Lignans from the Fruits of Melia toosendan and Their Agonistic Activities on Melatonin Receptor MT₁

Abstract

Investigation on the fruits of Melia toosendan afforded seven new lignans (1–7), along with seventeen known compounds (8–24). The structures of the new compounds, involving four neo-lignans (1–4), two sesquilignans (5–6), and a nor-lignan (7), were elucidated based on extensive spectroscopic analyses (high-resolution electrospray ionization mass spectra, ultraviolet, infrared, one-dimensional and two-dimensional nuclear magnetic resonance). Compound 24 exhibited activity on melatonin receptor type 1 with an agonistic rate of 57.77 % at 1.02 mM according to the assay on HEK293 cell lines in vitro.

Key words

Melia toosendan - Meliaceae - lignans - MT₁ receptor - agonistic activity

Introduction

The fruit of Melia toosendan Sieb. et Zucc. (Meliaceae), well-known as “Chuan-Lian-Zi” in Chinese, is an important traditional Chinese medicine (TCM) for the treatment of malaria, sting, stomachache, acute or chronic inflammations, and parasitosis [1], [2], [3], and possesses the property of “soothing liver-qi stagnation”, indicating the neuroprotective potency according to TCM theory [4]. Pharmacological studies have demonstrated that toosendanin is an active component of “Chuan-Lian-Zi” as a selective presynaptic blocker, effective antibotulismic agent, and proliferation inhibitor of various human cancer cells [1], [5]. In addition, “Chuan-Lian-Zi” is widely used as a botanical pesticide in agriculture due to the presence of limonoid-type triterpenoids [3], [6].

Previous research has mainly focused on the limonoid-type triterpenoids of M. toosendan, from which more than 50 cases have been isolated [3], [7], [8]. However, there have been only a few investigations of lignans, another main component of “Chuan-Lian-Zi”. Lignans as secondary metabolites derived from the shikimic acid pathway are widely distributed in plants and possess antitumor, antimitotic, antiviral, enzyme inhibitory, piscicidal, and antifungal activities [9], [10], [11]. Especially, it has been reported that lignans have neuroprotective activity in vitro and in vivo [12], [13], [14], [15].

Melatonin receptor type 1 (MT₁), a high-affinity G-protein-coupled receptor widely distributed in the brain, retina, cardiovascular system, immune system, reproductive system, kidneys, pancreas, and liver, is a very important target in the treatment of most melatonin-related nervous system diseases, including sleep disorders, depression, and anxiety syndromes [16], [17], [18]. Mediation on the activation of MT₁ is an effective therapy for these psychiatric problems, based on which several agonistic drugs (circadin, ramelteon, agomelatine, etc.) have been developed successfully, along with many potential candidates undergoing clinical trials [18].

In order to clarify the neuroprotective components of the entitled plant, the bioassay-guided investigation on the EtOAc part of the fruit of M. toosendan led to the isolation of seven new lignans (1–7; [Fig. 1]) and seventeen known ones (8–24; Fig. 1S, Supporting Information). Among them, compounds 8–23 were isolated from the fruits of M. toosendan for the first time. Their structural elucidation and biological evaluation are reported in this paper.

Fig. 1 The structures of compounds 1–7. (Color figure available online only.)

Results and Discussion

Compound 1 showed a quasi-molecular ion peak at m/z 425.1585 [M + Na]⁺ in the positive HRESIMS, indicating a molecular formula of C₂₂H₂₆O₇ with ten degrees of unsaturation. Its IR spectrum suggested the groups of hydroxyl (3428 cm⁻¹), α,β-unsaturated carbonyl (1666 cm⁻¹), aromatic rings (1597, 1511, and 1460 cm⁻¹), and ether bonds (1273, 1130, and 1033 cm⁻¹). The ¹H NMR spectrum showed an ethoxy group at δ 3.41 (1H, dq, J = 9.5, 7.2 Hz), 3.36 (1H, dq, J = 9.5, 7.2 Hz), and 1.14 (3H, t, J = 7.2 Hz), two methoxy groups at δ 3.80 (3H, s) and 3.77 (3H, s), and six aromatic protons in two ABX spin systems, δ 6.94 (1H, d, J = 1.8 Hz), 6.69 (1H, d, J = 8.1 Hz), and 6.80 (1H, dd, J = 8.1, 1.8 Hz), and δ 7.18 (1H, d, J = 1.9 Hz), 6.95 (1H, d, J = 8.4 Hz), and 7.12 (1H, dd, J = 8.4, 1.9 Hz), suggesting the presence of two 1,3,4-trisubstituted phenyl rings. The signals at δ 9.56 (1H, d, J = 7.8 Hz), 7.54 (1H, d, J = 15.8 Hz), and 6.64 (1H, dd, J = 15.8, 7.8 Hz) were owed to a conjugated formyl group with a trans-form double bond. Its ¹³C NMR (DEPT) spectrum was similar with that of threo-guaiacylglycerol-β-coniferyl aldehyde ether (15) [19] except for the additional ethoxy group and the obvious downfield shift of C-7 from δ _C 73.4 to 82.5, suggesting that 1 was the 7-O-ethyl derivative of 15. This deduction was supported by the correlations of δ _H 4.46 (H-7)/δ _C 65.6 (C-1″) and δ _H 3.41, 3.36 (H-1″)/δ _C 82.5 (C-7) in the HMBC spectrum ([Fig. 2]). Previous reports revealed that compounds with a threo-guaiacylglycerol unit could be distinguished from its erythro isomer with the chemical shifts of C-8 and the ³ J _H,H coupling constants of the H-7/H-8 protons. The chemical shifts of C-8 in the threo-form were slightly lower than its erythro isomer, and the J _7,8 value in the threo-form was larger than the erythro-form [20], [21], [22], [23], [24]. Thus, the threo-configuration of the C-7/C-8 system was indicated by the J _7,8 value of 7.1 Hz, and compound 1 was assigned as threo-guaiacylethoxyglycerol-β-O-4′-coniferyl aldehyde ether.

Fig. 2 The key ¹H-¹H COSY and HMBC correlations of compounds 1–7. (Color figure available online only.)

Compound 2 had a molecular formula of C₂₂H₂₆O₇ based on the positive HRESIMS at m/z 425.1589 [M + Na]⁺. It was proposed to be the isomer of 1 due to their similar ¹H NMR and ¹³C NMR (DEPT) spectral data. Detailed analyses of its ¹H-¹H COSY and HMBC spectra verified that compound 2 possessed the same planar structure with 1. The upfield shift of H-9 from δ 3.86 (1H, dd, J = 9.6, 3.7 Hz, H-9a) and 3.84 (1H, dd, J = 9.6, 5.7 Hz, H-9b) in compound 1 to δ 3.67 (1H, dd, J = 10.4, 3.1 Hz, H-9a) and 3.49 (1H, dd, J = 10.4, 4.3 Hz, H-9b) in 2, along with the upfield shift of C-8 from δ _C 85.3 in 1 to 84.6 in 2, suggested the changes of the stereochemistry. In contrast to the J _7,8 value of 7.1 Hz in 1, the obvious small value of 4.2 Hz suggested the erythro-configuration [20], [21], [22], [23], [24]. Therefore, compound 2 was characterized as erythro-guaiacylethoxyglycerol-β-O-4′-coniferyl aldehyde ether.

Compound 3 was determined to be C₂₀H₂₄O₇ on the basis of the positive HRESIMS at m/z 399.1435 [M + Na]⁺. Its ¹H- and ¹³C-NMR data ([Tables 1] and [3]) were similar with those of compound 1, except for the absence of two vinylic carbons and the upfield shift of the aldehyde group from δ _C 196.1 in 1 to 192.9 in 3, which suggested the aldehyde group was directly linked to C-1′. This deduction was confirmed by HMBC correlations ([Fig. 2]) from δ _H 9.73 (H-7′) to δ _C 111.5 (C-2′) and 127.2 (C-6′). The threo-configuration was indicated by the J _7,8 value of 7.1 Hz. Thus, compound 3 was elucidated as threo-guaiacylethoxyglycerol-β-O-4′-guaiacyl aldehyde ether.

Table 1  ¹H-NMR (600 MHz) data for compounds 1–4 in CD₃OD (δ in ppm, J in Hz).

No.	1	2	3	4
2	6.94 d (1.8)	6.96 d (1.8)	6.94 d (1.8)	6.96 d (1.7)
5	6.69 d (8.1)	6.76 d (8.1)	6.67 d (8.0)	6.75 d (8.0)
6	6.80 dd (8.1, 1.8)	6.80 dd (8.1, 1.8)	6.80 dd (8.0, 1.8)	6.81 dd (8.0, 1.7)
7	4.46 d (7.1)	4.52 d (4.2)	4.46 d (7.1)	4.53 d (4.5)
8	4.54 ddd (7.1, 5.7, 3.7)	4.51 ddd (4.3, 4.2, 3.1)	4.64 ddd (7.1, 5.7, 3.6)	4.62 ddd (6.2, 4.5, 3.5)
9a	3.86 dd (9.6, 3.7)	3.67 dd (10.4, 3.1)	3.89 dd (11.4, 3.6)	3.68 dd (12.0, 3.5)
9b	3.84 dd (9.6, 5.7)	3.49 dd (10.4, 4.3)	3.85 dd (11.4, 5.7)	3.53 dd (12.0, 6.2)
2′	7.18 d (1.9)	7.28 d (1.9)	7.35 d (1.9)	7.45 d (1.8)
5′	6.95 d (8.4)	7.08 d (8.4)	7.07 d (8.3)	7.19 d (8.1)
6′	7.12 dd (8.4, 1.9)	7.19 dd (8.4, 1.9)	7.38 dd (8.3, 1.9)	7.46 dd (8.1, 1.8)
7′	7.54 d (15.8)	7.59 d (15.8)	9.73 s	9.78 s
8′	6.64 dd (15.8, 7.8)	6.68 dd (15.8, 7.8)
9′	9.56 d (7.8)	9.59 d (7.8)
1″a	3.41 dq (9.5, 7.2)	3.42 dq (9.5, 7.2)	3.41 dq (9.5, 7.2)	3.41 dq (9.5, 7.2)
1″b	3.36 dq (9.5, 7.2)	3.36 dq (9.5, 7.2)	3.36 dq (9.5, 7.2)	3.35 dq (9.5, 7.2)
2″	1.14 t (7.2)	1.08 t (7.2)	1.14 t (7.2)	1.06 t (7.2)
3-OMe	3.77 s	3.82 s	3.77 s	3.81 s
3′-OMe	3.80 s	3.90 s	3.81 s	3.92 s

Table 2  ¹H-NMR (600 MHz) data for compounds 5–7 in CD₃OD (δ in ppm, J in Hz).

No.	5	6	7
2	6.97 d (1.8)	6.97 d (1.8)	6.94 d (1.8)
5	6.84 d (8.1)	6.77 d (8.1)	6.66 d (8.1)
6	6.78 dd (8.1, 1.8)	6.73 dd (8.1, 1.8)	6.75 dd (8.1, 1.8)
7	5.73 d (6.9)	4.90 d (3.6)	3.16 dd (10.8, 4.4)
8	3.68 ddd (6.9, 5.1, 3.6)	4.22 ddd (5.6, 3.8, 3.6)	4.45 ddd (4.4, 4.2, 0.5)
9a	3.91 dd (9.4, 3.6)	3.89 dd (12.2, 5.6)	4.42 dd (9.4, 4.2)
9b	3.89 dd (9.4, 5.1)	3.56 dd (12.2, 3.8)	3.99 dd (9.4, 0.5)
2′	6.94 d (1.3)	6.65 d (1.9)	6.82 d (1.8)
5′			6.71 d (8.1)
6′	6.96 d (1.3)	6.65 d (1.9)	6.70 dd (8.1, 1.8)
7′	5.55 d (4.7)	4.83 d (6.9)	5.13 d (10.8)
8′	3.52 ddd (5.2, 4.7, 3.4)	2.35 m
9′a	3.82 dd (12.9, 3.4)	3.86 dd (10.8, 5.5)
9′b	3.79 dd (12.9, 5.2)	3.68 dd (10.8, 3.2)
2″	7.48 d (1.6)	6.50 brs
6″	7.55 d (1.6)	6.50 brs
7″a	9.81 s	2.92 dd (13.4, 5.9)
7″b		2.50 dd (13.4, 10.4)
8″		2.71 m
9″a		4.01 dd (9.6, 2.6)
9″b		3.76 dd (9.6, 6.4)
3-OMe	3.87 s	3.82 s	3.82 s
3′-OMe	3.95 s	3.81 s	3.75 s
3″-OMe	3.84 s	3.81 s
5′-OMe		3.81 s
5″-OMe		3.81 s

Table 3  ¹³C-NMR (150 MHz) data for compounds 1–7 in CD₃OD (δ _C values).

No.	1	2	3	4	5	6	7
1	131.4 s	131.3 s	131.6 s	131.6 s	134.3 s	133.8 s	133.8 s
2	112.0 d	112.5 d	111.9 d	112.4 d	110.5 d	111.3 d	114.7 d
3	149.1 s	148.8 s	149.1 s	148.8 s	144.2 s	148.7 s	148.9 s
4	147.5 s	147.4 s	147.6 s	147.4 s	146.2 s	146.8 s	146.7 s
5	116.0 d	115.6 d	116.0 d	115.6 d	114.4 d	115.7 d	115.9 d
6	121.4 d	122.0 d	121.4 d	122.0 d	114.8 d	120.6 d	123.9 d
7	82.5 d	81.7 d	82.5 d	81.6 d	89.3 d	74.0 d	60.2 d
8	85.3 d	84.6 d	85.1 d	84.3 d	53.8 d	87.4 d	76.0 d
9	62.4 t	62.5 t	62.3 t	62.5 t	63.3 t	61.6 t	77.1 t
1′	129.1 s	129.1 s	131.3 s	131.1 s	133.0 s	141.0 s	128.6 s
2′	112.7 d	112.7 d	111.5 d	111.4 d	109.1 d	104.0 d	111.0 d
3′	151.7 s	151.7 s	151.7 s	151.7 s	144.9 s	154.4 s	148.8 s
4′	153.6 s	152.8 s	156.3 s	155.5 s	148.2 s	134.8 s	147.3 s
5′	117.2 d	117.1 d	116.0 d	115.8 d	129.7 s	154.4 s	116.0 d
6′	124.5 d	124.3 d	127.2 d	127.0 d	118.3 d	104.0 d	120.5 d
7′	155.6 d	155.5 d	192.9 d	192.9 d	87.9 d	84.0 d	84.8 d
8′	127.6 d	127.6 d			53.0 d	54.2 d
9′	196.1 d	196.1 d			63.1 t	60.6 t
1″	65.6 t	65.3 t	65.6 t	65.3 t	131.4 s	132.8 s
2″	15.6 q	15.6 q	15.5 q	15.6 q	112.5 d	106.8 d
3″					147.7 s	149.3 s
4″					154.1 s	135.8 s
5″					129.2 s	149.3 s
6″					120.9 d	106.8 d
7″					191.3 d	34.1 t
8″						43.8 d
9″						73.7 t
3-OMe	56.3 q	56.3 q	56.3 q	56.3 q	55.3 q	56.3 q	56.5 q
3′-OMe	56.6 q	56.6 q	56.5 q	56.4 q	55.4 q	56.7 q	56.3 q
5′-OMe						56.7 q
3″-OMe					55.0 q	56.6 q
5″-OMe						56.6 q

Compound 4 had a quasi-molecular ion peak at m/z 399.1405 [M + Na]⁺ (calcd. for 399.1414) in HRESIMS, suggesting a molecular formula of C₂₀H₂₄O₇ with nine degrees of unsaturation. Comparing the ¹H NMR and ¹³C NMR (DEPT) spectral data with those of compound 3, the upfield shift of H-9 from δ 3.89 (1H, dd, J = 11.4, 3.6 Hz, H-9a) and 3.85 (1H, dd, J = 11.4, 5.7 Hz, H-9b) in 3 to δ 3.68 (1H, dd, J = 12.0, 3.5 Hz, H-9a) and 3.53 (1H, dd, J = 12.0, 6.2 Hz, H-9b) in 4, along with the upfield shifts of C-7 (from 82.5 in 3 to 81.6 in 4) and C-8 (from 85.1 in 3 to 84.3 in 4), suggested that compound 4 should be the stereoisomer of 3. The erythro-configuration was concluded by the J _7,8 value of 4.5 Hz. Hence, compound 4 was elucidated as erythro-guaiacylethoxy glycerol-β-O-4′-guaiacyl aldehyde ether.

Compound 5 had a molecular formula of C₂₈H₂₈O₉ with 15 degrees of unsaturation based on its [M + H]⁺ ion peak at m/z 509.1778 (calcd. for 509.1806) in HRESIMS. The functional groups of hydroxyl (3442 cm⁻¹), aromatic rings (1516, 1493, and 1452 cm⁻¹), and ether bonds (1276, 1136, and 1032 cm⁻¹) were deduced from the IR spectrum. The ¹H NMR spectrum showed three methoxyls at δ 3.95, 3.87, and 3.84 (each 3H, s), one 1,3,4-trisubstituted phenyl ring at δ 6.97 (1H, d, J = 1.8 Hz, H-2), 6.84 (1H, d, J = 8.1 Hz, H-5), and 6.78 (1H, dd, J = 8.1, 1.8 Hz, H-6), and two 1,3,4,5-substituted phenyl rings at δ 7.55, 7.48, 6.96, and 6.94 (each 1H). Three methoxy groups, eleven quaternary carbons, twelve methines, and two methylenes observed in the ¹³C NMR (DEPT) spectrum indicated a sesquilignan skeleton. The proton resonances at δ _H 7.55 (H-6″), 7.48 (H-2″), 6.96 (H-6′), 6.94 (H-2′), 5.73 (H-7), and 5.55 (H-7′), together with the carbon resonances at δ _C 154.1 (C-4″), 148.2 (C-4′), 129.7 (C-5′), 129.2 (C-5″), 89.3 (C-7), 87.9 (C-7′), 53.8 (C-8), and 53.0 (C-8′), suggested the presence of two dihydrobenzofuran moieties. The correlations from H-2′ and H-6′ to C-7′, and from H-7′ to C-2′ and C-6′ in HMBC ([Fig. 2]) indicated the two dihydrobenzofuran moieties were linked by the C_1′-C_7′ bond. The linkage of a guaiacyl moiety with C-7 was further confirmed by the correlations from H-2 and H-6 to C-7, together with the correlations from H-7 to C-2 and C-6 in the HMBC spectrum. Compared with the coupling constant (6.9 Hz) of J _7′,8′ in rosalaevin A (8) [25], the trans-orientation of H-7 and H-8 was evident from the J _7,8 value of 6.9 Hz, while the cis-configuration of H-7′ and H-8′ was verified by the J _7′,8′ value of 4.7 Hz. Thus, the structure of 5 was finally determined as trans-2-guaiacyl-3-hydroxymethyl-5-(cis-3′-hydroxymethyl-5′-formyl-7′-methoxybenzofuranyl)-7-methoxybenzofuran.

Compound 6 had a molecular formula of C₃₂H₄₀O₁₂ with 13 degrees of unsaturation determined by its quasi-molecular ion peak at m/z 639.2400 [M + Na]⁺ (calcd. for 639.2412) in the positive HRESIMS. The sesquilignan structure was evident from the existence of one 1,3,4-trisubstituted aromatic ring at δ 6.97 (1H, d, J = 1.8 Hz, H-2), 6.77 (1H, d, J = 8.1 Hz, H-5), and 6.73 (1H, dd, J = 8.1, 1.8 Hz, H-6), and two 1,3,4,5-substituted phenyl rings (δ 6.65 and 6.50, each 2H) in the ¹H NMR spectrum ([Table 2]). Comparing the ¹H and ¹³C NMR (DEPT) data with those of rosalaevins A (8), the presence of an additional methoxyl at C-5″ in 6 was confirmed by the downfield shift of C-5″ from δ _C 114.4 in 8 to 149.3 in 6, and the correlation of δ _H 3.81 to δ _C 149.3 in the HMBC spectrum ([Fig. 2]). The J _7,8 (3.6 Hz) and J _7′,8′ (6.9 Hz) values indicated the 7,8-erythro configuration and 7′,8′-trans orentation [25]. Furthermore, the absence of a ROESY correlation of H-7′/H-8″ together with the correlations of H-7′/H-8′/H-9′, H-7′/H-7″ (δ 2.50), H-8′/H-8″ and H-7″/H-9′ (δ 3.86) ([Fig. 3]) indicated the trans-orentation of H-7′ and H-8″. Hence, the structure of 6 was deduced as erythro-guaiacylglycerol-β-O-4′-(+)-5,5′-dimethoxylariciresinol ether.

Fig. 3 The key correlations in ROESY experiments of compounds 6–7. (Color figure available online only.)

Compound 7 was obtained as pale yellow oil, and had a molecular formula of C₁₈H₂₀O₆ by the negative HRESIMS at m/z 331.1157 [M – H]⁻, requiring nine degrees of unsaturation. The hydroxy (3494 and 3444 cm⁻¹), aromatic (1609, 1517 and 1456 cm⁻¹), and ether bond (1274, 1233, 1124, and 1034 cm⁻¹) groups were revealed in the IR spectrum. In the ¹H NMR spectrum, signals of two 1,3,4-trisubstituted aromatic groups at δ 6.94 (1H, d, J = 1.8 Hz, H-2), 6.66 (1H, d, J = 8.1 Hz, H-5) and 6.75 (1H, dd, J = 8.1, 1.8 Hz, H-6), δ 6.82 (1H, d, J = 1.8 Hz, H-2′), 6.71 (1H, d, J = 8.1 Hz, H-5′) and 6.70 (1H, dd, J = 8.1, 1.8 Hz, H-6′), together with signals of a substituted epoxybutyl unit, were unambiguously designated by ¹H-¹H COSY and HSQC experiments ([Fig. 2]). The correlations from H-2 and H-6 to C-7, from H-7 to C-2 and C-6, from H-2′ and H-6′ to C-7′, and from H-7′ to C-2′ and C-6′ in the HMBC experiment suggested that two 4-hydroxy-3-methoxybenzyl units were located at C-7 and C-7′, respectively. Therefore, the planar structure of 7 was established as norlignan from the ¹H and ¹³C NMR data ([Tables 2] and [3]). The 7,8-cis and 7,7′-trans orentation were indicated by the J _7,8 (4.4 Hz) and J _7,7′ (10.8 Hz) values, which were confirmed by the ROESY experiment ([Fig. 3]). Finally, compound 7 was elucidated as (2R*,3R*,4S*)-2,3-diguaiacyl-4-hydroxyl tetrahydrofuran.

The known lignans were identified as rosalaevins A (8) [25], ficusesquilignans A (9) [26], buddlenol E (10) [27], 5′-demethoxybuddlenol E (11) [28], leptolepisol D (12) [24], lyoniside (13) [29], picrasmalignan A (14) [30], threo-guaiacylglycerol-β-coniferyl aldehyde ether (15) [19], erythro-guaiacylglycerol-β-coniferyl aldehyde ether (16) [19], threo-guaiacylglycerol-β-coniferyl ether (17) [31], erythro-guaiacylglycerol-β-coniferyl ether (18) [31], erythro-dihydroxydehydrodiconifery alcohol (19) [20], vladinol D (20) [32], (−)-secoisolariciresinol (21) [33], ficusal (22) [26], (2S)-3,3-diguaiacyl-1,2-propanediol (23) [34], and (+)-pinoresinol (24) [19] by comparing their spectroscopic data with the literature.

Seven new compounds (1–7) and the abundant known (+)-pinoresinol (24) were evaluated for agonistic activity on human melatonin receptor MT₁ on HEK293 cell lines in vitro. As shown in [Table 4], (+)-pinoresinol (24), the major lignan, exhibited the strongest agonistic effect among the tested compounds with an agonistic rate of 57.77 % at the concentration of 1.02 mM. Compounds 7 and 2 possessed moderate agonistic activities on MT₁ with agonistic rates of 37.55 % and 21.16 % at about 1.00 mM, respectively. Compounds 1 and 5 showed weak agonistic activities on MT₁, while no obvious effects for compounds 3, 4, and 6 were detected at the tested concentration.

Table 4 Agonistic activities of compounds 1–7 and 24 on MT₁ ^a, 5-HT_1A ^b, and 5-HT_2C ^c receptors.

Compounds	Test concentration (mM)	Agonistic rated (%)
		MT1	5-HT1A	5-HT2C
a The positive control was melatonin (EC50 0.54 nM); b the positive control was 5-hydroxytryptamine (EC50 43.28 nM); c the positive control was 5-hydroxytryptamine (EC50 0.94 nM); d the agonistic rate was the percentage versus the control (normalized to 100 %) and was the average of two independent tests; e no agonistic activity was detected
1	0.91	4.28	0.64	6.94
2	0.99	21.16	5.07	9.80
3	0.97	NDe	ND	5.08
4	0.95	ND	3.49	11.39
5	1.05	8.04	ND	10.28
6	0.92	ND	7.73	5.25
7	0.90	37.55	ND	7.89
24	1.02	57.77	14.88	7.40

Pharmacological studies demonstrated that serotonin (5-hydroxytryptamine, 5-HT) receptors, especially the subtypes of 5-HT_1A and 5-HT_2C, were closely involved in the pathogenesis and treatment of psychiatric problems such as depression and anxiety disorders [35], [36]. Consequently, these compounds were further assayed for the agitating activities on 5-HT_1A and 5-HT_2C receptors on HEK293 cell lines in vitro. However, no meaningful agonistic activities were observed at the concentration of about 1 mM ([Table 4]).

Different from the previous research, this investigation was focused on the lignans and resulted in seven new together with seventeen known ones. A bioassay on HEK293 cell lines in vitro revealed that (+)-pinoresinol (24) exhibited a moderate activity against MT₁ mediation. This study may provide another view for understanding the active components of “Chuan-Lian-Zi”.

Materials and Methods

General

Melting points (m. p.) were measured by an SGW®X-4B melting point apparatus. UV spectra were recorded on a UV-2401A spectrophotometer with methanol (MeOH) as the solvent. Optical rotations were obtained on a Jasco model 1020 polarimeter in MeOH solution. IR spectra were collected on a Bio-Rad FTS-135 spectrometer with KBr pellets. HRESIMS data were acquired on a liquid chromatography-mass spectrometry-ion trap-time-of-flight (LCMS-IT-TOF). 1D and 2D NMR experiments were carried out using Advance III-600 NMR spectrometers with tetramethylsilane (TMS) as an internal standard. Semipreparative HPLC was performed on a Newstyle™ (pump: NP-7000 serials, detector: NU-3000 serials) with a reversed-phase (RP) C₁₈ column (9.4 × 250 mm, 5 µm). Silica gel (200–300 mesh), MCI gel CHP-20P, and Sephadex LH-20 were used for column chromatography (CC). Thin-layer chromatography (TLC) was conducted on silica gel GF₂₅₄ plates and the spots were visualized under UV light or by spraying with 10 % H₂SO₄ in 95 % ethanol (EtOH) followed by heating. Melatonin (purity > 99 %) and serotonin hydrochloride (purity > 99 %) were purchased from Alfa Aesar as positive controls.

Plant material

The fruit of M. toosendan Sieb. et Zucc. was purchased from Jvhuacun traditional Chinese medicine market, Kunming, Yunnan Province of China, in July 2012, and identified by Prof. Dr. Li-Gong Lei, Kunming Institute of Botany (KIB), Chinese Academy of Sciences (CAS). A voucher specimen (No. 2012–07–18) was deposited in the Laboratory of Antivirus and Natural Medicinal Chemistry, KIB, CAS.

Extraction and isolation

The air-dried and powdered fruit of M. toosendan (6 kg) was extracted with 90 % EtOH at room temperature three times, each for 24 h. All the extracts were combined and condensed in vacuo and partitioned between EtOAc and H₂O to afford EtOAc part 125 g.

The EtOAc part was subjected to silica gel column chromatograph (SiCC, 900 g, 14 × 65 cm) eluted with H₂O-MeOH-CHCl₃ (0 : 0 : 100, 0 : 5 : 95, 0 : 10 : 90, 2 : 20 : 80, v/v/v, each 15 L) to afford Frs. 1–5 based on the TLC characteristics. According to our in vitro bioassay (Table 1S, Supporting Information), Fr. 3 (71.5 g) was further separated by SiCC to obtain Frs. 3–1 ~ 3–6. Fr. 3–1 (11.1 g) was fractionated by MCI CHP-20P gel CC to get Frs. 3–1–1 ~ 3–1–5. Fr. 3–1–3 was subjected to repeated SiCC and further purified by semipreparative HPLC (Zorbax-C₁₈, 5.0 µm, 9.4 × 250 mm, UV detection at λ _max = 210, 254 nm) using MeOH-H₂O (18 : 82, flow rate 3 mL/min) to obtain compound 7 (6 mg, t _R = 26 min, purity > 98 % detected by HPLC). Fr. 3–2 (4.5 g) was partitioned by repeated SiCC to obtain Frs. 3–2–2–1 ~ 3–2–2–5. Fr. 3–2–2–1 was purified by semipreparative HPLC with MeOH-H₂O (65 : 35, flow rate 2.5 mL/min) to afford compounds 3 (5 mg, t _R = 36 min, purity > 97 %) and 4 (5 mg, t _R = 38 min, purity > 96 %). Fr. 3–2–2–2 was purified by Sephadex LH-20 CC to get compound 24 (910 mg, purity > 95 %). Fr. 3–2–2–3 was purified by semipreparative HPLC with MeOH-H₂O (60 : 40, flow rate 3 mL/min) to obtain compounds 1 (7 mg, t _R = 31 min, purity > 96 %) and 2 (8 mg, t _R = 32.5 min, purity > 96 %). Fr. 3–3 (10.2 g) was subjected to MCI CHP-20P gel CC, SiCC, and Sephadex LH-20 CC, respectively, and finally purified by semipreparative HPLC to yield known compounds 17 (12 mg), 18 (10 mg), and 22 (30 mg). Fr. 3–4 (3.9 g) was successively partitioned by MCI CHP-20P gel CC, SiCC, and semipreparative HPLC to obtain known compounds 15 (21 mg), 16 (30 mg), and 23 (27 mg). Fr. 3–5 (2.7 g) was repeatedly isolated to yield compounds 9 (30 mg), 10 (11 mg), 13 (10 mg), and Fr. 3–5–2–5–3 (51 mg), which was further separated by semipreparative HPLC eluted with MeCN-H₂O (28 : 72, flow rate 3 mL/min) to yield compounds 5 (7 mg, t _R = 19 min, purity > 97 %) and 14 (9 mg). Fr. 3–6 (7.8 g) was partitioned by repeated CC and finally purified by semipreparative HPLC eluted with MeCN-H₂O (28 : 72, flow rate 3 mL/min) to yield compounds 6 (4 mg, t _R = 17.5 min, purity > 97 %) and 8 (9 mg). Fr. 4 (14 g) was repeatedly isolated to afford compound 19 (13 mg) and Fr. 5 (7.8 g) was partitioned to obtain compounds 11 (5 mg), 12 (13 mg), and 21 (3 mg), respectively (the detailed isolation procedure is provided in Supporting Information). The purities of all known compounds were > 95 % as determined by HPLC.

threo-Guaiacylethoxyglycerol-β-O-4′-coniferyl aldehyde ether (1): pale yellow amorphous powder, m. p. 59–61 °C; [α]_D ²⁰: − 3.93 (c 0.10, MeOH); UV (MeOH) λ _max (log ε): 337 (4.21) nm; IR (KBr) v _max 3428, 1666, 1597, 1511, 1460, 1273, 1130, 1033, 867, 816 cm⁻¹; ¹H (600 MHz) and ¹³C NMR (150 MHz) data see [Tables 1] and [3]; (+)HRESIMS m/z 425.1585 [M + Na]⁺ (calcd. for C₂₂H₂₆O₇Na, 425.1571).

erythro-Guaiacylethoxyglycerol-β-O-4′-coniferyl aldehyde ether (2): pale yellow amorphous powder, m. p. 43–45 °C; [α]_D ²⁰: − 6.13 (c 0.10, MeOH); UV (MeOH) λ _max (log ε): 337 (3.89) nm; IR (KBr) v _max 3441, 1661, 1597, 1511, 1454, 1272, 1130, 1032, 875, 819 cm⁻¹; ¹H (600 MHz) and ¹³C NMR (150 MHz) data see [Tables 1] and [3]; (+)HRESIMS m/z 425.1589 [M + Na]⁺ (calcd. for C₂₂H₂₆O₇Na, 425.1571).

threo-Guaiacylethoxyglycerol-β-O-4′-guaiacyl aldehyde ether (3): pale yellow oil; [α]_D ²⁰: − 4.52 (c 0.09, MeOH); UV (MeOH) λ _max (log ε): 309 (3.50), 279 (3.67), 228 (3.85) nm; IR (KBr) v _max 3449, 1673, 1594, 1510, 1463, 1273, 1130, 1033, 867, 814 cm⁻¹; ¹H (600 MHz) and ¹³C NMR (150 MHz) data see [Tables 1] and [3]; (+)HRESIMS m/z 399.1435 [M + Na]⁺ (calcd. for C₂₀H₂₄O₇Na, 399.1414).

erythro-Guaiacylethoxyglycerol-β-O-4′-guaiacyl aldehyde ether (4): pale yellow oil; [α]_D ²⁰: − 9.07 (c 0.09, MeOH); UV (MeOH) λ _max (log ε): 309 (3.57), 279 (3.71), 228 (3.88) nm; IR (KBr) v _max 3450, 1677, 1596, 1508, 1466, 1271, 1136, 1031, 868, 815 cm⁻¹; ¹H (600 MHz) and ¹³C NMR (150 MHz) data see [Tables 1] and [3]; (+)HRESIMS m/z 399.1405 [M + Na]⁺ (calcd. for C₂₀H₂₄O₇Na, 399.1414).

trans-2-Guaiacyl-3-hydroxymethyl-5-(cis-3′-hydroxymethyl-5′-formyl-7′-methoxybenzofuranyl)-7-methoxybenzofuran (5): pale yellow amorphous powder, m. p. 67–69 °C; [α]_D ²⁰: − 10.38 (c 0.07, MeOH); UV (MeOH) λ _max (log ε): 378 (2.36), 288 (4.10), 235 (4.33) nm; IR (KBr) v _max 3442, 1631, 1516, 1493, 1452, 1276, 1136, 1032 cm⁻¹; ¹H (600 MHz) and ¹³C NMR (150 MHz) data see [Tables 2] and [3]; (+)HRESIMS m/z 509.1778 [M + H]⁺ (calcd. for C₂₈H₂₉O₉, 509.1806).

erythro-Guaiacylglycerol-β-O-4′-(+)-5,5′-dimethoxylariciresinol ether (6): pale yellow amorphous powder, m. p. 92–94 °C; [α]_D ²⁰: − 9.00 (c 0.20, MeOH); UV (MeOH) λ _max (log ε): 378 (2.74), 279 (3.73) nm; IR (KBr) v _max 3441, 3433, 1620, 1517, 1462, 1274, 1121, 1035, 877, 857 cm⁻¹; ¹H (600 MHz) and ¹³C NMR (150 MHz) data see [Tables 2] and [3]; (+)HRESIMS m/z 639.2400 [M + Na]⁺ (calcd. for C₃₂H₄₀O₁₂Na, 639.2412).

(2R*,3R*,4S*)-2,3-Diguaiacyl-4-hydroxyl tetrahydrofuran (7): pale yellow oil; [α]_D ²⁰: − 5.25 (c 0.08, MeOH); UV (MeOH) λ _max (log ε): 281 (3.88), 228 (4.26) nm; IR (KBr) v _max 3494, 3444, 1609, 1517, 1456, 1274, 1233, 1124, 1034, 874, 817 cm⁻¹; ¹H (600 MHz) and ¹³C NMR (150 MHz) data see [Tables 2] and [3]; (−)HRESIMS m/z 331.1157 [M – H]⁻ (calcd. for C₁₈H₁₉O₆, 331.1187).

Agonistic activity assay

The agonistic rates of the test compounds were performed according to our previous report [37].

Supporting information

1D and 2D NMR spectra as well as HRESIMS spectra of the seven new compounds, detailed extraction and isolation, and the procedure of the bioassay are available as Supporting Information.

Acknowledgements

This work was supported by the National Science Foundation of China for Distinguished Young Scholars (No. 81 025 023), the National Natural Science Foundation of China (No. 81 202 436), and the Youth Innovation Promotion Association, CAS. The authors sincerely thank the staff of the analytical group of the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, CAS.

Conflict of Interest

The authors declare no conflict of interest.

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