TWI515004B - Novel biphenyl compounds extracted from Magnolia officinalis and their bioactive components and their preparation methods - Google Patents

Description

Novel biphenyl compound extracted from Magnolia officinalis and biological active ingredient and preparation method thereof

The present invention relates to a magnified extraction of a novel biphenyl compound and a biologically active ingredient and a preparation method thereof; more specifically, a novel biphenyl compound is analyzed from Magnolia: 5 ' -allyl-5-( 1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),5,5 ' -diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ) and 5,5 ' -diallyl-2-(3- Analysis system for methylbut-2-enyloxy)biphenyl-2 ' -ol( 3 ). In addition, the results of the anti-inflammatory activity test showed 5 ' -allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),honokiol( 5 ),randainal( 7 ),magnaldehyde D ( 8 ), and randaiol( 9 ) have good inhibitory activity on the production of superoxide anion (O ₂ ^‧- ) induced by fMLP/CB (IC _{50 )} 4.42 μg/mL). In addition, 5 ' -allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),randainal( 7 ),magnaldehyde D( 8 ), and randaiol( 9 ) for fMLP/ The release of elastase induced by CB has a good inhibitory effect (IC ₅₀ 2.35 μg/mL).

Magnolia is a deciduous tree distributed in the Yangtze River basin and southern China. The bark of Magnolia is also known as Houpo. It is a precious Chinese herbal medicine and rare and protected plant in Chinese herbal medicines. It has been recorded in the past genus, bitter and warm. Yuan, dampness, phlegm, phlegm, phlegm, phlegm and diuresis, mainly used to treat gastrointestinal discomfort, cough, asthma, thrombotic stroke, anxiety, lack of spirit; Magnolia has inhibition of gastric ulcer, anti-allergy, cells Toxicity, muscle relaxation, anti-oxidation, vascular relaxation, neurotrophic, anti-fungal, etc.

The inventor of the present invention has been engaged in medical related research for many years, and has a considerable degree of understanding of medical related knowledge. From the above analysis, it is known that magnolia is quite precious, the material cost is high, and the therapeutic effect is wide. The inventors believe that the therapeutic effect of high-value medicinal materials should be more than that. Therefore, there is still a new therapeutic function for the study of Magnolia.

In view of this, the inventor of the present invention has carried out in-depth research on the above-mentioned research and development experience in the related fields, and has searched for a new method for extracting compounds according to the foregoing, and has completed the present invention after a long period of hard research and multiple tests.

The main object of the present invention is to provide a novel biphenyl compound and a biologically active ingredient and a preparation method thereof, and to analyze three kinds of novel biphenyl compounds and compounds having anti-inflammatory activity by various experimental data.

A. Using the extraction, chromatography, purification and structural resolution steps described in the present invention, three new compounds were found: 5 ' -allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),5 , 5 ' -diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ) and 5,5 ' -diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2 ' -ol( 3 ).

B. Experimental data for three new compounds are as follows: 5 ' -Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ):colorless oil;UV(MeOH)λ _max (log ε)209( 4.55), 255 (3.82), 295 (3.90) nm; IR (neat) υ _max 3521 (OH), 1639, 1431, 990, 916 (-CH=CH ₂ ), 1607, 1488, 818 ( ₁ , ₂ , 4- Trisubstituted benzene) cm ^-1 ; ¹ H NMR (CDCl ₃ , 400 MHz): δ 3.34 (2H, d, J = 6.8 Hz, H-7 ' ), 5.05 (1H, ddt, J = 10.4, 1.6, 1.6 Hz, H-9 ' ), 5.08 (1H, ddt, J = 17.2, 1.6, 1.6 Hz, H-9 ' ), 5.12 (1H, br s, D ₂ O exchangeable, OH), 5.55 (1H, ddd, J = 10.4, 0.8, 0.8 Hz, H-3 " ), 5.70 (1H, ddd, J = 17.2, 0.8, 0.8 Hz, H-3 " ), 5.96 (1H, ddd, J = 11.2, 10.4, 6.8 Hz, H -8 ' ), 6.09 (1H, ddd, J = 17.2, 10.4, 6.0 Hz, H-2 " ), 6.48 (1H, br d, J = 6.0 Hz, H-1 " ), 6.89 (1H, d, J = 8.0 Hz, H-3 ' ), 6.90 (1H, dd, J = 8.0, 2.0 Hz, H-4), 6.90 (1H, d, J = 2.0 Hz, H-6), 6.91 (1H, d , J = 8.0 Hz, H-3), 7.02 (1H, d, J = 2.0 Hz, H-6 ' ), 7.05 (1H, dd, J = 8.0, 2.0 Hz, H-4 ' ); ¹³ C NMR (CDCl ₃ , 100MHz): δ 39.4 (C-7 ' ), 108.8 (C-4), 109.5 (C-6), 110.7 (C-1 " ), 115.7 (C-3 ' ), 115.7 (C- 9 ' ), 122.1 (C-3 " ), 122.2 (C-3), 127.6 (C-1 ' ), 129.0 (C-4 ' ), 130.2 (C-6 ' ), 130.7 (C-1) , 132.0 (C-2 " ), 132.2 (C-5 ' ), 137.7 (C-8 ' ), 147.3 (C-5), 148.4 (C-2), 150.7 (C-2 ' ); ESIMS m/ z 321[M+Na] ⁺ ;HRESIMS m/z 321.1107[M+Na] ⁺ (calcd for C ₁₈ H ₁₈ O ₄ Na, 321.1103).

5,5 ' -Diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ):yellowish oil;UV(MeOH)λ _max (log ε)205(4.72),286(3.91)nm;IR(neat) υ _max 3407(OH), 1640, 1418, 995, 916 (-CH=CH ₂ ), 1608, 1496, 819 (1, ₂ , 4-trisubstituted benzene) cm ^-1 ; ¹ H NMR (CDCl ₃ , 400 MHz): δ 3.41 (2H, br d, J = 5.2 Hz, H-7 ' ), 3.43 (2H, br d, J = 5.6 Hz, H-7), 4.60 (2H, ddd, J = 5.2, 1.6, 1.6 Hz, H-1 " ), 5.10 (1H, ddt, J = 10.4, 1.6, 1.6 Hz, H-9 ' ), 5.11 (1H, ddt, J = 10.4, 1.6, 1.2 Hz, H-9), 5.13 (1H , ddt, J =17.2, 1.6, 1.6 Hz, H-9 ' ), 5.13 (1H, ddt, J = 17.2, 1.6, 1.2 Hz, H-9), 5.27 (1H, ddt, J = 10.4, 1.6, 1.6Hz, H-3 "), 5.35 (1H, ddt, J = 17.2,1.6,1.6Hz, H-3"), 5.99 (1H, ddd, J = 17.2,10.4,5.2Hz, H-2 ") , 6.01 (1H, ddd, J = 11.2, 10.4, 5.2 Hz, H-8 ' ), 6.04 (1H, ddd, J = 17.2, 10.4, 5.6 Hz, H-8), 6.42 (1H, br s, D ₂ O exchangeable, OH-2 ' ), 7.00 (1H, d, J = 8.0 Hz, H-3), 7.01 (1H, d, J = 8.0 Hz, H-3 ' ), 7.12 (1H, d, J =2.0 Hz, H-6 ' ), 7.15 (1H, dd, J = 8.0, 2.0 Hz, H-4 ' ), 7.20 (1H, dd, J = 8.0, 2.4 Hz, H-4), 7.21 (1H , d, J = 2.4 Hz, H-6); ¹³ C NMR (CDCl ₃ , 100 MHz): δ 39.3 (C-7), 39.4 (C-7 ' ), 70.2 (C-1 " ), 113.6 (C -3), 115.5 (C-9 ' ), 115.8 (C-9), 117.6 (C-3 ' ), 118.2 (C-3 " ), 126.3 (C-1 ' ), 127.8 (C-1), 129.0 (C-4), 129.2 (C-4 ' ), 131.2 (C-6 ' ), 132.3 (C-2 " ), 132.6 (C-5), 132.6 (C-6), 134.1 (C-5 ' ), 137.3 (C-8), 137.8 (C -8 ' ), 152.0(C-2 ' ), 152.8(C-2); ESIMS m/z 329[M+Na] ⁺ ;HRESIMS m/z 329.1515[M+Na] ⁺ (calcd for C ₂₁ H ₂₂ O ₂ Na, 329.1517).

5,5 ' -Diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2 ' -ol( 3 ):colorless oil;UV(MeOH)λ _max (log ε)206(4.73),287(3.89) Nm;IR(neat)υ _max 3380(OH),1674(-C=C(Me) ₂ ),1640,1414,993,914 (-CH=CH ₂ ),1607,1496,822 (1,2,4- Trisubstituted benzene) cm ^-1 ; ¹ H NMR (CDCl ₃ , 600 MHz): δ 1.64 (1H, d, J = 1.2 Hz, H-5 " ), 1.74 (1H, d, J = 1.2 Hz, H-4 " ), 3.39 (2H, br d, J = 7.2 Hz, H-7 ' ), 3.40 (2H, br d, J = 7.2 Hz, H-7), 4.57 (1H, d, J = 6.6 Hz, H- 1 " ), 5.06 (1H, ddd, J = 10.4, 1.6, 1.6 Hz, H-9 ' ), 5.08 (1H, ddt, J = 10.4, 1.6, 1.6 Hz, H-9), 5.10 (1H, ddt , J =17.2, 1.6, 1.6 Hz, H-9), 5.10 (1H, ddt, J = 17.2, 1.6, 1.6 Hz, H-9 ' ), 5.40 (1H, tqq, J = 6.6, 1.2, 1.2 Hz , H-2 " ), 5.98 (1H, ddd, J = 11.2, 10.4, 7.2 Hz, H-8 ' ), 6.00 (1H, ddd, J = 11.2, 10.4, 7.2 Hz, H-8), 6.70 ( 1H, s, D ₂ O exchangeable, OH-2 ' ), 6.97 (1H, d, J = 8.0 Hz, H-3 ' ), 6.99 (1H, d, J = 8.4 Hz, H-3), 7.09 ( 1H,d, J =1.8 Hz, H-6 ' ), 7.11 (1H, dd, J = 8.0, 1.8 Hz, H-4 ' ), 7.17 (1H, d, J = 2.4 Hz, H-6), 7.18 (1H, dd, J = 8.4, 2.4 Hz, H-4); ¹³ C NMR (CDCl ₃ , 150 MHz): δ 18.1 (C-5 " ), 25.7 (C-4 " ), 39.4 (C-7) ), 39.5 (C-7 ' ), 66.7 (C-1 " ), 114.1 (C-3) , 115.4 (C-9 ' ), 115.8 (C-9), 117.9 (C-3 ' ), 118.8 (C-2 " ), 126.7 (C-1 ' ), 128.3 (C-1), 128.9 (C -4), 129.2 (C-4 ' ), 131.2 (C-6 ' ), 132.3 (C-5), 132.7 (C-6), 134.1 (C-5 ' ), 137.4 (C-8), 137.9 (C-8 ' ), 139.0 (C-3 " ), 152.3 (C-2 ' ), 152.9 (C-2); ESIMS m/z 357 [M+Na] ⁺ ; HRESIMS m/z 357.1828 [M+ Na] ⁺ (calcd for C ₂₃ H ₂₆ O ₂ Na, 357.1830).

C. Magnolia-separated compound, which produces superoxide anion (O ₂ ^‧- ) by inhibiting human neutrophils induced by foryl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB) The effect of elastase was evaluated to evaluate its anti-inflammatory activity. The results of the anti-inflammatory activity test showed 5 ' -allyl-5-(1-hydroxyallyloxy)-biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),honokiol( 5 ),randainal( 7 ),magnaldehyde D( 8 ) and randaiol( 9 ) have good inhibitory activity on the production of superoxide anion (O ₂ ^‧- ) induced by fMLP/CB (IC _{50 )} 4.42 μg/mL). In addition, 5 ' -allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),randainal( 7 ),magnaldehyde D( 8 ), and randaiol( 9 ) for fMLP/ The release of elastase induced by CB has a good inhibitory effect (IC ₅₀ 2.35 μg/mL).

D. Among all the isolated compounds, Randainal ( 7 ) and randaiol ( 9 ) are the most effective compounds, and the IC ₅₀ values for inhibiting the release of elastase induced by fMLP/CB and the production of superoxide anion are 1.21 ± 0.12 and 0.39 ± 0.06 μg/mL. These two compounds have the potential to be researched and developed as anti-inflammatory drugs.

In order to have a more detailed understanding of the features and structures of the present invention, please refer to Annex I and Annex II; Annex I is a structural diagram of the compound of the present invention, and Annex II is a data chart of the analysis results of the compound of the present invention.

The method for preparing a novel biphenyl compound is extracted from the magnolia of the present invention, and the steps are as follows:

A. The dried bark bark was dried and dried to obtain a total of 5.0 kg, which was extracted three times with methanol, and the obtained extract was concentrated under reduced pressure to give MeOH (d) 510 g, followed by EtOAc:H ₂ O (1:1, v /v) EtOAc (EtOAc) (EtOAc) The H ₂ O layer extract was further distributed with n -BuOH (1:1, v/v) to obtain n -BuOH layer extract (Fr. B, 177 g) and H ₂ O layer extract (Fr. C). , 206g).

B. The EtOAc layer extract (Fr. A, 118 g) from step A was separated by column chromatography (silica gel, 70-230 mesh, 4.9 kg), starting with n- hexane, gradually increasing EtOAc and MeOH. To increase its polarity, a total of 10 fractions were obtained: Fr.A1 (2L, n- hexane), A2 (7L, n- hexane/EtOAc, 30:1), A3 (7L, n- hexane/EtOAc, 20:1 ), A4 (6L, n- hexane/EtOAc, 10:1), A5 (5L, n- hexane/EtOAc, 5:1), A6 (6L, n- hexane/EtOAc, 3:1), A7 (5L) , n- hexane/EtOAc, 2:1), A8 (6L, n- hexane/EtOAc, 1:1), A9 (6L,EtOAc), A10 (6L, MeOH).

C. Fraction A2 (11.2 g) from step B was separated by column chromatography (silica gel, 230-400 mesh, 505 g), and washed with n- hexane/acetone (40:1) to obtain 16 partitions. (each 1.0L, Fr.A2-1~A2-16). Fr. A2-3 (185 mg) was purified by preparative TLC (silica gel, CHCl ₃ /EtOAc, 60:1) to give (-)-monoterpenylmagnolol ( 6 ) (20.5 mg, _Rf = 0.90). Fr A2-4 (150 mg) was purified by preparative TLC (silica gel, CHCl ₃ /EtOAc, 60:1) to afford randaiol ( 9 ) (3.8 mg, _Rf = 0.55). Fr A2-6 (165 mg) was purified by preparative TLC (silica gel, n- hexane/acetone, 5:1) to give 5,5 ' -diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 )( 4.2 mg, R _f = 0.55). A2-9 (185 mg) was purified by preparative TLC (silica gel, n- hexane/acetone, 10:1) to give 5,5 ' -diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2 ' -ol (3) (4.5mg) ( R f = 0.60). Take A2-11 (145mg) (silica gel n -hexane / acetone,, 5: 1) preparative TLC purification to give 5 '-allyl-5- (1- hydroxyallyloxy) biphenyl-2,2' -diol (1 ) (3.7 mg) ( R _f = 0.36).

D. Fraction A3 (10.5g) from step B was separated by column chromatography (silica gel, 230-400 mesh, 475 g), and washed with n- hexane/acetone (3:1) to obtain 13 partitions. (each 1.2L, Fr.A3-1~A3-13). Fr.A3-2 (148 mg) was purified by preparative TLC (silica gel, n- hexane/acetone, 6:1) to obtain a mixture of β-sitostenone ( 10 ) and stigmasta-4, 22-dien-3-one ( 11 ) (6.7 mg) ( R _f = 0.58). A3-4 (235 mg) was washed with MeOH and filtered and then recrystallised from MeOH to give a mixture of β-sitosterol ( 12 ) and stigmasterol ( 13 ) (55 mg). Fr A3-6 (272 mg) was filtered with n- hexane elution and then recrystallized from CH ₂ Cl ₂ to give magnolol ( 4 ) (95 mg).

E. Fraction A4 (9.5 g) from step B was separated by column chromatography (silica gel, 230-400 mesh, 420 g), and washed with n- hexane/acetone (3:1) to obtain 11 fractions. (each 1.0L, Fr.A4-1~A4-11). Take Fr.A4-5 (145mg) preparative TLC to _{(silica gel, CHCl 3 / MeOH} , 25: 1) was purified to give randainal (7) (4.6mg) ( R f = 0.26). Take Fr.A4-6 (138mg) to _{preparative TLC (silica gel, CHCl 3} / MeOH, 25: 1) was purified to give magnaldehyde D (8) (3.8mg) (R f = 0.24). Take Fr.A4-7 (220mg) to preparative TLC (silica gel, n -hexane / acetone, 3: 1) was purified to give honokiol (5) (90mg) ( R f = 0.36).

F. Fraction A7 (9.8 g) from step B was separated by column chromatography (silica gel, 230-400 mesh, 440 g), and washed with n- hexane/acetone (2:1) to obtain 14 partitions. (each 900mL, Fr.A7-1~A7-14). Fr.A7-5 (190 mg) was purified by preparative TLC (silica gel, n- hexane/acetone, 3:1) to give a mixture of 3β-hydroxystigmast-5-en-7-one ( 14 and 3β-hydroxystigmasta-5). , 22-dien-7-one ( 15 ) (6.3 mg).

The purified compound obtained in the above step was tested for anti-inflammatory activity as shown in the following steps:

First, the experimental materials:

(A) Compounds isolated from the upper part of Zeeland, Taiwan

(B)human neutrophils

(C)HBSS: Hank’s buffered saline solution

(D)fMLP: formyl-L-methionyl-L-leucyl-L-phenylalanine

(E)CB: cytochchalasin B

(F)SOD: superoxide dismutase

(G)ferricytochrome c

Second, the experimental method: (A) Preparation of human neutrophils

Healthy blood donors from about 20-30 years old (normal work and banned drugs for more than two weeks), vacuum The sterile blood collection tube collects blood in the elbow vein, about 30-80 ml. The neutrophils were separated by Ficoll gradient centrifugation method as follows: 25 ° C, 650 g, male whole blood was centrifuged for 10 min, female whole blood was centrifuged for 8 min, the supernatant was removed, and the lower layer of blood cells and 3% dextran solution were removed. Mix in an equal volume and let stand at room temperature for 30 min. The upper layer containing abundant neutrophils was covered in a 50 ml centrifuge tube of an equal volume of Histopaque-1077 solution, and centrifuged at 400 g for 20 min at 20 ° C to obtain a pellet. The remaining red blood cells are broken by a method of hemolysis of a low-tension solution. Finally, the supernatant was removed by centrifugation at 4 ° C, and the separated neutrophils were suspended in Hank's buffered saline solution (HBSS) in an ice bath.

(B) Determination of superoxide anion radical (O ₂ ^‧- ) release

A suspension of neutrophils (6 × 10 ⁵ cells / ml) containing 0.5 mg / ml of ferricytochrome c , 1 mM CaCl ₂ and 1 mM MgCl ₂ was preheated for 5 minutes to reach 37 ° C, and the drug to be tested was added. After 5 minutes, fMLP (0.1 μM)/cyto-chalasin B (CB, 1 μg/ml) was further added for 10 minutes. The absorbance was measured at a wavelength of 550 nm using an ultraviolet spectrophotometer. In the experiment, the amount of superoxide radical released by neutrophils (extinction coefficient 21.1/mM/cm) was evaluated by superoxide dismutase (SOD, 100 U/ml) to inhibit ferricytochrome c reduction.

△A ₅₅₀ =extinction coefficient×1×△C

△C=△A ₅₅₀ /(21.1/mM/cm×1cm)

△C=△A ₅₅₀ × 47.4nmol/ml

Among them, the absorbance of the sample of ^ΔA _{550 to} be tested is subtracted from the absorbance of the SOD inhibition group, and ΔC is the O ₂ ^‧ yield of the sample to be tested.

(C) Determination of elastase release

The suspension of neutrophils contains Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (Ms-Ala-Ala-Pro-Val-pNA), preheated for 5 minutes to reach 37 ° C, and added to the drug to be tested 5 After a minute, the fMLP/CB reaction was further added for 10 minutes. The absorbance was measured at a wavelength of 405 nm using an ultraviolet spectrophotometer.

Third, the experimental principle:

Using fMLP/CB (formyl-L-methionyl-L-leucyl-L-phenylalanine/cyto-chalasin B) to induce neutrophil to produce superoxide anion and elastase, and super The production of oxygen anions and the release of elastase are associated with the development of inflammatory diseases. Therefore, the anti-inflammatory activity of the isolated compound can be evaluated by the inhibitory effect of fMLP/CB on induction of superoxide anion production and elastase release.

Fourth, statistical analysis:

The experimental results were presented as mean ± standard deviation (SEM) (n = 4), and compared using "Student's t test", a probability of 0.05 or less was considered statistically significant.

V. Results and discussion

The chemical constituents of magnolia bark were studied. At present, three new biphenyl compounds were isolated from the thick bark: 5 ' -allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),5,5 ' -diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ), and 5,5 ' -diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2 ' -ol ( 3 ), and 12 known compounds ( 4-15 ) (Fig. 1) (Annex I). The above compounds were analyzed by various experimental data (including 2D-NMR: NOESY, COSY, HMBC, HSQC, etc.) and the data in the related literature were confirmed.

Compound structure identification

Three are new biphenyl compounds ( 1-3 ) with UV, IR, ESI-MS, HR-ESI-MS, NMR ( ¹ H-NMR, ¹³ C-NMR, DEPT, COSY, NOESY, HSQC, HMBC.. ..), ..... and other experimental materials determine the chemical structure. Twelve known compounds ( 4-15 ), including 4 neolignans: magnolol ( 4 ), honiciol ( 5 ), (-)-monoterpenylmagnolol ( 6 ), randainal ( 7 ); 2 norlignans: magnaldehyde D ( 8 ) And randaiol( 9 ); and 6 steroids: a mixture of β-sitostenone( 10 ) and stigmasta-4,22-dien-3-one( 11 ), a mixture of β-sitosterol( 12 ) and stigmasterol( 13 ) And a mixture of 3β-hydroxystigmast-5-en-7-one( 14 ) and 3β-hydroxystigmasta-5,22-dien-7-one( 15 ), compared with the literature, ¹ H-NMR, IR, UV, MS and other data, the results are consistent and determine its structure.

2. Biological activity research

Inflammation is the normal defense response of the human body. In order to combat the foreign pathogens, the protective mechanism is characterized by red, hot, swollen, painful symptoms. When white blood cells fight against foreign pathogens, high-oxidation free radicals are used, which not only kills foreign pathogens, but also the surrounding normal tissues or cells are often affected. Once the DNA of a normal cell is damaged, the gene will mutate and affect the growth and differentiation of the cell. If mutations are made in important genes, the chances of developing malignant tumors increase. Since chronic inflammation is the stimulation of normal cells over the years, chromosome variation has been accumulating, eventually leading to cancer, or the immune system may also cause many chronic degenerative diseases to start due to long-term load.

Research indicates that about 30% of cancers are associated with chronic inflammation or chronic infections; especially myocardial infarction, diabetes, Alzheimer's disease, cancer, allergic and autoimmune diseases, and more and more The evidence shows that it is closely related to chronic inflammation. For example, some people with heart attacks actually have low cholesterol and atherosclerosis caused by chronic inflammation on the blood vessel wall. Block flaking, initiation of coagulation mechanism, occlusion of coronary artery is the cause of myocardial infarction; chronic inflammation of the liver becomes liver cancer, which is caused by the attack of the immune system; cervical cancer is also caused by the inflammatory reaction against human papillomavirus in vivo. Gastrointestinal reflux causes esophageal inflammation and is also prone to esophageal cancer. Therefore, in theory, if the inflammatory response can be blocked, the cancer cells can be significantly inhibited.

When human neutrophils are stimulated and activated, superoxide anion (O ₂ ^‧- ), hydrogen peroxide, and hydroxyl radicals are released. [(Reactive oxygen species (ROS)], and release of elastase (elastase), β-glucuronidase, lysozyme, PAF and other media, and these substances are related to inflammatory diseases such as: rheumatoid arthritis, kidney skein Nephritis, skin disease, ischemic reperfusion injury, myocardial infarction, chronic lung obstruction and asthma. Inhibition of neutrophilic leukemia or inappropriate activation is thought to improve these inflammatory diseases. Therefore, new treatment and prevention of inflammation The development of drugs for diseases has been a hot topic in the research of the pharmaceutical industry. Studies on anti-inflammatory active ingredients isolated from natural materials in the past have shown that flavonoids, terpenoids and lignans have good anti-inflammatory activity, so they are In vitro screening of anti-inflammatory tests, selection of plants with better anti-inflammatory activity than "good" for component separation and pharmacological evaluation , Developing new anti-inflammatory drugs is a viable path.

A compound isolated from the bark of bark, which produces superoxide anion (O ₂ ^‧- ) and elasticity by inhibiting formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB) in human neutrophils The effect of protease (elastase) to assess its anti-inflammatory activity. Anti-inflammatory activity data is listed in Table 1. Diphenyleneiodonium and phenylmethylsulfonyl fluoride were treated as positive controls for [O ₂ ^‧- production] and [elastase release], respectively. For the results of the anti-inflammatory test, the following seven conclusions can be summarized:

(a) 5 ' -Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),honokiol( 5 ),randainal( 7 ),magnaldehyde D( 8 ), and Randaiol ( 9 ) Good inhibitory activity (IC ₅₀ ) for the production of superoxide anion (O ₂ ^‧- ) induced by fMLP/CB 4.42 μg/mL).

(b) 5 ' -Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ),magnolol( 4 ),randainal( 7 ),magnaldehyde D( 8 ), and randaiol( 9 ) for fMLP /CB induces the release of elastase, which has a good inhibitory effect (IC ₅₀ 2.35 μg/mL).

(c) 5,5 ' -Diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ) (10 μg/ml) induces the release of elastase from human neutrophils in the presence of cytochalasin B (CB). 5 ' -Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 3 ) (10 μg/ml) alone induces the release of superoxide anion from human neutrophils without the addition of fMLP/CB ( O ₂ ^‧- ) and elastase (elastase). Without the addition of fMLP/CB, Honokiol( 5 ) (10 μg/ml) alone induced the release of elastase from human neutrophils.

(d) In the 5 ' -allylbiphenyl-2,2 ' -diol analogs ( 1 , 4 , 6-9 ), compounds 1 , 4 and 7-9 , for the production and elasticity of superoxide anion induced by fMLP/CB The release of protease has significant inhibitory activity. For inhibition of superoxide anion production, the order of activity intensity is 9 (with 5-hydroxy group) > 4 (with 5-allyl group) > 7 (with( E )-3-oxoprop-1-enyl group at C- 5)> 8 (with 5-formyl group) > 1 (with 1-hydroxyallyloxy group at C-5). For inhibition of elastase release, the order of activity intensity is 7 (with( E )-3-oxoprop-1-enyl group at C-5)> 1 (with 1-hydroxyallyloxy group at C-5)≒ 9 (with 5-hydroxy group)> 4 (with 5-allyl group)> 8 (with 5-formyl group). In contrast, compound 6 (with a(1 S ,6 R )-6-isopropyl-3-methylcyclohex-2-enyl group at C-3 ' and an allyl group at C-5) is not active.

(e) Honokiol ( 5 ) (C-4 at OH) is more effective at inhibiting the production of superoxide anion than its similar compound magnolol( 4 ) (C-2 at OH).

(f) Among all the isolated compounds, Randainal ( 7 ) and randaiol ( 9 ) were the most potent compounds, and their IC ₅₀ values for inhibiting the release of elastase induced by fMLP/CB and the production of superoxide anion were 1.21, respectively. ±0.12 and 0.39±0.06 μg/mL.

Claims (3)

A method for preparing a novel biphenyl compound extracted from Magnolia officinalis is prepared by the following steps: A. A total of 5.0 kg of dried bark bark is dried and extracted three times with methanol, and the obtained extract is concentrated under reduced pressure. after afford 510g MeOH extract, followed by _{EtOAc: H 2 O (1:} 1, v / v) was partitioned to obtain EtOAc layer was withdrawn, dried EtOAc layer was concentrated under reduced pressure to give after extraction (Fr.A, 118g) Then, the H ₂ O layer extract was further distributed with n -BuOH (1:1, v/v) to obtain n -BuOH layer extract (Fr. B, 177 g) and H ₂ O layer extract (Fr. C, 206 g); B. The EtOAc layer extract (Fr. A, 118 g) from step A was separated by column chromatography (silica gel, 70-230 mesh, 4.9 kg), starting from n- hexane, gradually EtOAc and MeOH were added to increase the polarity. A total of 10 fractions were obtained: Fr.A1 (2L, n- hexane), A2 (7L, n- hexane/EtOAc, 30:1), A3 (7L, n- hexane/ EtOAc, 20:1), A4 (6L, n- hexane/EtOAc, 10:1), A5 (5L, n- hexane/EtOAc, 5:1), A6 (6L, n- hexane/EtOAc, 3:1 , A7 (5L, n- hexane/EtOAc, 2:1), A8 (6L, n- hexane/EtOAc, 1:1), A9 (6L, EtOAc), A10 (6L, MeOH); Step B of Fraction A2 (11.2g) through the column layer Separation (silica gel, 230-400mesh, 505g) , in n -hexane / acetone (40: 1 ) for punching Shi, to give 16 partition (each 1.0L, Fr.A2-1 ~ A2-16) , Fr Purified and purified by preparative TLC (silica gel, CHCl ₃ /EtOAc, 60:1) to afford randaiol( 9 ) (3.8 mg, R _f = 0.55), Fr A2-6 (165 mg) by preparative TLC (silica gel, n- hexane/acetone, 5:1) purified and purified to obtain 5,5 ' -diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ) (4.2 mg, R _f =0.55), A2-9 (185 mg) was purified by preparative TLC (silica gel, n- hexane/acetone, 10:1) to give 5,5 ' -diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2 ' - ol (3) (4.5mg) ( R f = 0.60), taking A2-11 (145mg) to preparative TLC (silica gel, n -hexane / acetone, 5: 1) was purified to give 5 '-allyl-5- (1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 )(3.7mg)( R _f =0.36); D. Fraction A3 (10.5g) from step B is separated by column chromatography (silica gel, 230-400 mesh, 475 g), washed with n- hexane/acetone (3:1) to obtain 13 divisions (each 1.2L, Fr.A3-1~A3-13), Fr A3-6 (272mg) After filtration with n- hexane washing, recrystallization from CH ₂ Cl ₂ To magnolol ( 4 ) (95 mg); E. Fraction A4 (9.5 g) from step B was separated by column chromatography (silica gel, 230-400 mesh, 420 g) to n- hexane/acetone (3:1) Rinse and obtain 11 fractions (each 1.0L, Fr.A4-1~A4-11), and take Fr.A4-5 (145mg) to preparative TLC (silica gel, CHCl ₃ / MeOH, 25:1) Purified and purified to obtain randainal ( 7 ) (4.6 mg) ( R _f = 0.26), and purified by preparative TLC (silica gel, CHCl ₃ /MeOH, 25:1) to obtain magnaldehyde D. ( 8 ) (3.8 mg) ( R _f = 0.24), and purified by preparative TLC (silica gel, n- hexane/acetone, 3:1) to obtain hnokiol ( 5 ) (90 mg). ) ( R _f =0.36). A novel biphenyl compound extracted from Magnolia officinalis, which is selected from the group consisting of: 5 ' -Allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ) and 5,5 ' -Diallyl-2-(allyloxy)biphenyl-2 ' -ol( 2 ) and 5,5 ' -Diallyl-2-(3-methylbut-2-enyloxy)biphenyl-2 ' -ol( 3 ). A bioactive component of a novel biphenyl compound extracted from Magnolia officinalis, the 5 ' -allyl-5-(1-hydroxyallyloxy)biphenyl-2,2 ' -diol( 1 ) for formyl-L-methionyl-L-leucyl-L -phenylalanine/cytochalasin B (fMLP/CB) induces superoxide anion (O ₂ ^‧- ) and elastase (elastasc) in human neutrophils, with IC ₅₀ values of 4.42 ± 0.24 and 1.45, respectively. ±0.20 μg/mL.