Stilbenes with anti-inflammatory and cytotoxic activity from the rhizomes of Bletilla ochracea Schltr

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Fitoterapia

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Stilbenes with anti-in fl ammatory and cytotoxic activity from the rhizomes of Bletilla ochracea Schltr

Jin-Yu Li

^a,b

, Meng-Ting Kuang

^a,b

, Liu Yang

^a

, Qing-Hua Kong

^a

, Bo Hou

^a,b

, Zhen-Hua Liu

^a,b

, Xiao-Qian Chi

^a,b

, Ming-Yan Yuan

^a

, Jiang-Miao Hu

^a,⁎

, Jun Zhou

^a,⁎

aState Key Laboratory of Phytochemistry and Plant Resources in West China, Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People's Republic of China

bUniversity of Chinese Academy of Sciences, Beijing 100049, People's Republic of China

A R T I C L E I N F O

Keywords:

Bletilla ochracea Anti-inflammation Cytotoxic activity Dihydrophenanthrenofuran

A B S T R A C T

Four new dihydrophenanthrenofuran, bleochranols A–D (1–4), along with 21 known compounds including phenanthrenes (5–14) and bibenzyls (15–25) were isolated and elucidated from the rhizomes ofBletilla ochracea.

Combination of 1D/2D NMR techniques and the Electronic Circular Dichroism (ECD) spectroscopy based on the empirical helicity rules, chemical structure of those isolates were determined. All the compounds were evaluated for cytotoxicity against HL-60, SMMC-7721, A-549, MCF-7 and SW480 human cancer cell lines by MTS assay and anti-inflammatory activity by nitric oxide (NO) production in LPS-stimulated RAW 264.7 macrophages. Among the 25 tested compounds, bleochranol A (1) showed remarkable cytotoxic activity against HL-60, A-549, and MCF-7 with IC50values of 0.24 ± 0.03, 3.51 ± 0.09 and 3.30 ± 0.99μM respectively. The anti-inflammatory assay showed that compound12exhibited most potential activity against NO production in RAW 264.7 mac- rophages with IC502.86 ± 0.17μM. The results indicated that the main chemical constituents ofB. ochracea were phenanthrene and bibenzyl and similar to that ofB. striata.

1. Introduction

The genus Bletilla (Orchidaceae) is mainly distributed in China, North Korea, Japan and Myanmar. The rhizomes of someBletillaspecies were used in folk and traditional medicine ("Bai-Ji" in Chinese) for the treatment of bleeding, burns, esophagitis, erosive gastritis and ulcera- tive carbuncle [1,2]. Biological source for the herbal medicine "Bai-Ji" is mainly from threeBletillaspecies:B. striataRchb. f.,B. ochraceaSchltr andB. formosanaSchltr. Previous investigations mainly focused onB.

striataand resulted in the isolation of phenanthrenes, biphenanthrenes, bibenzyls, flavonoids, cyanidin glycosides, triterpenoids, steroidal sa- ponins, and polysaccharides [1,3–5]. Pharmacological researches have demonstrated thatBletillaspecies possessed a variety of biological ac- tivities including anti-inflammatory, antitumor, antimicrobial, anti- fungal, antiallergic, and spasmolyticetc [1,2,6–8]. The abundant re- sources of B. ochracea mainly distributed in southwest of China, especially in Yunnan Province, which also used as "Bai-Ji" in most parts of southern China for the treatment of pulmonary tuberculosis, pneu- monorrhagia, and malignant ulcers [9]. However, to the best of our knowledge, Chemical and pharmaceutical research of the rhizomes of B. ochraceahas not yet been done in-depth [9,10]. With our continual

research of chemical constituents and pharmacological usage with herbal medicine of Orchidaceae, chemical research of the rhizome ofB.

ochraceawas carried out in this work with a view to explore anti-in- flammation and anti-tumor ingredients. The results may give us con- crete evidence to support the ethnopharmacological usage of B.

ochracea, especially on anti-inflammation and anti-tumor.

2. Materials and methods 2.1. General experimental procedures

Optical rotations were measured on a JASCO P-1020 digital po- larimeter (Horibia, Tokyo, Japan).UV sepectra were obtained on a Shimadzu UV-2401 PC spectrometer (Shimadzu, Kyoto, Japan). IR (KBr) spectra were recorded on a Bio-Rad FTS-135 spectrometer (Bio- Rad, Hercules, California USA). NMR spectra were performed on Bruker DRX-500 and AVANCE III-600 spectrometer with tetramethylsilane (TMS) as an internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. MS and HR-ESI-MS were per- formed on a VG-Auto-spec-3000 spectrometer. Semi-preparative HPLC was carried out on an Agilent 1100 HPLC with a ZORBAX SB-C18

https://doi.org/10.1016/j.fitote.2018.02.007

Received 22 November 2017; Received in revised form 29 January 2018; Accepted 3 February 2018

⁎Corresponding authors.

E-mail addresses:hujiangmiao@mail.kib.ac.cn(J.-M. Hu),jzhou@mail.kib.ac.cn(J. Zhou).

0367-326X/ © 2018 Elsevier B.V. All rights reserved.

Please cite this article as: Li, J.-Y., Fitoterapia (2018), https://doi.org/10.1016/j.fitote.2018.02.007

(9.4 × 250 mm, Agilent, USA) column. Column chromatography (CC) was performed on silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical Co., Ltd, Qingdao, China), Lichroprep RP-18 (43–63μm, Merck, Darmstadt, Germany), YMC*-GEL ODS-A (12 nm, S- 50μm, YMC, Japan) and Sephadex LH-20 (Amersham Biosciences AB, Uppsala, Sweden). Fractions were monitored by TLC plates (Si gel G and GF254, Qingdao Haiyang Chemical Co., Ltd, Qingdao, China) and spots were visualized by heating silica gel plates sprayed with 10%

H2SO4in EtOH.

2.2. Plant material

Rhizomes of B. ochracea were collected in Zhaotong Country of Yunnan province, People's Republic of China, in December 2015, and identified by Dr. Zhi-Kun Wu, Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (NO.20151203) was de- posited at the State key Laboratory of Phytochemistry and Plant Resource in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

2.3. Extraction and isolation

The air-dried and powdered rhizomes ofB. ochracea(8.0 kg) were extracted three times with 80% EtOH (25 L × 3) at room temperature and concentrated at vacuum to yield crude extract, which was sus- pended in H2O and then extracted by EtOAc (4 L × 4) andn-BuOH (4 L × 4) successively. The EtOAc fraction (270.0 g) was chromato- graphed on a silica gel (2.9 kg, 100–200 mesh) column eluted with CHCl3–MeOH gradient system (50:1–4:1) to afford fractions A–I.

Fraction A (60.5 g) was further fractionated over Medium Pressure Liquid Chromatography (MPLC, ODS), eluting with 30–100%

MeOH–H2O (100 min, flow rate 10 mL min⁻¹) to afford nine

subfractions (A1~A9). Fr.A2 (5.3 g) was applied to Sephadex LH-20 (CHCl3–MeOH 1:1) to provide Fr.A2–1 ~ Fr.A2–5. Fr.A2–2 (1.2 g) was further chromatographed over silica gel using petroleum ether-acetone (3:1–1:1) as solvent to afford24(960.3 mg). Fr.A2–3 (4.2 g) was ap- plied to a silica gel using petroleum ether-acetone (2:1) as eluent to yield 25 (2.6 g). Fr.A2–4 (420.0 mg) was separated over silica gel eluting with CHCl3–MeOH (20:1–10:1), followed by semi-preparative HPLC (51% CH3CN–H2O) to obtain11 (22.3 mg,tR= 10.5 min),13 (4.1 mg, tR= 15.2 min) and 14 (8.9 mg, tR= 17.1 min). Fr.A2-5 (376.5 mg) was applied to Sephadex LH-20 (MeOH–H2O 90:10) and then further to semi-preparative HPLC with 40% CH3CN–H2O to pro- vide 12 (46.5 mg, tR= 11.5 min). Fr.A4 (4.2 g) was purified by Sephadex LH-20 (CHCl3–MeOH 1:1) to give seven fractions Fr.A4- 1 ~ Fr.A4-7. Fr.A4-2 (354.6 mg) was subjected to silica gel CC eluted with a CHCl3–MeOH gradient (25:1–10:1) and then purified by semi- preparative HPLC (40% CH3CN–H2O) to afford 10 (9.9 mg, tR= 13.4 min). Fr.A4-4 (520.0 mg) was applied to silica gel (CHCl3–MeOH 20:1–10:1), and then further to semi-preparative HPLC with 51% CH3CN–H2O to provide6(16.5 mg,tR= 13.3 min) and23 (13.0 mg,tR= 16.5 min). Fr.A6 (3.6 g) was applied to Sephadex LH-20 (MeOH–H2O 90:10), silica gel column (CHCl3–MeOH, 20:1–10:1) and then to semi-preparative HPLC (52% CH3CN–H2O) to afford 20 (10.1 mg,tR= 15.6 min),21(42.1 mg,tR= 17.3 min) and22(4.9 mg, tR= 18.6 min). Fraction B (27.2 g) was subjected to Sephadex LH-20 column (MeOH–H2O 90:10) yielding B1 ~ B10. Subfraction B6 (4.6 g) was separated by Sephadex LH-20 (MeOH–H2O 90:10) to give seven fractions B6-1 ~ B6-7. Fr.B6-2 (567.6 mg) was subjected to silica gel CC eluted with a CHCl3–MeOH gradient (15:1–1:1), to yield fractions B6-2- 1 ~ B6-2-8. Fr.B6-2-3 (182.5 mg) was purified over repeated Sephadex LH-20 (MeOH–H2O 90:10), followed by semi-preparative HPLC 51%

CH3CN–H2O to obtain compounds 7 (13.2 mg, tR= 11.7 min), 2 (5.2 mg, tR= 13.5 min) and 4 (2.4 mg, tR= 17.2 min). Similarly,

Table 1

1H NMR data for compounds1–4(CD3OD,δin ppm,Jin Hz).

Position 1^a 2^a 3^a 4^b

2 5.53, d, 5.6 5.43, d, 5.5 5.48, d, 5.6 5.40, d, 6.8

3 3.58, m 3.46, m 3.46, m 3.69, m

4 5

7.75, s 8.05, s 8.01, s 8.02, s

6 6.35, d, 2.3 6.39, d, 2.3 6.51, s 6.53, s

8 6.29, d, 2.3 6.30, d, 2.3

9 2.69–2.70, m 2.62–2.65, m mmmmm 2.49–2.51, m 2.54–2.56, m

10 2.69–2.70, m 2.62–2.65, m 2.49–2.51, m 2.54–2.56, m

11 6.65, s 6.66, s 6.64, s 6.63, s

2′ 6.30, d, 2.0 6.95, d, 1.8 6.66, s 6.64, d, 1.6

5′ 6.66, d, 8.2 6.76, d, 8.1 6.78, 8.1

6′ 6.56, dd, 8.2, 2.0 6.82, dd, 8.1, 1.8 6.66, s 6.83, dd, 8.1,

1.6

7′ 2.99, m 3.85, 3.76, m 3.86, 3.75, m 4.40, 4.34, m

2″ 6.93, d, 8.6 6.94, d, 8.8

3″ 6.62, d, 8.6 6.64, d, 8.8

4″ 6.31, d, 1.8

5″ 6.62, d, 8.6 6.64, d, 8.8

6″ 6.42, d, 1.8 6.93, d, 8.6 6.94, d, 8.8

7″ 3.92, s 3.94, s

8″ 6.53, d, 1.8

10″ 6.58, dd, 8.2, 1.8

11″ 6.99, dd, 8.2, 7.7

12″ 6.50, d, 7.7

5-OCH3 3.67, s 3.81, s 3.81, s 3.83, s

3′-OCH3OCH3OCH3OCH3 3.66, s 3.81, s 3.80, s 3.82, s

5′-OCH3 3.80, s

5″-OCH3 3.68, s

α 2.72, m

α′ 2.57, m

7′-OAc 2.03, s

aNMR date was recorded in 500 Hz.

bNMR date was recorded in 600 Hz.

purification of fraction B6–2-5 (182.5 mg) applied to semi-preparative HPLC with 52% CH3CN–H2O afforded 3 (7.6 mg, tR= 14.2 min).

Subfraction B9 (4.1 g) was separated by Sephadex LH-20 (MeOH) to give compound8(16.8 mg). Fraction C (31.0 g) was subjected to MPLC (ODS), eluted with gradient MeOH–H2O (40–70%, 250 min,flow rate 10 mL min⁻¹), to afford eight subfractions, C1 ~ C8. Subfractions C3 (2.6 g) was purified over repeated Sephadex LH-20 (MeOH–H2O 90:10) to yield9 (163.6 mg). Fractions C4 (11.2 g) was applied to repeated Sephadex LH-20 (MeOH–H2O, 90:1), then further separated on silica gel CC eluted with gradient CHCl3–MeOH (15:1–4:1) to afford 15 (970 mg), 18(860.0 mg) and 19(13.9 mg). Fractions C6 (4.7 g) was separated on repeated Sephadex LH-20 (CHCl3-MeOH, 1:1) to give seven fractions (C6-1 ~ C6-7). Fr·C6-3 (461.0 mg) was applied to silica gel column (CHCl3–MeOH 20:1), Sephadhex LH-20 (MeOH–H2O 90:10) and then further purified by semi-preparative HPLC 48% CH3CN–H2O to obtain16(22.1 mg,tR= 12.4 min) and17(53.2 mg,tR= 15.6 min).

Fr·C6-4 (263.2 mg) was applied to Sephadhex LH-20 and further by semi-preparative HPLC using 45% CH3CN–H2O as solvent to give compounds5(16.8 mg,tR= 14.3 min) and1(6.0 mg,tR= 15.4 min).

Theflow rate for Sephadex LH-20 column was 0.5 mL min⁻¹and for semi-preparative HPLC was 2 mL min⁻¹, together with the detection wavelength for HPLC at 280 nm.

2.3.1. Bleochranol A (1)

Brown amorphous powder; [α]D25 –11.8182 (c 0.11, MeOH). UV (MeOH)λmax(logε): 298.5 (4.26), 282.5 (4.37), 204.5 (4.98) nm; IR (KBr):νmax= 3425, 2936, 1605, 1516, 1461, 1271, 1216, 1194, 1156, 1087, 981 cm⁻¹;¹H and¹³C NMR data: seeTables 1 and 2. Positive HR- ESIMS:m/z685.2192 [M + K]⁺(calcd 685.2198 for [C40H38O8K]⁺).

2.3.2. Bleochranol B (2)

Brown amorphous powder; [α]D25 –16.2963 (c 0.09, MeOH). UV (MeOH)λmax(logε): 297.5 (4.15), 282.5 (4.23), 205.5 (4.65) nm; IR (KBr):νmax= 3425, 2935, 1612, 1517, 1463, 1271, 1214, 1159, 1032, 982 cm⁻¹;¹H and¹³C NMR data: seeTables 1 and 2. HR-ESIMS:m/z 459.1199 [M + K]⁺(calcd 459.1204 for [C25H24O6K]⁺).

2.3.3. Bleochranol C (3)

Brown amorphous powder; [α]D25 –16.3333 (c 0.10, MeOH). UV (MeOH)λmax(logε): 303.5 (3.88), 282.0 (3.96), 209.5 (4.51) nm; IR (KBr): νmax= 3432, 2934, 1621, 1513, 1464, 1384, 1321, 1217, 1113 cm⁻¹;¹H and¹³C NMR data: seeTables 1 and 2. HR-ESIMS:m/z 595.1725 [M + K]⁺(calcd 595.1725 for [C33H32O8K]⁺).

2.3.4. Bleochranol D (4)

Brown amorphous powder; [α]D25 –16.9231 (c 0.07, MeOH). UV (MeOH)λmax(logε): 303.0 (3.88), 282.4 (4.00), 204.0 (4.49) nm; IR (KBr):νmax= 3423, 2937, 1613, 1600, 1514, 1465, 1320, 1218, 1112, 982 cm⁻¹;¹H and¹³C NMR data: seeTables 1 and 2. HR-ESIMS:m/z 567.2047 [M−H]⁻(calcd 567.2044 for [C34H31O8]⁻).

2.4. Cytotoxicity assay

Five human cancer cell lines: human myeloid leukemia HL-60, he- patocellular carcinoma SMMC-7721, human lung cancer A-549, mam- mary cancer MCF-7 and human colon cancer SW480 cells were used in the cytotoxic assay. All the cells were cultured in RPMI-1640 (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA), in 5% CO2at 37 °C. The cytotoxicity assay was conducted by the MTS assay (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4- sulfopheny)-2H-tetrazolim) method in 96-well microplates. Briefly, 100μL of adherent cells was seeded into each well of 96-well cell cul- ture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, with an initial density 1 × 10⁵cells/mL in 100μL of medium. The tumor cells were exposed to the tested compounds dissolved in DMSO at various con- centrations in triplicate for 48 h, with cisplatin (Sigma, USA) as a po- sitive control. After compound treatment, cell viability was detected and cell growth curve was graphed. The IC50values were calculated by Reed and Muench's method.

2.5. Anti-inflammatory assay

Murine monocytic RAW 264.7 cells were seeded in 96-well plates (2 × 10⁵cells) containing RPMI-1640 (Hyclone) with 10% FBS in 5%

CO2 at 37 °C. After 24 h preinubation, cells were treated with com- pounds (25μM) dissolved in DMSO, in the prenence of 1μg/mL LPS for 18 h. NO production in each well was assessed by adding 100μL of Giress regent (reagent A and reagent B, respectively; Sigma, USA) to 100μL of supernatant from LPS treated or LPS- and compounds-treated cells in triplicate. After 5 min incubation, the absorbance was measured at 570 nm with a 2014 Envision Multilable Plate Reader (PerkinElmer life Sciences, Inc., Boston, MA, USA).^L-NMMA was used as a positive control.

3. Result and discussion

The isolation of ethanol extract of rhizomes ofB. ochraceaafforded 25 phenanthrenes and bibenzyls, including four new Table 2

13C NMR spectroscopic data of compounds1–4(CD3OD,δin ppm,Jin Hz).

Position 1^a 2^a 3^a 4^b

2 90.9, d 88.7, d 88.8, d 89.3, d

3 52.6, d 55.2, d 55.9, d 51.8, d

3a 127.2, s 125.8, s 125.6, s 125.0, s

4 5

125.4, d 125.4, d 125.6, d 125.6, d

4a 127.2, s 127.3, s 127.7, s 128.0, s

4b 117.9, s 116.9, s 117.6, s 117.5, s

5 160.3, s 159.1, s 157.2, s 157.4, s

6 99.2, d 99.3, d 99.1, d 99.2, d

7 157.5, s 157.6, s 155.8, s 156.0, s

8 108.2, d 108.3, d 118.5, s 118.6, s

8a 141.9, s 142.0, s 141.1, s 141.2, s

9 31.9, t 31.8, t 31.0, t 31.2, t

10 31.5, t 31.5, t 27.5, t 27.6, t

10a 139.6, s 140.3, s 140.2, s 140.7, s

11 108.8, d 108.9, d 108.5, d 108.9, d

11a 157.8, s 158.9, s 158.9, s 158.9, s

1′ 135.6, s 135.3, s 134.6, s 134.5, s

2′ 109.2, d 109.0, d 103.9, d 110.6, d

3′ 148.8, s 149.1,s 149.3, s 149.3, s

4′ 146.9, s 147.9, s 155.8, s 147.8, s

5′ 115.9, d 116.2, d 149.3, s 116.3, d

6′ 118.9, d 119.6, d 103.9, d 120.1, d

7′ 32.2, t 65.6, t 65.6, t 67.3, t

1″ 143.7, s 133.8, s 133.9, s

2″ 117.9, s 130.0, d 130.2, d

3″ 158.3, s 115.8, d 116.0, d

4″ 97.9, d 156.0, s 156.2, s

5″ 159.1, s 115.8, d 116.0, d

6″ 109.8, d 130.0, d 130.2, d

7″ 144.7, s 31.2, t 31.4, t

8″ 116.2, d

9″ 158.5, s

10″ 113.8, d

11″ 130.3, d

12″ 120.6, d

5-OCH3 55.8, q 55.2, q 55.9, q 56.1, q

3′-OCH3 56.2, q 56.3, q 56.7, q 56.2, q

5′-OCH3 56.7, q

5″-OCH3 55.8, q

α 38.5, t

α′ 38.7, t

7′-OAc 172.9, s

20.9, q

aNMR date was recorded in 125 Hz;^bNMR date was recorded in 150 Hz.

dihydrophenanthrenofurans (1–4) (Fig. 1). The known compounds 5–25 were identified as (2,3-trans)-3-[2-hydroxy-6-(3-hydro- xyphenethyl)-4-methoxybenzyl]-2-(4-hydroxy-3-methoxyphenyl)-10- methoxy-2,3,4,5-tetrahydrophenanthro[2,1-b]furan-7-ol (5) [11], pleionesin C (6) [12], (2,3-trans)-2-(4-hydroxy-3-methoxy-phenyl)-3- hydroxymethyl-10-methoxy-2,3,4,5-tetrahydro-phenanthro[2,1-b]

furan-7-ol (7) [11], blestriaren A (8) [13], 1-(4-hydroxybenzyl)-4- methoxy-9,10-dihydrophenanthrene-2,7-diol (9) [14], 3-(4-hydro- xybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol (10) [15], 4- methoxy-9,10-dihydrophenan-threne-2,7-diol (11) [14], 4-methox- yphenanthrene-2,7-diol (12) [15], 2,4-dimethoxyphenanthrene-3,7- diol (13) [16], 3,4-dimethoxyphenanthrene-2,7-diol (14) [16], 3,3′- dihydroxy-2,6-bis(p-hydroxybenzyl)-5-methoxybenzyl (15) [17], 2,6- bis(p-hydroxybenzyl)-3,5′-dimethoxy-3-hydroxybibenzyl (16) [17], ar- undinin (17) [18], 3′,5-dihydroxy-2-(4-hydroxybenzyl)-3-methox- ybenzyl (18) [19], 3,3′-dihydroxy-2-(4-hydroxybenzyl)-5-methox- ybenzyl (19) [19], 3,3′-dimethoxy-5-hydroxy-2-(p-hydroxybenzyl) bibenzyl (20) [20], gymcononopin D (21) [21], 2-(p-hydroxybenzyl)-3- hydroxy-5-methoxybibenzyl (22) [22], 5-hydroxy-2-(p-hydroxybenzyl)- 3-methoxybenzyl (23) [23], 3′-O-methylbatatasin III (24) [24] and batatasin III (25) [15] by comparing their spectral data with those in the literatures. In addition, compounds5–7were isolated fromBletilla genus for thefirst time.

Compound1was obtained as brown amorphous powder. The mo- lecular formula was determined to be C40H38O8 from its positive HRESIMS ([M + K]⁺m/z685.2192, calcd as 685.2198), corresponding to 22 degrees of unsaturation. In the¹H NMR spectrum (Table 1), it could be seen multiplet signals for two methylene hydrogen at δH

2.69–2.70 (4H, m, H-9, H-10) and another two methylene signals atδH

2.72 (2H, m, H-α) and 2.57 (2H, m, H-α′), together with 13 aromatic

proton signals. The¹³C NMR spectra data (Table 2) showed the pre- sences of 30 aromatic carbons including one oxygenated methane, one methane, one oxygenated methylene, five methylenes and three methoxyls. Consulting to all of those NMR data, it could be indicated that there are two skeleton fragments, including 2-(4′-hydroxy-3′- methoxyphenyl)-3-hydroxymethyl-5-methoxy-2,3,9,10-tetrahydro-phe- nanthro[2,3-b]furan-7-ol unit, similar to shanciol H [12] and a bibenzyl moiety identical to that of25. The dihydrophenanthrenofuran and bi- benzyl moieties linked via the C-7′–C-2″bond wasfinally made by the HMBC (Fig. 2) correlations from H-7′(δH2.99, m) to C-2 (δC90.9), C-3 (δC52.6), C-2″(δC117.9) and C-1″(δC143.7). Thetransconfiguration of H-2 and H-3 at the dihydrofuran ring was determined by coupling constant of 5.6 Hz [25], which was further conformed by the correla- tion between H-7′and H-2 observed in the ROESY spectrum (Fig. 2) [11,12]. Thus, compound1was established, and named bleochranol A.

Compound2was obtain as brown amorphous powder and exhibited a molecular formula of C25H24O6from its HRESIMS ([M + K]⁺m/z 459.1199, calcd 459.1204), requiring 14 degrees of unsaturation. The

1H NMR spectrum (Table 1) of 2 showed characteristic of a dihy- drophenanthrene skeleton with multiplet signals atδH2.62–2.65 (4H, m, H-9, H-10), together with four aromatic proton signals at [δH8.05 (1H, s, H-4), 6.39 (1H, d,J= 2.3 Hz, H-6), 6.30 (1H, d,J= 2.3 Hz, H-8) and 6.66 (1H, s, H-11)]. Three aromatic proton signals in a typical ABX spin systems [δH6.95 (1H, d,J= 1.8 Hz, H-2′), 6.76 (1H, d,J= 8.1 Hz, H-5′) and 6.82 (1H, dd,J= 8.1, 1.8 Hz, H-6′)], suggested the presence of a 1,3,4-trisubstuted phenyl ring. Comparing the NMR data of com- pound2with that of shanciol H [12], it could be inferred that the acetyl group at C-7′of shanciol H absented. The molecular weight and cor- relations in HMBC (Fig. 2) spectra from H-7′(δH3.85, 3.76, m) to C-2 (δC88.7), C-3 (δC55.2) and C-3a (δC125.8) certificated the structure of Fig. 1.Structures of compounds1–25.

2. The relative configuration of H-2 and H-3 at the dihydrofuran ring was determined to betransby correlation between H-7'and H-2 in the ROESY experiment (Fig. 2) [11,12]. Therefore, compound2was de- termined, named as bleochranol B.

Compound3 was isolated as brown amorphous powder, and the molecular formula of C33H32O8could be inferred on basis of the posi- tive-ion HRESIMS ([M + K]⁺595.1725; calcd 595.1729), with 18 de- grees of unsaturation. The¹H NMR spectrum of3(Table 1) revealed the presence ap-hydroxybenzyl group [δH6.93 (2H, d,J= 8.6 Hz, H-2″, 6″) and 6.62 (2H, d,J= 8.6 Hz, H-3″, 5″)] and one 1,3,4,5-substuted phenyl ringδH6.66 (2H, s, H-2′, 6′). Detailed comparing of the¹H and

13C NMR spectral data (Tables 1 and 2) of3with that of2, it's clear that these two compounds were similar excepting the presence of ap-hy- droxybenzyl group and a methoxyl group together with the absence of two aromatic protons for compound3. The correlations between H-7″

(δH3.92, 2H, s) with C-7 (δC155.8), C-8 (δC118.5) and C-8a (δC141.1) from the HMBC (Fig. 2) spectrum indicated thep-hydroxybenzyl group was located at C-8 of compound3. The signals of two methoxyl groups [δH3.80 (6H, s);δC56.7] and two protons [δH6.66 (2H, s);δC103.9] in the NMR spectral data suggested the phenyl moiety in3was substituted symmetrically with one hydroxyl and two methoxyl groups. Thetrans relationship between H-2 and H-3 was determined by comparison of the coupling constant of 5.6 Hz, the ROESY spectrum (Fig. 2) correlation between H-7′ and H-2 [11,12] determined it further. Based on the above evidences, compound3was determined, named as bleochranol C.

Compound4was get as brown amorphous powder and possessed the molecular formula of C34H32O8 by the negative HRESIMS

([M−H]⁻567.2047; calcd for 567.2044), indicating 19 degrees of unsaturation. The¹H NMR spectroscopic data (Table 1) of4revealed a p-hydroxybenzyl moiety [δH6.94 (2H, d,J= 8.8 Hz, H-2″, 6″) and 6.64 (2H, d, J= 8.8 Hz, H-3″, 5″)] as compound3 (Table 1). Otherwise, signals for an acetyl group (δH2.03;δC172.9 and 20.9) were observed and a methoxyl group was replaced by an aromatic proton in4. It could be inferred that the acetyl group was located at C-7′based on the de- shielded of H-7′resonance (ΔδH+ 0.59) and the HMBC (Fig. 1) cor- relation from H-7′ to the acetyl carbonyl. In the ROESY spectrum (Fig. 2), the interaction between H-2 and H-7′revealed that the relative stereochemistry at the dihydrofuran ring astrans[11,12]. Accordingly, the structure of compound4was elucidated, and named bleochranol D.

The EtOAc fraction and compounds1–25were screened for their cytotoxicity against HL-60, SMMC-7721, A-549, MCF-7 and SW480 human cell lines using the MTS assay in vitro, with cisplatin as the positive control. Most compounds showed cytotoxic activity and the results could be seen as shown inTable 3. The EtOAc fraction demon- strated inhibitory activity of five tumor cell lines with IC50 values ranging from 15.01 ± 0.52 to 36.63 ± 1.56μg/mL (Table 3). Among all the tested compounds, bleochranol A (1) showed remarkable cyto- toxic activity against allfive tumor cell lines, especially HL-60, A-549, and MCF-7 cell lines with IC50values at 0.24 ± 0.03, 3.51 ± 0.09 and 3.30 ± 0.99μM respectively. Comparing the effects of compound1 with that of5, it could be seen that the [2,3-b]furan fragment in di- hydrophenanthrenofuran might increases the cytotoxic activity. Com- pounds6and7exhibited selective significant cytotoxic activity against HL-60 cell line with IC50values of 3.67 ± 0.24 and 10.25 ± 0.52μM together with better cytotoxicity of6than7, indicating that the acetyl Fig. 2.The key HMBC ( ) and ROESY ( ) correlation of1–4.

group at C-7′might increases the cytotoxic activity. Compounds13and 14exhibited selective cytotoxic activity against SMMC-7721 cell line with IC50values of 20.80 ± 0.55 and 7.56 ± 0.31μM indicated that the oxygen substitutions at C-1–C-3 of the phenanthrene are very im- portant for the tumor cell line cytotoxicity and methoxy at C-2 will be better.

It's well known that inflammation is a precursor for many cancers, and NO is an essential component of the host innate andinflammation response to variety of pathogens [26]. The EtOAc fraction and all the compounds were sent for scanning against NO production in LPS-acti- vated RAW 264.7 macrophages by MTS assay. As a result (Table 4), the EtOAc fraction showed weaker inhibitory activity at 45.85 ± 2.21%

(concentration in 20μg/mL). Compounds 10–12 exhibited most po- tential activity with IC50 values of 8.17 ± 0.64, 8.81 ± 0.46 and 2.86 ± 0.17μM, respectively. Compounds6,7,14and15possessed moderated activities at inhibiting NO production with IC50value ran- ging from 17.41 ± 0.20 to 22.29 ± 0.48μM. Other tested compounds showed no activity.

In conclusions, our study demonstrated that phenanthrences and bizbenzyls are main compounds of the rhizomes ofB. ochracea. Twenty- five phenanthrences and bizbenzyls, including four new dihyophenan- throfurans (1–4) were isolated from the tubers ofB. ochracea. It is no- teworthy that the dihyophenanthrofurans were first reported from Bletillaspecies. Among them, the structures of compounds1and5were unusual as dimers, possessing a dihydrophenanthrenofuran unit

connecting to a bibenzyl unit. According to the cytotoxicity assay, the results showed that most compounds had cytotoxic activity againstfive tumor cell lines. Among isolates, compound1 showed the strongest activity against HL-60, A-549, and MCF-7 with IC50values ranging from 0.24 ± 0.03 to 3.51 ± 0.09μM. The anti-inflammatory assay showed that compound 12 (IC50 2.86 ± 0.17μM) exhibited most potential activity against NO production in RAW 264.7 macrophages. These re- sults provided evidences for traditional use ofB. ochraceaas "Bai-Ji".

Conflict of interest statement

The authors declare no competingfinancial interests.

Acknowledgments

This work was supported by grants from the National Key Research and Development Program of China (2017YFD0201402) and Yunnan Province (2015HB093, 2015FB168 and 2017ZF003). The authors are grateful to the staffs of the analytical group at State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, for measuring the spectral data.

Appendix A. Supplementary data

1D and 2D NMR, HRESIMS IR and CD spectra of compounds1–4are available as Supporting Information. Supplementary data associated with this article can be found in the online version, at doi:https://doi.

org/10.1016/j.fitote.2018.02.007.

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Table 3

Cytotoxicity of EtOAc fraction (IC50μg/mL) and Compounds1–25(IC50μM).

Compounds^c HL-60^ab SMMC-7721^ab A-549^ab MCF-7^ab SW480^ab

EtOAc extract 23.55 ± 1.00 24.89 ± 0.16 32.28 ± 1.23 15.01 ± 0.52 36.63 ± 1.56

1 0.24 ± 0.03 12.22 ± 0.26 3.51 ± 0.09 3.30 ± 0.99 12.97 ± 0.34

2 > 40 > 40 34.87 ± 0.40 29.07 ± 1.34 > 40

3 15.05 ± 0.33 19.85 ± 0.42 19.16 ± 0.41 18.84 ± 0.47 18.61 ± 0.68

4 10.65 ± 0.09 17.95 ± 0.44 18.32 ± 0.44 17.62 ± 0.81 18.60 ± 0.99

5 12.22 ± 0.12 14.70 ± 0.11 15.64 ± 0.31 8.39 ± 0.48 17.13 ± 0.06

6 3.67 ± 0.24 > 40 31.46 ± 1.41 > 40 > 40

7 10.25 ± 0.52 > 40 > 40 > 40 > 40

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Compounds^b NO^a

6 18.29 ± 2.17

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aCompounds1–25and positive controls were expressed as values inμM.

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