Synthesis and Biological Activities of Tyrosol Phenolic Acid Ester Derivatives

Article abstract of DOI:10.1007/s10600-019-02889-z

Sixteen tyrosol derivatives were synthesized and characterized by NMR and HR-MS. The antioxidant activity of those compounds was evaluated using four different assays. The results showed that some target compounds displayed better antioxidant activity tha

Full text of DOI:10.1007/s10600-019-02889-z

DOI 10.1007/s10600-019-02889-z
Chemistry of Natural Compounds, Vol. 55, No. 6, November, 2019
SYNTHESIS AND BIOLOGICAL ACTIVITIES OF TYROSOL
PHENOLIC ACID ESTER DERIVATIVES
1,2
1
1
1,3
Hao Zang, Peng Shen, Qian Xu, Luyun Zhang,
1,3
3*
1*
Guangqing Xia, Jiaming Sun, Junyi Zhu,
and Xiaohong Yang
2*
Sixteen tyrosol derivatives were synthesized and characterized by NMR and HR-MS. The antioxidant activity
of those compounds was evaluated using four different assays. The results showed that some target compounds
displayed better antioxidant activity than L-ascorbic acid and Trolox. Five target compounds exhibited
more potent α-glucosidase inhibition activity (18.1–56.7 μM) than acarbose (60.9 μM). Eight target
compounds showed some anticholinesterase activities.
Keywords: tyrosol, phenolic acid, ester derivatives, synthesis, biological activity.
Epidemiological studies in Mediterranean countries, where the traditional diet is rich in olive oil and unsaturated fatty
acids, have shown that there is a low incidence of degenerative diseases, such as heart disease and tumors [1–3]. Tyrosol is one
of the major natural phenolic antioxidant contained in olive oil [4] and has been reported to possess various physiological
activities via its potent antioxidant activity [5]. Tyrosol shows antigenotoxic activity and prevents apoptosis in keratinocytes
[6]. It also prevents Alzheimer′s disease and Parkinson′s disease by antagonizing β-amyloid (Aβ) and inhibiting apoptosis of
dopaminergic neurons [7, 8]. Tyrosol can also effectively increase lifespan [9], protect the heart [10], and prevent cancer [11].
Despite these remarkable properties, tyrosol is still seldom used as dietary supplement or stabilizer in foods and cosmetics.
Phenolic acids have attracted widespread research attention because of their broad range of biological activities and
their potential health benefits. Our previous work [12] found that the phenolic hydroxyl group is vital to the activity of the
compound, so we study some compounds that contain tyrosol linked with phenolic acids through the ester bond. Most of the
phenolic acids we selected are famous natural products such as gallic acid, caffeic acid, sinapic acid, and so on. The list also
contains a well-known antioxidant such as Trolox. The antioxidant activity of the target compounds may be higher than
tyrosol and phenolic acids themselves. Based on the antioxidant activity of tyrosol as an antioxidant, this study reports the
synthesis of 16 tyrosol ester derivatives (3a–3p).All of the compounds were analyzed for their antioxidant activity, hypoglycemic
activity, and anticholinesterase activity.
The desired tyrosol esters 3a–3p were obtained in moderate to good yields (Scheme 1). In the experiment, the target
compound 3a was synthesized using compounds 1 and 2a as an example. The synthetic conditions of compound 3a were
discussed. Initially, we use DCC and DMAP as a condensing agent and found that there is serious side effect and the
yield is low. It is considered that tyrosol has an alcoholic hydroxyl group and a phenolic hydroxyl group. The phenolic
acids also have a phenolic hydroxyl group, all of them are involved in the esterification reaction, and the selectivity of the
reaction is poor. When we use TPP and DIAD according to the reference [13], the side effect was significantly reduced, and the
yield was significantly improved; however, the reaction still produces a by-product, and its polarity is close to the target
compound. The use of a silica gel column to purify is difficult, so we used Sephadex LH-20 gel to obtain the target compound.
1) Tonghua Normal University, School of Pharmacy and Medicine, Green Medicinal Chemistry Laboratory, 134002,
Tonghua, P. R. China, e-mail: zanghao_1984@163.com; swx0527@163.com; 2) Jilin University, School of Pharmacy, 130021,
Changchun, P. R. China, e-mail: xiaohongyang88@126.com; 3) Changchun University of Chinese Medicine, Jilin Ginseng
Academy, 130117, Changchun, P. R. China, e-mail: sun_jiaming2000@163.com. Published in Khimiya Prirodnykh Soedinenii,
No. 6, November–December, 2019, pp. 899–904. Original article submitted September 18, 2018.
0009-3130/19/5506-1043 ©2019 Springer Science+Business Media, LLC
1043

TABLE 1. Antioxidant Activity of Compounds 3a–3p (IC , μM)
50
Compound
DPPH
ABTS
FRAP, mmol/g
·OH
> 100
> 100
> 100
> 2500
> 2500
> 2500
404.1 5.3
> 2500
> 2500
797.3 5.6
> 2500
> 2500
1352.8 0.8
675.1 8.3
530.5 18.6
> 2500
486.7 11.6
1505.8 5.1
705.3 1.3
> 2500
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
1
89.66 1.38
44.32 0.56
20.93 1.95
8.81 0.27
39.15 5.00
12.78 2.95
6.54 0.95
> 200
11.81 0.44
9.17 2.20
8.29 1.56
4.51 0.20
15.68 1.83
14.32 1.10
26.12 0.32
15.56 1.20
28.49 3.20
27.34 3.63
16.71 3.07
8.21 0.06
8.53 0.06
11.23 0.13
65.30 0.03
8.21 0.03
18.89 0.29
58.27 0.16
7.83 0.03
7.96 0.03
43.80 0.19
52.65 0.38
52.67 0.60
8.54 0.10
55.31 0.79
24.39 0.13
22.76 0.33
8.78 0.10
17.29 0.24
32.46 0.03
8.75 0.11
> 100
37.91 0.19
1.81 0.17
> 100
> 100
22.65 0.04
1.66 0.13
1.13 0.03
> 100
5.02 0.14
55.92 0.15
8.39 0.35
> 100
L-Ascorbic acid
–
9.86 0.33
2.50 0.02
Trolox
652.4 7.5
3
O
O
1
a
α
OH
OH
+
HO
OH
R
7
O
R
β
5
1
2a-p
3a-p
β'
8
β'
1'
3'
H CO
3
1'
1'
H CO 3'
3
1'
H CO
3
1'
1'
5'
R =
HO
α'
α'
HO
HO
HO
HO
HO
HO
5'
5'
5'
OCH
3
OH
d
f
a
b
e
c
1'
1'
HO
HO
5'
3'
HO
HO
1'
HO 5'
1'
1'
1'
5'
OH
HO
OH
OH
5'
OH
5'
OH
OH
g
j
h
i
k
l
14
12
11
9
β'
β'
9
13
1'
HO
HO
1'
H CO
3
O
1'
8
α'
8
α'
1'
HO
HO
HO 3'
5'
5'
5'
10
p
n
m
o
OCH
3
8
a. TPP-DIAD-THF, r.t., 48 h
Scheme 1
We applied the method to successfully synthesized other target compounds. The method has the advantages of short steps,
good selectivity, and high yield.
The radical scavenging activity of tyrosol esters (3a–3p) in comparison with compound 1, L-ascorbic acid, and
Trolox determined by the DPPH assays is shown in Table 1. The antioxidant activity of 3a–3p varied depending on the
number of free hydroxyl groups contained in the molecule, showing a direct relationship with the antioxidant power.
In particular, compounds containing the ortho-diphenolic structure are more active than L-ascorbic acid in antioxidant activity,
such as 3d, 3g, 3k, 3l, and 3n. Compound 3p is also better than L-ascorbic acid because it bears the Trolox moieties.
Compounds 3g, 3k, and 3l are better than Trolox. Compounds 3h–3j all contain two non-adjacent phenolic hydroxyl groups,
but they did not perform well. Finally, other tyrosol derivatives (3a, 3b, 3c, 3e, 3f, 3m, and 3o) showed lower radical scavenging
activities than L-ascorbic acid. So the ortho-diphenolic structure is crucial for antioxidant activity. Compound 1 showed lower
activity in DPPH assays.
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TABLE 2. α-Glucosidase Inhibition Activity of Compounds 3a–3p
Compound
Compound
IC50, μM
IC50, μM
3a
3b
3d
3e
3f
3g
3h
3i
42.6 0.3
439.1 8.6
384.2 5.0
19.0 0.3
146.8 2.2
56.7 0.9
48.9 0.5
203.6 1.3
3j
3k
3l
3m
3n
245.8 3.8
138.5 1.9
88.8 0.5
100.2 4.8
18.1 0.1
729.6 6.1
250.9 3.4
60.9 1.0
3o
3p
Acarbose
TABLE 3. Anticholinesterase Activity of Compounds 3a–3p (IC , μM)
50
Compound
AChE
BChE
Compound
AChE
BChE
3b
3d
3f
3g
3h
3j
3k
3l
828.0 17.7
> 1000
846.7 15.5
219.7 10.6
535.0 12.8
912.1 8.8
363.3 7.6
> 1000
690.2 7.9
> 1000
205.4 6.6
> 1000
634.2 9.8
0.1 0.0
511.6 5.9
865.0 6.5
586.7 4.6
3.6 0.1
Donepezil
·+
The ABTS assay is a method widely used for measuring the radical-scavenging activity of antioxidants. The radical
scavenging activity of tyrosol esters (3a–3p) in comparison with compound 1, L-ascorbic acid, and Trolox determined by
·+
ABTS assays is shown in Table 1. Compounds 3c, 3d, 3f, 3g, 3i, 3j, 3k, 3l, 3m, 3n, 3o, and 3p showed higher antioxidant
capacity, ranging from IC = 4.51 to 26.12 μM, than L-ascorbic acid (IC = 27.34 μM). Compounds 3d, 3f, 3g, 3i, 3j, 3k, 3l,
50
50
3m, 3n, and 3p are better than Trolox (16.71 μM). Compared to L-ascorbic acid, compound 1 showed similar activity in ABTS
assays.
Results of the reducing power of tyrosol esters (3a–3p) evaluated by the FRAP assay (expressed in millimoles of
Fe(II) per gram) in comparison with compound 1, L-ascorbic acid, and Trolox are summarized in Table 1. Compounds 3d, 3f,
3g, 3j, 3k, 3l, 3n, 3o, and 3p (18.89–65.30 mmol/g) showed higher antioxidant potency than L-ascorbic acid (17.29 mmol/g),
and 3d, 3g, 3j, 3k, 3l, and 3n are better than Trolox (32.46 mmol/g). Compound 1 showed lower activity in FRAP assays.
The radical scavenging activity of tyrosol esters (3a–3p) in comparison with compound 1 and Trolox determined by
hydroxyl radical scavenging assays is shown in Table 1. Compounds 3d, 3l, and 3n exhibited more potent hydroxyl radical
scavenging activity (IC = 404.1–530.5 μM) than Trolox (652.4 μM). Compound 1 showed lower activity in hydroxyl radical
50
scavenging assays.
Results of the α-glucosidase inhibition activity of tyrosol esters (3a–3p) evaluated by the α-glucosidase inhibition
assay in comparison with compound 1 and acarbose are summarized in Table 2. Compounds 3a, 3e, 3g, 3h, and 3n
(18.1–56.7 μM) showed higher inhibition potency than acarbose (60.9 μM). Compounds 1 and 3c (IC > 800 μM) showed
50
lower inhibition activity.
The anticholinesterase activity of tyrosol esters (3a–3p) in comparison with compound 1 and donepezil determined
by cholinesterase inhibition assays is shown in Table 3. Compounds 3b, 3f, 3g, 3h, 3j, and 3l showed weaker AChE inhibitory
activities, ranging from IC = 205.4 to 846.7 μM, than donepezil (IC = 0.1 μM). Compounds 3b, 3d, 3g, 3j, 3k, and 3l
50
50
showed weaker BChE inhibitory activities, ranging from IC = 363.3 to 912.1 μM, than donepezil (IC = 3.6 μM).
50
50
Compounds 3b, 3g, 3j, and 3l showed inhibitory effects on both cholinesterases, and compounds 1, 3a, 3c, 3e, 3i, and 3m–3p
(IC > 1000 μM) showed lower activity in cholinesterase inhibition assays.
50
Generally, free radicals play an important role in a number of biological processes. Many of these are necessary for
life. ROS are formed as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and
homeostasis [14]. However, because of their reactivity, these same free radicals can participate in unwanted side reactions,
resulting in cell damage. Excessive amounts of these free radicals can lead to cell injury and death, which is recognized as a
leading cause of a variety of chronic diseases such as atherosclerosis, diabetes, and dyslipidemia [15]. It is well known that
1045

antioxidants can scavenge free radicals and ROS and protect the body from oxidative damage. Dietary antioxidants have been
investigated for potential effects on neurodegenerative diseases such as Alzheimer′s disease [16] and Parkinson′s disease [17].
To some extent, this study indicates that tyrosol ester derivatives are all potent antioxidants; they also have hypoglycemic
activity and anticholinesterase activity, which are potential functional chemicals beneficial for human health worthy of further
investigation.
EXPERIMENTAL
General.All manipulations were conducted with a standard Schlenk tube under N . Unless otherwise noted, materials
2
obtained from commercial suppliers were used without further purification. Yeast α-glucosidase (EC 3.2.1.20), electric eel
acetylcholinesterase (AChE, Type-VI-S, EC 3.1.1.7), horse serum butyrylcholinesterase (BChE, EC 3.1.1.8), and s-butyrylthiocholine
chloride were purchased from the supplier (Sigma-Aldrich). Tyrosol, 98%, triphenylphosphine (TPP, 98%),
diisopropyl azodicarboxylate (DIAD, 97%), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium (ABTS, 98%),
2,4,6-tri(2-pyridyl)-s-triazine (TPTZ, 98%), and acetylthiocholine iodide (ATCI, 98%) were purchased from the supplier
(Energy Chemical). Acarbose (98%) was from Ark Pharm. p-Nitrophenyl-α-D-glucopyranoside (pNPG, 99%) were from
ACROS. Donepezil hydrochloride (98%) was from Adamas. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox, 98%) was purchased from the supplier (TCI). 1,1-Diphenyl-2-picrylhydrazyl (DPPH, 95%) and 5,5′-dithiobis-(2-
nitrobenzoic acid) (DTNB, 99%) were purchased from the supplier (Alfa Aesar).
Sephadex LH-20 (GE) was used for column chromatography, with MeOH as eluent. Chemical reactions were monitored
by thin-layer chromatography (TLC) on silica gel precoated F plates. Developed plates were visualized by ultraviolet light
254
1
13
(254 nm). H and C NMR spectra were measured on an AV-600 Spectrometer (Bruker, Germany) using tetramethylsilane
(TMS) as internal standard. High-Resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on an
Aglient 6520 Q-TOF spectrometer (Agilent, USA). Melting points were determined in open capillary tubes and the temperature
was uncorrected.
Synthesis of the Target Compounds. The synthesis of compounds 3a–3f was reported in our previous work [12].
Synthesis of Target Compounds 3g–3p. Tyrosol (1, 0.4 mmol, 1.0 equiv), organic acids 2g–2p (0.4 mmol, 1.0 equiv),
and TPP (0.4 mmol, 1.0 equiv) were placed in a dry standard Schlenk tube under N . Dry THF (1.0 mL) was added, followed
2
by the addition of DIAD (0.4 mmol, 1.0 equiv) at 0°C. The reaction mixture was stirred at room temperature for 48 h, and the
reaction was monitored with TLC. The crude reaction mixture was purified by column chromatography on Sephadex LH-20 to
afford the corresponding product.
1
4-Hydroxyphenethyl 3,4-Dihydroxybenzoate (3g). C H O , white solid, yield 69%; mp 178–179°C. H NMR
15 14
5
spectrum (600 MHz, DMSO-d , δ, ppm, J/Hz): 9.44 (3H, s, 3′, 4′, 4-OH), 7.34 (1H, d, J = 2.0, H-2′), 7.29 (1H, dd, J = 8.2, 2.0,
6
H-6′), 7.08 (2H, d, J = 8.4, H-2, 6), 6.80 (1H, d, J = 8.2, H-5′), 6.70 (2H, d, J = 8.4, H-3, 5), 4.30 (2H, t, J = 6.8, H-α), 2.86 (2H,
13
t, J = 6.8, H-β). C NMR spectrum (150 MHz, DMSO-d , δ, ppm): 165.6 (C-7), 155.8 (C-4), 150.4 (C-4′), 145.0 (C-3′), 129.8
6
(C-2, 6), 128.1 (C-1), 121.8 (C-1′), 120.7 (C-6′), 116.3 (C-5′), 115.3 (C-2′), 115.2 (C-3, 5), 65.0 (C-α), 33.7 (C-β).
–
HR-ESI-MS m/z 273.0821 [M – H] .
1
4-Hydroxyphenethyl 2,4-Dihydroxybenzoate (3h). C H O , white solid, yield 67%; mp 100–102°C. H NMR
15 14
5
spectrum (600 MHz, DMSO-d , δ, ppm, J/Hz): 10.72 (1H, s, 2′-OH), 10.45 (1H, s, 4′-OH), 9.24 (1H, s, 4-OH), 7.59 (1H, d,
6
J = 8.8, H-6′), 7.08 (2H, d, J = 8.4, H-2, 6), 6.70 (2H, d, J = 8.4, H-3, 5), 6.37 (1H, dd, J = 8.8, 2.4, H-5′), 6.29 (1H, d, J = 2.4,
13
H-3′), 4.38 (2H, t, J = 6.8, H-α), 2.89 (2H, t, J = 6.8, H-β). C NMR spectrum (150 MHz, DMSO-d , δ, ppm): 169.1 (C-7),
6
164.3 (C-4′), 162.8 (C-2′), 155.9 (C-4), 131.5 (C-6′), 129.8 (C-2, 6), 127.8 (C-1), 115.2 (C-3, 5), 108.4 (C-5′), 103.9 (C-1′),
–
102.5 (C-3′), 65.5 (C-α), 33.5 (C-β). HR-ESI-MS m/z 273.0786 [M – H] .
1
4-Hydroxyphenethyl 3,5-Dihydroxybenzoate (3i). C H O , white solid, yield 78%; mp 125–127°C. H NMR
15 14
5
spectrum (600 MHz, DMSO-d , δ, ppm, J/Hz): 9.61 (2H, s, 3′, 5′-OH), 9.21 (1H, s, 4-OH), 7.08 (2H, d, J = 7.4, H-2, 6), 6.79
6
13
(2H, s, H-2′, 6′), 6.69 (2H, d, J = 7.4, H-3, 5), 6.43 (1H, s, H-4′), 4.33 (2H, s, H-α), 2.87 (2H, s, H-β). C NMR spectrum
(150 MHz, DMSO-d , δ, ppm): 165.7 (C-7), 158.5 (C-3′, 5′), 155.8 (C-4), 131.5 (C-1), 129.8 (C-2, 6), 128.0 (C-1′), 115.2
6
+
(C-3, 5), 107.2 (C-4′), 107.1 (C-2′, 6′), 65.4 (C-α), 33.6 (C-β). HR-ESI-MS m/z 297.0411 [M + Na] .
1
4-Hydroxyphenethyl 2,5-Dihydroxybenzoate (3j). C H O , white solid, yield 82%; mp 91–92°C. H NMR
15 14
5
spectrum (600 MHz, DMSO-d , δ, ppm, J/Hz): 9.94 (1H, s, 2′-OH), 9.23 (1H, s, 5′-OH), 9.22 (1H, s, 4-OH), 7.12 (1H, d,
6
J = 3.0, H-6′), 7.10 (2H, d, J = 8.4, H-2, 6), 6.97 (1H, dd, J = 8.9, 3.0, H-4′), 6.81 (1H, d, J = 8.9, H-3′), 6.70 (2H, d, J = 8.4,
13
H-3, 5), 4.42 (2H, t, J = 6.8, H-α), 2.91 (2H, t, J = 6.8, H-β). C NMR spectrum (150 MHz, DMSO-d , δ, ppm): 168.8 (C-7),
6
1046

155.9 (C-4), 153.3 (C-2′), 149.5 (C-5′), 129.8 (C-2, 6), 127.7 (C-1), 123.9 (C-6′), 118.1 (C-4′), 115.2 (C-3, 5), 114.0 (C-3′),
+
112.3 (C-1′), 65.9 (C-α), 33.4 (C-β). HR-ESI-MS m/z 297.0411 [M + Na] .
1
4-Hydroxyphenethyl 2,3-Dihydroxybenzoate (3k). C H O , white solid, yield 86%; mp 110–111°C. H NMR
15 14
5
spectrum (600 MHz, DMSO-d , δ, ppm, J/Hz): 10.42 (1H, s, 2′-OH), 9.39 (1H, s, 3′-OH), 9.22 (1H, s, 4-OH), 7.19 (1H, d,
6
J = 7.9, H-6′), 7.09 (2H, d, J = 8.2, H-2, 6), 7.02 (1H, d, J = 7.9, H-4′), 6.74 (1H, t, J = 7.9, H-5′), 6.69 (2H, d, J = 8.2, H-3, 5),
13
4.43 (2H, t, J = 6.8, H-α), 2.92 (2H, t, J = 6.8, H-β). C NMR spectrum (150 MHz, DMSO-d , δ, ppm): 169.4 (C-7), 155.9
6
(C-4), 149.6 (C-2′), 146.1 (C-3′), 129.8 (C-2, 6), 127.7 (C-1), 120.7 (C-4′), 119.4 (C-6′), 118.9 (C-5′), 115.2 (C-3, 5), 113.0
+
(C-1′), 66.0 (C-α), 33.4 (C-β). HR-ESI-MS m/z 297.0409 [M + Na] .
1
4-Hydroxyphenethyl 3,4,5-Trihydroxybenzoate (3l). C H O , white solid, yield 40%; mp 207–209°C. H NMR
15 14
6
spectrum (600 MHz, DMSO-d , δ, ppm, J/Hz): 9.21 (3H, s, 3′, 5′, 4-OH), 8.92 (1H, s, 4′-OH), 7.07 (2H, d, J = 8.4, H-2, 6),
6
13
6.92 (2H, s, H-2′, 6′), 6.69 (2H, d, J = 8.4, H-3, 5), 4.28 (2H, t, J = 6.8, H-α), 2.85 (2H, t, J = 6.8, H-β). C NMR spectrum
(150 MHz, DMSO-d , δ, ppm): 165.8 (C-7), 155.8 (C-4), 145.5 (C-3′, 5′), 138.4 (C-4′), 129.8 (C-2, 6), 128.1 (C-1), 119.4 (C-1′),
6
+
115.1 (C-3, 5), 108.5 (C-2′, 6′), 65.0 (C-α), 33.7 (C-β). HR-ESI-MS m/z 313.0544 [M + Na] .
4-Hydroxyphenethyl 3-(4-Hydroxyphenyl)propanoate (3m). C H O , white solid, yield 85%; mp 122–123°C.
17 18
4
1
H NMR spectrum (600 MHz, DMSO-d , δ, ppm, J/Hz): 9.19 (1H, s, 4-OH), 9.15 (1H, s, 4′-OH), 6.99 (2H, d, J = 8.0, H-2, 6),
6
6.95 (2H, d, J = 8.1, H-2′, 6′), 6.68 (2H, d, J = 8.0, H-3, 5), 6.65 (2H, d, J = 8.1, H-3′, 5′), 4.11 (2H, t, J = 6.8, H-α), 2.77–2.65
13
(4H, m, H-β, 9), 2.50 (2H, t, J = 7.1, H-8). C NMR spectrum (150 MHz, DMSO-d , δ, ppm): 172.2 (C-7), 155.8 (C-4), 155.6
6
(C-4′), 130.5 (C-1′), 129.7 (C-2, 6), 129.1 (C-2′, 6′), 127.9 (C-1), 115.1 (C-3, 5, 3′, 5′), 64.7 (C-α), 35.6 (C-8), 33.5 (C-β), 29.5
+
(C-9). HR-ESI-MS m/z 309.0756 [M + Na] .
4-Hydroxyphenethyl (E)-3-(3,4-Dihydroxyphenyl)-acrylate (3n). C
H O , white solid, yield 59%;
17 16 5
1
mp 173–174°C. H NMR spectrum (600 MHz, DMSO-d , δ, ppm, J/Hz): 9.23 (3H, s, 3′, 4′, 4-OH), 7.45 (1H, d, J = 15.9, H-β′),
6
7.06 (2H, d, J = 8.2, H-2, 6), 7.04 (1H, d, J = 1.2, H-2′), 6.99 (1H, d, J = 8.1, 1.2, H-6′), 6.76 (1H, d, J = 8.1, H-5′), 6.69 (2H,
13
d, J = 8.2, H-3, 5), 6.23 (1H, d, J = 15.9, H-α′), 4.24 (2H, t, J = 6.8, H-α), 2.82 (2H, t, J = 6.8, H-β). C NMR spectrum
(150 MHz, DMSO-d , δ, ppm): 166.5 (C-7), 155.8 (C-4), 148.4 (C-4′), 145.6 (C-3′), 145.1 (C-β′), 129.8 (C-2, 6), 128.0 (C-1),
6
125.5 (C-1′), 121.3 (C-6′), 115.7 (C-5′), 115.1 (C-3, 5), 114.8 (C-α′), 113.9 (C-2′), 64.7 (C-α), 33.7 (C-β). HR-ESI-MS
+
m/z 323.0539 [M + Na] .
4-Hydroxyphenethyl (E)-3-(4-Hydroxy-3,5-dimethoxyphenyl)acrylate (3o). C H O , colorless oil, yield 46%.
19 20
6
1
H NMR spectrum (600 MHz, DMSO-d , δ, ppm, J/Hz): 9.20 (1H, s, 4-OH), 8.95 (1H, s, 4′-OH), 7.53 (1H, d, J = 15.9, H-β′),
6
7.07 (2H, d, J = 8.4, H-2, 6), 7.02 (2H, s, H-2′, 6′), 6.70 (2H, d, J = 8.4, H-3, 5), 6.50 (1H, d, J = 15.9, H-α′), 4.27 (2H, t,
13
J = 6.8, H-α), 3.80 (6H, s, H-8, 9), 2.83 (2H, t, J = 6.8, H-β). C NMR spectrum (150 MHz, DMSO-d , δ, ppm): 166.5 (C-7),
6
155.8 (C-4), 148.0 (C-3′, 5′), 145.4 (C-β′), 138.3 (C-4′), 129.7 (C-2, 6), 127.9 (C-1), 124.3 (C-1′), 115.1 (C-3, 5), 114.8 (C-α′),
+
106.2 (C-2′, 6′), 64.6 (C-α), 56.1 (C-8, 9), 33.7 (C-β). HR-ESI-MS m/z 367.0749 [M + Na] .
4-Hydroxyphenethyl 6-Hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxylate (3p). C H O , white solid,
22 26
5
1
yield 91%; mp 137–139°C. H NMR spectrum (600 MHz, DMSO-d , δ, ppm, J/Hz): 9.20 (1H, s, 4-OH), 7.45 (1H, s, 3′-OH),
6
6.94 (2H, d, J = 8.4, H-2, 6), 6.66 (2H, d, J = 8.4, H-3, 5), 4.23–4.08 (2H, m, H-α), 2.75–2.62 (2H, m, H-β), 2.49–2.43 (1H, m,
H-10a), 2.26–2.23 (1H, m, H-10b), 2.22–2.14 (1H, m, H-9a), 2.07 (3H, s, H-14), 2.03 (3H, s, H-12), 1.96 (3H, s, H-13),
13
1.76–1.71 (1H, m, H-9b), 1.45 (3H, s, H-11). C NMR spectrum (150 MHz, DMSO-d , δ, ppm): 172.8 (C-7), 155.9 (C-4),
6
145.7 (C-6′), 144.7 (C-3′), 129.7 (C-2, 6), 127.9 (C-1), 122.6 (C-5′), 120.9 (C-4′), 120.2 (C-2′), 116.4 (C-1′), 115.1 (C-3, 5),
76.4 (C-8), 65.5 (C-α), 33.5 (C-β), 30.2 (C-10), 25.2 (C-9), 20.3 (C-11), 12.7 (C-14), 11.8 (C-12), 11.7 (C-13). HR-ESI-MS
+
m/z 763.2607 [2M + Na] .
DPPH Assay. DPPH assay was performed according to the method as previously described with slight modifications
[18, 19]. Each sample in DMSO solution (100 μL) was added to 100 μL of DPPH methanol solution (50 μM). The solution
was vortexed in 96-well plates for 10 s and then left at room temperature for 20 min in the dark. The absorbance of the
resulting solution was measured at 492 nm on a microplate spectrophotometer (BioTek). L-Ascorbic acid and Trolox were
used as positive references. IC values (concentration required to scavenge 50% DPPH radicals present in the test solution)
50
were calculated and expressed as means SD in micromoles.
ABTS Free Radical Cation (ABTS ) Assay. The ABTS scavenging activity of the sample was assayed following
procedures previously described with slight modifications [19]. ABTS solution was produced by reacting 7 mM ABTS water
·+
·+
·+
solution (10 mL) with 2.6 mM potassium persulfate (10 mL). The mixture was allowed to stand in the dark at room temperature
·+
for 12–16 h before use. The ABTS solution was diluted with methanol to provide an absorbance of 0.70 0.02 at 734 nm.
·+
Then each sample in DMSO solution (5 μL) was added to 200 μL of diluted ABTS solution. The solution was vortexed in
1047

96-well plates for 10 s and then left at room temperature for 20 min in the dark. The absorbance of the resulting solution was
measured at 734 nm on a microplate spectrophotometer (BioTek). L-Ascorbic acid and Trolox were used as positive references.
IC values were calculated and expressed as means SD in micromoles.
50
Ferric Reducing Antioxidant Power (FRAP) Assay. Ferric reducing ability of the sample was conducted according
to procedures previously described with slight modifications [19, 20]. FRAP reagent was made freshly by mixing 300 mM
acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM HCl, and 20 mM aqueous FeCl solution in a 10:1:1 (v/v) ratio. Each
3
sample in DMSO solution (5 μL) was added to 180 μL of FRAP reagent and vortexed in 96-well plates for 10 s and then
incubated at 37°C for 30 min in the dark. The absorbance was determined at 595 nm using a microplate spectrophotometer
(BioTek). L-Ascorbic acid and Trolox were used as positive references. FeSO was used for a calibration curve. FRAP values
4
were calculated and expressed as means SD in millimoles of Fe(II) per gram.
Hydroxyl Radical (·OH)Assay. Hydroxyl radical scavenging activity was assayed according to the method previously
described with slight modifications [21]. Each sample in DMSO solution (50 μL) was treated with 3 mM FeSO solution
4
(50 μL) and 3 mM H O solution (50 μL), vortexed in 96-well plates, and left to stand for 10 min; 6 mM salicylic acid solution
2
2
(50 μL) was added. The reaction mixtures were vortexed and the plates incubated at room temperature for 30 min in the dark.
The absorbance of the resulting solution was measured at 492 nm on a microplate spectrophotometer (BioTek). Trolox was
used as a positive reference. IC values (concentration required to scavenge 50% hydroxyl radicals present in the test solution)
50
were calculated and expressed as means SD in micromoles.
α-Glucosidase Inhibition Assay. α-Glucosidase was assayed according to the method previously described with
slight modifications [22]. Each sample in DMSO solution (20 μL) was added to 100 μL of α-glucosidase solution (pH 6.9,
0.1 U/mL, in 0.1 M phosphate buffer). The reaction mixtures were incubated at 25°C for 10 min. Then 50 μL pNPG solution
(pH 6.9, 5 mM, in 0.1 M phosphate buffer) was added to each well, and the reaction mixtures were incubated at 25°C for 5 min.
Before and after incubation, the absorbance was recorded at 405 nm on a microplate spectrophotometer (BioTek).
Acarbose was used as a positive reference.
Acetylcholinesterase Inhibition Assay. Acetylcholinesterase (AChE) inhibitory activities were measured according
to the method previously described with slight modifications [23]. Each sample in 10% DMSO solution (20 μL) was added to
120 μL phosphate buffer (pH 8.0, 0.1 M) and 20 μL of acetylcholinesterase solution (pH 8.0, 0.8 U/mL, in 0.1 M phosphate
buffer). The reaction mixtures were incubated at 25°C for 15 min. Then 20 μL ATCI solution (pH 8.0, 1.78 mM, in 0.1 M
phosphate buffer) and 20 μL DTNB solution (pH 8.0, 1.25 mM, in 0.1 M phosphate buffer) were added to each well, and the
reaction mixtures were incubated at 25°C for 5 min. Before and after incubation, the absorbance was recorded at 405 nm on a
microplate spectrophotometer (BioTek). Donepezil was used as a positive reference.
Butyrylcholinesterase InhibitionAssay. Butyrylcholinesterase (BChE) inhibitory activities were measured according
to the method previously described with slight modifications [23]. Each sample in 10% DMSO solution (20 μL) was added to
120 μL phosphate buffer (pH 8.0, 0.1 M) and 20 μL of butyrylcholinesterase solution (pH 8.0, 0.8 U/mL, in 0.1 M phosphate
buffer). The reaction mixtures were incubated at 25°C for 15 min. Then 20 μL butyrylthiocholine chloride solution (pH 8.0,
0.4 mM, in 0.1 M phosphate buffer) and 20 μL DTNB solution (pH 8.0, 1.25 mM, in 0.1 M phosphate buffer) were added to
each well, and the reaction mixtures incubated at 25°C for 5 min. Before and after incubation, the absorbance was recorded at
405 nm on a microplate spectrophotometer (BioTek). Donepezil was used as a positive reference.
The α-glucosidase, acetylcholinesterase, and butyrylcholinesterase inhibitory activity was expressed as % inhibition
and was calculated as follows:
%Inhibition = (1 – ΔA
/ΔA
) × 100%.
sample
control
All the experiments were carried out in triplicate, and the data were analyzed using SPSS software (Version 22.0) and
Origin software (Version 8.0).
ACKNOWLEDGMENT
This work was supported by the Science and Technology Development Funds of Jilin Province (No. 20160520044JH)
and the Science and Technology Projects of Administration of Traditional Chinese Medicine of Jilin Province (No. 2017110).
In addition, Hao Zang and Peng Shen contributed equally to this work.
1048

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