U.S. patent application number 17/235927 was filed with the patent office on 2021-08-05 for medicine with dicaffeoylquinic acid from artemisia selengensis for treating gout.
This patent application is currently assigned to Huazhong Agricultural University. The applicant listed for this patent is Huazhong Agricultural University, Zhiren (Wuhan) Health Industry Co., Ltd.. Invention is credited to Weiwei Cao, Siyi Pan, Ting Wu, Xiaoyun Xu.
Application Number | 20210236451 17/235927 |
Document ID | / |
Family ID | 1000005527197 |
Filed Date | 2021-08-05 |
United States Patent
Application |
20210236451 |
Kind Code |
A1 |
Xu; Xiaoyun ; et
al. |
August 5, 2021 |
Medicine with dicaffeoylquinic acid from Artemisia selengensis for
treating gout
Abstract
1,4-dicaffeoylquinic acid is used as a xanthine oxidase
inhibitor and in preparation of a medicament for treating gout. The
present invention is the first to isolate and identify five
dicaffeoylquinic acid compounds from Artemisia selengensis leaves.
The compound not only has an ability of inhibiting xanthine oxidase
activity, but also has the function of anti-gout inflammation,
which can be better used for treating gout. The dicaffeoylquinic
acid is derived from natural plants and has the advantages of being
safe and non-toxic, and having fewer side effects.
Inventors: |
Xu; Xiaoyun; (Wuhan, CN)
; Cao; Weiwei; (Wuhan, CN) ; Wu; Ting;
(Whuan, CN) ; Pan; Siyi; (Wuhan, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Huazhong Agricultural University
Zhiren (Wuhan) Health Industry Co., Ltd. |
Wuhan
Wuhan |
|
CN
CN |
|
|
Assignee: |
Huazhong Agricultural
University
Zhiren (Wuhan) Health Industry Co., Ltd.
|
Family ID: |
1000005527197 |
Appl. No.: |
17/235927 |
Filed: |
April 20, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
16566886 |
Sep 11, 2019 |
11020367 |
|
|
17235927 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/216 20130101;
A61K 2236/333 20130101 |
International
Class: |
A61K 31/216 20060101
A61K031/216 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 10, 2019 |
CN |
201910496409.X |
Claims
1. A xanthine oxidase inhibitor, comprising an active ingredient of
a dicaffeoylquinic acid.
2. The xanthine oxidase inhibitor, as recited in claim 1, wherein
the dicaffeoylquinic acid is 1,4-dicaffeoylquinic acid,
3,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid,
3,5-dicaffeoylquinic acid, or 4,5-dicaffeoylquinic acid.
3. The xanthine oxidase inhibitor, as recited in claim 1, wherein
the dicaffeoylquinic acid is extracted from Artemisia selengensis
leaves.
Description
CROSS REFERENCE OF RELATED APPLICATION
[0001] This is a continuation application of U.S. patent
application Ser. No. 16/566,886, filed on Sep. 11, 2019; and claims
the priority of Chinese Patent Application CN 201910496409.X, filed
to the China National Intellectual Property Administration (CNIPA)
on Jun. 10, 2019, the entire content of which are incorporated
hereby by reference.
BACKGROUND OF THE PRESENT INVENTION
Field of Invention
[0002] The present invention relates to a pharmaceutical use of
dicaffeoylquinic acid, and more particularly to a medicine for
treating hyperuricemia and gout, and belongs to a field of
botanical medicine.
Description of Related Arts
[0003] Gout is a disease in which uric acid is deposited in tissues
due to an increase in blood uric acid or a decrease in renal
excretion of uric acid caused by a disorder of sputum metabolism.
Hyperuricemia is the biochemical basis of gout, and gout has become
a common disease that threatens human health such as hypertension,
diabetes, and atherosclerosis. Xanthine oxidase (XOD) is a key
enzyme in the metabolism of sputum. It can catalyze the production
of uric acid from jaundice and hypoxanthine. The increase of XOD
activity will cause an increase in uric acid level, leading to
hyperuricemia. When too much uric acid in the body is deposited in
the form of crystals in the synovial membrane and interstitial
fluid of the joint, it will cause a series of inflammatory
reactions and cause gouty arthritis. Therefore, inhibition of
xanthine oxidase can prevent hyperuricemia, reduce the production
of uric acid, and thus prevent the occurrence of gout inflammation.
Conventionally, western medicine for treating hyperuricemia and
gout has great side effects such as gastrointestinal discomfort as
well as liver and kidney damage, and some natural products such as
polyphenols, flavonoids, and triterpenes have the advantages of
multiple targets and low toxic side effects in the treatment of
gout.
[0004] Artemisia selengensis leaves are rich in a variety of active
ingredients such as polyphenols. It has been reported that
caffeoylquinic acid in the leaves of Artemisia selengensis can
inhibit the activity of XOD, but polyphenols as secondary
metabolites of plants have wide varieties. Factors such as climate
and geographic location also affect the varieties of secondary
metabolites of plants. Therefore, it is necessary to excavate more
natural products that inhibit xanthine oxidase and relieve gout
inflammation from the vegetable processing by-product of Artemisia
selengensis leaves.
SUMMARY OF THE PRESENT INVENTION
[0005] Since conventional drugs for treating hyperuricemia and gout
inflammation have many side effects, an object of the present
invention is to provide a dicaffeoylquinic acid compound derived
from Artemisia selengensis leaves as a xanthine oxidase inhibitor
and a medicine for treating gout by using the same.
[0006] The object of the present invention is achieved by the
following technical solutions.
[0007] First, Artemisia selengensis leaves are extracted with 50%
ethanol, petroleum ether, and ethyl acetate to obtain the ethyl
acetate fraction of the Artemisia selengensis leaves. The ethyl
acetate fraction is then subjected to high performance liquid
chromatography quadrupole time-of-flight mass spectrometry
(HPLC-Q/TOF-MS) to identify five dicaffeoylquinic acid compounds in
the ethyl acetate fraction. Finally, IC.sub.50 values of the
above-mentioned dicaffeoylquinic acid compound in inhibiting
xanthine oxidase are determined by an enzyme kinetic method, and a
gout inflammation model is established by using THP-1 cells
stimulated by lipopolysaccharide and sodium urate, so as to
determine effects of the above five kinds of dicaffeoylquinic acid
compounds on gout inflammation.
[0008] Results shows that the five dicaffeoylquinic acid compounds
isolated from the Artemisia selengensis leaves are
1,4-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid,
1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and
4,5-dicaffeoylquinic acid, all of which have certain inhibition of
xanthine oxidase activity, indicating uric acid lowering function
and potential for treating hyperuricemia, wherein
1,4-dicaffeoylquinic acid is first reported in the Artemisia
selengensis leaves, and ability thereof for inhibiting the xanthine
oxidase activity is significantly higher than those of the other
four compounds.
[0009] At the same time, the five kinds of the dicaffeoylquinic
acid, except the 3,5-dicaffeoylquinic acid, can all inhibit
increase of IL-1.beta. secretion level of THP-1 cells induced by
lipopolysaccharide and sodium urate. IL-1.beta. secretion
inhibiting functions of 1,4-dicaffeoylquinic acid and
1,5-dicaffeoylquinic acid are better than those of other
dicaffeoylquinic acids, indicating anti-gout inflammation functions
and potential to alleviate clinical symptoms of gouty
arthritis.
[0010] Using 1,4-dicaffeoylquinic acid for treating gout can reduce
the production of uric acid from the source as well as effectively
alleviate the symptoms of inflammation, and has better curative
effect and application value.
[0011] The present invention has advantages as follows.
[0012] The present invention is the first to isolate and identify a
dicaffeoylquinic acid compound, 1,4-dicaffeoylquinic acid, from
Artemisia selengensis leaves. The compound not only has a
significant ability of inhibiting xanthine oxidase activity, but
also has the function of anti-gout inflammation, which can be
better used for treating gout.
[0013] The present invention combines a leaching method with
liquid-liquid extraction, and uses a semi-preparative
high-performance liquid chromatography to separate the chemical
components in the Artemisia selengensis leaves. Compared with
chemical synthesis methods, the present invention has the
advantages of simple operation, low cost and high efficiency.
[0014] The dicaffeoylquinic acid of the present invention is
derived from natural plants and has the advantages of being safe
and non-toxic, and having fewer side effects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGURE is a liquid chromatogram of an ethyl acetate fraction
of Artemisia selengensis leaves and five mixed standards; wherein
A: mixed standards, B: ethyl acetate fraction of Artemisia
selengensis leaves.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0016] Embodiment 1: Extraction and Identification of
Dicaffeoylquinic Acid
[0017] Artemisia selengensis leaves were dried in an oven at
60.degree. C. for 20 h, and pulverized through a 60-mesh sieve to
obtain powder of the Artemisia selengensis leaves.
[0018] The powder of the Artemisia selengensis leaves was extracted
with 50% (by volume) ethanol, and a ratio of material to liquid was
1:15. The mixture was stirred for 1 hour at room temperature,
extracted twice and filtered wherein the filtrate was combined.
After removing ethanol by rotary evaporation, a concrete of the
Artemisia selengensis leaves was obtained.
[0019] The concrete was extracted with petroleum ether and ethyl
acetate in sequence to obtain an extract of the Artemisia
selengensis leaves. The ethyl acetate extract layer was rotary
evaporated to remove ethyl acetate, and lyophilized to obtain
powder of the ethyl acetate fraction of the Artemisia selengensis
leaves.
[0020] The ethyl acetate fraction of the above-mentioned Artemisia
selengensis leaves was placed in a 5 mg/mL methanol solution. An
Agilent HPLC-Q/TOF-MS was used to identify monomer composition
which was compared to standards. Mobile phases were A: 0.1% formic
acid and B: acetonitrile. Gradient elution conditions were: 0-10
min, 10% B; 10-15 min, 15% B; 15-17 min, 18% B; 17-37 min, 18% B;
37-42 min, 50% B; 42-48 min, 65% B; 48-52 min, 80% B; 52-58 min,
80% B; 58-62 min, 10%, B. Mass spectrometry conditions were: ESI
source, negative ion mode to acquisition signal, scan range
100-1100 m/z, capillary voltage, 3.5 kV; nebulizer gas pressure, 50
psi; dry gas flow rate, 10.0 L/min; desolvation temperature,
325.degree. C. Fragator voltage was: 175 V. Collision energy was:
10-40 V.
[0021] It can be seen from FIGURE that the retention times of the
five peaks of the ethyl acetate fraction of the Artemisia
selengensis leaves are the same as the retention times of the mixed
standards, respectively, which are 1,4-dicaffeoylquinic acid
(1,4-diCQA), 3,4-dicaffeoylquinic acid (3,4-diCQA),
1,5-dicaffeoylquinic acid (1,5-diCQA), 3,5-dicaffeoylquinic acid
(3, 5-diCQA), and 4,5-dicaffeoylquinic acid (4,5-diCQA).
[0022] It can be seen from Table 1 that mass spectrometry fragment
information of the five peaks of the ethyl acetate fraction of the
Artemisia selengensis leaves meets fragment information of the five
standards, and the retention times are the same as shown in FIGURE,
which proves that the Artemisia selengensis leaves contain these
five compounds, wherein a content of the 1,4-dicaffeoylquinic acid
in the ethyl acetate fraction of the Artemisia selengensis leaves
is 0.39%. The 1,4-dicaffeoylquinic acid has not been reported in
the Artemisia selengensis leaves, and the present invention is the
first to report that the 1,4-dicaffeoylquinic acid is present in
the Artemisia selengensis leaves.
TABLE-US-00001 TABLE 1 HPLC-Q/TOF-MS identification of chemical
constituents in ethyl acetate fraction of Artemisia selengensis
leaves RT [M-H].sup.- MS.sup.2 m/z Molecular Content Peak (t/min)
m/z (% base peak) formula compounds (g/100 gdw) 1 31.029 515.1198
353 (100), C.sub.25H.sub.24O.sub.12 1,4-diCQA 0.39 .+-. 0.09 299
(26.7), 203 (49.0) 2 32.162 515.1162 353 (100),
C.sub.25H.sub.24O.sub.12 3,4-diCQA 1.39 .+-. 0.07 335 (20.3), 179
(72.0) 3 34.876 515.1197 353 (100), C.sub.25H.sub.24O.sub.12
1,5-diCQA 6.85 .+-. 0.26 191 (44.9), 179 (6.1), 4 35.949 515.1208
353 (100), C.sub.25H.sub.24O.sub.12 3,5-diCQA 13.06 .+-. 0.54 191
(16.8), 179 (9.4) 5 41.900 515.1100 353 (100),
C.sub.25H.sub.24O.sub.12 4,5-diCQA 4.72 .+-. 0.17 191 (18.6), 179
(51.6)
[0023] Embodiment 2: Inhibition of XOD Enzyme Activity by
Dicaffeoylquinic Acid
[0024] Twenty .mu.L of the above identified five monomeric compound
solutions from the Artemisia selengensis leaves and positive
control (allopurinol) were pipetted into enzyme labels before
adding 100 .mu.L of XOD enzyme solution (0.02 U/mL), and vibrating
in a 37.degree. C. microplate reader for 10 s to incubate for 2
min. Forty .mu.L of xanthine solution (1 mmol/L) was added with a
spray gun to measure absorbance every 20 s for 15 times. Change of
absorbance in the reaction system at 295 nm within 5 min was
K.sub.1, and the change in PBS control was K.sub.0.
The inhibition rate(%)of the samples on XOD enzyme activity=(1
-K.sub.1/K.sub.0).times.100%.
[0025] As can be seen from Table 2, among the five compounds,
1,4-diCQA has the lowest IC.sub.50 value and is lower than that of
the allopurinol (positive drug). This indicates that 1,4-diCQA in
the Artemisia selengensis leaves has a good ability to inhibit XOD
enzyme activity, and it can be applied to product development for
preventing hyperuricemia.
TABLE-US-00002 TABLE 2 IC.sub.50 of five monomeric compounds in
ethyl acetate fraction of Artemisia selengensis leaves in
inhibiting XOD IC.sub.50/(.mu.M) 3,5-diCQA 288.87 .+-. 21.78
3,4-diCQA 260.23 .+-. 18.35 4,5-diCQA 233.12 .+-. 10.90 1,4-diCQA
7.36 .+-. 0.63 1,5-diCQA 121.52 .+-. 2.56 allopurinol (positive
drug) 12.15 .+-. 0.32
[0026] Embodiment 3: Effect of Dicaffeoylquinic Acid on Relieving
Gout Inflammation
[0027] THP-1 suspension cells were inoculated into a 24-well plate.
When the density reached 80%, a phorbol ester (PMA) solution with a
final concentration of 100 ng/mL was added. After 12 hours of PMA
treatment, THP-1 cells differentiated into adherent macrophages.
Culture medium was aspirated before washing the cells with PBS,
completely replacing the culture medium to culture for 12 h before
carrying out a dosing treatment. One hundred .mu.mol/L of the above
identified monomer compounds were pre-incubated for 12 h, then
lipopolysaccharide with a final concentration of 1 .mu.g/mL was
added and acted for 2 h, and finally a sodium urate solution with a
final concentration of 200 .mu.g/mL was added and acted for 4 h.
The control group did not add drugs, and did not stimulate the
cells with lipopolysaccharide and sodium urate. The model group was
pre-incubated without drugs, and cells were stimulated with
lipopolysaccharide and sodium urate. Finally, the cell supernatant
was collected, centrifuged, and IL-10 content in the supernatant
was measured by using an ELISA kit.
[0028] As can be seen from Table 3, lipopolysaccharide and sodium
urate led to a significant increase in secretion level of
IL-1.beta. in the model group. Except 3,5-dicaffeoylquinic acid,
the other four types of dicaffeoylquinic acid can all inhibit the
increase in the level of IL-1.beta.. There was no significant
difference in the increase of IL-1.beta. caused by 1,4-diCQA and
1,5-diCQA in inhibiting lipopolysaccharide and sodium urate, but
their inhibition of IL-1.beta. secretion was superior to those of
other dicaffeoylquinic acid. It indicates that 1,4-diCQA can be
applied to product development against gout inflammation.
TABLE-US-00003 TABLE 3 Effects of dicaffeoylquinic acid on
secretion of IL-1.beta. in THP-1 cells stimulated by
lipopolysaccharide and sodium urate IL-1.beta. (pg/mL) Control
group 214.83 .+-. 11.92.sup.a Model group 8747.09 .+-. 975.97.sup.e
3,5-diCQA 8850.00 .+-. 190.20.sup.e 3,4-diCQA 7088.57 .+-.
621.91.sup.d 4,5-diCQA 5572.09 .+-. 632.81.sup.c 1,4-diCQA 4647.67
.+-. 607.03.sup.bc 1,5-diCQA 4176.74 .+-. 1214.41.sup.b
[0029] From the results of the above embodiments, the present
invention identifies a novel compound 1,4-dicaffeoylquinic acid in
the Artemisia selengensis leaves by HPLC-Q/TOF-MS, which not only
has a good inhibition of xanthine oxidase, but also can
significantly inhibit the gout inflammation caused by sodium urate,
which indicates that 1,4-dicaffeoylquinic acid of the Artemisia
selengensis leaves can be developed into a product for preventing
hyperuricemia and gout.
* * * * *