Bioactive Constituents from the Aerial Parts of Lippia triphylla

Abstract

Five new compounds, lippianosides A (1), B (2), C (3), D (4), and E (5), along with 26 (6–31) 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 17–30 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.

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 (1–5), along with 26 known ones (6–31) 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 (1–5) (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 17–30 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), ${\left[\mathsf{\alpha }\right]}_D^{25}$ +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 δ_H 4.56 (H-7) and δ_H 2.40 (H-8′) and 3.75 (H-9a); δ_H 1.75 (H-8) and δ_H 3.97 (H-7′); and δ_H 2.40 (H-8′) and δ_H 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 (1–5) obtained from L. triphylla.

Figure 1. The new compounds (1–5) obtained from L. triphylla.

Figure 2. The known compounds (6–31) obtained from L. triphylla.

Figure 2. The known compounds (6–31) obtained from L. triphylla.

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.

Table 1. The ¹H- and ¹³C-NMR data of 1.

No.	1 a		1 b
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	133.2	-	134.8	-
2	110.4	6.88 (d, 2.0)	111.6	6.97 (d, 1.5)
3	147.2	-	149.0	-
4	145.5	-	147.2	-
5	115.0	6.71 (d, 8.0)	116.1	6.77 (d, 8.0)
6	118.5	6.77 (dd, 2.0, 8.0)	120.3	6.86 (dd, 1.5, 8.0)
7	82.7	4.56 (d, 7.5)	85.3	4.76 (d, 7.0)
8	49.7	1.75 (m)	51.6	1.96 (m)
9	69.5	3.75 (m, overlapped)	70.4	3.17 (dd, 4.0, 10.0)
		4.05 (dd, 4.5, 9.0)		3.55 (dd, 6.5, 10.0)
3-OCH3	55.5	3.76 (s)	56.7	3.86 (s)
1′	130.7	-	132.7	-
2′	110.9	6.76 (d, 2.0)	112.1	6.79 (d, 1.5)
3′	147.5	-	149.3	-
4′	145.9	-	147.6	-
5′	115.0	6.71 (d, 8.0)	116.2	6.76 (d, 8.5)
6′	120.2	6.64 (dd, 2.0, 8.0)	121.8	6.70 (dd, 1.5, 8.0)
7′	84.5	3.97 (d, 7.5)	87.1	4.01 (d, 9.0)
8′	48.5	2.40 (m)	50.3	2.52 (m)
9′	68.7	3.11 (dd, 9.0, 9.0)	71.8	3.97 (dd, 9.0, 9.0)
		3.46 (dd, 4.0, 9.0)		4.18 (dd, 4.0, 9.0)
3′-OCH3	55.7	3.73 (s)	56.6	3.82 (s)
7′-OCH3	55.4	3.07 (s)	56.7	3.16 (s)
1′′	102.8	3.98 (d, 7.5)	104.5	4.02 (d, 8.0)
2′′	73.4	2.91 (dd, 7.5, 9.0)	75.2	3.11 (dd, 8.0, 9.0)
3′′	76.6	3.11 (dd, 9.0, 9.0)	78.2	3.29 (dd, 9.0, 9.0)
4′′	69.8	3.03 (dd, 9.0, 9.0)	71.7	3.28 (dd, 9.0, 9.0)
5′′	76.7	3.01 (m)	77.9	3.16 (m)
6′′	60.9	3.45 (dd, 5.5, 12.5)	62.8	3.66 (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.

Table 1. The ¹H- and ¹³C-NMR data of 1.

No.	1 a		1 b
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	133.2	-	134.8	-
2	110.4	6.88 (d, 2.0)	111.6	6.97 (d, 1.5)
3	147.2	-	149.0	-
4	145.5	-	147.2	-
5	115.0	6.71 (d, 8.0)	116.1	6.77 (d, 8.0)
6	118.5	6.77 (dd, 2.0, 8.0)	120.3	6.86 (dd, 1.5, 8.0)
7	82.7	4.56 (d, 7.5)	85.3	4.76 (d, 7.0)
8	49.7	1.75 (m)	51.6	1.96 (m)
9	69.5	3.75 (m, overlapped)	70.4	3.17 (dd, 4.0, 10.0)
		4.05 (dd, 4.5, 9.0)		3.55 (dd, 6.5, 10.0)
3-OCH3	55.5	3.76 (s)	56.7	3.86 (s)
1′	130.7	-	132.7	-
2′	110.9	6.76 (d, 2.0)	112.1	6.79 (d, 1.5)
3′	147.5	-	149.3	-
4′	145.9	-	147.6	-
5′	115.0	6.71 (d, 8.0)	116.2	6.76 (d, 8.5)
6′	120.2	6.64 (dd, 2.0, 8.0)	121.8	6.70 (dd, 1.5, 8.0)
7′	84.5	3.97 (d, 7.5)	87.1	4.01 (d, 9.0)
8′	48.5	2.40 (m)	50.3	2.52 (m)
9′	68.7	3.11 (dd, 9.0, 9.0)	71.8	3.97 (dd, 9.0, 9.0)
		3.46 (dd, 4.0, 9.0)		4.18 (dd, 4.0, 9.0)
3′-OCH3	55.7	3.73 (s)	56.6	3.82 (s)
7′-OCH3	55.4	3.07 (s)	56.7	3.16 (s)
1′′	102.8	3.98 (d, 7.5)	104.5	4.02 (d, 8.0)
2′′	73.4	2.91 (dd, 7.5, 9.0)	75.2	3.11 (dd, 8.0, 9.0)
3′′	76.6	3.11 (dd, 9.0, 9.0)	78.2	3.29 (dd, 9.0, 9.0)
4′′	69.8	3.03 (dd, 9.0, 9.0)	71.7	3.28 (dd, 9.0, 9.0)
5′′	76.7	3.01 (m)	77.9	3.16 (m)
6′′	60.9	3.45 (dd, 5.5, 12.5)	62.8	3.66 (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 (${\left[\mathsf{\alpha }\right]}_D^{25}$ −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: δ_H 4.98 (H-1) and δ_C 145.0 (C-8), 152.9 (C-3); δ_H 3.13 (H-5) and δ_C 145.0 (C-8), 171.0 (C-11); δ_H 2.69 (H-9) and δ_C 113.3 (C-4), 128.9 (C-7); δ_H 4.19, 4.25 (H₂-10) and δ_C 46.7 (C-9), 128.9 (C-7), 145.0 (C-8); δ_H 4.73 (H-1′) and δ_C 98.9 (C-1); δ_H 4.40, 4.44 (H₂-6′) and δ_C 169.1 (C-9′′). Finally, in the NOESY spectrum, the NOE correlations between δ_H 4.98 (H-1) and δ_H 1.96 (Hα-6); δ_H 3.13 (H-5) and δ_H 2.69 (H-9) and 2.75 (Hβ-6); and δ_H 2.75 (Hβ-6) and δ_H 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.

Table 2. The ¹H- and ¹³C-NMR data of 2 in CD₃OD.

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
1	98.9	4.98 (d, 8.0)	3′	77.8	3.43 (dd, 8.5, 9.0)
3	152.9	7.49 (s)	4′	71.9	3.37 (dd, 9.0, 9.0)
4	113.3	-	5′	75.7	3.56 (m)
5	37.1	3.13 (q like, 8.0, 8.0)	6′	64.4	4.40 (dd, 6.0, 12.0)
6	39.9	1.96 (br. dd, ca. 8, 16)			4.44 (dd, 2.5, 12.0)
		2.75 (br. dd, ca. 8, 16)	1′′	127.7	-
7	128.9	5.76 (br. s)	2′′	111.6	7.17 (br. s)
8	145.0	-	3′′	149.4	-
9	46.7	2.69 (dd, 8.0, 8.0)	4′′	150.7	-
10	61.6	4.19 (d, 14.0)	5′′	116.5	6.81 (d, 8.0)
		4.25 (d, 14.0)	6′′	124.3	7.05 (br. d, ca. 8)
11	171.0	-	7′′	147.1	7.59 (d, 16.0)
1′	100.7	4.73 (d, 7.5)	8′′	115.3	6.35 (d, 16.0)
2′	74.8	3.27 (dd, 7.5, 8.5)	9′′	169.1	-
			3′′-OCH3	56.5	3.88 (s)

Table 2. The ¹H- and ¹³C-NMR data of 2 in CD₃OD.

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
1	98.9	4.98 (d, 8.0)	3′	77.8	3.43 (dd, 8.5, 9.0)
3	152.9	7.49 (s)	4′	71.9	3.37 (dd, 9.0, 9.0)
4	113.3	-	5′	75.7	3.56 (m)
5	37.1	3.13 (q like, 8.0, 8.0)	6′	64.4	4.40 (dd, 6.0, 12.0)
6	39.9	1.96 (br. dd, ca. 8, 16)			4.44 (dd, 2.5, 12.0)
		2.75 (br. dd, ca. 8, 16)	1′′	127.7	-
7	128.9	5.76 (br. s)	2′′	111.6	7.17 (br. s)
8	145.0	-	3′′	149.4	-
9	46.7	2.69 (dd, 8.0, 8.0)	4′′	150.7	-
10	61.6	4.19 (d, 14.0)	5′′	116.5	6.81 (d, 8.0)
		4.25 (d, 14.0)	6′′	124.3	7.05 (br. d, ca. 8)
11	171.0	-	7′′	147.1	7.59 (d, 16.0)
1′	100.7	4.73 (d, 7.5)	8′′	115.3	6.35 (d, 16.0)
2′	74.8	3.27 (dd, 7.5, 8.5)	9′′	169.1	-
			3′′-OCH3	56.5	3.88 (s)

Lippianoside C (3), ${\left[\mathsf{\alpha }\right]}_D^{25}$ −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 δ_H 4.36 (H-1′) and δ_C 70.7 (C-1); δ_H 5.19 (H-1′′) and δ_C 81.7 (C-3′); and δ_H 4.92 (H-4′) and δ_C 168.4 (C-9′′′), and the connectivities of the above-mentioned moieties were elucidated. Finally, in the NOESY experiment, the NOE correlations observed between δ_H 2.39 (H₂-4) and δ_H 3.56, 3.87 (H₂-1) as well as δ_H 5.47 (H-2) and δ_H 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 (${\left[\mathsf{\alpha }\right]}_D^{25}$ −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.

Figure 6. The main ¹H-¹H COSY and HMBC correlations of 4.

Figure 6. The main ¹H-¹H COSY and HMBC correlations of 4.

Table 3. The ¹H- and ¹³C-NMR data of 3 in CD₃OD.

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
1	70.7	3.56 (m) 3.87 (m)	2′′	72.4	3.91 (dd, 1.0, 3.0)
2	134.6	5.47 (m)	3′′	72.1	3.57 (dd, 3.0, 9.5)
3	125.9	5.38 (m)	4′′	73.8	3.27 (dd, 9.5, 9.5)
4	28.8	2.39 (q like, ca. 7)	5′′	70.4	3.58 (m)
5	21.6	2.08 (m)	6′′	18.5	1.08 (d, 6.5)
6	14.7	0.97 (t, 7.5)	1′′′	127.1	-
1′	104.2	4.36 (d, 8.0)	2′′′	131.4	7.46 (d, 8.5)
2′	76.2	3.38 (dd, 8.0, 8.5)	3′′′	117.0	6.80 (d, 8.5)
3′	81.7	3.82 (dd, 8.5, 9.0)	4′′′	161.6	-
4′	70.7	4.92 (dd, 9.0, 9.0)	5′′′	117.0	6.80 (d, 8.5)
5′	76.1	3.54 (m)	6′′′	131.4	7.46 (d, 8.5)
6′	62.4	3.52 (dd, 6.0, 12.0)	7′′′	147.7	7.66 (d, 16.0)
		3.62 (br. d, ca. 12)	8′′′	114.8	6.34 (d, 16.0)
1′′	103.1	5.19 (d, 1.0)	9′′′	168.4	-

Table 3. The ¹H- and ¹³C-NMR data of 3 in CD₃OD.

No.	δC	δH (J in Hz)	No.	δC	δH (J in Hz)
1	70.7	3.56 (m) 3.87 (m)	2′′	72.4	3.91 (dd, 1.0, 3.0)
2	134.6	5.47 (m)	3′′	72.1	3.57 (dd, 3.0, 9.5)
3	125.9	5.38 (m)	4′′	73.8	3.27 (dd, 9.5, 9.5)
4	28.8	2.39 (q like, ca. 7)	5′′	70.4	3.58 (m)
5	21.6	2.08 (m)	6′′	18.5	1.08 (d, 6.5)
6	14.7	0.97 (t, 7.5)	1′′′	127.1	-
1′	104.2	4.36 (d, 8.0)	2′′′	131.4	7.46 (d, 8.5)
2′	76.2	3.38 (dd, 8.0, 8.5)	3′′′	117.0	6.80 (d, 8.5)
3′	81.7	3.82 (dd, 8.5, 9.0)	4′′′	161.6	-
4′	70.7	4.92 (dd, 9.0, 9.0)	5′′′	117.0	6.80 (d, 8.5)
5′	76.1	3.54 (m)	6′′′	131.4	7.46 (d, 8.5)
6′	62.4	3.52 (dd, 6.0, 12.0)	7′′′	147.7	7.66 (d, 16.0)
		3.62 (br. d, ca. 12)	8′′′	114.8	6.34 (d, 16.0)
1′′	103.1	5.19 (d, 1.0)	9′′′	168.4	-

Table 4. The ¹H- and ¹³C-NMR data of 4.

No.	4 a		4 b
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	38.8	-	37.9	-
2	42.0	1.38 (dd, 5.0, 13.0)	42.4	1.72 (dd, 3.0, 12.5)
		1.74 (dd, 13.0, 13.0)		2.20 (dd, 12.5, 12.5)
3	68.1	3.71 (ddd, 4.0, 5.0, 13.0)	67.1	4.12 (ddd, 4.0, 5.5, 12.5)
4	73.1	3.73 (br. d, ca. 4)	72.3	4.19 (br. d, ca. 4)
5	128.1	-	128.4	-
6	143.7	-	141.6	-
7	25.6	1.99 (ddd, 5.0, 13.0, 13.0)	25.0	2.11 (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)
8	38.3	1.57 (m), 1.66 (m)	37.9	1.65 (m), 1.85 (m)
9	76.1	3.90 (m)	74.8	4.14 (m)
10	19.8	1.20 (d, 6.0)	19.9	1.31 (d, 6.5)
11	27.7	1.05 (s)	27.4	1.03 (s)
12	30.0	1.07 (s)	29.6	1.04 (s)
13	18.5	1.78 (s)	18.5	1.97 (s)
1′	102.3	4.35 (d, 7.5)	102.4	4.93 (d, 7.5)
2′	75.2	3.16 (dd, 7.5, 8.5)	75.2	4.04 (dd, 7.5, 8.5)
3′	78.2	3.36 (dd, 8.5, 9.0)	78.6	4.29 (dd, 8.5, 9.0)
4′	71.9	3.30 (dd, 9.0, 9.0)	71.9	4.23 (dd, 9.0, 9.0)
5′	77.9	3.26 (m)	78.3	3.97 (m)
6′	63.0	3.67 (dd, 5.5, 12.0)	63.0	4.36 (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.

Table 4. The ¹H- and ¹³C-NMR data of 4.

No.	4 a		4 b
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	38.8	-	37.9	-
2	42.0	1.38 (dd, 5.0, 13.0)	42.4	1.72 (dd, 3.0, 12.5)
		1.74 (dd, 13.0, 13.0)		2.20 (dd, 12.5, 12.5)
3	68.1	3.71 (ddd, 4.0, 5.0, 13.0)	67.1	4.12 (ddd, 4.0, 5.5, 12.5)
4	73.1	3.73 (br. d, ca. 4)	72.3	4.19 (br. d, ca. 4)
5	128.1	-	128.4	-
6	143.7	-	141.6	-
7	25.6	1.99 (ddd, 5.0, 13.0, 13.0)	25.0	2.11 (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)
8	38.3	1.57 (m), 1.66 (m)	37.9	1.65 (m), 1.85 (m)
9	76.1	3.90 (m)	74.8	4.14 (m)
10	19.8	1.20 (d, 6.0)	19.9	1.31 (d, 6.5)
11	27.7	1.05 (s)	27.4	1.03 (s)
12	30.0	1.07 (s)	29.6	1.04 (s)
13	18.5	1.78 (s)	18.5	1.97 (s)
1′	102.3	4.35 (d, 7.5)	102.4	4.93 (d, 7.5)
2′	75.2	3.16 (dd, 7.5, 8.5)	75.2	4.04 (dd, 7.5, 8.5)
3′	78.2	3.36 (dd, 8.5, 9.0)	78.6	4.29 (dd, 8.5, 9.0)
4′	71.9	3.30 (dd, 9.0, 9.0)	71.9	4.23 (dd, 9.0, 9.0)
5′	77.9	3.26 (m)	78.3	3.97 (m)
6′	63.0	3.67 (dd, 5.5, 12.0)	63.0	4.36 (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 (δ_C 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: δ_H 1.92 (Heq-2) and δ_H 1.37 (H₃-11) and 4.32 (H-3); δ_H 4.32 (H-3) and δ_H 1.37 (H₃-11) and 1.47 (H₃-13); and δ_H 5.89 (H-7) and δ_H 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.

Table 5. The ¹H- and ¹³C-NMR data of 5.

No.	5 a		5 b
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	37.1	-	35.4	-
2	49.9	1.33 (dd, 12.0, 12.0)	49.1	1.21 (dd, 12.5, 12.5)
		1.92 (ddd, 2.5, 5.0, 12.0)		1.79 (ddd, 3.0, 4.5, 12.0)
3	63.8	4.32 (m)	61.0	4.15 (m)
4	48.1	1.37 (dd, 12.0, 12.0)	45.9	1.19 (dd, 12.0, 12.0)
		2.48 (ddd, 2.5, 4.5, 12.0)		2.38 (ddd, 3.0, 5.0, 12.0)
5	78.7	-	76.9	-
6	141.6	-	117.4	-
6	119.1	-	117.4	-
7	101.4	5.89 (s)	99.8	5.86 (s)
8	213.0	-	210.6	-
9	200.7	-	197.5	-
10	26.7	2.19 (s)	26.2	2.12 (s)
11	30.1	1.37 (s)	29.0	1.29 (s)
12	32.5	1.15 (s)	31.7	1.05 (s)
13	26.6	1.47 (s)	26.3	1.33 (s)
1′	98.7	4.52 (d, 7.5)	96.8	4.36 (d, 7.0)
2′	75.3	3.14 (dd, 7.5, 9.0)	73.6	2.91 (dd, 7.0, 8.0)
3′	78.6	3.35 (dd, 9.0, 9.0)	77.3	3.16 (dd, 8.0, 9.0)
4′	71.7	3.25 (dd, 9.0, 9.0)	70.1	3.01 (dd, 9.0, 9.0)
5′	77.8	3.22 (m)	76.6	3.06 (m)
6′	62.9	3.61 (dd, 5.5, 12.0)	61.1	3.37 (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.

Table 5. The ¹H- and ¹³C-NMR data of 5.

No.	5 a		5 b
	δC	δH (J in Hz)	δC	δH (J in Hz)
1	37.1	-	35.4	-
2	49.9	1.33 (dd, 12.0, 12.0)	49.1	1.21 (dd, 12.5, 12.5)
		1.92 (ddd, 2.5, 5.0, 12.0)		1.79 (ddd, 3.0, 4.5, 12.0)
3	63.8	4.32 (m)	61.0	4.15 (m)
4	48.1	1.37 (dd, 12.0, 12.0)	45.9	1.19 (dd, 12.0, 12.0)
		2.48 (ddd, 2.5, 4.5, 12.0)		2.38 (ddd, 3.0, 5.0, 12.0)
5	78.7	-	76.9	-
6	141.6	-	117.4	-
6	119.1	-	117.4	-
7	101.4	5.89 (s)	99.8	5.86 (s)
8	213.0	-	210.6	-
9	200.7	-	197.5	-
10	26.7	2.19 (s)	26.2	2.12 (s)
11	30.1	1.37 (s)	29.0	1.29 (s)
12	32.5	1.15 (s)	31.7	1.05 (s)
13	26.6	1.47 (s)	26.3	1.33 (s)
1′	98.7	4.52 (d, 7.5)	96.8	4.36 (d, 7.0)
2′	75.3	3.14 (dd, 7.5, 9.0)	73.6	2.91 (dd, 7.0, 8.0)
3′	78.6	3.35 (dd, 9.0, 9.0)	77.3	3.16 (dd, 8.0, 9.0)
4′	71.7	3.25 (dd, 9.0, 9.0)	70.1	3.01 (dd, 9.0, 9.0)
5′	77.8	3.22 (m)	76.6	3.06 (m)
6′	62.9	3.61 (dd, 5.5, 12.0)	61.1	3.37 (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 1–31 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 1–31 was 30 μM.

Figure 8. Cell survival rate of 1–31 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 1–31 was 30 μM.

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 20–22, all isolates displayed inhibitory effects on TG accumulation in FFA induced HepG2 cells.

Figure 9. TG accumulation inhibitory effects of 1–31 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 1–31 was 10 μM, and oristat was 0.5 μM.

Figure 9. TG accumulation inhibitory effects of 1–31 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 1–31 was 10 μM, and oristat was 0.5 μM.

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; ${\left[\mathsf{\alpha }\right]}_D^{25}$ +7.9° (c = 0.61, MeOH); CD (c = 0.00166 M, MeOH) Δε (λ nm) −121.7 (201), −5.9 (228); UV (MeOH) λ_max (log ε) 203 (4.89), 229 (4.07), 279 (3.70); IR (KBr) ν_max 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; ${\left[\mathsf{\alpha }\right]}_D^{25}$ −8.9° (c = 0.79, MeOH); UV (MeOH) λ_max (log ε) 218 (4.21), 231 (4.20), 286 (3.92, sh), 323 (4.09); IR (KBr) ν_max 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; ${\left[\mathsf{\alpha }\right]}_D^{25}$ −61.4° (c = 0.37, MeOH); UV (MeOH) λ_max (log ε) 312 (4.21); IR (KBr) ν_max 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; ${\left[\mathsf{\alpha }\right]}_D^{25}$ −62.1° (c = 0.86, MeOH); IR (KBr) ν_max 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; ${\left[\mathsf{\alpha }\right]}_D^{25}$ −87.0° (c = 0.20, MeOH); UV (MeOH) λ_max (log ε) 229 (3.62); IR (KBr) ν_max 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 1–5: A solution of compounds 1–5 (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 1–5 present in the aqueous layer was carried out by comparison of its retention time and optical rotation with those of authentic sample, t_R: (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.