LU502965B1 - A method for extracting flavonoid compounds from folium cortex eucommiae - Google Patents

A method for extracting flavonoid compounds from folium cortex eucommiae Download PDF

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LU502965B1
LU502965B1 LU502965A LU502965A LU502965B1 LU 502965 B1 LU502965 B1 LU 502965B1 LU 502965 A LU502965 A LU 502965A LU 502965 A LU502965 A LU 502965A LU 502965 B1 LU502965 B1 LU 502965B1
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cortex eucommiae
folium cortex
seh
formula
flavonoids
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LU502965A
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French (fr)
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Jinming Gao
Yulan Du
Junmian Tian
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Univ Northwest A&F
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/40—Separation, e.g. from natural material; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans
    • C07H17/07—Benzo[b]pyran-4-ones

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to flavonoids isolated from folium cortex eucommiae and their applications. The structural formula of the compound is shown in formula I, and the extraction raw material of the related compound is folium cortex eucommiae. The related compound is verified to have soluble epoxide hydrolase (sEH) inhibitory activity, and has the advantages of good water solubility, small toxic and side effects, high content, wide distribution and the like. Based on the action principle of sEH, the compound provided by the invention can be used for preventing and treating various cardiovascular diseases.

Description

A METHOD FOR EXTRACTING FLAVONOID COMPOUNDS FROM 7502965
FOLIUM CORTEX EUCOMMIAE
TECHNICAL FIELD
The invention relates to pharmaceutical chemistry, in particular to seven flavonoids isolated from folium cortex eucommiae and their inhibitors verified as soluble epoxide hydrolase.
BACKGROUND
Cardiovascular diseases have become one of the serious threats to human health. With the development of science and technology, people have made great progress in treating cardiovascular diseases. However, a large number of patients with cardiovascular diseases still die every year because they can't get effective treatment. Therefore, human beings have an urgent need for new therapeutic approaches and drugs for cardiovascular diseases.
Soluble epoxide hydrolase (sEH) is an enzyme widely existing in mammals. Recently, it has been found that sEH is closely related to hypertension, end organ injury, myocardial hypertrophy and ischemia/reperfusion injury, and it is likely to become a new target for the prevention and treatment of cardiovascular diseases (Hum Mol Genet, 2006,15 (10): 1640-1649; J Int Pathol Clinical Med, 2008, 28(5):443-446). Therefore, inhibition of sHE enzyme activity can be used as a potential therapeutic approach to relieve and treat various cardiovascular diseases such as hypertension, terminal organ injury, myocardial hypertrophy, diabetes and so on, as well as anti-inflammatory and analgesic effects.
Urea-based sEH inhibitors have developed rapidly in the past ten years. Relevant research results show that these ingredients have the effects of anti-hypertension, anti- inflammation and protecting the heart, kidney and brain from damage. However, the characteristics of poor solubility and rapid degradation also limit the application prospects of these compounds to a great extent. In addition, some sEH inhibitors can be rapidly metabolized in liver microsomes. Later, the sEH inhibitors based on urea are improved from the aspects of pharmacokinetics and bioavailability, but there are still 502965 some shortcomings such as slow absorption, short half-life and poor water solubility.
In 1951, the former Soviet Union studied that Eucommia ulmoides could regulate blood pressure. In 1976, a professor at the University of Wisconsin in the United States thought that the mechanism of lowering blood pressure of Eucommia ulmoides is the effect of pinoresinin diglucoside. Since then, Japanese studies have found that geniposide and geniposide in folium cortex eucommiae are effective components for lowering blood pressure. Chinese scientists think that the medicinal active ingredients of folium cortex eucommiae are the same as the bark of folium cortex eucommiae, their medicinal functions are basically the same, and the mechanism of lowering blood pressure of folium cortex eucommiae is the dilating effect of its active ingredients on coronary arteries. Up to now, there are no reports about SEH inhibitors in folium cortex eucommiae.
SUMMARY
The technical problem to be solved by this invention lies in overcoming the above shortcomings, and researching and designing flavonoid compounds which can inhibit the activity of sEH, have good water solubility and low toxicity.
The flavonoid compound provided by the invention has a structural formula as shown in formula I:
HO, eu. OL Au J om, on © formula I;
In the formula, Rı and R> can be as given in table 1.
Table 1: R1 and R; of formula I 7502965
C18 ghiccsids Fi
Ea aylopyrandsyl-{$ > Zlaluonside OH
Eu rutin Ci
BLAAG M H
FLAS Ghuncaids +
EU-43 rutinonite 3
The flavonoids are extracted from folium cortex eucommiae.
Another object of the present invention is to provide an application of the above flavonoids as sEH inhibitors.
Another object of the present invention is to provide the application of the flavonoids in preparing drugs for preventing and treating cardiovascular diseases.
According to the invention, seven flavonoid components with sEH inhibitory activity are separated from folium cortex eucommiae. This kind of ingredients has the advantages of good water solubility, low toxic and side effects, high content and wide distribution. The activity test results showed that flavonoids from folium cortex eucommiae could inhibit sEH activity. Based on the action principle of sEH, the sEH inhibitor in folium cortex eucommiae provided by the invention can be used for preventing and treating various cardiovascular diseases.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 is the effect of flavonoids in folium cortex eucommiae on the hydrolysis substrate
PHOME by sEH (sEH) at 37°C.
Fig. 2 shows EU-26 double reciprocal curve of Michaelis equation.
Fig. 3 shows double reciprocal curve of RU-40 Michaelis equation.
Fig. 4 shows EU-26 Dixon curve. 17008965
Fig. 5 shows RU-40Dixon curve.
Fig. 6 is an extraction process diagram of flavonoids in the present invention.
DESCRIPTION OF THE INVENTION
As shown in Fig. 6, the preparation method of flavonoids provided by the invention is as follows: (1) freshly picked folium cortex eucommiae are fully dried in the shade indoors; (2) crushing, percolating and extracting with 50% methanol until the effluent is colorless; (3) mixing extractive solutions, and concentrating under reduced pressure to obtain extract; (4) the extract is suspended with pure water at the ratio of kg/L 1:10, and extracted with equal volume of n-butanol for 3 times; concentrate that water phase and the n-butanol phase respectively, and recover the solvent; (5) the water phase is mixed with 100-200 mesh silica gel according to the mass ratio of 1:3, and chloroform: methanol (5:1 to 1:1) is used as the mobile phase for elution; collecting the yellow part of sulfuric acid ethanol, and combining; (6) continue to use AB-8 macroporous adsorption resin, silica gel, reversed-phase silica gel, gel and HPLC for purification; finally, EU-6 and EU-7 are separated; (7) dissolving the obtained n-butanol phase with a small amount of water, and separating with AB-8 macroporous adsorption resin; eluting with water, 10% methanol, 30% methanol, 50% methanol, 75% methanol and 100% methanol in sequence; collect 30% to 75% fraction, and separate and purify by silica gel, gel, reversed-phase silica gel and HPLC; finally, Eu-18, EU-23, EU-26, EU-40 and EU-43 are separated.
The structural identification results of flavonoids of the invention are as follows:
EU-26, yellow solid, ESI-MS m/z: 301 [M-HT, *C-NMR is shown in table 2, "H-NMR is shown in table 3;
EU-18, yellow solid, ESI-MS m/z: 465 [M-H], C-NMR is shown in table 2, "H-NMR is shown in table 3;
. LU502965
EU-6, yellow solid, ESI-ms m/z: 619 [M+NA]*,see table 2 for ’c-NMR and table 3 for "H-NMR;
EU-7, yellow solid, ESI-ms m/z: 633 [m+na]*,see table 2 for *c-NMR and table 3 for "H-NMR; 5 EU-40, yellow solid, ESI-MS m/z: 285 [M-HT, *C-NMR is shown in table 2, "'H-NMR is shown in table 3;
EU-23, yellow solid, ESI-MS m/z: 447 [M-HT, >*C-NMR is shown in table 2, 'H-NMR is shown in table 3;
EU-43, yellow solid, ESI-ms m/z: 617 [M+NAT",see table 2 for *C-NMR and table 3 for 'H-NMR.
Table 2 *C NMR data of flavonoids from folium cortex eucommiae
EU-26 EU-18 EU-6 EU-7 EU-40 EU-23 FEU-43 2 146.7 1585 1583 1585 1480 1585 161.2 3 135.7 1356 1352 1356 137.1 135.3 135.5 4 1758 1795 1796 1794 177.3 1795 1794 5 160.7 163.1 163.2 1629 162.5 163.0 162.9 6 98.2 99.9 997 1000 993 99.9 100.0 7 1640 166.0 1658 166.1 1657 166.1 166.1 8 933 94.7 946 949 94.5 94.8 95.0 9 156.1 1591 1584 1593 1582 159.1 158.5 10 102.9 105.7 1058 1056 104.5 105.7 105.6 1’ 1219 123.2 123.3 123.1 123.7 1228 122.7 2 1150 1176 1173 117.7 1307 1323 132.4 3 145.0 1459 1460 1458 1163 116.1 116.1 4 1476 1499 1497 1498 160.5 161.5 1594 5’ 1156 1160 116.1 116.1 1163 116.1 116.1 6’ 1200 123.1 1234 123.6 130.7 1323 132.4 1” 104.3 100.9 104.7 1042 104.4 2” 75.2 82.3 75.7 75.7 75.8 3” 78.1 782 78.2 78.0 78.1 4” 71.2 71.1 71.4 71.4 71.4 5” 78.4 770 772 78.4 77.2 6” 62.6 624 68.5 62.6 68.6 1°” 105.3 1024 102.4 2°” 749 72.1 72.1 3” 78.3 72.2 72.3 4”? 71.0 73.9 73.9 5°” 66.6 69.7 69.7 6” 17.9 17.9
. . . LU502965
Table 3 1H NMR data of flavonoids from folium cortex eucommiae
EU-26 EU-18 EU-6 EU-7 EU-40 EU-23 EU-43 6.40, d,J = 6.22,dJ= 625,d,J 621,dJ= 6.19,d,7 6 6.21,s 6.20, br 14 Hz 2.0 Hz = 2.0 Hz 1.7 Hz = 1.3 Hz 6.18,d,J = 641,d,J= 641,d,J 643,d,J= 6.40, d,J 8 6.40,s 6.39, br 1.4 Hz 1.7 Hz =20Hz 1.5 Hz =13Hz 7.68, d, J = 773,d,J = 7.67 7.71,d, J= 8.09,d,J 8.06,d,J 8.06,d J 67, m 1.8 Hz 2.1 Hz 1.7 Hz =86Hz =86Hz =82Hz 3° 6.92,d, 7 690,d,J 6.89,d,J =88Hz =87Hz =83Hz 5 6.88, d, J = 6.89, d,J = 6.90 d,J 6.87 d, J = 6.92,d, 7 690,d,J 6.89,d,J 8.5 Hz 8.5 Hz = 8.2 Hz 8.4 Hz =88Hz =87Hz =83Hz 6 7.54, dd,J = 7.60, dd, J = 267 7.64, dd, J= 8.09,d,J 806,d,J 8.06,d,J 67, m 8.5, 1.9 Hz 8.5, 2.1 Hz 8.5, 1.8 Hz =86Hz =86Hz =82Hz 1” 5.27, d, J = 551,d,J 5.08, d, J= 525.d,J 5.12,d,J 7.6 Hz =75Hz 7.6 Hz =71Hz =62Hz 481,d,J 1” 4.53, s 4.52, 5 = 7.0 Hz
The inventors examined the inhibitory activity of flavonoids of the present invention on sEH: 5 Table 4 Inhibitory activity of flavonoids of the present invention on sEH at 100 M
SEH inhibitory activity
EU-26 16.7+0.2
EU-18 30.1+2.5 37.4+0.7 31.3+0.3
EU-40 19.3+1.3
EU-23 30.9+2.2
EU-43 40.4+5.9
The results shown in Table 4 show that flavonoids from folium cortex eucommiae have certain inhibitory activity on sEH.
Further activity test results are shown in Table 5:
Table 5 LU502965
SFH inhibitory activity
Compound ———
IC50 (M) kinetic mode (ki, JM)
EU-26 22.5+0.9 Non-competitive (18.2)
EU-18 39.6+1.4 NT
EU-6 52.4415 N.T
EU-7 48.3+£1.7 N.T
EU-40 31.3£2.6 Non-competitive (27.5)
EU-23 49 8+£5.2 N.T
EU-43 61.9+1.5 NT
AUDA 109+12nM
AUDA is the positive control; Ki represents the inhibition constant; NT means not detected.
The activity test results show that the compound of the invention has a certain degree of inhibitory activity on sEH hydrolase, and there is a certain relationship between the activity and the structural characteristics. Among them, EU-26 (quercetin) and EU-40 (kaempferol) are non-competitive inhibitors, and their inhibition constants are 18.2 M and 27.5 M, respectively.

Claims (4)

CLAIMS LU502965
1. Isolated flavonoid compound, characterized in that the structural formula of the flavonoid compound is shown in formula I: By OH HG iy A —- IS LAs RL or a formula I; in the formula: Riis H, Ry is OH; when Rı is glucoside, Ra is OH; when Rı is xylopyranosyl-(1—2)glucoside, Ra is OH; Rı is rutinoside, Ra is OH; Riish, Rois H; when Rj is glucoside, R is H; when R is rutinoside, Ra is H.
2. The flavonoid compound according to claim 1, characterized in that the extraction raw material of the flavonoid compound is folium cortex eucommiae.
3. An application of flavonoid compound according to claim 1 as inhibitor of SEH.
4. An application of the flavonoid compound according to claim 1 in preparing medicines for preventing and treating cardiovascular diseases.
LU502965A 2022-10-27 2022-10-27 A method for extracting flavonoid compounds from folium cortex eucommiae LU502965B1 (en)

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