Chemical constituents from Bletilla striata and their NO production suppression in RAW 264.7 macrophage cells

Article abstract of DOI:10.1080/10286020.2017.1339696

A novel glucoside bletilloside A (1) was isolated from the tubers of Bletilla striata, together with seven known compounds (2–8). Their structures were determined on the basis of extensive spectroscopic analyses. All compounds were evaluated for the inhib

Full text of DOI:10.1080/10286020.2017.1339696

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Chemical constituents from Bletilla striata and
their NO production suppression in RAW 264.7
macrophage cells
Yang Zhao, Jun-Jie Niu, Xiao-Chen Cheng, Yu-Xin Lu, Xiao-Feng Jun, Xi-Rong
Zhao, Qing-Lin Zhang & Chu-Tse Wu
To cite this article: Yang Zhao, Jun-Jie Niu, Xiao-Chen Cheng, Yu-Xin Lu, Xiao-Feng Jun, Xi-
Rong Zhao, Qing-Lin Zhang & Chu-Tse Wu (2017): Chemical constituents from Bletilla striata
and their NO production suppression in RAW 264.7 macrophage cells, Journal of Asian Natural
Products Research, DOI: 10.1080/10286020.2017.1339696
To link to this article: http://dx.doi.org/10.1080/10286020.2017.1339696
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Date: 16 June 2017, At: 07:15

Journal of asian natural Products research, 2017
https://doi.org/10.1080/10286020.2017.1339696
Chemical constituents from Bletilla striata and their NO
production suppression in RAW 264.7 macrophage cells
Yang Zhaoa, Jun-Jie Niua,b, Xiao-Chen Chenga, Yu-Xin Lua, Xiao-Feng Juna,
Xi-Rong Zhaob, Qing-Lin Zhanga and Chu-Tse Wua
adepartment of experimental hematology, Beijing institute of radiation Medicine, Beijing 100850, china;
bcollege of life science and food engineering, huaiyin institute of technology, huai’an 223003, china
ABSTRACT
ARTICLE HISTORY
received 8 february 2017
accepted 5 June 2017
A novel glucoside bletilloside A (1) was isolated from the tubers of
Bletilla striata, together with seven known compounds (2–8). Their
structures were determined on the basis of extensive spectroscopic
analyses. All compounds were evaluated for the inhibition on NO
production effects in RAW 264.7 macrophage cells, while militarine
(4) and dactylorhin A (5) exhibited moderate inhibitory effects.
KEYWORDS
Glucosides; Bletilla striata;
orchidaceae; no production
1. Introduction
e tubers of Bletilla striata, a traditional Chinese medicine known as “Bai-ji”, have been
widely used in the treatment for hematemesis, hemoptysis, traumatic bleeding, chapped
skin, and ulcerative carbuncle [1]. Previous phytochemical investigations on B. striata
resulted in the isolation of bibenzyls [2–4], phenanthrenes [5–8], triterpenoids [9,10], ster-
oids [11,12], and glucosyloxybenzyl 2-isobutylmalates [13,14]. eir biological activities
include cytotoxicity [4,11,12], antimicrobial [7], and anti-inflammation [12,14].
In our research on chemical constituents of its tubers, a new glucoside bletillo-
side A (1), together with seven known compounds shancigusin I (2), bleformin J (3),
militarine (4), dactylorhin A (5), 1-(4-β-D-glucopyranosyloxybenzyl) 4-methoxyl
CONTACT Qing-lin Zhang
qinglz@yahoo.com; chu-tse Wu
wuct@bmi.ac.cn
the supplemental data for this article can be accessed at https://doi.org/10.1080/10286020.2017.1339696.
© 2017 informa uK limited, trading as taylor & francis Group

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Y. ZHAO ET AL.
(2R)-2-hydroxyisobutylmalate (6), 5-methoxybibenzyl-3, 3′-di-O-β-D-glucopyranoside
(7), and gastrodin (8) (Figure 1), was observed. Herein, we report the isolation, structural
elucidation, and biological activity of these compounds.
2. Results and discussion
e molecular formula (C29H36O15) of 1 was determined by the HR-ESI-MS and NMR data,
requiring 12 degrees of unsaturation. e IR absorption bands suggested the presence of OH
group (3422 cm−1) and α, β-unsaturated ester group (1701 and 1637 cm−1). Analyses of NMR
data revealed 1 as the analog of known compound shancigusin I (2), which was isolated
from Pleione yunnanensis [15]. e distinction was attributed to the substituted benzene
ring in cinnamoyl moiety, in which the 1H-NMR spectrum (Table 1) showed an ABX system
[δH 7.33 (1H, d, J = 1.7 Hz), 7.57 (1H, d, J = 8.7 Hz), 7.22 (1H, d, J = 1.7, 8.7 Hz)], and one
methoxyl group at δH 3.78 (3H, s), establishing the benzene ring to be methoxy-substituted,
which was supported by the HMBC correlations (Figure 2). e NOE correlation from H-2
(δH 7.33) to the methoxyl group (δH 3.78) and H-8 (δH 6.70) indicated the methoxy group
was located at C-3 (Figure 3). e sugar moieties were confirmed as β-D-glucose by the
coupling constants (7.2 and 7.2 Hz, respectively) and comparison with the authentic sugar
by the acid hydrolysis. us, compound 1 was determined as bletilloside A.
e known compounds bleformin J (3) [16], militarine (4) [17], dactylorhin A (5) [18],
1-(4-β-D-glucopyranosyloxybenzyl) 4-methoxyl (2R)-2-isobutylmalate (6) [19], 5-methox-
ybibenzyl-3, 3′-di-O-β-D-glucopyranoside (7) [20], and gastrodin (8) [21], were identified
by comparison of their spectroscopic data with reported values.
All the compounds were evaluated for their suppressive activity against the LPS-induced
NO production in RAW 264.7 macrophage cells. As NO plays a pivotal role in inflamma-
tion, the steroidal anti-inflammatory drug dexamethasong (Dex) was selected as positive
control. e cytotoxicity of compounds 1–8 (0–240 μM) showed no cell viability on RAW
264.7 macrophage over 24 h using an MTT method. erefore, the suppressive effects of
4 and 5 on NO production could not be ascribed to cytotoxicity, and exhibited moderate
inhibitory NO production effects with IC50 values of 48.5 and 51.7 μM, respectively (Table 2).
Figure 1. structures of compounds 1–3.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1h and 13c nMr spectral data for compound 1 in pyridine-d5 (at 600 and 125 Mhz, resp.).
δC(ppm)
δH(ppm)
1
2
3
129.2, c
112.1, ch
150.4,c
7.33, d (1.7)
4
5
6
7
8
9
1′
2′,6′
3′,5′
4′
145.6, c
116.3, ch
123.1, ch
145.6, ch
118.0, ch
167.4, c
130.8, c
130.7, ch
117.3,ch
158,9,c
7.57, d (8.7)
7.22, dd (1.7, 8.7)
7.94, d (15.9)
6.70, d (15.9)
7.46, d (8.7)
7.40, d (8.7)
7′
66.3,ch2
102.1,ch
75.3,ch
78.9,ch
71.6,ch
79.4,ch
62.7,ch2
102.4,ch
75.1,ch
78.9,ch
71.5,ch
79.3,ch
62.7,ch2
56.3, ch3
5.33, s
1″
2″
3″
4″
5″
6″
1″′
2″′
3″′
4″′
5″′
6″′
3-och3
5.77, d (7.2)
4.36–4.38, m
4.35–4.37, m
4.34–4.36, m
4.13–4.15, m
4.43, m; 4.56, m
5.68, d (7.2)
4.36–4.38, m
4.35–4.37, m
4.34–4.36, m
4.13–4.15, m
4.43, m; 4.56, m
3.78, s
note: 1.93–1.95, m.
Figure 2. Key hMBc correlations of compound 1.
Figure 3. Key noesY correlations of compound 1.

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Y. ZHAO ET AL.
Table 2. inhibition effects of 1–8 on no production.
Comp.
IC50 (μM)
CC50 (μM)
1
>70
>70
>70
48.5 0.7
51.7 1.1
>70
>70
>70
68 1.2
>240
>240
>240
>240
>240
>240
>240
>240
>240
2
3
4
5
6
7
8
dex
3. Experimental
3.1. General experimental procedures
Optical rotations were measured using an Autopol III automatic polarimeter (Rudolph
Research Co., NJ, USA). UV spectra were recorded on Shimadzu UV2550 (Shimadzu, Kyoto,
Japan). IR spectra were measured on a ermo Nicolet Nexus 470 FT-IR spectrometer
1
(MT, USA). e H NMR and 13C NMR spectra were recorded on JEOL ECX 600 MHz
spectrometer (JEOL Ltd., Tokyo, Japan) or Bruker Avance-400FT NMR spectrometer
(Bruker, Billerica, German) using TMS as an internal standard. HRESIMS were obtained
on a Bruker APEX IV 70 ev FT-MS spectrometer and on a ermo DFS spectrometer using a
matrix of 3-nitrobenzyl alcohol.D101 macroporous resin (Mitsubishi Chemical Co., Japan),
Sephadex LH-20 (Pharmacia, Uppsala, Sweden), and silica gel (200–300 mesh, Qingdao
Marine Chemistry Co. Ltd., Qingdao, China) were used for column chromatography. And
HF254 silica gel plates (Qingdao Marine Chemistry Co. Ltd., Qingdao, China) were used
for TLC. HPLC was performed on Alltech 426 pump employing a UV detector, and the
Prevail C18 column (semi-preparative, 5 μm) was purchased from Pharmacia. e murine
macrophage cell line RAW 264.7 was obtained from the Cell Resource Center, IBMS, CAMS/
PUMC (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffered
saline (PBS), and fetal bovine serum (FBS) were purchased from Gibco ibcogle (Invitrogen,
NY, USA). 96-well plates were purchased from Corning Costar (Corning Inc., NY, USA).
Dimethylsulphoxide (DMSO), LPS, and MTT were purchased from Sigma–Aldrich Co. (St.
Louis, MO, USA). Dexamethasong was obtained from Tianjin Jiaozuo Co. (Tianjin China),
and Griess reagent was purchased from applygen (Beijing, China).
3.2. Plant materials
e tubers of Bletilla striata were collected from Shaoxing, Zhejiang Province and identified
by Professor Qing-Lin Zhang from the Department of Experimental Hematology, Beijing
Institute of Radiation Medicine, Beijing, China. A voucher specimen (No. BS20150824)
was deposited in Beijing Institute of Radiation Medicine.
3.3. Extraction and isolation
e dried and powdered tubers of B. striata (1.5 kg) were refluxed with 95% EtOH (15 L×3
times, 3 h for each). e extract was evaporated to dryness under vacuum to afford a crude
residue (50 g), which was subjected to D101 macroporous resin column chromatography

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
(CC) eluted with aqueous EtOH (0, 20, 40, and 60%) to afford four fractions (FA–FD). FB
was further purified on a Sephadex LH-20 column to give 8 (980.2 mg). FC (15.2 g) was
further fractionated on an ODS column (3 × 30 cm), eluted with a constant gradient of
MeOH/H2O (20%), to obtain five portions (FC1–FC5). FC1 (1.2 g) was further fractionated
on an ODS column with a constant gradient of MeOH /H2O (30%), followed by preparative
RP-C18 HPLC, to give 5 (30.3 mg). FC2 (2.5 g) was further fractionated on an ODS column
with a constant gradient of MeOH /H2O (32%), followed by preparative RP-C18 HPLC
(flow rate 2 ml/min, UV detection at λ = 300 nm), to give 1 (5.8 mg, tR = 22.3 min) and 3
(34.0 mg, tR = 31.5 min). FC3 (450 mg) was further fractionated on an ODS column with a
gradient of MeOH/H2O (from 30 to 50%), followed by preparative RP-C18 HPLC, to give
2 (6.8 mg, tR = 19.6 min) and 7 (6.8 mg, tR = 21.5 min). FC4 (4.5 g) was further purified on
a Sephadex LH-20 column to give 4 (1.9 g) and 6 (10.5 mg).
3.3.1. Bletilloside A (1)
Amorphous power; [ꢀ]2D5 − 40.8 (c 0.05, MeOH); UV (MeOH) λ
(log ε) 223 (4.26)
max
304 (4.24) nm; IR (KBr) vmax 3422, 2923, 1701, 1637, 1509, 1458, 1384, 1258, 1234, 1162,
1072 cm−1; 1H and 13C NMR spectral data, see Table 1; HRESIMS: m/z 647.1939 [M + Na]+
(calcd for C29H36O15Na, 647.1952).
3.4. Acid hydrolysis of 1
Compound 1 (1.0 mg) was heated in 2 mol/L trifluoroacetic acid (TFA) (1 ml) for 2 h in
screen-cap vial. Afer cooling, 1 ml of EtOAc was added into the hydrolysate. e aque-
ous layer was evaporated to dryness with ethanol under vacuum until TFA was removed.
e residues were determined by comparison with D-glucose using TLC (CHCl3: MeOH:
H2O = 3: 2:0.2, visualization with ethanol-5% H2SO4 spraying). In addition, the specific
rotation of sugar derived from 1 ([ꢀ]2D5 + 79.8) was in accordance with that of D-glucose
([ꢀ]2D5 + 96.8) and opposite to that of L-glucose [ꢀ]2D5 − 79.8), indicating the sugar of 1 to
be in the D-form.
3.5. Cell viability
Cell viability was evaluated by MTT assay. RAW 264.7 macrophage cell was seeded at
2 × 105 cells/ml in 96-well plates containing 100 μl of DMEM (Dulbecco’s modified Eagle’s
medium) (supplemented with 10% FBS (Fetal bovine serum), 100 units/ml penicillin,
and 100 μg/ml streptomycin sulfate), and cultured in a 37 °C humidified atmosphere of
5% CO2. Afer overnight incubation, the cells were treated with LPS (1 μg/ml) in the absence
or presence of the test compounds at different concentrations and the plates were incubated
for 24 h. e cells were then incubated with an MTT solution for 4 h at 37 °C under 5% CO2.
e medium was removed and DMSO (150 μl) was added to each well. e absorbance was
measured at 492 nm by a Microplate Reader (Multiskan MK3, ermo).
3.6. NO inhibition assay
Using the Griess method, the nitrite concentration was determined in the culture superna-
tants. e cells were planted at 2 × 105 cells/ml in 96-well plates and incubated overnight.

6
Y. ZHAO ET AL.
Afer treatment with various concentrations of tested compounds for 1 h, cells were treated
with LPS (1 μg/ml) for 24 h. Dex served as a positive control. e culture supernatant
(100 μl) was moved into another 96-well plate, where nitrite reacted with the standard
Griess reagent (100 μl) for 15 min. e optical density of each well was measured at 492 nm
by a Microplate Reader.
Disclosure statement
No potential conflict of interest was reported by the authors.
References
[1] X.R. He, X.X. Wang, J.C. Fang, Z.F. Zhao, L.H. Huang, H. Guo, and X.H. Zheng, J. Ethnopharmacol.
195, 20 (2017).
[2] J.Q. Feng, R.J. Zhang, and W.M. Zhao, Helv. Chim. Acta. 91, 520 (2008).
[3] G.X. Han, L.X. Wang, Z.B. Gu, and W.D. Zhang, Acta Pharm. Sin. 37, 194 (2002).
[4] K.W. Woo, J.E. Park, S.U. Choi, K.H. Kim, and K.R. Lee, Nat. Prod. Sci. 20, 91 (2014).
[5] M. Yamaki, T. Kato, L. Bai, K. Inoue, and S. Takagi, Phytochemistry 34, 535 (1993).
[6] M. Yamaki, B. Li, T. Kato, K. Inoue, and S. Takagi, Phytochemistry 32, 427 (1993).
[7] C.D. Qian, F.S. Jiang, H.S. Yu, Y. Shen, Y.H. Fu, D.Q. Cheng, L.S. Gan, and Z.S. Ding, J. Nat.
Prod. 78, 939 (2015).
[8] A.J. Sun, S.Q. Pang, and G.Q. Wang, Chin. J. Pharm. 47, 35 (2016).
[9] M. Yamaki, C. Honda, T. Kato, L. Bai, and S. Takagi, Nat. Med. 51, 493 (1997).
[10] L. Yang, C. Peng, C.W. Meng, C.J. He, X.H. Li, L. Guo, Q.M. Zhou, and L. Xiong, Biochem. Syst.
Ecol. 57, 238 (2014).
[11] J.E. Park, K.W. Woo, S.U. Choi, J.H. Lee, and K.R. Lee, Helv. Chim. Acta. 97, 56 (2014).
[12] W. Wang and H. Meng, Fitoterapia 101, 12 (2014).
[13] G.X. Han, L.X. Wang, W.D. Zhang, M.L. Wang, H.T. Liu, and J.H. Xiao, Acad. J. Second. Mil.
Med. Univ. 23, 1029 (2002).
[14] L. Yue, W. Wang, Y. Wang, T. Du, W.P. Shen, H.L. Tang, Y. Wang, and H.P. Yin, Int. J. Biol.
Macromol. 89, 376 (2016).
[15] H.L. Dong, H.Q. Liang, C.L. Wang, S.X. Guo, and J.S. Yang, Magn. Reson. Chem. 51, 371 (2013).
[16] C.W. Lin, T.L. Hwang, F.A. Chen, C.H. Huang, H.Y. Hung, and T.S. Wu, J. Nat. Prod. 79, 1911
(2016).
[17] Y.W. Leong, C.C. Kang, L.J. Harrison, and A.D. Powell, Phytochemistry 44, 157 (1997).
[18] H. Kizu, E. Kaneko, and T. Tomimori, Chem. Pharm. Bull. 47, 1618 (1999).
[19] Y. Wang, S.H. Guan, Y.H. Meng, Y.B. Zhang, C.R. Cheng, Y.Y. Shi, R.H. Feng, F. Zeng, Z.Y. Wu,
J.X. Zhang, M. Yang, X. Liu, Q. Li, X.H. Chen, K.S. Bi, and D.A. Guo, Phytochemistry 94, 268
(2013).
[20] N. Baek, S.Y. Choi, J.K. Park, S.W. Cho, E.M. Ahn, S.G. Jeon, B.R. Lee, J.H. Bahn, Y.K. Kim, and
I.H. Shon, Arch. Pharm. Res. 22, 219 (1999).
[21] J. Liu, Z.B. Yu, Y.H. Ye, and Y.W. Zhou, Acta. Pham. Sin. 43, 181 (2008).