English

• 1.   Introduction

  Traditional Chinese medicine, Fructus carotae, it's the ripe fruit of plant Daucus carota(umbelliferae). This plant is widely distributed in Jiangsu, Hubei, Sichuan, Zhejiang, Anhui and Guizhou provinces of China.^[1] It has been used as a herbal medicine for the treatment of ancylostomiasis, dropsy, chronic kidney diseases and bladder afflictions.^[2] Modern pharmacological research displays that Fructus carotae has the activities of insecticidal, antibacterial, antidiarrheal and anti-inflammatory.^[3-5] Previous phyto- chemical investigations on the Fructus carotae indicated the presence of flavonoids, ^[6] coumarins and sesquter- penoids.^{[7, 8]} As part of an ongoing research program to isolate and determine structures of secondary metabolites from traditional Chinese medicine, ^[9] we sequentially performed a phytochemical study on Fructus carotae.^[10] As a result, two new sesquterpenoids and five known analogues were obtained (Figure 1). Their structures were identified as 7-ethoxy-4(15)-oppositen-1β-ol (1), 11-acet- oxy-8β-ange- loyloxy-15-methoxy-4α, 5α-epoxyarbutane-3-one (2), 11- acetoxy-8β-propionyl-4-guaien-3-one (3), ^[11] 1-oxo-5α, 7αH-eudesma-3-en-15-al (4), ^[12] 1β-hydroxy-4(15), 7-eude- smadiene (5), ^[13] 1β-hydroxy-4(15), 5E, 10(14)-germacra- triene (6), ^[14] and 1β-hydroxy-4(15), 5-eudesmadiene (7).^[14] Compounds 3, 4, 6 and 7 were isolated from the Daucus for the first time, and compound 5 was isolated from this plant for the first time.

  Figure 1

  

  Figure 1.  Structures of compounds 1~7

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  2.   Results and discussion

  2.1   Structure identification of compound 1

  Compound 1 was obtained as a colorless oil. The molecular formula C₁₇H₃₀O₂ was established by HR-ESIMS (m/z 267.23183 [M+H]⁺), indicating three degrees of unsaturations. The IR spectrum showed absorption band due to hydroxyl group (3395 cm^-1). The ¹H NMR spectrum (Table 1) showed the presence of four methyl signals at δ_H 0.63 (s, 3H), 0.92 (d, J＝6.7 Hz, 3H), 0.87 (d, J＝6.7 Hz, 3H), and 1.04 (t, J＝7.0 Hz, 3H), an exocyclic methylene group at δ_H 4.77 (br s, 1H) and 4.83 (br s, 1H)^[15], an oxygenated methyne proton signal at δ_H 3.54 (dd, J＝11.3 Hz, 4.8 Hz, 1H), an oxygenated methylene proton signal at δ_H 3.40 (m, 2H). The ¹³C NMR and heteronuclear single quantum correlation (HSQC) spectra showed 17 carbon resonances due to two olefinic carbon signals at δ_C 145.8, 107.5, two oxygenated tertiary carbons at δ_C 89.4, 79.4, an oxygenated secondary carbon at δ_C 68.0, three tertiary carbons at δ_C 55.5, 39.4, 31.5, four secondary carbon at δ_C 32.1, 35.1, 26.5, 37.3, and four methyl carbon signals at δ_C 12.5, 15.7, 16.4, 21.0. The ¹H NMR and ¹³C NMR spectra were very similar to those of 7-methoxy-4(15)-oppositen- 1β-ol^[16] except for the replacement of the methoxy group by the ethoxy group at C-7. The structure of compound 1 was further confirmed by 2D NMR. The heteronuclear multiple-bond correlations (HMBC) of H-7 to C-1', H-1' to C-7, H-1' to C-2' established the ethoxy group located at C-7. In its nuclear overhauser effect spectroscopy (NOESY), the cross-peaks of H-1/H-5 and H-6/H-11 confirmed the relative configuration of 1-OH (cis) with 11-Me, 11-Me(trans) with H-5. Accordingly, the structure of compound 1 was established and named as 7-ethoxy- 4(15)-oppositen-1β-ol.

  Table 1

  

  Table 1.  ¹H NMR and ¹³C NMR (400/100 MHz, CDCl₃) data of compounds 1 and 2

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  No.	1			2
  	δH (J in Hz)	δC		δH (J in Hz)	δC
  1	3.54 (dd, 11.3, 4.8)	79.4		2.14 (overlapped)	45.5
  2	1.44 (m), 1.75 (m)	32.1		2.14 (overlapped)	37.7
  3	2.27 (m), 2.02 (m)	35.1			209.4
  4		145.8			68.3
  5	1.80 (m)	55.5			73.3
  6	2.36 (m)	39.4		2.44 (dd, 14.3, 11.5) β, 1.75 (d, 14.3) α	26.4
  7	2.82 (dd, 8.1, 3.6)	89.4		2.81 (br d, 11.5)	45.3
  8	1.33 (m), 1.83 (m)	26.5		5.57 (m)	70.3
  9	1.65 (m), 1.31 (m)	37.3		1.51 (overlapped), 2.28 (ddd, 15.4, 6.8, 4.4)	42.3
  10		49.6		2.01 (m)	30.6
  11	1.77 (m)	31.5			84.2
  12	0.92 (d, 6.7)	16.4		1.44 (s)	24.6
  13	0.87 (d, 6.7)	21.0		1.40 (s)	24.8
  14	0.63 (s)	12.5		0.88 (d, 6.7)	21.6
  15	4.77 (br. s), 4.83 (br s)	107.5		3.93 (d, 11.3), 3.61 (d, 11.3)	66.0
  1'	3.40 (m)	68.0			166.9
  2'	1.04 (t, 7.0)	15.7			127.8
  3'				6.08 (q, 7.1)	139.2
  4'				2.00 (d, 7.1)	16.0
  5'				1.87 (s)	20.9
  1"					170.5
  2"				1.96 (s)	22.7
  15-OCH3				3.39 (s)	60.0

  2.2   Structure identification of compound 2

  Compound 2 was obtained as a colorless oil, with the molecular formula of C₂₃H₃₄O₇, which was deduced by HR-ESIMS (m/z 440.26428 [M+NH₄]⁺), with seven double bond equivalents. The IR spectrum showed absorption band due to carbonyl group (1734 cm^-1). The ¹H NMR (Table 1) indicated six methyl group signals at δ_H 0.87 (d, J＝6.7 Hz, 3H), 2.00 (d, J＝7.1 Hz, 3H), 1.87 (s, 3H), 1.40 (s, 3H), 1.44 (s, 3H), 1.96 (s, 3H), a methoxy group signal at δ_H 3.39 (s, 3H), an oxygenated methyne proton signal at δ_H 5.57 (m, 1H), an oxygenated methylene proton signal at 3.93, 3.61 (each d, J＝11.3 Hz, 1H), an olefinic proton signal at δ_H 6.08 (q, J＝7.1 Hz, 1H). The ¹³C NMR and HSQC spectra showed 23 carbon resonances due to three carbonyl signals at δ_C 209.4, 170.5, 166.9, two olefinic carbon signals at δ_C 127.8, 139.2, three oxygenated quaternary carbons at δ_C 84.2, 73.3, 68.3, an oxygenated tertiary carbon at δ_C 70.3, an oxygenated secondary carbon at δ_C 66.0, a methoxy carbon at δ_C 60.0, three tertiary carbons at δ_C 45.5, 45.3, 30.6, three secondary carbon at δ_C 37.7, 26.4, 42.3, and six methyl carbon signals at δ_C 24.8, 24.6, 22.7, 21.6, 20.9, 16.0. These NMR data above indicated that compound 2 has an arbutane skeleton with angeloyloxy and acetoxy.^[10] Detailed analysis of the NMR and HSQC spectra suggested that the structure of compound 2 was very similar to those of torilin^[17] except for the replacement of the double bond by the epoxy group at C-4 and C-5. The HMBC correlations of OMe to C-15 showed the methoxy group located at C-15. The HMBC correlations of H-15 to C-4, H-2 to C-4, H-2 to C-5, H-7 to C-5, H-6 to C-5, established the presence of epoxy group at C-4 and C-5. In its NOESY experiment, the cross-peaks of H-15/H_α-6, H_α-6/H-7, and H-7/H-8 displayed that epoxy group and angeloyloxy group existed in the same face of the molecule. The NOESY correlations of H-1/H-14 and H-7/H-10 suggested that H-1, 14-Me and the substitutional group of C-7 were in the same face of the molecule. Therefore, the structure of compound 2 was unambiguously established as 11-acetoxy-8β-angeloyloxy-15-methoxy- 4α, 5α-epoxyarbutane-3-one.

  Figure 2

  

  Figure 2.  Key HMBC correlations () of compounds 1 and 2

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  Figure 3

  

  Figure 3.  Key NOESY correlations () of compounds 1 and 2

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  3.   Conclusions

  Seven compounds were obtained from the Chinese traditional medicine, Fructus carotae. Compounds 1 and 2 were new sesquterpenoids compounds, compounds 4~7 were eudesmane sesquterpenoids, compounds 3, 4, 6 and 7 were isolated from the Daucus for the first time, compound 5 was isolated from this plant for the first time.

  4.   Experimental

  4.1   Instruments and reagents

  IR spectra were measured on an FTIR-850 spectrometer (KBr) (Perkin-Elmer, Waltham, USA). 1D NMR and 2D NMR spectra were recorded on an Agilent DD2400-MR instrument using TMS as the internal reference. HR-ESIMS were measured on a MicrO TOF-Q Ⅱ mass spectrometer (Bruker, Karlsruhe, Germany). Column chromatography was performed using 300~400 mesh silica gel (Qingdao Marine Chemical Inc., Qingdao, China). Semi-preparative HPLC was performed on LC3000 system (Beijing Chuang Xing Tong Heng Science and Technology Co., Ltd., Beijing, China) equipped with ODS column (5 μm, i.d. 10 mm×250 mm) (YMC, Kyoto, Japan).

  4.2   Plant materials

  Fructus carotae was collected from medicinal materials market in July 2016, and identified as the ripe fruit of plant Daucus carota by associate professor Nie (Zunyi Medical University). A voucher specimen (No. 20151108) was deposited with the Herbarium of the School of Pharmacy, Zunyi Medical Universty.

  4.3   Extraction and separation

  The air-dried Fructus carotae (5.0 kg) were extraction with 95% ethanol (25 L) for four times, the ethanol extract was evaporated under reduced pressure to obtain a crude extract (420 g), which was further suspended in water and extracted with petroleum ether, EtOAc and n-BuOH, successively. The EtOAc extract (30 g) was subjected to silica gel column chromatography (80 mm×600 mm, 400 g, 300~400 mesh), eluted with a gradient of petroleum ether-EtOAc to yield 6 fractions (Fr.1~Fr.6). Fr.1 was subjected to silica gel column chromatography (80 mm×600 mm, 180 g, 300~400 mesh), eluted with a gradient of petroleum ether-EtOAc to yield 8 subfractions (Fr.1.1~Fr.1.8). Fr.1.1 was purified by semi-preparative HPLC with acetonitrile-H₂O (V:V＝98:2) to yield 4 fractions (Fr.1.1.1~Fr.1.1.4). Fr.1.1.4 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝75:25, 5.0 mL/min, 230 nm) to afford compound 1 (R_t＝17.9 min, 10.6 mg). Fr.1.1.3 was purified by semi-preparative HPLC with acetonitrile-H₂O (V:V＝98:2, 2.0 mL/min, 230 nm) to afford compound 6 (R_t＝7.5 min, 110 mg). Fr.1.1.2 was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝72:28, 4 mL/min, 230 nm) to afford compound 4 (R_t＝13.0 min, 50 mg). Fr.1.4 was further purified by semi-preparative HPLC with acetonitrile-H₂O (V:V＝90:10, 4 ml/min, 230 nm) to afford compound 5 (R_t＝15.7 min, 40 mg). Fr.1.5 was isolated by SephadexLH-20 column chromatography (MeOH) to afford 6 fractions (Fr.1.5.1~Fr.1.5.6). Fr.1.5.2 was purified by semi-preparative HPLC with MeOH-H₂O (V:V＝89:11) to give 3 subfractions (Fr.1.5.2A~Fr.1.5.2C). Fr.1.5.2C was purified by semi-preparative HPLC with MeOH-H₂O (V:V＝89:11, 4.0 mL/min, 230 nm) to afford compound 7 (R_t＝22.3 min, 80 mg). Fr.3 was isolated by Sephadex LH-20 (MeOH) to give 2 fractions (Fr.3.1~Fr.3.2). Fr.3.1 was purified by semi-preparative HPLC with MeOH-H₂O (V:V＝80:20) to give 6 subfractions (Fr.3.1A~Fr.3.1.F). Fr.3.1B was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝70:30, , 5 mL/min, 230 nm) to afford compound 3 (R_t＝38.9 min, 13 mg). Fr.3.1D was further purified by semi-preparative HPLC with MeOH-H₂O (V:V＝85:15, 4 mL/min, 230 nm) to afford compound 2 (R_t＝17.4 min, 7.1 mg).

  7-Ethoxy-4(15)-oppositen-1β-ol (1): colorless oil; [α]_D²⁰－85 (c 0.04, MeOH); ¹H NMR and ¹³C NMR data, see Table 1; IR (KBr) ν_max: 3395, 2925, 1460, 1377, 1080, 1019, 888 cm^-1; HR-ESI-MS calcd for C₁₇H₃₁O₂ [M+H]⁺ 267.2318, found 267.2319.

  11-Acetoxy-8β-angeloyloxy-15-methoxy-4α, 5α-epoxy- arbutane-3-one (2): colorless oil; [α]_D²⁰ －82 (c 0.04, MeOH); UV (MeOH) λ_max [lg ε/(L·mol^-1·cm^-1)] 209 (1.82) nm; ¹H NMR and ¹³C NMR data see Table 1; IR (KBr)ν_max: 2928, 1734, 1459, 1369, 1233, 1120, 607 cm^-1; HR-ESI-MS calcd for C₂₃H₃₈O₇N [M+NH₄]⁺ 440.2643, found 440.2643.

  Supporting Information   1D NMR and 2D NMR spectra of compounds 1 and 2. These materials can be downloaded for free from our website (http://sioc-journal.cn/).

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• 

  Figure 1  Structures of compounds 1~7

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  Figure 2  Key HMBC correlations () of compounds 1 and 2

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  Figure 3  Key NOESY correlations () of compounds 1 and 2

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  Table 1.  ¹H NMR and ¹³C NMR (400/100 MHz, CDCl₃) data of compounds 1 and 2

  No.	1			2
  	δH (J in Hz)	δC		δH (J in Hz)	δC
  1	3.54 (dd, 11.3, 4.8)	79.4		2.14 (overlapped)	45.5
  2	1.44 (m), 1.75 (m)	32.1		2.14 (overlapped)	37.7
  3	2.27 (m), 2.02 (m)	35.1			209.4
  4		145.8			68.3
  5	1.80 (m)	55.5			73.3
  6	2.36 (m)	39.4		2.44 (dd, 14.3, 11.5) β, 1.75 (d, 14.3) α	26.4
  7	2.82 (dd, 8.1, 3.6)	89.4		2.81 (br d, 11.5)	45.3
  8	1.33 (m), 1.83 (m)	26.5		5.57 (m)	70.3
  9	1.65 (m), 1.31 (m)	37.3		1.51 (overlapped), 2.28 (ddd, 15.4, 6.8, 4.4)	42.3
  10		49.6		2.01 (m)	30.6
  11	1.77 (m)	31.5			84.2
  12	0.92 (d, 6.7)	16.4		1.44 (s)	24.6
  13	0.87 (d, 6.7)	21.0		1.40 (s)	24.8
  14	0.63 (s)	12.5		0.88 (d, 6.7)	21.6
  15	4.77 (br. s), 4.83 (br s)	107.5		3.93 (d, 11.3), 3.61 (d, 11.3)	66.0
  1'	3.40 (m)	68.0			166.9
  2'	1.04 (t, 7.0)	15.7			127.8
  3'				6.08 (q, 7.1)	139.2
  4'				2.00 (d, 7.1)	16.0
  5'				1.87 (s)	20.9
  1"					170.5
  2"				1.96 (s)	22.7
  15-OCH3				3.39 (s)	60.0

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•