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Extraction, isolation and identification of shikonofuran a and shikonofuran e extraction


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Extraction, isolation and identification of shikonofuran A and shikonofuran E

Extraction

The WCL cells (50 g dry weight) were cultured in improved LS liquid medium in the dark at 25 ℃ for 14 days, incubated with MeJA for 6-8 days, and extracted with methanol.


Isolation

The concentrated methanol extracts were preliminarily purified by silica-gel column chromatography. Two fractions were isolated and prepared by semi-preparative HPLC with the YMC-Pack ODS column. All the agent grade chemicals were purchased from Fisher Chemical Co. (Thermo Fisher Scientific, Massachusetts, USA). Column chromatography was performed using 230–400 mesh silica gels (Kieselgel 60, Merck, Germany). Hypersil ODS (Thermo Fisher Scientific, Massachusetts, USA) and YMC-Pack ODS (YMC, Kyoto, Japan) were used for column chromatography.

The methanol extraction was concentrated on an evaporating dish, and 500 mL was chromatographed on a silica gel using 2 L mixtures of methanol-water of decreasing polarity (30:70-40:60-50:50-60:40-70:30-100:0), yielding 6 fractions. Each fraction was divided into eight sub-fractions. The sub-fractions 4-6, 4-7, and 4-8 were exposed to column chromatography on a silica gel eluted with methanol-water (60:40, v/v). The sub-fractions 5-1 and 5-2 eluted with methanol-water (70:30, v/v) harbored compound I, and the sub-fractions 5-8, 6-1, and 6-2 harbored compound II. The sub-fractions 4-6, 4-7, 4-8, 5-1, and 5-2 were separated and purified by chromatography on a YMC-Pack ODS column and preparative-HPLC to yield compound I (10.2 mg). Compound II (20.5 mg) was isolated from the 5-8, 6-1, and 6-2 sub-fractions using preparative-HPLC (Fukui et al. 1992).
Identification

F
ig. 1
: Structures of shikonofuran A (compound I) and shikonofuran E (compound II)

To confirm the structures of compounds I and II that accumulated in the treated WCL, we extracted and isolated these compounds; they were identified using 1HNMR, 13CNMR and mass-spectrometric techniques as shikonofuran A and shikonofuran E, respectively (Fig. 1). 1H and 13CNMR data were obtained on a Varian 600 MHz (Varian, Salt Lake City, America) spectrometer in CD3OD and tetramethylsilane as an internal standard. The ESI mass spectra were scanned using an Agilent 6410 (Agilent, California, America) with the ESI in negative mode.

Shikonofuran A: amorphous white powder, ESI-MS (negative) m/z 315.1[M–H]-, molecular formula: C18H20O5. 1H-NMR (600 MHz, CD3OD) δ 7.45 (1H, s, H-10), 7.16 (1H, d, J = 3.0 Hz, H-2), 6.97 (1H, s, H-8), 6.71 (1H, d, J = 9.0 Hz, H-5), 6.56 (1H, dd, J = 9.0, 3.0 Hz, H-6), 5.73 (1H, t, J = 7.2 Hz, H-13), 5.11 (1H, t, J = 7.2 Hz, H-11), 2.62 (1H, dt, J = 14.4, 7.2 Hz, Hβ-12), 2.52 (1H, dt, J = 14.4, 7.2 Hz, Hα-12), 2.04 (3H, s, -COOCH3), 1.67 (3H, s, H-15), 1.61 (3H, s, H-16); 13C-NMR (150 MHz, CD3OD) δ 149.7 (C-1), 108.2 (C-2), 118.0 (C-3), 146.6 (C-4), 114.6 (C-5), 116.1 (C-6), 151.3 (C-7), 111.0 (C-8), 118.7 (C-9), 138.2 (C-10), 68.9 (C-11), 33.2 (C-12), 126.7 (C-13), 134.4 (C-14), 19.7 (C-15), 16.6 (C-16), 166.2 (-COOCH3), 24.5 (-COOCH3); The spectral data presented above are consistent with the published results (Yoshizaki et al. 1982).

Shikonofuran E: pale yellow oil, ESI-MS (negative) m/z 355.1[M–H]-, molecular formula: C21H24O5. 1H-NMR (600 MHz, CD3OD) δ 7.44 (1H, s, H-10), 7.16 (1H, d, J = 3.0 Hz, H-2), 6.98 (1H, s, H-8), 6.71 (1H, d, J = 9.0 Hz, H-5), 6.56 (1H, dd, J = 9.0, 3.0 Hz, H-6), 5.76 (1H, t, J = 7.2 Hz, H-13), 5.71 (1H, t-like, J = 1.2 Hz, H-1′), 5.13 (1H, t, J = 7.2 Hz, H-11), 2.62 (1H, dt, J = 14.4, 7.2 Hz, Hβ-12), 2.53 (1H, dt, J = 14.4, 7.2 Hz, Hα-12), 2.16 (3H, d, J = 1.2 Hz, H-3′), 1.91 (3H, d, J = 0.6 Hz, H-4′), 1.68 (3H, s, H-15), 1.63 (3H, s, H-16); 13C-NMR (150 MHz, CD3OD) δ 149.6 (C-1), 108.3 (C-2), 118.1 (C-3), 146.6 (C-4), 114.6 (C-5), 116.2 (C-6), 151.2 (C-7), 111.1 (C-8), 118.8 (C-9), 138.0 (C-10), 67.9 (C-11), 33.4 (C-12), 127.0 (C-13), 134.3 (C-14), 19.0 (C-15), 16.6 (C-16), 115.5 (C-1′), 157.2 (C-2′), 26.0 (C-3′), 24.6 (C-4′), 166.2 (-COO-); The spectral data presented above are consistent with the published results (Kim et al. 2012; Yazaki et al. 1986; Yoshizaki et al. 1982)


SPECTROGRAMS
Shikonofuran A 1H-NMR



Shikonofuran A 1H-NMR



Shikonofuran A 13C-NMR



Shikonofuran A 13C-NMR



Shikonofuran E 1H-NMR



Shikonofuran E 1H-NMR



Shikonofuran E 13C-NMR


MS of Shikonofuran A



MS of Shikonofuran E




References
Fukui H, Tani M, Tabata M (1992) An unusual metabolite, dihydroechinofuran, released from cultured cells of Lithospermum erythrorhizon. Phytochemistry 31(2):519-521

Kim JY, Jeong HJ, Park JY, Kim YM, Park SJ, Cho JK, Park KH, Ryu YB, Lee WS (2012) Selective and slow-binding inhibition of shikonin derivatives isolated from Lithospermum erythrorhizon on glycosyl hydrolase 33 and 34 sialidases. Bioorgan Med Chem 20(5):1740-1748



Yazaki K, Fukui H, Tabata M (1986) Isolation of the intermediates and related metabolites of shikonin biosynthesis from Lithospermum erythrorhizon cell cultures. Chem Pharm Bull 34(5):2290-2293

Yoshizaki F, Hisamichi S, Kondo Y, Sato Y, Nozoe S (1982) Studies on Shikon. III. New Furylhydroquinone Derivatives, Shikonofurans A, B, C, D and E, from Lithospermum erythrorhizon Sieb. et Zucc. Chem Pharm Bull 30(12):4407-4411


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