiO2 gel and Sephadex LH-20 CC to give nepetin (16, 12.0 mg). Fraction 6 (1.2 g) was subjected to ODS, Sephadex LH-20 CC, and purified by PHPLC to yield benzyl alcohol O-β-d-glucopyranoside (30, 5.2 mg) and icariside H1 (31, 4.3 mg). Fraction 7 (7.3 g) was separated by PHPLC and Sephadex LH-20 CC with different analysis conditions to afford lippianosides B (2, 19.0 mg) and C (3, 5.0 mg), martynoside (10, 7.0 mg), isomartynoside (11, 7.0 mg), nepitrin (17, 12.0 mg), dehydrodiconiferyl glucosides D (18, 20.0 mg) and E (19, 13.0 mg), (+)-lariciresinol-9-O-β-d-glucopyranoside (20, 28.0 mg), (+)-pinoresinol 4-O-β-d-glucoside (21, 6.0 mg), and dihydrovomifoliol-O-β-d-glucopyranoside (22, 40.0 mg). Fraction 8 (5.8 g) was purified by PHPLC, and as a result, lippianosides A (1, 14.0 mg), D (4, 10.1 mg), and E (5, 11.0 mg), jionoside C (6, 8.4 mg), and turpinionoside D (23, 7.2 mg) were given. Fraction 11 (8.6 g) was separated by PHPLC and ODS CC to yield trans-acteoside (7, 17.3 mg), isoverbascoside (8, 55.1 mg), cis-acteoside (9, 36.1 mg), β-hydroxyacteoside (12, 8.3 mg), campneoside I (13, 60.6 mg), and cistanoside F (14, 70.4 mg).

The above-mentioned CHCl3 layer (85.0 g) was subjected to Silica gel CC (CHCl3 → CHCl3–MeOH (100:1 → 100:3 → 100:5 → 100:7, v/v) → MeOH) to give 13 fractions (Fr. 1′–13′). Fraction 5 (29.3 g) was purified by Sephadex LH-20 and Silica gel CC, along with PHPLC to afford jaceosidin (15, 11.2 mg), 9-hydroxymegastigm-5-en-4-one (24, 2.4 mg), (−)-loliolide (25, 13.1 mg), (6S)-3,7-dimethyl-7-hydroxy-2(Z)-octen-6-olide (27, 11.5 mg), ursolic acid (28, 20.9 mg), and avicennone A (29, 5.4 mg).

Lippianoside A (1): White powder; [ α ] D 25 +7.9° (c = 0.61, MeOH); CD (c = 0.00166 M, MeOH) Δε (λ nm) −121.7 (201), −5.9 (228); UV (MeOH) λmax (log ε) 203 (4.89), 229 (4.07), 279 (3.70); IR (KBr) νmax 3384, 2930, 1603, 1514, 1451, 1263, 1163, 1064, 1039, 833 cm−1; 1H- and 13C-NMR (CD3OD) data see Table 1; Positive-ion mode HR-Q-TOF-ESI-MS m/z 575.2113 (calcd for C27H36O12Na [M + Na]+, 575.2099).

Lippianoside B (2): White powder; [ α ] D 25 −8.9° (c = 0.79, MeOH); UV (MeOH) λmax (log ε) 218 (4.21), 231 (4.20), 286 (3.92, sh), 323 (4.09); IR (KBr) νmax 3552, 2940, 2840, 1690, 1596, 1517, 1448, 1161, 1025, 944, 897 cm−1. 1H- and 13C-NMR (CD3OD) data see Table 2. Negative-ion mode HR-Q-TOF-ESI-MS m/z 549.1604 [M − H] (calcd for C26H29O13, 549.1614).

Lippianoside C (3): White powder; [ α ] D 25 −61.4° (c = 0.37, MeOH); UV (MeOH) λmax (log ε) 312 (4.21); IR (KBr) νmax 3384, 2930, 1701, 1603, 1514, 1263, 1163, 1064, 1039, 833 cm−1; 1H- and 13C-NMR (CD3OD) data see Table 3. Positive-ion mode HR-Q-TOF-ESI-MS m/z 557.2267 (calcd for C27H38O12Na [M + Na]+, 557.2255).

Lippianoside D (4): White powder; [ α ] D 25 −62.1° (c = 0.86, MeOH); IR (KBr) νmax 3360, 2964, 2929, 1621, 1377, 1262, 1073, 1026, 875 cm−1; 1H- and 13C-NMR (CD3OD) data see Table 4; Positive-ion mode HR-Q-TOF-ESI-MS m/z 413.2158 (calcd for C19H34O8Na [M + Na]+, 413.2146).

Lippianoside E (5): White powder; [ α ] D 25 −87.0° (c = 0.20, MeOH); UV (MeOH) λmax (log ε) 229 (3.62); IR (KBr) νmax 3350, 2915, 2848, 1665, 1405, 1248, 1070, 1070, 1036 cm−1; 1H- and 13C-NMR (DMSO-d6) data see Table 5; Positive-ion mode HR-Q-TOF-ESI-MS m/z 425.1720 (calcd for C19H30O9Na [M + Na]+, 425.1782).

Acid Hydrolysis of 15: A solution of compounds 15 (each 2.0 mg) in 1 M HCl (1.0 mL) was heated under reflux for 3 h. After cooling, the reaction mixture was extracted with EtOAc. The aqueous layer was subjected to HPLC analysis under the following conditions, respectively: HPLC column, Kaseisorb LC NH2-60-5, 4.6 mm i.d. 250 mm (Tokyo Kasei Co., Ltd., Tokyo, Japan); detection, optical rotation (Shodex OR-2 (Showa Denko Co., Ltd., Tokyo, Japan); mobile phase, CH3CN–H2O (75:25, v/v); flow rate 0.7 mL/min). Identification of l-rhamnose (i) from 3; and D-glucose (ii) from 15 present in the aqueous layer was carried out by comparison of its retention time and optical rotation with those of authentic sample, tR: (i) 7.5 min (l-rhamnose, negative optical rotation); and (ii) 14.1 min (d-glucose, positive optical rotation).

Enzymatic hydrolysis of 4 with β-glucosidase: A solution of 4 (7.0 mg) in H2O (2.5 mL) was treated with β-glucosidase (5.0 mg, Almond, Sigma-Aldrich, Co., St. Louis, MO, USA), and the solution was stirred at 37 °C for 20 h. After cooling, the reaction mixture was extracted with EtOAc. The EtOAc solvent was removed under reduced pressure to give (3S,4R,9R)-3,4,9-trihydroxymegastigman-5-ene.

Mitochondrial oxidative stress protective effects assay: Antimycin A was used to induce mitochondrial oxidative stress. Briefly, L6 cells (Cell Resource Center, IBMS, CAMS/PUMC, Beijing, China) were plated at a density of 5 × 104 cells/well in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Scientific, Logan, UT, USA) supplemented with 10% calf serum (Thermo Scientific) in a 96-well plate and were incubated at 37 °C for 24 h. Cells were treated with or without 30 μM sample DMSO solution (final DMSO concentration was 0.5%). One hour later, medium was removed and 20 μg/mL antimycin A (Sigma Co. Ltd.) in 200 μL DMEM was added to each well. The MTT assay was performed 24 h later to detect the cell survival rate. Resveratrol was used as positive control.

TG accumulation inhibitory effects assay: The hepatic cell line HepG2 (IBMS, CAMS/PUMC, Beijing, China) was maintained in high glucose Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin under a humidified atmosphere of 5% CO2 in air. After growth to 80% confluence, cells were seeded at 4 × 104 cells/mL on a 48-well dish. After 24 h incubation, the medium was switched to high glucose MEM and supplemented with 10% FBS and 0.2 mM oleic acid sodium salt, together with sample DMSO solution (final concentration of DMSO was less than 0.1%). After 48 h incubation, the amount of intracellular triglycerides was determined with a Triglycerides kit (BioSino Bio-technology and Science Inc., Beijing, China) after cell lysis. Orlistat was used as positive control.

Statistical analysis: Values are expressed as mean ± SD. Analyses on the grouped data were performed using SPSS 11.0. Significant differences between means were evaluated by one-way analysis of variance (ANOVA) and Tukey’s Studentized range test was used for post hoc evaluations. A p value of <0.05 was considered to indicate statistical significance.

4. Conclusions

In summary, five new, along with 26 known, compounds were identified from the 95% EtOH extract of L. triphylla aerial parts collected from Rwanda, Africa. Their structures were elucidated by chemical and spectroscopic methods. Among the known compounds, 11, and 17–30 were isolated from Lippia genus for the first time. In addition, 12, 13, and 16 were obtained from this species for the first time. All compounds were tested for their antioxidant and triglyceride accumulation inhibition effects in L6 cells and HepG2 cells, respectively. The results indicated that, except for 1, 2 and 20, all compounds isolated from L. triphylla displayed significant protective effects against antimycin A-induced L6 cell injury at 30 µM, and 21 showed the strongest protective activity. Meanwhile, 1–11, 14–19, and 23–26 displayed inhibitory effects on TG accumulation in FFA induced HepG2 cells. Our study provides partial scientific support for the development and utilization of L. triphylla aerial parts.

Acknowledgments

Part of this research was supported by Programs for New Century Excellent Talents in University (NCET-12-1069), Tianjin Innovative Research Team in University (TD12-5033), and Changjiang Scholars and Innovative Research Team in University (PCSIRT IRT_14R41).

Author Contributions

Yi Zhang and Tao Wang designed the research and wrote the manuscript; Yue Chen, Shiyu Wang, Tingting Wang, Yongzhe Dong, and Lu Qu performed the experimental work; and Nan Li perfected the language. All authors discussed, edited and approved the final version.

Conflicts of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Samples of all the compounds are available from the authors.
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