CN107011140A - A kind of monoterpenes compound and preparation method and application - Google Patents
A kind of monoterpenes compound and preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of monoterpenes compound and preparation method and application.Described monoterpenes compound is using chenopodium ambrosiodies leaf as raw material, extracted through medicinal extract, organic solvent extraction, silica gel column chromatography, the column chromatographies of Sephadex LH 20, high pressure liquid chromatography it is isolated, compound molecule formula C10H16O2, it is named as the ketone of 4 hydroxyl, 4 isopropyl, 2 methyl, 2 cyclohexene 1（4‑hydroxy‑4‑isopropyl‑2‑methyl‑2–cyclohexen‑1‑one）, with following structure：.Preparation method is using chenopodium ambrosiodies leaf as raw material, extracted through medicinal extract, organic solvent extraction, silica gel column chromatography, the column chromatographies of Sephadex LH 20, high pressure liquid chromatography separation obtain.Described application is application of the described monoterpenes compound in prevention and/or treatment anti-inflammatory drug is prepared.Monoterpenes compound of the present invention has carried out extracorporeal anti-inflammatory active testing, experimental result shows that good suppression lipopolysaccharides (LPS) inducing mouse macrophage (RAW264.7) produces the effect of nitric oxide (NO), and the lead compound of application value can be provided with for medical industry.
Description
Technical field
The invention belongs to effective ingredients in plant extractive technique field, and in particular to a kind of monoterpenes compound and its preparation side
Method and application.
Background technology
Inflammation is body the stimulation of various pro-inflammatory cytokines and damage are produced it is a kind of main non-specific to defend to be adapted to
Property immune response.The pathogenic process of many diseases all with inflammation-related, clinically common rheumatoid, arthritis, asthma, wound
Wound, burn, septic shock, heart failure etc. have inflammatory mediator（Cell factor）Participation.Anti-inflammatory drug can block inflammation
The generation or release of medium, suppress inflammatory reaction, so as to treat the medicine of the disease relevant with inflammatory mediator.Current pin both at home and abroad
Treatment to active chronic inflammation is main based on antibiotics, although the treatment of these medicines can receive good effect,
But because of being continuously increased for antibody-resistant bacterium, there is increasing case although with antibiotic therapy, the state of an illness but cannot be controlled effectively,
And it is of common occurrence that antibiotic produces the case of side effect in clinical practice.Caused by the virus infection of segmental inflammation disease system,
Antibiotic is substantially invalid.In recent years, sight is gradually turned to aboundresources, pure natural Chinese herbal medicine, nationality by China and foreign countries researcher
Medicine, it is desirable to therefrom find new anti-inflammatory drug.Sinalbin, forulic acid, rattletop are found that from Chinese herbal medicine, ethnic drug at present
A series of natural anti-inflammatory drugs such as glycosides, quercitin, kuh-seng alkaloid, rheum emodin, decanoy acetaldehyde, celastrin.These crude drugs
The good anti-inflammatory activity that thing is shown increasingly proves that it is a very promising direction that anti-inflammatory drug is screened from natural products.
Chenopodium ambrosiodies (Chenopodium ambrosioides L.) is Chenopodiaceae (Chenopodiaceae) genus Chenopodium, not
Goose pin is careless for name, red Herba Lycopi, stinkweed, hookworm grass, lousewort etc., be Yi nationality, distributed over Yunnan, Sichuan and Guizhou's common drug among the people.All herbal medicine, there is wind-dispelling, dehumidifying,
Desinsection, antipruritic, the effect of stimulating the menstrual flow, relieve pain, for treat skin arthralgia pain due to rheumatism, hookworm, roundworm, dysmenorrhoea, amenorrhoea, skin eczema and
The diseases such as snake bite and insect sting.China's genus Chenopodium has 19 kinds of original seeds and 2 subspecies, is distributed mainly on salt-soda soil and dry area in north china.
The genus Chenopodium chemical composition is mainly containing type compounds such as volatile oil, sesquiterpene, steroidal, flavones, saponin(es.At present to native chaste tree
The pharmacology activity research report of mustard plant and its chemical composition is less.The present invention is attempted to having the change of antiinflammatory action in chenopodium ambrosiodies
Compound is furtherd investigate, to find anti-inflammatory activity natural products therein, for screening efficiently, the anti-inflammatory drug of low toxicity provide according to
According to.
The content of the invention
The first object of the present invention is to provide a kind of monoterpenes compound；Second purpose is the monoterpenes described in offer
The preparation method of compound；3rd purpose is the application of the monoterpenes compound described in offer.
The first object of the present invention is achieved in that described monoterpenes compound is the warp using chenopodium ambrosiodies leaf as raw material
Medicinal extract extraction, organic solvent extraction, silica gel column chromatography, Sephadex LH-20 column chromatographies, high pressure liquid chromatography are isolated,
Compound molecule formula C10H16O2, it is named as 4- hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone（4-hydroxy-4-
isopropyl-2-methyl-2-cyclohexen-1-one）, with following structure：
。
The second object of the present invention is achieved in that using chenopodium ambrosiodies leaf as raw material, is extracted through medicinal extract, organic solvent extracts
Take, the separation of silica gel column chromatography, Sephadex LH-20 column chromatographies, high pressure liquid chromatography is obtained, be specially：
A, medicinal extract are extracted：Chenopodium ambrosiodies leaf dried and crushed is obtained to the particle of 0.1 ~ 0.2cm particle sizes, chenopodium ambrosiodies leaf weight is added
5 ~ 10 times of the ethanol solution of concentration expressed in percentage by volume 85 ~ 100% is in refluxing extraction 3 ~ 5 times at 64 ~ 74 DEG C of temperature, and 1.5 ~ 3h, is closed every time
And extract solution, filter, filtrate decompression is concentrated into proportion and obtains medicinal extract a for 1.1 ~ 1.3；
B, organic solvent extraction：The water of 3 ~ 5 times of medicinal extract a weight will be added in medicinal extract a, successively with the petroleum ether isometric with water,
Ethyl acetate and n-butanol solvent are extracted, every kind of solvent extraction 5 ~ 7 times, boil off solvent respectively obtain petroleum ether extract b,
Acetic acid ethyl ester extract c and n-butyl alcohol extract d；
C, silica gel column chromatography：
1）By silicagel column on acetic acid ethyl ester extract b, dress post silica gel is 100 ~ 200 mesh, and consumption is 8 ~ 10 times of extract b weight,
It is 50 with volume ratio:1~0:1 chloroform-methanol organic solvent gradient elution, collects gradient eluent, concentration, is monitored through TLC,
Merge identical part；
2）By 1）In with 50:The eluent that the chloroform-methanol organic solvent of 1 proportioning is afforded is concentrated to relative density
1.1 ~ 1.3 obtain concentrate e, then upper silicagel column, and dress post silica gel is 200 ~ 300 mesh, and consumption is 8 ~ 10 times of concentrate e volumes,
With volume ratio 100:1~10:1 chloroform-methanol organic solvent is eluted, concentration, is monitored through TLC, is merged identical part；
D, by 2）In with 50:The eluent that the chloroform-methanol organic solvent of 1 proportioning is afforded is concentrated to relative density
Isolated and purified, produced described with Sephadex LH-20 column chromatographies and half preparative high-performance liquid chromatographic instrument respectively after 1.1 ~ 1.3
Monoterpenes compound 4-hydroxy base -4- isopropyl -2- methyl -2- cyclohexene -1- ketone（4-hydroxy-4- isopropyl-2-
methyl-2-cyclohexen-1-one）.
The third object of the present invention is achieved in that described monoterpenes compound is preparing prevention and/or treatment inflammation
Application in disease drug.
Monoterpenes compound of the present invention has good suppression lipopolysaccharides（LPS）Inducing mouse macrophage
（RAW264.7）Produce nitric oxide（NO）Effect, the lead compound of application value can be provided with for medical industry.
Brief description of the drawings
Fig. 1 is the structural formula of the compounds of this invention；
Fig. 2 is the compounds of this invention high resolution mass spectrum（HRESI-MS)；
Fig. 3 is the proton nmr spectra of the compounds of this invention（1H NMR)；
Fig. 4 is the carbon-13 nmr spectra of the compounds of this invention（13C NMR)；
Fig. 5 is the HSQC Correlated Spectroscopies of the compounds of this invention；
Fig. 6 is the HMBC Correlated Spectroscopies of the compounds of this invention；
Fig. 7 is the COSY Correlated Spectroscopies of the compounds of this invention.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is further illustrated, but the present invention is not subject in any way
Limitation, based on present invention teach that any conversion or replacement made, belong to protection scope of the present invention.
Monoterpenes compound of the present invention is using chenopodium ambrosiodies leaf as raw material, is extracted through medicinal extract, organic solvent is extracted, silicon
Plastic column chromatography, Sephadex LH-20 column chromatographies, high pressure liquid chromatography are isolated, compound molecule formula C10H16O2, life
Entitled 4- hydroxyls -4- isopropyls -2- methyl -2- cyclohexene -1- ketone（4-hydroxy-4- isopropyl-2-methyl-2-
cyclohexen-1-one）, with following structure：
。
The preparation method of monoterpenes compound of the present invention, is using chenopodium ambrosiodies leaf as raw material, is extracted through medicinal extract, organic
Solvent extraction, silica gel column chromatography, Sephadex LH-20 column chromatographies, high pressure liquid chromatography separation are obtained, and are specially：
A, medicinal extract are extracted：Chenopodium ambrosiodies leaf dried and crushed is obtained to the particle of 0.1 ~ 0.2cm particle sizes, chenopodium ambrosiodies leaf weight is added
5 ~ 10 times of the ethanol solution of concentration expressed in percentage by volume 85 ~ 100% is in refluxing extraction 3 ~ 5 times at 64 ~ 74 DEG C of temperature, and 1.5 ~ 3h, is closed every time
And extract solution, filter, filtrate decompression is concentrated into proportion and obtains medicinal extract a for 1.1 ~ 1.3；
B, organic solvent extraction：The water of 3 ~ 5 times of medicinal extract a weight will be added in medicinal extract a, successively with the petroleum ether isometric with water,
Ethyl acetate and n-butanol solvent are extracted, every kind of solvent extraction 5 ~ 7 times, boil off solvent respectively obtain petroleum ether extract b,
Acetic acid ethyl ester extract c and n-butyl alcohol extract d；
C, silica gel column chromatography：
1）By silicagel column on acetic acid ethyl ester extract b, dress post silica gel is 100 ~ 200 mesh, and consumption is 8 ~ 10 times of extract b weight,
It is 50 with volume ratio:1~0:1 chloroform-methanol organic solvent gradient elution, collects gradient eluent, concentration, is monitored through TLC,
Merge identical part；
2）By 1）In with 50:The eluent that the chloroform-methanol organic solvent of 1 proportioning is afforded is concentrated to relative density
1.1 ~ 1.3 obtain concentrate e, then upper silicagel column, and dress post silica gel is 200 ~ 300 mesh, and consumption is 8 ~ 10 times of concentrate e volumes,
With volume ratio 100:1~10:1 chloroform-methanol organic solvent is eluted, concentration, is monitored through TLC, is merged identical part；
D, by 2）In with 50:The eluent that the chloroform-methanol organic solvent of 1 proportioning is afforded is concentrated to relative density
Isolated and purified, produced described with Sephadex LH-20 column chromatographies and half preparative high-performance liquid chromatographic instrument respectively after 1.1 ~ 1.3
Monoterpenes compound 4-hydroxy base -4- isopropyl -2- methyl -2- cyclohexene -1- ketone（4-hydroxy-4- isopropyl-2-
methyl-2-cyclohexen-1-one）.
Filtering described in step A is filtered through 80 ~ 120 μm of filter paper.
Being concentrated under reduced pressure described in step A be below temperature 50 C under conditions of carried out with Rotary Evaporators decompression it is dense
Contracting.
The solvent that boils off described in step B is to boil off solvent using Rotary Evaporators.
Step C 1）Described in chloroform-methanol organic solvent volume proportion be 50:1、25:1、20:1、15:1、10:1、
7:1、5:1、2:1 and 0:1.
Step C 2）Described in chloroform-methanol organic solvent volume proportion be 100:1、50:1、25:1、20:1、15:1
With 10:1.
Isolating and purifying described in D steps is by 2）In with 50:The chloroform-methanol organic solvent of 1 proportioning elute
To eluent be concentrated into after relative density 1.1 ~ 1.3 through Sephadex LH-20 column chromatographys, purified using methanol as mobile phase
Afterwards, through half preparative high-performance liquid chromatographic, using volume proportion as 80:20 methanol-water is mobile phase, flow velocity 2.5ml/min, 250
× 10mm, 5 μm of C18 is stationary phase, and UV-detector Detection wavelength is 210nm, each μ L of sample introduction 50, collects 28 ~ 32min's
Chromatographic peak, it is repeatedly cumulative after be evaporated, obtain described monoterpenes compound 4-hydroxy base -4- isopropyl -2- methyl -2- cyclohexene -
1- ketone（4-hydroxy-4-isopropyl-2-methyl-2- cyclohexen-1-one）.
The application of monoterpenes compound of the present invention is that described monoterpenes compound is preparing prevention and/or treated
Application in anti-inflammatory drugs.
Application of the described monoterpenes compound in prevention and/or treatment active chronic inflammation medicine is prepared.
So that case is embodied, the present invention will be further described below：
Embodiment 1
Material source：Chenopodium ambrosiodies is adopted in Kunming, Yunnan, is accredited as through national medicine institute of Yunnan Institute for nationalities associate professor Yang QingsongChenopodium ambrosioides L., Saving specimen is in national medicine institute of Yunnan Institute for nationalities specimen museum.
A. crush：By 6Kg chenopodium ambrosiodies leaf dried and crusheds into the particle of particle diameter 0.10cm sizes, chenopodium ambrosiodies powder is obtained, it is standby；
B. refluxing extraction：By obtained chenopodium ambrosiodies powder refluxing extraction 4 times at a temperature of 65 DEG C in step a, 2 hours every time,
Extracted every time with the ethanol of the concentration of 30Kg 85%, merge ethanol extract, it is standby；
C, concentration：Ethanol extract made from step b is filtered through 80-120 urn filters and at a temperature of 50 DEG C with rotation
Evaporimeter is concentrated under reduced pressure, and when to proportion being 1.2, obtains medicinal extract 580g, standby；
D, extraction：580g medicinal extract is suspended in 1800ml water, successively with 1800ml petroleum ethers, 1800ml ethyl acetate and
1800ml n-butanols are extracted, every kind of solvent extraction 5 times, then are concentrated under reduced pressure with Rotary Evaporators, respectively petroleum ether, acetic acid
Ethyl ester and n-butyl alcohol extract 90 g, 131g, 230g.
E. separate:By obtained acetic acid ethyl ester extract in step d（130g）Through 100-200 mesh silica gel column chromatographies, use
Volume ratio 50:1、25:1、20:1、15:1、10:1、7:1、5:1、2:1、0:1 chloroform-methanol gradient elution obtains Fr.1-
Fr.9 totally 9 components, Fr.1 is 27.6g, and Fr.2 is 5.7g, and Fr.3 is 4.4g, and Fr.4 is 6.3g, and Fr.5 is 9.1g, and Fr.6 is
11.2g, Fr.7 are 15.3g, and Fr.8 is 8.8g, and Fr.9 is 34.7g, Fr.1 (27.1 g) through 200-300 mesh silica gel column chromatographies,
With volume ratio 100:1、50:1、25:1、20:1、15:1 and 10:1 chloroform-methanol gradient elution obtains Fr.1-1-Fr.1-6 and is total to
6 components, Fr. 1-1 are 5.4g, and Fr. 1-2 are 6.2g, and Fr. 1-3 are 3.3g, and Fr. 1-4 are 1.8g, and Fr. 1-5 are
2.8g, Fr. 1-6 are 4.1g, and Fr.1-2 is obtained after purification through Sephadex LH-20 column chromatographys using methanol as mobile phase
Fr.A-C totally 3 components, Fr. A are 0.9 g, and Fr. B are 2.6 g, and Fr. C are 1.8 g, and Fr. B prepare efficient liquid phase through half
Prepared by chromatograph, using volume proportion as 80:20 methanol-water is mobile phase, flow velocity 2.5ml/min, 250 × 10mm, 5 μm
C18 be stationary phase, UV-detector Detection wavelength be 210 nm, each μ L of sample introduction 50, collect 28 ~ 32min chromatographic peak, it is many
It is secondary it is cumulative after be evaporated, prepare target compound 4- hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone (4-
hydroxy-4-isopropyl-2- methyl-2-cyclohexen-1-one) (17 mg)。
Embodiment 2
A. crush：By 6Kg chenopodium ambrosiodies leaf dried and crusheds into the particle of particle diameter 0.15cm sizes, chenopodium ambrosiodies powder is obtained, it is standby；
B. refluxing extraction：By obtained chenopodium ambrosiodies powder refluxing extraction 4 times at a temperature of 70 DEG C in step a, 1.5 is small every time
When, extracted every time with the ethanol of the concentration of 36Kg 88%, merge ethanol extract, it is standby；
C, concentration：Ethanol extract made from step b is filtered through 80-120 urn filters and at a temperature of 50 DEG C with rotation
Evaporimeter is concentrated under reduced pressure, and when to proportion being 1.1, obtains medicinal extract 576g, standby；
D, extraction：576g medicinal extract is suspended in 2300ml water, successively with 2300ml petroleum ethers, 2300ml ethyl acetate and
2300ml n-butanols are extracted, every kind of solvent extraction 5 times, then are concentrated under reduced pressure with Rotary Evaporators, respectively petroleum ether, acetic acid
Ethyl ester and n-butyl alcohol extract 87 g, 136g, 219g.
E. separate:By obtained acetic acid ethyl ester extract in step d（136g）Through 100-200 mesh silica gel column chromatographies, use
Volume ratio 50:1、25:1、20:1、15:1、10:1、7:1、5:1、2:1、0:1 chloroform-methanol gradient elution obtains Fr.1-
Fr.9 totally 9 components, Fr.1 is 28.3g, and Fr.2 is 5.4g, and Fr.3 is 4.2g, and Fr.4 is 6.0g, and Fr.5 is 8.7g, and Fr.6 is
13.2g, Fr.7 are 16.2g, and Fr.8 is 8.5g, and Fr.9 is 30.9g, Fr.1 (28.3 g) through 200-300 mesh silica gel column chromatographies,
With volume ratio 100:1、50:1、25:1、20:1、15:1 and 10:1 chloroform-methanol gradient elution obtains Fr.1-1-Fr.1-6 and is total to
6 components, Fr. 1-1 are 5.7g, and Fr. 1-2 are 5.8g, and Fr. 1-3 are 3.9g, and Fr. 1-4 are 1.6g, and Fr. 1-5 are
3.1g, Fr. 1-6 are 4.4g, and Fr.1-2 is obtained after purification through Sephadex LH-20 column chromatographys using methanol as mobile phase
Fr.A-C totally 3 components, Fr. A are 0.8 g, and Fr. B are 2.5 g, and Fr. C are 1.9 g, and Fr. B prepare efficient liquid phase through half
Prepared by chromatograph, using volume proportion as 80:20 methanol-water is mobile phase, flow velocity 2.5ml/min, 250 × 10mm, 5 μm
C18 be stationary phase, UV-detector Detection wavelength be 210 nm, each μ L of sample introduction 50, collect 28 ~ 32min chromatographic peak, it is many
It is secondary it is cumulative after be evaporated, prepare target compound 4- hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone (4-
hydroxy-4-isopropyl-2-methyl -2-cyclohexen-1-one) (18 mg)。
Embodiment 3
A. crush：By 6Kg chenopodium ambrosiodies leaf dried and crusheds into the particle of particle diameter 0.2cm sizes, chenopodium ambrosiodies powder is obtained, it is standby；
B. refluxing extraction：By obtained chenopodium ambrosiodies powder refluxing extraction 4 times at a temperature of 74 DEG C in step a, 2.5 is small every time
When, extracted every time with the ethanol of the concentration of 42Kg 92%, merge ethanol extract, it is standby；
C, concentration：Ethanol extract made from step b is filtered through 80-120 urn filters and at a temperature of 50 DEG C with rotation
Evaporimeter is concentrated under reduced pressure, and when to proportion being 1.2, obtains medicinal extract 588g, standby；
D, extraction：588g medicinal extract is suspended in 2900ml water, successively with 2900ml petroleum ethers, 2900ml ethyl acetate and
2900ml n-butanols are extracted, every kind of solvent extraction 5 times, then are concentrated under reduced pressure with Rotary Evaporators, respectively petroleum ether, acetic acid
Ethyl ester and n-butyl alcohol extract 92 g, 133g, 232g.
E. separate:By obtained acetic acid ethyl ester extract in step d（133g）Through 100-200 mesh silica gel column chromatographies, use
Volume ratio 50:1、25:1、20:1、15:1、10:1、7:1、5:1、2:1、0:1 chloroform-methanol gradient elution obtains Fr.1-
Fr.9 totally 9 components, Fr.1 is 27.7g, and Fr.2 is 5.2g, and Fr.3 is 4.1g, and Fr.4 is 6.4g, and Fr.5 is 8.3g, and Fr.6 is
19.9g, Fr.7 are 16.6g, and Fr.8 is 7.9g, and Fr.9 is 30.7g, Fr.1 (27.7 g) through 200-300 mesh silica gel column chromatographies,
With volume ratio 100:1、50:1、25:1、20:1、15:1 and 10:1 chloroform-methanol gradient elution obtains Fr.1-1-Fr.1-6 and is total to
6 components, Fr. 1-1 are 4.7g, and Fr. 1-2 are 5.2g, and Fr. 1-3 are 3.6g, and Fr. 1-4 are 1.8g, and Fr. 1-5 are
3.4g, Fr. 1-6 are 5.6g, and Fr.1-2 is obtained after purification through Sephadex LH-20 column chromatographys using methanol as mobile phase
Fr.A-C totally 3 components, Fr. A are 0.6 g, and Fr. B are 2.5 g, and Fr. C are 1.6 g, and Fr. B prepare efficient liquid phase through half
Prepared by chromatograph, using volume proportion as 80:20 methanol-water is mobile phase, flow velocity 2.5ml/min, 250 × 10mm, 5 μm
C18 be stationary phase, UV-detector Detection wavelength be 210 nm, each μ L of sample introduction 50, collect 28 ~ 32min chromatographic peak, it is many
It is secondary it is cumulative after be evaporated, prepare target compound 4- hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone (4-
hydroxy -4-isopropyl-2-methyl -2-cyclohexen-1-one) (19 mg)。
Embodiment 4
A. crush：By 6Kg chenopodium ambrosiodies leaf dried and crusheds into the particle of particle diameter 0.2cm sizes, chenopodium ambrosiodies powder is obtained, it is standby；
B. refluxing extraction：By obtained chenopodium ambrosiodies powder refluxing extraction 4 times at a temperature of 72 DEG C in step a, 3.0 is small every time
When, extracted every time with the ethanol of the concentration of 48Kg 95%, merge ethanol extract, it is standby；
C, concentration：Ethanol extract made from step b is filtered through 80-120 urn filters and at a temperature of 50 DEG C with rotation
Evaporimeter is concentrated under reduced pressure, and when to proportion being 1.1, obtains medicinal extract 590g, standby；
D, extraction：590g medicinal extract is suspended in 2950ml water, successively with 2950ml petroleum ethers, 2950ml ethyl acetate and
2950ml n-butanols are extracted, every kind of solvent extraction 5 times, then are concentrated under reduced pressure with Rotary Evaporators, respectively petroleum ether, acetic acid
Ethyl ester and n-butyl alcohol extract 95 g, 135g, 227g.
E. separate:By obtained acetic acid ethyl ester extract in step d（135g）Through 100-200 mesh silica gel column chromatographies, use
Volume ratio 50:1、25:1、20:1、15:1、10:1、7:1、5:1、2:1、0:1 chloroform-methanol gradient elution obtains Fr.1-
Fr.9 totally 9 components, Fr.1 is 29.7g, and Fr.2 is 5.1g, and Fr.3 is 4.4g, and Fr.4 is 6.2g, and Fr.5 is 8.0g, and Fr.6 is
18.9g, Fr.7 are 15.6g, and Fr.8 is 7.6g, and Fr.9 is 31.3g, Fr.1 (29.7 g) through 200-300 mesh silica gel column chromatographies,
With volume ratio 100:1、50:1、25:1、20:1、15:1 and 10:1 chloroform-methanol gradient elution obtains Fr.1-1-Fr.1-6 and is total to
6 components, Fr. 1-1 are 5.3g, and Fr. 1-2 are 4.9g, and Fr. 1-3 are 3.5g, and Fr. 1-4 are 1.7g, and Fr. 1-5 are
3.6g, Fr. 1-6 are 5.8g, and Fr.1-2 is obtained after purification through Sephadex LH-20 column chromatographys using methanol as mobile phase
Fr.A-C totally 3 components, Fr. A are 0.7 g, and Fr. B are 2.3 g, and Fr. C are 1.5 g, and Fr. B prepare efficient liquid phase through half
Prepared by chromatograph, using volume proportion as 80:20 methanol-water is mobile phase, flow velocity 2.5ml/min, 250 × 10mm, 5 μm
C18 be stationary phase, UV-detector Detection wavelength be 210 nm, each μ L of sample introduction 50, collect 28 ~ 32min chromatographic peak, it is many
It is secondary it is cumulative after be evaporated, prepare target compound 4- hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone (4-
hydroxy -4-isopropyl-2-methyl -2-cyclohexen-1-one) (20 mg)。
Embodiment 5
A. crush：By 6Kg chenopodium ambrosiodies leaf dried and crusheds into the particle of particle diameter 0.15cm sizes, chenopodium ambrosiodies powder is obtained, it is standby；
B. refluxing extraction：By obtained chenopodium ambrosiodies powder refluxing extraction 4 times at a temperature of 68 DEG C in step a, 2.0 is small every time
When, extracted every time with the ethanol of the concentration of 60Kg 100%, merge ethanol extract, it is standby；
C, concentration：Ethanol extract made from step b is filtered through 80-120 urn filters and at a temperature of 50 DEG C with rotation
Evaporimeter is concentrated under reduced pressure, and when to proportion being 1.2, obtains medicinal extract 595g, standby；
D, extraction：595g medicinal extract is suspended in 3000ml water, successively with 3000ml petroleum ethers, 3000ml ethyl acetate and
3000ml n-butanols are extracted, every kind of solvent extraction 5 times, then are concentrated under reduced pressure with Rotary Evaporators, respectively petroleum ether, acetic acid
Ethyl ester and n-butyl alcohol extract 98 g, 136g, 217g.
E. separate:By obtained acetic acid ethyl ester extract in step d（136g）Through 100-200 mesh silica gel column chromatographies, use
Volume ratio 50:1、25:1、20:1、15:1、10:1、7:1、5:1、2:1、0:1 chloroform-methanol gradient elution obtains Fr.1-
Fr.9 totally 9 components, Fr.1 is 30.4g, and Fr.2 is 4.7g, and Fr.3 is 4.6g, and Fr.4 is 6.0g, and Fr.5 is 7.8g, and Fr.6 is
18.7g, Fr.7 are 15.5g, and Fr.8 is 7.9g, and Fr.9 is 32.2g, Fr.1 (30.4 g) through 200-300 mesh silica gel column chromatographies,
With volume ratio 100:1、50:1、25:1、20:1、15:1 and 10:1 chloroform-methanol gradient elution obtains Fr.1-1-Fr.1-6 and is total to
6 components, Fr. 1-1 are 5.5g, and Fr. 1-2 are 4.7g, and Fr. 1-3 are 3.8g, and Fr. 1-4 are 1.6g, and Fr. 1-5 are
3.4g, Fr. 1-6 are 5.7g, and Fr.1-2 is obtained after purification through Sephadex LH-20 column chromatographys using methanol as mobile phase
Fr.A-C totally 3 components, Fr. A are 0.6 g, and Fr. B are 2.3 g, and Fr. C are 1.4 g, and Fr. B prepare efficient liquid phase through half
Prepared by chromatograph, using volume proportion as 80:20 methanol-water is mobile phase, flow velocity 2.5ml/min, 250 × 10mm, 5 μm
C18 be stationary phase, UV-detector Detection wavelength be 210 nm, each μ L of sample introduction 50, collect 28 ~ 32min chromatographic peak, it is many
It is secondary it is cumulative after be evaporated, prepare target compound 4- hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone (4-
hydroxy -4-isopropyl-2-methyl -2-cyclohexen-1-one) (21 mg)。
Embodiment 6
4- hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone (4-hydroxy -4- prepared by Example 1
Isopropyl-2-methyl-2-cyclohexen-1-one), it is colorless oil；Assay method is：With nuclear magnetic resonance, knot
Close other spectroscopic technique identification structures.
[α]23.6 D 0 (c=0.23, MeOH)；IR (KBr) is composed 3442,3386,3132,1676,1634
cm-1There is absorption, show there is carboxyl, hydroxyl and double bond in molecule.HRESI-MS (accompanying drawing 2) shows its quasi-molecular ion peak m/z
168.1151 [M]+(calcd. 168.1150), determines that its molecular formula is C with reference to NMR spectrums10H16O2, degree of unsaturation is 3.1H
(accompanying drawing 3, attribution data is shown in Table in 1) display molecule 3 methyl signals, respectively δ to NMRH 1.76 (d, J = 1.4 Hz,
), 1.00 H-7 (d, J=6.9 Hz, H-9) and 0.96 (d, J=6.9 Hz, H-10), there is 1 three substitution double bond letter
Number (δ 6.62 (s, H-15)); 13(accompanying drawing 4, attribution data is shown in Table in 1) display molecule 10 carbon to C NMR, wherein there is 3
Quaternary carbon (contain 1 carbonyl carbon, 1 olefinic carbon and 1 company's oxygen quaternary carbon, its chemical displacement value is respectively δ 202.2,136.1 and 72.8),
2 methines (δ 150.8 and 38.2), 2 methylene (δ 34.8 and 31.5), and 3 methyl (δ 17.8,16.9 and
16.0).With reference to HSQC Correlated Spectroscopies (accompanying drawing 5), and documents data are it can be confirmed that the compound and known compound 4-
Hydroxy-2,4-dimethyl-2-cyclohexen-1-one structure are similar, and difference is that 4 carbon in molecule connect one
Isopropyl, rather than methyl.Further through HMBC related (accompanying drawing 6) and COSY correlations (accompanying drawing 7) and with document (Ochoa ME,
Arias MS, Aguilar R, Delgado F, Tamariz J. Captodative olefin 3-p-
nitrobenzoyloxy-3-buten-2-one as a Diels-Alder ketene equivalent for the
synthesis of hydroxycyclohexenones. Tetrahedron. 1999,55:14535-14546) contrast card
It is real, the structure of compound is finally given confirmation, be named as 4- hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone (4-
hydroxy-4-isopropyl-2-methyl-2-cyclohexen-1-one)。
Confirm that the compound is noval chemical compound by SCIFINDER database retrievals and literature query.
The compounds of this invention of table 11H- and13C-NMR (CDCl3) data.
Embodiment 7
Compound prepared by Example 2, is colorless oil；By in embodiment 6 method carry out structure determination, as a result for：
Its structure be the same as Example 6, molecular formula is C10H16O2.Confirm that compound prepared by embodiment 2 is described monoterpenes compound 4-
Hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone (4-hydroxy-4-isopropyl-2-methyl-2-
cyclohexen-1-one)。
Embodiment 8
Compound prepared by Example 3, is colorless oil；By in embodiment 6 method carry out structure determination, as a result for：
Its structure be the same as Example 6, molecular formula is C10H16O2.Confirm that compound prepared by embodiment 3 is described monoterpenes compound 4-
Hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone (4- hydroxy-4-isopropyl-2-methyl-2-
cyclohexen-1-one)。
Embodiment 9
Compound prepared by Example 4, is colorless oil；By in embodiment 6 method carry out structure determination, as a result for：
Its structure be the same as Example 6, molecular formula is C10H16O2.Confirm that compound prepared by embodiment 4 is described monoterpenes compound 4-
Hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone (4- hydroxy-4-isopropyl-2-methyl-2-
cyclohexen-1-one)。
Embodiment 10
Compound prepared by Example 5, is colorless oil；By in embodiment 6 method carry out structure determination, as a result for：
Its structure be the same as Example 6, molecular formula is C10H16O2.Confirm that compound prepared by embodiment 5 is described monoterpenes compound 4-
Hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone (4- hydroxy-4-isopropyl-2-methyl-2-
cyclohexen-1-one)。
The compounds of this invention anti-inflammatory activity of embodiment 11 is detected
Any monoterpenes compound prepared by Example 1 ~ 5 carries out anti-inflammatory activity detection experiment, and test situation is as follows：
The suppression work that the compounds of this invention is generated to NO is evaluated using the LPS screening models for inducing RAW264.7 cells to produce NO
With：
(1) preparation of reagent：D-Hank ' s cushioning liquid：Weigh NaCl 8.0 g, KCl 0.4 g, Na2HPO4•12H2O
0.134 g、KH2PO4 0.06 g、NaHCO30.35 g, the g of phenolphthalein 0.02, adding the mL of ultra-pure water about 800 makes complete molten, regulation
PH to 7.0-7.2, ultra-pure water is settled to 1 L, filters, packing, and damp and hot (121 oC, 20 min) sterilizing, 4 oC storages are standby.
PBS cushioning liquid：Weigh NaCl 8.0 g, KCl 0.2 g, KH2PO4 0.27 g、Na2HPO4•12H2O 3.58
G, adding the mL of ultra-pure water about 800 makes complete molten, adjusts pH to 7.2-7.4, ultra-pure water is settled to 1 L, filters, packing is damp and hot to go out
Bacterium, 4 oC storages are standby.
Pancreatin：Pancreatin 1.25 g, EDTA-Na20.1 g, 500 mL is settled to PBS cushioning liquid, all operations are in ice
It is upper to complete.
Compound stock solutions：Compound is configured to 100 mmol/L mother liquor, -20 oC storages by solvent of DMSO.
(2) inoculating cell：The mouse macrophage (RAW264.7) in exponential phase is taken, with containing 10% hyclone
DMEM nutrient solutions cell suspension is made, with 8*104/ hole is inoculated in 96 orifice plates, per the μ L of hole 100, in constant incubator culture
24h。
(3) compound to be detected is added：Experiment is divided into blank group (DMSO+ nutrient solutions), LPS groups, and (LPS+DMSO+ is cultivated
Liquid), positive drug group (L-NMMA+LPS+DMSO+ nutrient solutions) and compound group (various dose compound+LPS+ nutrient solutions),
Compound group is divided into 100,25,6.25,1.56,0.39 μM of five dosage group；It is careful to suction out after residual nutrient solution, according to difference
Different reagents and nutrient solution, the μ L of cumulative volume 100 are given in packet respectively；3 multiple holes of every group of setting, each group DMSO contents are controlled
System is 1 ‰.Continue to cultivate 24h.
(4) NO content detections：Careful Aspirate supernatant 50L, adds Griess I liquid 50L, and 10 min are shaken in shaking table,
The μ L of Griess II liquid 50 are added, continues to shake 10min, extinction is read at the nm of all-wave length enzyme-linked immunosorbent assay instrument 540
Value, according to NO2 —Content standard curve (being made according to Griess kit specifications) calculates NO contents.With IBM SPSS
Statistics 21 and GraphPad Prism 5 carries out one-way analysis of variance to data, calculates IC50Deng.
Calculate the compounds of this invention LPS is induced RAW264.7 cells produce NO half-inhibition concentration for 16.8 ±
4.1 μ g/mL (positive control L- monomethyl arginines (L-NMMA）, IC50For 15.6 ± 3.3 μ g/mL), show the present invention's
Compound has preferable anti-inflammatory activity.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modification equivalent substitution made within refreshing and principle and improvement etc., should be included in the scope of the protection.
Claims (10)
1. a kind of monoterpenes compound, it is characterised in that described monoterpenes compound is using chenopodium ambrosiodies leaf as raw material, through medicinal extract
Extraction, organic solvent extraction, silica gel column chromatography, Sephadex LH-20 column chromatographies, high pressure liquid chromatography are isolated, the change
Adduct molecule formula C10H16O2, it is named as 4- hydroxyl -4- isopropyl -2- methyl -2- cyclohexene -1- ketone（4-hydroxy-4-
isopropyl-2-methyl-2-cyclohexen-1-one）, with following structure：
。
2. a kind of preparation method of the monoterpenes compound described in claim 1, it is characterised in that be using chenopodium ambrosiodies leaf as raw material,
Extracted through medicinal extract, organic solvent extraction, silica gel column chromatography, Sephadex LH-20 column chromatographies, high pressure liquid chromatography separation obtain,
Specially：
A, medicinal extract are extracted：Chenopodium ambrosiodies leaf dried and crushed is obtained to the particle of 0.1 ~ 0.2cm particle sizes, chenopodium ambrosiodies leaf weight is added
5 ~ 10 times of the ethanol solution of concentration expressed in percentage by volume 85 ~ 100% is in refluxing extraction 3 ~ 5 times at 64 ~ 74 DEG C of temperature, and 1.5 ~ 3h, is closed every time
And extract solution, filter, filtrate decompression is concentrated into proportion and obtains medicinal extract a for 1.1 ~ 1.3；
B, organic solvent extraction：The water of 3 ~ 5 times of medicinal extract a weight will be added in medicinal extract a, successively with the petroleum ether isometric with water,
Ethyl acetate and n-butanol solvent are extracted, every kind of solvent extraction 5 ~ 7 times, boil off solvent respectively obtain petroleum ether extract b,
Acetic acid ethyl ester extract c and n-butyl alcohol extract d；
C, silica gel column chromatography：
1）By silicagel column on acetic acid ethyl ester extract b, dress post silica gel is 100 ~ 200 mesh, and consumption is 8 ~ 10 times of extract b weight,
It is 50 with volume ratio:1~0:1 chloroform-methanol organic solvent gradient elution, collects gradient eluent, concentration, is monitored through TLC,
Merge identical part；
2）By 1）In with 50:The eluent that the chloroform-methanol organic solvent of 1 proportioning is afforded is concentrated to relative density
Concentrate e is obtained for 1.0 ~ 1.3, then upper silicagel column, dress post silica gel is 200 ~ 300 mesh, consumption is concentrate e volumes 8 ~ 10
Times, with volume ratio 100:1~10:1 chloroform-methanol organic solvent is eluted, concentration, is monitored through TLC, is merged identical part；
D, by 2）In with 50:The eluent that the chloroform-methanol organic solvent of 1 proportioning is afforded is concentrated to relative density
To be isolated and purified respectively with Sephadex LH-20 column chromatographies and half preparative high-performance liquid chromatographic instrument after 1.0 ~ 1.3, produce described
Monoterpenes compound 4-hydroxy base -4- isopropyl -2- methyl -2- cyclohexene -1- ketone（4-hydroxy-4-isopropyl-2-
methyl-2-cyclohexen-1 -one）.
3. preparation method according to claim 2, it is characterised in that the filtering described in step A is through 80 ~ 120 μm of filter
Paper is filtered.
4. preparation method according to claim 2, it is characterised in that being concentrated under reduced pressure described in step A is in temperature 50 C
It is concentrated under reduced pressure under conditions of below with Rotary Evaporators.
5. preparation method according to claim 2, it is characterised in that the solvent that boils off described in step B is steamed using rotation
Hair instrument boils off solvent.
6. preparation method according to claim 2, it is characterised in that step C 1）Described in chloroform-methanol organic solvent
Volume proportion be 50:1、25:1、20:1、15:1、10:1、7:1、5:1、2:1 and 0:1.
7. preparation method according to claim 2, it is characterised in that step C 2）Described in chloroform-methanol organic solvent
Volume proportion be 100:1、50:1、25:1、20:1、15:1 and 10:1.
8. preparation method according to claim 2, it is characterised in that isolating and purifying described in D steps is by 2）In with
50:The eluent that the chloroform-methanol organic solvent of 1 proportioning is afforded is passed through after being concentrated into relative density 1.1 ~ 1.3
Sephadex LH-20 column chromatographys, using methanol as mobile phase after purification, through half preparative high-performance liquid chromatographic, using volume proportion as
80:20 methanol-water is mobile phase, and the C18 that 2.5ml/min, 250 × 10mm, 5 μm of flow velocity is stationary phase, UV-detector inspection
Survey wavelength is 210nm, each μ L of sample introduction 50, collects 28 ~ 32min chromatographic peak, is evaporated after repeatedly adding up, obtains described monoterpene
Class compound 4-hydroxy base -4- isopropyl -2- methyl -2- cyclohexene -1- ketone（4-hydroxy-4-isopropyl-2-methyl-
2-cyclohexen-1-one）.
9. the application of the monoterpenes compound described in a kind of claim 1, it is characterised in that described monoterpenes compound is in system
It is standby to prevent and/or treat the application in anti-inflammatory drugs.
10. the application of monoterpenes compound according to claim 9, it is characterised in that described monoterpenes compound is in system
It is standby to prevent and/or treat the application in active chronic inflammation medicine.
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| CN110143991A (en) * | 2019-06-21 | 2019-08-20 | 中国科学院新疆理化技术研究所 | Monocyclic monoterpene glucoside compound and its preparation method and application |
| CN113336627A (en) * | 2021-05-21 | 2021-09-03 | 沈阳药科大学 | Monoterpene compound and preparation method and application thereof |
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| CN105152921B (en) * | 2015-10-22 | 2017-03-08 | 云南民族大学 | A kind of tricyclic diterpene class compound and preparation method and application |
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| CN110143991A (en) * | 2019-06-21 | 2019-08-20 | 中国科学院新疆理化技术研究所 | Monocyclic monoterpene glucoside compound and its preparation method and application |
| CN110143991B (en) * | 2019-06-21 | 2022-06-24 | 中国科学院新疆理化技术研究所 | Monocyclic monoterpene glucoside compound and preparation method and application thereof |
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