CN102249862A - Application of phenol compounds in preparation of anti-complement medicines - Google Patents
Application of phenol compounds in preparation of anti-complement medicines Download PDFInfo
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Abstract
The invention belongs to the field of traditional Chinese medicine pharmacy, and relates to phenol compounds represented by a formula I and novel purposes of the compounds in the preparations of anti-complement medicines. According to the invention, phenol active substances are separated from n-butyl alcohol site of ethanol extract of commelinaceae commelina commelina communis Linn., ethyl acetate site of ethanol extract of boraginaceae arnebia arnebia euchroma (Royal) Johnst dried root, and n-butyl alcohol site of ethanol extract of polygonaceae fagopyrum fagopyrum dibotrys (D. Don) Hara dried rhizome. As a result of in vitro anti-complement activity selection experiments, the compounds have substantial inhibitive effects against classical pathways and alternative pathways of complement systems. CH50 of the inhibitive effects of the compounds against classical pathways of complement systems are 41+-8mug/ml to 278+-11mug/ml. AP 50 of the inhibitive effects of the compounds against alternative pathways of complement systems are 39+-5mug/ml to 761+-110mug/ml. The compounds can be applied in the preparation of anti-complement medicines.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, relate to phenol compound and the new purposes in the preparation anticomplement medicament thereof; Described phenol compound derives from Herba Commelinae, Asian puccoon or Wild Buckwheat Rhizome.
Background technology
Multiple diseases such as the excessive activation meeting initiating system lupus erythematosus of complement system, rheumatoid arthritis, adult respiratory distress syndrome.Anticomplement medicament research is the focus and emphasis of world's study of pharmacy for many years always.Therefore at present this type of disease is not still had the ideal medicine, be badly in need of efficient, low toxicity, single-minded novel complement inhibitor clinically.Directly the cost of research and development complement inhibitor is low from natural product, therefore and most of activeconstituentss can directly be digested and assimilated by body as the part of natural product, seek the new medicine with anticomplementary activity in recent years and be subjected to people and more and more pay close attention to from natural origin.Scholar both domestic and external separates from the natural product that comprises marine organisms and obtains a large amount of inhibiting monomeric compounds of complement system that have, for the research and development of anticomplement medicament provide wide prospect.
Commelianaceae (Commelinaceae) Herba Commelinae belongs to (Commelina) plant and mainly is distributed in the torrid zone, and the minority kind originates in subtropics and area, temperate zone.In homemade 13 belong to 49 kinds, be distributed in each province on the south the Changjiang river more, be Sheng especially with the southwest.Herba Commelinae is the herb that Herba Commelinae belongs to (Commelina) plant Herba Commelinae (Commelina communis Linn.), and flavor suffering warm in nature is specially gone into taste two warps, have clearing heat and detoxicating, cool blood, diuresis.Effect, clinically can be separately or share with other Chinese medicines be used for the treatment of that influenza, acute tonsillitis, pharyngitis, oedema, infantile pneumonia, urinary system infection, acute enteritis, icterohepatitis, hypertension, swollen joint are educated, dysentery, hordeolum, venomous snake bite, sore treat pyogenic infections, hemorrhoid etc. a series of with the caused disease of complement system excessive activation.Research to Herba Commelinae in recent years only concentrates in the separation and structure evaluation of chemical ingredients, therefrom separates having obtained some flavonoids, steroidal, terpenoid, but does not see the report to the inhibited compound of complement system up to now as yet.
Lithospermum euchromum Royle (Arnebia euchroma (Royle) Johnst) is Boraginaceae (Boraginaceae) Lithospermum (Arnebia) plant, claim Radix Arnebiae again, intend puccoon, main product is in Xinjiang, perennial herb, be distributed in ground such as the western part in north and south, the Tianshan Mountains slope in Xinjiang and Tibet and siberian, mountain gravel matter tailo, proluvial fan, meadow and the grassy marshland etc. that grow in 2500～4200 meters of height above sea level are located more.The dry root bitter of lithospermum euchromum Royle, cold in nature.The function that cooling and activating blood, clearing heat and detoxicating, laxation defaecation are arranged can be used for preventing measles, pyreticosis macula, jaundice, purpura, tells nosebleed hematuria, blood pouring, bloody flux, carbuncle sore tumefacting virus, erysipelas, eczema, burn, constipation with heat retention.In recent years, the basic substance research of the pharmacologically active of Asian puccoon, clinical application and drug effect thereof is more, and obtained a large amount of achievements, found to contain in the Asian puccoon multiple physiologically active ingredients such as naphthoquinones, benzoquinones, alkaloid, acidic polysaccharose, that these compositions have is antibiotic accordingly, anti-inflammatory, anticancer, antifertility, strengthening immunity, hypoglycemic, protect multiple effects such as liver.Research to Asian puccoon in recent years only concentrates in the separation and structure evaluation of chemical ingredients, therefrom separate having obtained compounds such as some benzoquinones classes, monoterpene phenol, but do not see phenol compound up to now as yet and the inhibited report of complement system.
Wild Buckwheat Rhizome is the dry rhizome of polygonaceae (Polygonaceae) Fagopyrum (Fagopyrum) plant Wild Buckwheat Rhizome (Fagopyrum dibotrys (D. Don) Hara).Belong to national II level and lay special stress on protecting wild plant, be born in wasteland, roadside, dark and damp ground, river bank.Mainly be distributed in national a plurality of provinces and regions, have clearing heat and detoxicating, the effects such as carbuncle, the sore of dispelling rheumatism, snake bite and insect sting that disappear of invigorating blood circulation.Cure mainly lung heat cough breathe heavily, swelling and pain in the throat, dysentery, rheumatism bi Zheng, wound and carbuncle cancer etc.Modern pharmacological research proves that it has effects such as anticancer and antibacterial.Chemical constitution study shows that wherein main component is the tannins compound, does not see the report of phenol compound and anticomplementary action thereof as yet.
Summary of the invention
The purpose of this invention is to provide new material, be specifically related to phenol compound, especially derive from the phenol compound in Herba Commelinae, Asian puccoon or the Wild Buckwheat Rhizome with anticomplementary activity, described phenol compound comprise methylphenol (
1), 6-methoxyl group-3,5-dimethyl-oxyhydroquinone (
2), oxyhydroquinone (
3), different Asian puccoon phenol (
4), the gentisinic acid pentyl ester (
5), the 4-methoxyl biphenyl (
6), 2,6-dimethoxy-3-methyl-Resorcinol (
7), 1,2,3, the 4-tetrahydroxy phenol (
8) and 2,4,6-trimethoxy-1,3, the 5-benzenetriol (
9).
Further purpose of the present invention provides the purposes of above-mentioned phenol compound in the preparation anticomplement medicament.
The present invention uses the modern pharmacology screening method, anticomplementary activity material in the plant amedica is studied, belonged to the n-butanol portion of (Commelina) plant Herba Commelinae (Commelina communis Linn.) herb ethanol extraction from Commelianaceae (Commelinaceae) Herba Commelinae, the n-butanol portion separation of the ethanol extraction of the dry rhizome of the ethyl acetate extract of Boraginaceae (Boraginaceae) Lithospermum (Arnebia) plant lithospermum euchromum Royle (Arnebia euchroma (Royle) Johnst) dry root ethanol extraction or polygonaceae (Polygonaceae) Fagopyrum (Fagopyrum) plant Wild Buckwheat Rhizome (Fagopyrum dibotrys (D. Don) Hara) obtains the phenol active substance and confirms that classical pathway and alternative pathway that it has pair complement system all have stronger restraining effect.
Active phenol's compounds of the present invention has formula
Chemical structure:
Wherein, R
1=OH, R
2=R
3=R
5=R
6=H, R
4=CH
3Or,
R
1=R
2=R
4=OH, R
3=R
5=CH
3, R
6=OCH
3Or,
R
1=R
2=R
4=OH, R
3=R
5=R
6=H; Or,
R
1=OH, R
4-R
6=OCH
2C (CH
3) CHCH
2CH
2C (CH
3) CHCH
2Or,
R
1=R
4=OH, R
2=COOC
5H
11, R
3=R
5=R
6=H; Or,
R
1=OCH
3, R
2=H, R
3=-C
6H
5Or,
R
1=R
4=OH, R
2=R
6=OCH
3, R
3=CH
3, R
5=H; Or,
R
1=R
2=R
3=R
4=OH, R
5=R
6=H; Or,
R
1=R
3=R
5=OH,?R
2=R
4=R
6=OCH
3?。
Above-mentioned phenol compound is worked as R
1=OH, R
2=R
3=R
5=R
6=H, R
4=CH
3The time, compound be p-methyl phenol (
1); Work as R
1=R
2=R
4=OH, R
3=R
5=CH
3, R
6=OCH
3The time, compound is a 6-methoxyl group-3,5-dimethyl-oxyhydroquinone (
2); Work as R
1=R
2=R
4=OH, R
3=R
5=R
6During=H, compound be oxyhydroquinone (
3); Work as R
1=OH, R
4-R
6=OCH
2C (CH
3) CHCH
2CH
2C (CH
3) CHCH
2The time, compound be different Asian puccoon phenol (
4); Work as R
1=R
4=OH, R
2=COOC
5H
11, R
3=R
5=R
6During=H, compound be the gentisinic acid pentyl ester (
5); Work as R
1=OCH
3, R
2=H, R
3=-C
6H
5The time, the 4-methoxyl biphenyl (
6); Work as R
1=R
4=OH, R
2=R
6=OCH
3, R
3=CH
3, R
5During=H, compound is 2,6-dimethoxy-3-methyl-Resorcinol (
7); Work as R
1=R
2=R
3=R
4=OH, R
5=R
6During=H, compound is 1,2,3, the 4-tetrahydroxy phenol (
8); Work as R
1=R
3=R
5=OH, R
2=R
4=R
6=OCH
3The time, compound is 2,4,6-trimethoxy-1,3, the 5-benzenetriol (
9).
Phenol compound of the present invention prepares by following method:
Get Herba Commelinae herb 14kg, with 95% ethanol room temperature cold soaking (50L * 5 time), united extraction liquid is concentrated into does not have the alcohol flavor, the extracting solution thin up is to 2L, use sherwood oil, ethyl acetate, n-butanol extraction (each 2L * 3 time) successively, merge butanol extraction liquid and be concentrated into to do and promptly get n-butyl alcohol extract 19.2g.N-butanol portion sample on dry method carries out silica gel column chromatography, with sherwood oil (60～90 ℃), sherwood oil (60～90 ℃)-acetone, acetone gradient elution, the gained flow point carries out silica gel column chromatography repeatedly with different eluents, SephadexLH-20 purifying and preparative chromatography, separate obtain the compound p-methyl phenol (
1), 6-methoxyl group-3,5-dimethyl-oxyhydroquinone (
2); Or,
Get Radix Arnebiae (Radix Lithospermi) meal 20 kg, under the room temperature with 95% ethanol after cold soaking, diacolation extract for several times repeatedly, decompression and solvent recovery, obtain medicinal extract 820 g, medicinal extract is suspended in the distilled water, with sherwood oil, ethyl acetate and n-butanol extraction, obtains acetic acid ethyl ester extract 420 g; Acetic acid ethyl ester extract 180 g are through silica gel column chromatography, with sherwood oil (60～90 ℃), sherwood oil (60～90 ℃)-acetone, acetone gradient elution, the gained flow point carries out silica gel column chromatography repeatedly with different eluents, SephadexLH-20 purifying and preparative chromatography, separate and obtain compound 1,2, the 4-benzenetriol (
3), different Asian puccoon phenol (
4), the gentisinic acid pentyl ester (
5) and the 4-methoxyl biphenyl (
6); Or,
Get Wild Buckwheat Rhizome rhizome meal 15kg, with ethanol room temperature cold soaking (50L * 5 time), united extraction liquid is concentrated into does not have the alcohol flavor, the extracting solution thin up is to 2L, use sherwood oil, ethyl acetate, n-butanol extraction (each 2L * 3 time) successively, merge butanol extraction liquid and be concentrated into to do and promptly get n-butyl alcohol extract 220 g; N-butanol portion sample on dry method carries out silica gel column chromatography, with chloroform, chloroform-methanol, methyl alcohol gradient elution, the gained flow point carries out silica gel column chromatography repeatedly with different eluents, SephadexLH-20 purifying and preparative chromatography, separate obtaining compound 2,6-dimethoxy-3-methyl-Resorcinol (
7), 1,2,3, the 4-tetrahydroxy phenol (
8) and 2,4,6-trimethoxy-1,3, the 5-benzenetriol (
9).
Wherein, p-methyl phenol (
1): white amorphous powder,
1H-NMR (400 MHz, Acetone-
d 6, ppm): δ 6.78 (2H, dd, 8.1,2.1Hz, H-2,6), 6.42 (2H, dd, 8.1,2.1Hz, H-3,5), 2.13 (3H, s ,-CH
3).
Wherein, 6-methoxyl group-3,5-dimethyl-oxyhydroquinone (
2): white amorphous powder,
1H-NMR (400 MHz, Acetone-
d 6, ppm): δ 3. 85 (3H, s ,-OCH
3), 2.17 (3H, s ,-CH
3), 2.15 (3H, s ,-CH
3).
Wherein, oxyhydroquinone (
3): white unformed powder,
1H NMR (400MHz, CDCl
3, ppm): δ 7.62 (1H, d,
J=8.0 Hz), 7.46 (1H, dd,
J=8.0,1.8 Hz), 6.90 (1H, d,
J=1.8 Hz).
Wherein, different Asian puccoon phenol (
4): white unformed powder,
1H NMR (400MHz, CDCl
3, ppm): δ 7.44 (1H, d,
J=3.0 Hz), 6.59 (1H, d,
J=8.5Hz), 6.55 (1H, dd,
J=3.0,8.5Hz), 5.67 (1H, brt,
J=7.0 Hz), 5.51 (1H, brt,
J=7.0 Hz), 3.30 (1H, brs), 3.07 (1H, brs), 2.34 (2H, brt,
J=7.0 Hz), 1.50 (3H, s), 1.24 (3H, s).
Wherein, the gentisinic acid pentyl ester (
5): white unformed powder,
1H NMR (400MHz, CDCl
3, ppm): δ 7.29 (1H, d,
J=2.1Hz), 7.16 (1H, dd,
J=8.2,2.1 Hz), 7.02 (1H, d,
J=8.1 Hz), 4.36 (2H, m), 1.87 (2H, m), 1.62 (2H, m), 1.33 (2H, m), 0.89 (3H, d,
J=6.9 Hz).
Wherein, the 4-methoxyl biphenyl (
6): white unformed powder,
1H NMR (400MHz, Acetone-
d 6 , ppm): δ 7.54-6.98 (9H, m), 3.83 (3H, s).
Wherein, 2,6-dimethoxy-3-methyl-Resorcinol (
7): white unformed powder,
1H-NMR (400 MHz, Acetone-
d 6, ppm): δ 7.59 (2H, brs), 3.85 (3H, s), 3.81 (3H, s), 2.56 (3H, s).
Wherein, 1,2,3, the 4-tetrahydroxy phenol (
8): white unformed powder,
1H NMR (400 MHz, Acetone-
d 6, ppm): δ 6.97 (2H, d,
J=8.0 Hz), and 6.33-6.20 (4H, s).
Wherein, 2,4,6-trimethoxy-1,3, the 5-benzenetriol (
9): white unformed powder,
1H NMR (400 MHz, Acetone-
d 6, ppm): δ 7.05 (3H, s), 3.84 (9H, s).
Above-mentioned phenol compound is through external classical pathway and bypass anticomplementary activity shaker test, and the result confirms that described compound all has remarkable restraining effect (as shown in table 1) to the classical pathway and the alternative pathway of complement system.Wherein, 1,2,3, the 4-tetrahydroxy phenol (
8) classical pathway and the alternative pathway of complement system all there is the strongest restraining effect, in the classical pathway, the required minimum trial-product concentration (CH of 50% haemolysis
50) be respectively 41 ± 8 μ g/ml, suitable with positive control heparin action intensity; In the alternative pathway, the required minimum trial-product concentration (AP of 50% haemolysis
50) be respectively 39 ± 5 μ g/ml, stronger than the effect of positive control heparin.
Table 1 be phenol compound (
1)～(
9) to complement system classical pathway and the inhibiting result of alternative pathway.
Table 1
| The compound title | CH 50（μg/ml） | AP 50（μg /ml） |
| P-methyl phenol ( 1) | 155 ± 20 | 761 ± 110 |
| 6-methoxyl group-3,5-dimethyl-oxyhydroquinone ( 2) | 49 ± 11 | 55 ± 18 |
| Oxyhydroquinone ( 3) | 49 ± 7 | 45 ± 14 |
| Different Asian puccoon phenol (4) | 128 ± 39 | 116± 28 |
| The gentisinic acid pentyl ester ( 5) | 113 ± 13 | - |
| The 4-methoxyl biphenyl ( 6) | 51 ± 8 | - |
| 2,6-dimethoxy-3-methyl-Resorcinol ( 7) | 59± 3 | 51 ± 8 |
| 1,2,3, the 4-tetrahydroxy phenol ( 8) | 41 ± 8 | 39 ± 5 |
| 2,4,6-trimethoxy-1,3, the 5-benzenetriol ( 9) | 278 ± 11 | 121 ±40 |
| Heparin (positive control) | 40±14 | 97±19 |
Description of drawings
Fig. 1 be phenol compound (
1)～(
9)The extraction separation schema.
Embodiment
The preparation of embodiment 1 phenol compound
Get Herba Commelinae herb 14kg, with ethanol room temperature cold soaking (50L * 5 time), united extraction liquid is concentrated into does not have the alcohol flavor, the extracting solution thin up is to 2L, use sherwood oil, ethyl acetate, n-butanol extraction (each 2L * 3 time) successively, merge butanol extraction liquid and be concentrated into to do and promptly get n-butyl alcohol extract 19.2g; N-butanol portion is through silica gel column chromatography, and with sherwood oil (60～90 ℃), sherwood oil-acetone, acetone gradient elution, stream part is with column chromatography repeatedly, SephadexLH-20 purifying and preparative chromatography obtain phenolic compound (
1) and (
2), concrete steps are as follows:
1. sherwood oil-acetone (5:1) wash-out gained stream part through column chromatography repeatedly, is eluent with the chloroform-methanol, and gradient elution, stream be part through the SephadexLH-20 purifying, methanol-eluted fractions, obtain compound (
1) (7 mg).The employing method of spectroscopy is analyzed, and its structure is defined as p-methyl phenol respectively.
2. sherwood oil-acetone (2:1) wash-out gained stream part is a developping agent with chloroform-methanol (5:1), preparative chromatography, obtain (
2) (4 mg); The employing method of spectroscopy is analyzed, and its structure is defined as 6-methoxyl group-3,5-dimethyl-oxyhydroquinone.
The preparation of embodiment 2 phenol compounds
Get Asian puccoon dry root 20kg, meal under the room temperature with 95% ethanol after cold soaking, diacolation extract for several times repeatedly, decompression and solvent recovery, obtain medicinal extract 820g, medicinal extract is suspended in the distilled water, with sherwood oil, ethyl acetate and n-butanol extraction, obtains acetic acid ethyl ester extract 420g; Get acetic acid ethyl ester extract 180g, through silica gel column chromatography, with sherwood oil (60～90 ℃), sherwood oil (60～90 ℃)-acetone, acetone gradient elution, the gained flow point carries out silica gel column chromatography repeatedly with different eluents, the SephadexLH-20 purifying separate with preparative chromatography obtain compound (
3), (
4), (
5) and (
6), concrete steps are as follows:
1. sherwood oil-acetone (3:1) wash-out gained flows part, and through silica gel column chromatography, chloroform-methanol (20:1) is an eluent, and stream part is through preparative chromatography, and (10:1) is developping agent with chloroform-methanol, obtains compound
3(10mg) and
4(7 mg).The employing method of spectroscopy is analyzed, and its structure is defined as oxyhydroquinone and different Asian puccoon phenol.
2. sherwood oil-acetone (3:1) wash-out gained flows part, and through silica gel column chromatography, chloroform-methanol (20:1) is an eluent, and stream part is through preparative chromatography, and (15:1) is developping agent with chloroform-methanol, obtains compound
5(5 mg); The employing method of spectroscopy is analyzed, and its structure is defined as the gentisinic acid pentyl ester.
3. sherwood oil-acetone (2:1) wash-out gained flows part, and through silica gel column chromatography, chloroform-methanol (20:1) is an eluent, and gained stream part is through SephadexLH-20 gel chromatography purifying, and methyl alcohol is eluent, obtains compound
6(9 mg).The employing method of spectroscopy is analyzed, and its structure is defined as the 4-methoxyl biphenyl.
The preparation of embodiment 3 phenol compounds
Get Wild Buckwheat Rhizome rhizome meal 15kg, with ethanol room temperature cold soaking (50L * 5 time), united extraction liquid is concentrated into does not have the alcohol flavor, the extracting solution thin up is to 2L, use sherwood oil, ethyl acetate, n-butanol extraction (each 2L * 4 time) successively, merge butanol extraction liquid and be concentrated into to do and promptly get n-butyl alcohol extract 790 g; Get n-butanol portion 220 g sample on dry method and carry out silica gel column chromatography, with chloroform, chloroform-methanol, methyl alcohol gradient elution, the gained flow point carries out silica gel column chromatography repeatedly with different eluents, SephadexLH-20 purifying and preparative chromatography, separate obtain compound (
7), (
8) and (
9).
1. chloroform-methanol (10:1) wash-out gained flows part, and through silica gel column chromatography, sherwood oil-acetone (4:1) is eluent, and stream part is through the SephadexLH-20 purifying, and (1:1) is eluent with chloroform-methanol, obtains compound
7(27mg).The employing method of spectroscopy is analyzed, and its structure is defined as 2,6-dimethoxy-3-methyl-Resorcinol.
2. chloroform-methanol (2:1) wash-out gained flows part, and through silica gel column chromatography, sherwood oil-acetone (1:1) is eluent, and stream part is eluent through the SephadexLH-20 purifying with methyl alcohol, obtains compound
8(8 mg).The employing method of spectroscopy is analyzed, and its structure is defined as 1,2,3, the 4-tetrahydroxy phenol.
3. chloroform-methanol (3:1) wash-out gained flows part, and through silica gel column chromatography, sherwood oil-acetone (3:1) is eluent, stream part is eluent through the SephadexLH-20 purifying with methyl alcohol, and gained stream part is through preparative chromatography, (2:1) is developping agent with chloroform-methanol, obtains compound
9(6 mg).The employing method of spectroscopy is analyzed, and its structure is defined as 2,4,6-trimethoxy-1,3,5-benzenetriol.
Embodiment 4 external anticomplement classical pathway tests
Get complement (guinea pig serum) 0.1ml, add BBS and be mixed with 1:5 solution, become 1:10,1:20,1:40,1:80,1:160,1:320 and 1:640 solution with the BBS two-fold dilution.Get each 0.1 ml of 1:1000 hemolysin, each concentration complement and 2% SRBC and be dissolved among the 0.3 ml BBS, mixing is put into the low-temperature and high-speed whizzer behind 37 oC water-baths, 30 min, centrifugal 10 min under 5000 rpm, 4 oC conditions.Get every pipe supernatant 0.2 ml respectively in 96 orifice plates, measure absorbancy at 405 nm.Experiment is provided with full haemolysis group (0.1 ml, 2% SRBC is dissolved in the 0.5 ml tri-distilled water) simultaneously.As full haemolysis standard, calculate hemolysis rate with the absorbancy of tri-distilled water haemolysis pipe.With the complement extent of dilution is X-axis, and the percentage of hemolysis that each weaker concn complement causes is the Y-axis mapping.Selection reaches the minimum complement concentration of similar high hemolysis rate as guaranteeing that system can the normal required critical complement concentration of haemolysis.Get the complement and the trial-product mixing of threshold concentration, behind pre-water-bath 10 min of 37 oC, add an amount of BBS, hemolysin and 2% SRBC.To put into the low-temperature and high-speed whizzer behind every pipe 37 oC water-baths 30 min, get every pipe supernatant 0.2 ml under 5000 rpm, the 4 oC conditions behind centrifugal 10 min respectively in 96 orifice plates, 405 nm measure absorbancy down.Experiment is provided with Chinese medicine control group, complement group and full haemolysis group simultaneously.Calculate hemolysis rate after the Chinese drug-treated group absorbance deducted corresponding Chinese medicine control group absorbance.As X-axis, the haemolysis inhibiting rate is mapped as Y-axis with Chinese medicine crude extract concentration.Calculate CH
50Value.
The result confirms that described phenol compound has remarkable restraining effect (as shown in table 1) to the classical pathway of complement system.Wherein, 1,2,3, the 4-tetrahydroxy phenol (
8) classical pathway of complement system there is the strongest restraining effect, wherein, the required minimum trial-product concentration (CH of 50% haemolysis
50) be respectively 41 ± 8 μ g/ml, suitable with positive control heparin action intensity.
Get complement (human serum) 0.2 ml, add the AP diluent preparing and become the 1:5 diluting soln, and the two-fold dilution becomes 1:10,1:20,1:40,1:80,1:160,1:320 and 1:640 solution.Get each concentration complement 0.15 ml, AP diluent 0.15 ml and 0.5% RE, 0.20 ml, mixing is placed into the low-temperature and high-speed whizzer behind 37 oC water-baths, 30 min, centrifugal 10 min under 5000 rpm, 4 oC conditions.Get every pipe supernatant 0.2 ml respectively in 96 orifice plates, measure absorbancy at 405 nm.Experiment is provided with full haemolysis group (0.20 ml, 0.5% RE is dissolved in the 0.3 ml tri-distilled water) simultaneously.As full haemolysis standard, calculate hemolysis rate with the absorbancy of tri-distilled water haemolysis pipe.With the complement extent of dilution is X-axis, and the percentage of hemolysis that each weaker concn complement causes is the Y-axis mapping.Selection reaches the minimum complement concentration of similar high hemolysis rate as guaranteeing that system can the normal required critical complement concentration of haemolysis.Get the complement and the trial-product mixing of definite threshold concentration, behind pre-water-bath 10 min of 37 oC, 1-2 adds an amount of 0.5% RE according to table.To be placed into the low-temperature and high-speed whizzer behind every pipe 37 oC water-baths 30 min, 5000 rpm, 4 oC get every pipe supernatant 0.2 ml respectively in 96 orifice plates behind centrifugal 10 min, and 405 nm measure absorbancy down.Experiment is provided with Chinese medicine control group, complement group and full haemolysis group simultaneously.Calculate hemolysis rate after the Chinese drug-treated group absorbance deducted corresponding Chinese medicine control group absorbance.As X-axis, the haemolysis inhibiting rate is mapped as Y-axis with Chinese medicine crude extract concentration.Calculate AP
50Value.
The result confirms that described phenol compound has remarkable restraining effect (as shown in table 1) to the alternative pathway of complement system.Wherein, 1,2,3, the 4-tetrahydroxy phenol (
8) alternative pathway of complement system there is the strongest restraining effect, wherein, the required minimum trial-product concentration (AP of 50% haemolysis
50) be respectively 39 ± 5 μ g/ml, stronger than the effect of positive control heparin.
The reagent that the present invention tests employing is techniques well known, and is commercially available.
Claims (7)
1. formula
Phenol compound,
（Ⅰ）
Wherein, R
1=OH, R
2=R
3=R
5=R
6=H, R
4=CH
3Or,
R
1=R
2=R
4=OH, R
3=R
5=CH
3, R
6=OCH
3Or,
R
1=R
2=R
4=OH, R
3=R
5=R
6=H; Or,
R
1=OH, R
4-R
6=OCH
2C (CH
3) CHCH
2CH
2C (CH
3) CHCH
2Or,
R
1=R
4=OH, R
2=COOC
5H
11, R
3=R
5=R
6=H; Or,
R
1=OCH
3, R
2=H, R
3=-C
6H
5Or,
R
1=R
4=OH, R
2=R
6=OCH
3, R
3=CH
3, R
5=H; Or,
R
1=R
2=R
3=R
4=OH, R
5=R
6=H; Or,
R
1=R
3=R
5=OH,?R
2=R
4=R
6=OCH
3?。
2. by the described phenol compound of claim 1, it is characterized in that, wherein, work as R
1=OH, R
2=R
3=R
5=R
6=H, R
4=CH
3The time, compound be p-methyl phenol (
1); Work as R
1=R
2=R
4=OH, R
3=R
5=CH
3, R
6=OCH
3The time, compound is a 6-methoxyl group-3,5-dimethyl-oxyhydroquinone (
2); Work as R
1=R
2=R
4=OH, R
3=R
5=R
6During=H, compound be oxyhydroquinone (
3); Work as R
1=OH, R
4-R
6=OCH
2C (CH
3) CHCH
2CH
2C (CH
3) CHCH
2The time, compound be different Asian puccoon phenol (
4); Work as R
1=R
4=OH, R
2=COOC
5H
11, R
3=R
5=R
6During=H, compound be the gentisinic acid pentyl ester (
5); Work as R
1=OCH
3, R
2=H, R
3=-C
6H
5The time, the 4-methoxyl biphenyl (
6); Work as R
1=R
4=OH, R
2=R
6=OCH
3, R
3=CH
3, R
5During=H, compound is 2,6-dimethoxy-3-methyl-Resorcinol (
7); Work as R
1=R
2=R
3=R
4=OH, R
5=R
6During=H, compound is 1,2,3, the 4-tetrahydroxy phenol (
8); Work as R
1=R
3=R
5=OH, R
2=R
4=R
6=OCH
3The time, compound is 2,4,6-trimethoxy-1,3, the 5-benzenetriol (
9).
3. the formula of claim 1
The preparation method of phenol compound, it is characterized in that, separate to obtain the phenol active substance from the n-butanol portion of the ethanol extraction of the dry rhizome of the ethyl acetate extract of the n-butanol portion of Herba Commelinae herb ethanol extraction, Asian puccoon dry root ethanol extraction or Wild Buckwheat Rhizome, it comprises step:
Get Herba Commelinae herb meal, with 95% ethanol room temperature cold soaking, united extraction liquid is concentrated into does not have the alcohol flavor, the extracting solution thin up, and successively with sherwood oil, ethyl acetate and n-butanol extraction merge butanol extraction liquid and are concentrated into the dried n-butanol portion that promptly gets; N-butanol portion sample on dry method carries out silica gel column chromatography, and with sherwood oil, 60～90 ℃, sherwood oil-acetone, acetone gradient elution, stream part are with column chromatography repeatedly, SephadexLH-20 purifying and preparative chromatography, separate obtain described compound (
1)(
2)Or,
Get Radix Arnebiae (Radix Lithospermi) meal 20 kg, under the room temperature with 95% ethanol after cold soaking, diacolation extract for several times repeatedly, decompression and solvent recovery, obtain medicinal extract 820 g, medicinal extract is suspended in the distilled water, with sherwood oil, ethyl acetate and n-butanol extraction, obtains acetic acid ethyl ester extract 420 g; Acetic acid ethyl ester extract 180 g are through silica gel column chromatography, with sherwood oil, 60～90 ℃, sherwood oil-acetone, acetone gradient elution, the gained flow point carries out silica gel column chromatography repeatedly with different eluents, SephadexLH-20 purifying and preparative chromatography, separate obtain compound (
3), (
4), (
5)(
6)Or,
Get Wild Buckwheat Rhizome rhizome meal 15kg, with ethanol room temperature cold soaking, united extraction liquid is concentrated into does not have the alcohol flavor, and the extracting solution thin up is to 2L, use sherwood oil, ethyl acetate, n-butanol extraction successively, merge butanol extraction liquid and be concentrated into dried n-butyl alcohol extract 790 g that promptly get; N-butanol portion 220 sample on dry method carries out silica gel column chromatography, and with chloroform, chloroform-methanol, methyl alcohol gradient elution, the gained flow point carries out silica gel column chromatography repeatedly with different eluents, SephadexLH-20 purifying and preparative chromatography, separate obtain compound (
7), (
8)(
9)
4. by the method for claim 3, it is characterized in that, the preparation compound (
1)(
2) in the step,Sherwood oil-acetone 5:1 wash-out gained stream part through column chromatography repeatedly, is eluent with the chloroform-methanol, and gradient elution, stream be part through the SephadexLH-20 purifying, methanol-eluted fractions, obtain compound (
1);
Sherwood oil-acetone 2:1 wash-out gained stream part is developping agent with chloroform-methanol 5:1, preparative chromatography, obtain compound (
2).
5. by the method for claim 3, it is characterized in that, preparation compound compound (
3), (
4), (
5)(
6) in the step,Sherwood oil-acetone 3:1 wash-out gained stream part, through silica gel column chromatography, chloroform-methanol 20:1 is an eluent, stream is developping agent with chloroform-methanol 10:1 part through preparative chromatography, obtain compound (
3)(
4)
Sherwood oil-acetone 3:1 wash-out gained stream part, through silica gel column chromatography, chloroform-methanol 20:1 is an eluent, stream is developping agent with chloroform-methanol 15:1 part through preparative chromatography, obtain compound (
5);
Sherwood oil-acetone 2:1 wash-out gained stream part, through silica gel column chromatography, chloroform-methanol 20:1 is an eluent, gained stream is part through SephadexLH-20 gel chromatography purifying, methyl alcohol is eluent, obtain compound (
6)
6. by the method for claim 3, it is characterized in that, the preparation compound (
7), (
8) and (
9)
Step in,
Chloroform-methanol 10:1 wash-out gained stream part, through silica gel column chromatography, sherwood oil-acetone 4:1 is an eluent, stream is eluent with chloroform-methanol 1:1 part through the SephadexLH-20 purifying, obtain compound (
7);
Chloroform-methanol 2:1 wash-out gained stream part, through silica gel column chromatography, sherwood oil-acetone 1:1 is an eluent, stream is eluent with methyl alcohol part through the SephadexLH-20 purifying, obtain compound (
8)
Chloroform-methanol 3:1 wash-out gained stream part, through silica gel column chromatography, sherwood oil-acetone 3:1 is an eluent, stream is eluent with methyl alcohol part through the SephadexLH-20 purifying, gained stream is developping agent with chloroform-methanol 2:1 part through preparative chromatography, obtain compound (
9)
7. the phenol compound of claim 1 is in the purposes of preparation in the anticomplement medicament.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116283510A (en) * | 2023-04-12 | 2023-06-23 | 辽宁中医药大学 | Novel phenol compound in purslane and extraction and separation method thereof |
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|---|---|---|---|---|
| CN1258566A (en) * | 1998-12-28 | 2000-07-05 | 北京燕山石油化工公司研究院 | Modified zeolite catalyst and its application in preparing 2-tert-butyl-4-methyl phenol |
| CN101347418A (en) * | 2007-07-20 | 2009-01-21 | 复旦大学 | Use of 8-0-4'type lignan in preparing anticomplement medicament |
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2011
- 2011-01-23 CN CN2011100243264A patent/CN102249862A/en active Pending
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|---|---|---|---|---|
| CN1258566A (en) * | 1998-12-28 | 2000-07-05 | 北京燕山石油化工公司研究院 | Modified zeolite catalyst and its application in preparing 2-tert-butyl-4-methyl phenol |
| CN101347418A (en) * | 2007-07-20 | 2009-01-21 | 复旦大学 | Use of 8-0-4'type lignan in preparing anticomplement medicament |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116283510A (en) * | 2023-04-12 | 2023-06-23 | 辽宁中医药大学 | Novel phenol compound in purslane and extraction and separation method thereof |
| CN116283510B (en) * | 2023-04-12 | 2024-03-01 | 辽宁中医药大学 | Novel phenol compound in purslane and extraction and separation method thereof |
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