Neuroprotective activity of different monosaccharide-modified gastrodin analogs

Article abstract of DOI:10.1002/jccs.201900223

Gastrodin is a very important and well-known bioactive glycoside compound in Chinese medicine. It is also known as a drug with neuroprotective function. Here, a practical diversified synthesis of a series of gastrodin analogs was reported, which involved four-step procedures consisting of bromination, oxidation, etherification, and reduction. Various gastrodin analogs were obtained in good yields. The compound 4c in this study has a good neuroprotective function: it can significantly downregulate tumor necrosis factor-α and inducible nitric oxide synthase protein levels. The results of this study can provide a research basis for the development of neuroprotective drugs.

Full text of DOI:10.1002/jccs.201900223

Received: 6 June 2019
Revised: 2 December 2019
Accepted: 23 December 2019
DOI: 10.1002/jccs.201900223
A R T I C L E
Neuroprotective activity of different
monosaccharide-modified gastrodin analogs
Kun-Lun Xu1,2 | Lan Yu1
1School of Pharmacy, Zunyi Medical
Abstract
University, Zunyi, China
2Zunyi Blood Center, Zunyi, China
Gastrodin is a very important and well-known bioactive glycoside compound
in Chinese medicine. It is also known as a drug with neuroprotective function.
Here, a practical diversified synthesis of a series of gastrodin analogs was
reported, which involved four-step procedures consisting of bromination, oxi-
dation, etherification, and reduction. Various gastrodin analogs were obtained
in good yields. The compound 4c in this study has a good neuroprotective
function: it can significantly downregulate tumor necrosis factor-α and induc-
ible nitric oxide synthase protein levels. The results of this study can provide a
research basis for the development of neuroprotective drugs.
Correspondence
Lan Yu, Department of School of
Pharmacy, Zunyi Medical University,
6 West Xue-Fu Road, Zunyi, Guizhou
563009, China.
Email: yulanzmu@126.com
Funding information
15851 Talent Elite Project in Zunyi City;
Key Laboratory of Ministry of Education
Open Fund Project, Grant/Award
Number: Qian Jiao He KY[2017]383;
National Natural Science Foundation of
China, Grant/Award Number: 21768006
K E Y W O R D S
diversified synthesis, gastrodin, glycosylation, neuroprotective
1 | INTRODUCTION
moiety is widely distributed in naturally occurring
products. In Figure 1, some of these gastrodin analogs
Gastrodin, also known as 4-(hydroxymethyl)phenyl-β-D-
glucopyranoside, is a well-known bioactive glycoside
component in the Chinese herb Gastrodia elata B1.[1–4]
This compound acts as a good Ca2+ channel blocker;
therefore, it has been widely used for the treatment of
nervous system, cardiovascular, and cerebrovascular dis-
eases, such as hypertension, stroke, dementia, migraine,
and hemiplegia.[5–7] In addition, gastrodin also exhibits a
variety of pharmacological values, including antioxidant
and radical scavenger, hypoxia tolerance, neuroprotective
and anticonvulsant effect, protection against cardiac hyper-
trophy, and fibrosis, as well as an antimyocardial ischemia
effect.[8–11] Recent studies have demonstrated that gas-
trodin can also suppress inflammatory response in septic
cardiac dysfunction and stimulating anticancer immune
response, as well as inhibiting cell growth of transplanted
H22 hepatic ascetic tumor.[12–16] Therefore, gastrodin ana-
logs have become a class of attractive targets for bio-
screening in drug discovery. Furthermore, gastrodin
are listed as representative examples.[17–20]
Typically, gastrodin was obtained by a time-consuming
and low-yielding solvent extraction process from G. elata
flower, but these traditional methods usually suffered from
high cost. Recently, a couple of practical synthetic
methods have been developed for its preparation using
2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide as
the glycosyl donor and p-hydroxybenzaldehyde or
4-hydroxybenzenemethanol as an accepter. Although
the bioactivities of gastrodin-derived analogs have been
screened by several research groups, the synthesis of
gastrodin analogs using other saccharides instead of glu-
cose has not been uncovered, and no bioscreening of
gastrodin analogs was reported.[14] Based on previous
reports, in this work, we chose 11 different saccharides
as acceptors to synthesize gastrodin analogs, with
corresponding gastrodin analogs obtained through a
four-step sequence. These compounds will be available
for possible bioscreening in the future.
© 2020 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J Chin Chem Soc. 2020;1–7.
http://www.jccs.wiley-vch.de
1

2
XU AND YU
FIGURE 1 Representative
natural products containing
gastrodin moiety
SCHEME 1 Reaction route to make gastrodin
2 | RESULTS AND DISCUSSION
to synthesize gastrodin analogs, we found that the use of
p-hydroxybenzaldehyde as a glycosyl acceptor is better
and more convenient than the other two acceptors (p-
cresol, 4-hydroxybenzenemethanol); due to only one
reactive position in the molecule, by reducing the car-
bonyl group to the hydroxy group, gastrodin analogs
can be easily obtained. Different gastrodin analogs
were synthesized when using other saccharides as
starting materials (Scheme 1).
To synthesize gastrodin molecule 4a, D-xylose 1a
was used to react with Ac2O in pyridine to generate
2,3,4-tri-O-acetyl-β-D-xylopyranose, followed by the
treatment with HBr that was dissolved in a mixture
Although several synthetic methods to synthesize gas-
trodin were reported, which focused only on gastrodin
itself, there is no report for the preparation of gastrodin
analogs by using different saccharides, and no one has
even bioscreened gastrodin analogs so far. In the gastrodin
case, p-cresol is an acceptor for glycosylation, and
2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide is a gly-
cosyl donor. In this work, we used different glycosyl
donors instead of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl
bromide to synthesize gastrodin analogs. After comparing
different existing methods by comparison experiments

XU AND YU
3
TABLE 1 Preparation of gastrodin analogs through a two-step procedure
Entry
Monosaccharide
Intermediate 2
Yield (%) (two steps)
1
70
2
3
72
78
4
74
5
6
44
74
7
8
68
78
solvent of acetic acid (AcOH)/CH2Cl2 (DCM) to give
2,3,4,6-tetra-O-acetyl-α-D-xylopyranosyl bromide 2a.
Scheme 2: D-xylose, L-arabinose, D-glucose, D-galactose, D-
ribose, D-arabinose, L-xylose, and L-fucose.
A
subsequent glycosylation of p-hydroxyben-
In order to synthesize different gastrodin analogs
more efficiently, here, we use a combinatorial method.
First, we commenced with the acetylation of different
saccharides in 11 reactors in a combinatorial syn-
thetic way. After completion, the removal of solvent
followed by recrystallization in ethyl acetate (EtOAc)
afforded the corresponding desired products in
90–98% yields. Subsequently, treatment of the acety-
lated products with HBr in DCM/AcOH provided the
corresponding bromination products 2a-h with good
overall efficiencies (44–78% for the two steps).
zaldehyde (made from 4-Hydroxybenzenemethanol)
was carried out in the presence of glycosyl donor 2a,
thus providing compound 3a. Next, 3a was reduced by
NaBH4 to give gastrodin molecule 4a. Although gas-
trodin bioactivities have been extensively explored by
several groups, its analogs remain underdeveloped, so
here, the synthesis of gastrodin analogs by using differ-
ent saccharides is very meaningful research (Table 1).
To synthesize gastrodin analogs, eight saccharides
including D- and L-pyranoses were chosen, as shown in

4
XU AND YU
SCHEME 2 Different
saccharides used for making
gastrodin analogs
FIGURE 2 Effect of gastrodin
1 and compound 4a-4h on mRNA
expression of TNF-α and iNOS. Data
represent the mean SD (n = 3)
With 2a-h in hand, p-hydroxybenzaldehyde reacted
with 2a-h in a combinatorial way in the presence of
tetrabutylammonium bromide and K2CO3 in CHCl3/
H2O. After completion, all the reaction mixtures were
extracted with EtOAc and concentrated in vacuum to give
the corresponding products, which were not purified for
further use. All products were reduced by NaBH4 in
CH3OH and purified by flash chromatography to give final
products 4a-h in two-step yields of 36–56%.
We first stimulated PC12 with never growth factor
(NGF) to obtain differentiated neuronal cells and then
added additional lipopolysaccharide (LPS) to obtain the
inflammatory model as previously described.[21] The cells
were then incubated with different compounds for 48 hr.
After treatment, cells were collected, and the messenger
RNA (mRNA) expression levels of inflammation-associated
tumor necrosis factor-α (TNF-α) and inducible nitric oxide
synthase (iNOS) were detected by quantitative real-time
polymerase chain reaction (qRT-PCR) experiments. As
shown in the results, the compound gastrodin and 4c can
significantly downregulate the mRNA expression levels of
TNF-α and iNOS, thereby playing a neuroprotective role.
For other compounds, we found that compound 4d had a
moderate effect, and all other compounds were inactive.
Meanwhile, we also investigated the effects of gastrodin
and compound 4c on the protein levels of TNF-α and iNOS
by western blotting. The results showed that compound 4c
could significantly downregulate the expression of TNF-α
FIGURE 3 Effect of gastrodin 1 and compound 4c on protein
expression of TNF-α and iNOS. (a) Representative Western blot
showing TNF-α and iNOS expression after LPS treatment.
Quantitative analyses of TNF-α and iNOS protein expression. (b, c)
Gray scale analysis results. Data represent the mean SD (n = 3).
β-actin was used as the control

XU AND YU
5
and iNOS. These results show that the compound 4c has a
good neuroprotective function by downregulating TNF-α
and iNOS protein levels. This result is consistent with previ-
ous reports in the literature (Figures 2 and 3).[22,23]
function, and the results of this work can provide a research
basis for the development of neuroprotective drugs
(Table 2).
In summary, the synthesis of gastrodin analogs was
reported in four steps by using different D-and L-pyranoses
as glycosyl donors instead of glucose; we have synthesized
eight gastrodin analogs in a combinatorial way, and differ-
ent gastrodin analogs were obtained in good yields. These
compounds have filled the gap left by other research groups.
In the inflammatory model of neuronal cells, compound 4c
can protect neurons by downregulating the expression of
the TNF-α and iNOS proteins. The gastrodin derivatives
synthesized in this work have a good neuroprotective
3 | EXPERIMENTAL SECTION
3.1 | General procedure for producing
(2R,3R,4S,5R)-2-bromotetrahydro-2H-pyran-
3,4,5-triyl triacetate 2a
D-Xylose (0.50 g, 3.3 mmol) was dissolved in pyridine
(4 ml), followed by the addition of acetic anhydride
(2.5 ml) at ice bath temperature. After stirring for half an
TABLE 2 The second two-step reactions to make gastrodin analogs
Entry
Intermediate 2
Expected product 4
Yield (%) (two steps)
1
40
2
3
50
54
4
52
5
6
36
48
7
8
44
56

6
XU AND YU
hour, the reaction was carried out at night. When thin
layer chromatography (TLC) analysis showed that the
reaction was over, the reaction solution was extracted
with water and EtOAc, and the organic layer was dried
and concentrated under vacuum; after crystallization, the
crude product was obtained.
The crude acetylated glucose was dissolved in CH2Cl2
(3 ml), followed by the addition of 33% HBr in acetic acid
(0.90 ml) at ice bath temperature. After stirring for 3 hr at
room temperature, when TLC analysis showed that the
reaction was over, the reaction solution was quenched
with water and extracted with CH2Cl2, and the organic
layer was dried and concentrated under vacuum and crys-
tallized in ether; the expected product 2a (0.78 g) was
obtained. Two-step yield is 70%. The same procedure was
used for 2b-h.
onto a polyvinylidene fluoride membrane. Then, mem-
branes were blocked with 5% skim milk for 2 hr before
being incubated with iNOS, TNF-α antibody at 4ꢀC over-
night, and secondary antibody for additional 2 hr at
room temperature. Quantitation of the bands were per-
formed by Image J (National Institutes of Health,
Bethesda, MD).
3.4 | qRT-PCR
PC12 cells were seeded on a polylysine-coated culture
plate at a density of 2.0 × 104 cells/cm2 for 18 hr. Then,
the cells were exposed to 100 ng/ml NGF in medium for
48 hr. For the model group, LPS (1 μg/ml in the cell cul-
ture medium) was added to stimulate cells for 24 hr.
After treatment by gastrodin 1 derivatives (100 μM in the
cell culture medium) for 48 hr, the cells were harvested
and lysed in TRIZOL (Synthgene) for total RNA extrac-
tion. Then, the RNA was converted to cDNA using a
cDNA Synthesis Kit according to the manufacturer's
instructions. RT-qPCR was performed with primers
specific for iNOS: 50-GCAAGCCCTCACCTACTTCC-30
(forward) 50-AACCTCTGCCTGTGCGTCT-30 (reverse),
TNF-α: 50-CAGGTTCCGTCCCTCTCATA-30 (forward)
and 50-TGCCAGTTCCACATCTCG-30(reverse), and
β-actin: 50-TCCTCCCTGGAGAAGAGCTAC-30 (for-
ward) and 50-TCCTGCTTGCTGATCCACAT-30(reverse).
β-actin was used as the control. The fluorescent signals
were collected, and Ct values of the sample were calcu-
lated. The mRNA levels were determined using the
3.2 | Cell culture
PC12 cells (purchased from ATCC) were cultured in
Dulbecco's Modified Eagle's Medium/Ham's Nutrient
Mixture F-12 supplemented with 1% streptomycin/peni-
cillin and 10% heat-inactivated fetal bovine serum in a
5% CO2 atmosphere at 37ꢀC. Heat inactivation was per-
formed by incubating the serum at 56ꢀC for 30 min. For
the differentiation of PC12 cells, the cells were seeded on
a polylysine-coated culture plate at a density of 2.0 × 104
cells/cm2 for 18 hr. Then, the cells were exposed to
100 ng/ml NGF in medium for 48 hr. LPS (1 μg/ml in the
cell culture medium) was added to the medium to stimu-
late cells for 24 hr, followed by gastrodin derivatives
(100 μM in the cell culture medium) treatment for 48 hr.
2
−44Ct method.
ACKNOWLEDGMENTS
This investigation was generously supported by funds
provided by National Natural Science Foundation of
China (No. 21768006), Key Laboratory of Ministry of
Education Open Fund Project (Qian Jiao He KY[2017]
383), and 15851 Talent Elite Project in Zunyi City.
3.3 | Western blot
PC12 cells were seeded on a polylysine-coated culture
plate at a density of 2.0 × 104 cells/cm2 for 18 hr. Then,
the cells were exposed to 100 ng/ml NGF in medium for
48 hr. For model group, LPS (1 μg/ml in the cell culture
medium) was added to stimulate cells for 24 hr. After
treatment with gastrodin 1 derivatives (100 μM in the
cell culture medium) for 48 hr, PC12 cells in plates
were washed by phosphate buffer saline and lysed
in radio immunoprecipitation assay lysis buffer
(synthgene, China); then, the cell lysates were cen-
trifuged at 12,000 rpm at 4ꢀC for 10 min. The superna-
tants were collected, and the protein concentrations were
determined using the BCA protein analysis kit (Beijing
Co Win). Each sample contained an equal amount of
protein gel separated in 10% sodium dodecyl sulfate-
polyacrylamide (SDS-polyacrylamide) and transferred
REFERENCES
[1] Q. H. Gong, J. S. Shi, D. L. Yang, B. Huang, X. L. Xie, Chin.
J. New Drugs Clin. Rem. 2011, 30(3), 176.
[2] J. Chen, Y. T. Gu, J. J. Xie, C. C. Wu, J. Xuan, W. J. Guo,
Y. Z. Yan, L. Chen, Y. S. Wu, X. L. Zhang, J. Xiao, X. Y. Wang,
Biomed. Pharmacother. 2017, 97, 642.
[3] H. Wen, C. Lin, L. Que, H. Ge, L. Ma, R. Cao, Y. Wan,
W. Peng, Z. Wang, H. Song, Eur. J. Med. Chem. 2008, 43
(1), 166.
[4] D. Murakami, Y. Shimada, S. Kamiya, K. Yamazaki,
Y. Makimura, K. Ito, N. Minamiura, K. Yamamoto, Arch. Bio-
chem. Biophys. 2008, 477(2), 299.
[5] Q. Zhang, Y. M. Yang, G. Y. Yu, J. Chinese 2008, 6(7), 695.
[6] X. Xu, Y. Lu, X. Bie, Planta Med. 2007, 73(7), 650.

XU AND YU
7
[7] X. Zeng, Y. Zhang, S. Zhang, X. Zheng, Biol. Pharm. Bull.
2007, 30(4), 801.
[19] E. Selenge, T. Murata, K. Kobayashi, J. Batkhuu, F. Yoshizaki,
J. Nat. Prod. 2013, 76(2), 186.
[8] C. L. Hsieh, S. Y. Chiang, K. S. Cheng, Y. H. Lin, N. Y. Tang,
C. J. Lee, C. Z. Pon, C. T. Hsieh, Am. J. Chin. Med. 2001, 29
(02), 331.
[9] W. Yong, T. R. Xing, S. Wang, L. Chen, P. Hu, C. C. Li, H. L. Wang,
M. Wang, J. T. Chen, D. Y. Ruan, Planta Med. 2009, 75, 1112.
[10] C. Shu, C. Chen, D.P. Zhang, H.P. Guo, H. Zhou, J. Zong,
Z. Y. Bian, X. Dong, J. Dai, Y. Zhang, Q. Z. Tang, Mol. Cell.
Biochem. 2012, 359(1–2), 9.
[20] C. Simmler, C. Antheaume, P. André, F. Bonté, A. Lobstein,
J. Nat. Prod. 2011, 74(5), 949.
[21] J. W. Wang, Y. M. Liu, X. F. Zhao, H. Zhang, Int.
J. Ophthalmol. 2017, 10, 1483.
[22] J. N. Dai, Y. Zong, L. M. Zhong, Y. M. Li, W. Zhang,
L. G. Bian, Q. L. Ai, Y. D. Liu, J. Sun, D. Lu, PLoS One 2011, 6
(7), e21891.
[23] J. N. Xiang, C. Y. Chen, L. H. Jiang, J. Carbohydr. Chem. 2016,
25, 595.
[11] E. K. Ahn, H. J. Jeon, E. J. Lim, H. J. Jung, E. H. Park,
J. Ethnopharmacol. 2007, 110(3), 476.
[12] P. Yang, Y. Han, L. Gui, J. Sun, Y. L. Chen, R. Song, J. Z. Guo,
Y. N. Xie, D. Lu, L. Sun, Biochem. Pharmacol. 2013, 85, 1124.
[13] G. Shu, T. Yang, C. Wang, H. Su, M. Xiang, Toxicol. Appl.
Pharmacol. 2013, 269(3), 270.
[14] X. Wang, M. M. Zhou, West China J. Pharm. Sci. 2003, 18, 269.
[15] X. C. Dai, X. Peng, S. F. Wu, W. S. Yang, Y. Mao, J. Yunnan
Univ. Nationalites 2004, 13(2), 83.
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
[16] A. Sharma, S. Gudala, S. R. Ambati, S. Penta, Y. Bomma,
V. R. Janapala, A. Jha, A. Kumar, J. Chin. Chem. Soc. 2018, 65
(7), 810.
[17] H. Liu, A. Zheng, H. Liu, H. Yu, X. Wu, C. Xiao, H. Dai,
F. Hao, L. Zhang, Y. Wang, H. Tang, J. Agric. Food Chem.
2012, 60(1), 129.
How to cite this article: Xu K-L, Yu L.
Neuroprotective activity of different
monosaccharide-modified gastrodin analogs.
J Chin Chem Soc. 2020;1–7. https://doi.org/10.
1002/jccs.201900223
[18] J. Zi, S. Li, M. Liu, M. Gan, S. Lin, W. Song, Y. Zhang, X. Fan,
Y. Yang, J. Zhang, J. Shi, D. di, J. Nat. Prod. 2008, 71(5), 799.