CN105669416B - A kind of guainane type sesquiterpenoid and preparation method and application - Google Patents
A kind of guainane type sesquiterpenoid and preparation method and application Download PDFInfo
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- CN105669416B CN105669416B CN201510759371.2A CN201510759371A CN105669416B CN 105669416 B CN105669416 B CN 105669416B CN 201510759371 A CN201510759371 A CN 201510759371A CN 105669416 B CN105669416 B CN 105669416B
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- C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
- C07C49/703—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups
- C07C49/723—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups polycyclic
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
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Abstract
The invention discloses a kind of guainane type sesquiterpenoid, its structural formula is as follows, and the invention also discloses the preparation method of the compound and its application in resisting rheumatoid disease arthritis drug is prepared.Present invention firstly discovers that activity of the guainane type sesquiterpenoid in synoviocytes in rheumatoid arthritis propagation is suppressed, available for preparing the arthritic medicine of resisting rheumatoid disease.
Description
Technical field
The present invention relates to technical field of phytochemistry, and in particular to a kind of guainane type sesquiterpenoid and its preparation
Method and application.
Background technology
Melon fragrance wood (Fissistigma oldhamii (Hemsl.) Merr.), the small fragrant flower rattan of alias, rattan longan, dalbergia wood rattan,
It is Annonaceae (Annonaceae) Fissistigma plant.The plant can be used for medicinal purpose, and stem, leaf can control fracture oedema, and complete stool can
To treat treating rheumatic ostealgia, it is numb in every limb etc..Report is had no from the isolated guaiaci lignum alkane type sesquiterpene compound of melon fragrance wood,
Also report of such compound in the application in suppressing synoviocytes in rheumatoid arthritis propagation is had no.
The content of the invention
The technical problems to be solved by the invention are to provide the guainane type sesquialter that separation is extracted in a kind of wood from melon fragrance
Terpene compound.
Present invention solves the technical problem that being to provide the preparation method of above-mentioned guainane type sesquiterpenoid.
The technical problem of the invention finally to be solved is to provide the application of above-mentioned guainane type sesquiterpenoid.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of guainane type sesquiterpenoid, its general structure is as follows：
Wherein R1、R2、R3、R4It is respectively and independently selected from as-OH or-CH2OH or-CH3Or-H.
Above-mentioned guainane type sesquiterpenoid, preferred structure formula is as follows：
The preparation method of above-mentioned preferred guainane type sesquiterpenoid, it comprises the following steps：
(1) melon Radix Fissistigmatis Oldhamii extract is prepared
The stem that melon fragrance wood is dried is carried with 30~95%v/v ethanol cold soaking either heat, is obtained extract solution, is concentrated under reduced pressure
Into paste, as melon Radix Fissistigmatis Oldhamii extract；
(2) isolate and purify
Above-mentioned melon Radix Fissistigmatis Oldhamii extract is diluted with water and is made after suspension, is extracted successively with petroleum ether, ethyl acetate,
Acetic acid ethyl acetate extract is condensed into medicinal extract, isolated through column chromatography, thin-layer chromatography, sieve chromatography, the compound is new
Compound and it is named as Fissistigmaterpene A.
In step (1), it is concentrated under reduced pressure, temperature is 30~70 DEG C, and pressure is -0.06~-0.15MPa, and preferable temperature is 45
DEG C, preferably pressure is -0.095MPa.
In step (2), the concentration, temperature is 30~70 DEG C, and pressure is -0.06~-0.15MPa, and preferable temperature is 45
DEG C, preferably pressure is -0.095MPa.
In step (2), the condition of column chromatography is：With 200~300 mesh silica gel upper props, ethyl acetate percentage by volume is
20% ethyl acetate-light petrol mixed solvent is eluant, eluent.
In step (2), thin-layer chromatography condition is：Ethyl acetate-light petrol using ethyl acetate percentage by volume as 25%
Mixed solvent is solvent.
In step (2), sieve chromatography condition is：Molecular sieve be Sephadex LH-20, using chloroform percentage by volume as
40% chloroform-methanol mixed solvent is eluant, eluent.
Application of the above-mentioned guainane type sesquiterpenoid in resisting rheumatoid disease arthritis drug is prepared is also in the present invention
Protection domain within.
Above-mentioned guainane type sesquiterpenoid is in suppression synoviocytes in rheumatoid arthritis hyperproliferation agent is prepared
Using also in protection scope of the present invention etc.
Wherein, described synovial cell is Primary rat synovial cell.
Beneficial effect：Present invention firstly discovers that guainane type sesquiterpenoid is suppressing rheumatoid arthritis synovial membrane
Activity in cell propagation, available for preparing the arthritic medicine of resisting rheumatoid disease.
Embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, without should be also without limitation on sheet described in detail in claims
Invention.
Embodiment 1：The preparation of sesquiterpenoids.
The stem 10Kg that melon fragrance wood is dried obtains extract solution, will carried routinely with 75%v/v alcohol steep 3 times 7 days every time
Take liquid to be concentrated under reduced pressure into paste, obtain melon Radix Fissistigmatis Oldhamii extract 1200g.Extract is diluted to suspension with distilled water (3L), according to
Secondary use petroleum ether (3L × 3 time), ethyl acetate extraction (3L × 3 time), medicinal extract (about 250g) is condensed into by acetic acid ethyl acetate extract,
Silica gel column chromatography is carried out, with petroleum ether-ethyl acetate mixed solvent (100:0―0:100, V/V) and acetate-methanol
(100:0―0:100, V/V) silica gel column chromatography is incrementally carried out by polarity, and cut is collected by each about 400mL.Examined by TLC
Survey the similar flow point of merging and be divided into 8 components, i.e. Fr.1-8.Fr.3 200~300 mesh silica gel upper props, ethyl acetate volume basis
The ethyl acetate-light petrol mixed solvent that number is 20% is that eluant, eluent is eluted, using ethyl acetate percentage by volume as 25%
Ethyl acetate-light petrol mixed solvent for solvent carry out thin-layer chromatography, according to chromatographic effect merge flow point obtain 3 components
Fr.3-1, Fr.3-2 and Fr.3-3.Fr.3-2 is elution by 40% chloroform-methanol mixed solvent of chloroform percentage by volume
Agent carries out obtaining component Fr.3-2-1, Fr.3-2-1 efficient liquid phase after molecular sieve Sephadex LH-20 column chromatographies remove depigmentaton
Carry out analysis preparation, eluant, eluent is acetonitrile and water its volume proportion is 2:3 obtain guainane type sesquiterpenoid
Fissistigmaterpene A。
Guainane type sesquiterpenoid Fissistigmaterpene A Structural Identification result is as follows：
Fissistigmaterpene A：For pale yellow oil.Ultraviolet 254nm has blackening, and 5% sulfuric acid-vanillic aldehyde shows
Color displaing amaranth.Optical activity isHR-ESI-MS m/z 251.1642[M-H]-, with reference to1H-NMR and13C NMR understand that molecular formula is C15H24O3, it is 4 to calculate degree of unsaturation.According to1H-NMR and DEPT is understood in compound
Contain 3 methyl signals [δH0.79 (d, J=6.8Hz), 0.81 (d, J=6.8Hz), 0.83 (s)]；According to13C-NMR and DEPT
Understand there is 3 methyl carbon, 6 methines, 4 methylene, 2 quaternary carbons in compound.Therefore, it is known that compound 1 is sesquiterpenoids
Compound.According to1H-1H COSY are understood：H-2/H-3/H-4/H-5 is related, and H-4/H-12/H-13 is related, hence, it can be determined that
Fragment C-2-C-3-C-4--C-5, C-4-C-11-C-12, C-11-C-3；H-8/H-9-H-10 is related, it may be determined that C-8-C-9-
C-10 fragments.The connection of the two fragments is confirmed by HMBC.In HMBC spectrums, H-15 and C-2/C-1/C-10/C-5
Correlation, what H-5 was connected by C-1 and C-6-C-7 respectively with C-6/C-7 related descriptions the two fragments.The relative structure of compound
Type determines that H-5 maximum coupling constant is 9.2Hz, is shown at H-4 and H-5 by NOESY determinations and coupling constant
The two hydrogen are in the same side to H-5 with H-10 related descriptions in rotary-inversion axis position, NOESY spectrums；H-4 and H-15 related descriptions H-4
The same side is in H-15.So far, the new sesquiterpenoid that the compound is guainane type is determined.
The compound 1 of table 11H-NMR and13C-NMR data
Embodiment 2：Pharmacological activity is tested
Experiment material
Cell：Synovial cell (Primary rat synovial cell).
Cell culture fluid：Primary rat synovial cell's culture medium.
Reagent：Tetrazolium bromide (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium
Bromide, MTT, Sigma are produced).Lactic dehydrogenase (lactate dehydrogenase, LDH) kit (is purchased from the green skies
Company).Methotrexate (MTX) (methot rexate, MTX, Shanghai Xinyi pharmaceutical factory Co., Ltd, lot number 20140307).
1.4 instrument：96 porocyte culture plates；Infinite 200Pro multi-function microplate readers.
Experimental method
Mtt assay determines inhibitory action of the medicine to synovial cell
The synovial cell of phase of taking the logarithm is seeded to 96 well culture plates (1 × 104Individual cells/well), be set to blank group (not to
Give medicine), methotrexate (MTX) group (methotrexate (MTX) for giving 1 μ g/mL), administration group (give the medicine of 10,50,100 μ g/mL concentration
Thing), culture is used to test after being incubated overnight.Cell is given after the medicine culture 48h of various concentrations, and 10 μ L concentration are added per hole and are
5mg/mL MTT, continue to cultivate after 4h, and careful inhale abandons culture supernatant, and 100 μ L DMSO dissolving first a ceremonial jade-ladle, used in libation crystallizations, room are added per hole
Temperature shakes 5min, after being completely dissolved, and detects optical density (OD values) at 570nm with ELIASA.Cell survival is calculated according to OD values
Rate.
LDH activity detection method surveys influence of the medicine to synovial cell's cell viability
The synovial cell of phase of taking the logarithm is seeded to 96 well culture plates (1 × 104Individual cells/well), be set to blank group (not to
Give medicine), methotrexate (MTX) group (methotrexate (MTX) for giving 1 μ g/mL), administration group (give the medicine of 10,50,100 μ g/mL concentration
Thing), culture is incubated overnight, after each group cell culture terminates, and takes cell supernatant, LDH detection kits detection LDH activity.LDH
Vigor calculate according to the following equation.
Statistical method
Experimental data is analyzed using the statistical softwares of SPSS 22.0.As a result mean ± standard deviation is usedRepresent, use Dan Yin
Plain variance (one-way ANOVA) analyzes group difference.With P<0.05 thinks with significant difference.
Experimental result
Mtt assay determines inhibitory action of the medicine to synovial cell
After various concentrations medicine culture 48h, mtt assay detection cel l proliferation result see the table below 2.As a result show
Fissistigmaterpene A have good inhibiting effect to synovial cell.
Inhibitory action of each medicine of table 2 to synovial cell
Note：Compared with blank group,*P ﹤ 0.05,**P ﹤ 0.01.
LDH activity detection method surveys influence of the medicine to synovial cell's cell viability
After various concentrations medicine culture 48h, LDH activity detection method detection cell viability result see the table below 3.As a result show
Show after the drug-treated of various concentrations, increase LDH activity (P in cell culture fluid<0.05 or P ＜ 0.01).
Effect of each medicine of table 3 to synovial cell's LDH activity
Note：Compared with blank group,*P ﹤ 0.05,**P ﹤ 0.01.
Result above shows that guaiane type sesquiterpenoid Fissistigmaterpene A have to synovial cell
Good inhibition, can improve LDH activity in cell culture fluid.Experiment shows sesquiterpenoid
Fissistigmaterpene A have good antirheumatic drug effect, can be used for the preparation of antirheumatic.
Claims (9)
1. a kind of guainane type sesquiterpenoid, its structural formula is as follows：
2. the preparation method of the guainane type sesquiterpenoid described in claim 1, it is characterised in that it includes following step
Suddenly：
(1) melon Radix Fissistigmatis Oldhamii extract is prepared
The stem that melon fragrance wood is dried is carried with 30~95%v/v ethanol cold soaking either heat, is obtained extract solution, is concentrated under reduced pressure into cream
Shape, as melon Radix Fissistigmatis Oldhamii extract；
(2) isolate and purify
Above-mentioned melon Radix Fissistigmatis Oldhamii extract is diluted with water and is made after suspension, is extracted successively with petroleum ether, ethyl acetate, by second
Acetoacetic ester extract is condensed into medicinal extract, isolated through column chromatography, thin-layer chromatography, sieve chromatography.
3. the preparation method of guainane type sesquiterpenoid according to claim 2, it is characterised in that step (1)
In, it is concentrated under reduced pressure, temperature is 30~70 DEG C, pressure is -0.06~-0.15MPa.
4. the preparation method of guainane type sesquiterpenoid according to claim 2, it is characterised in that step (2)
In, the concentration, temperature is 30~70 DEG C, and pressure is -0.06~-0.15MPa.
5. the preparation method of guainane type sesquiterpenoid according to claim 2, it is characterised in that step (2)
In, the condition of column chromatography is：With 200~300 mesh silica gel upper props, ethyl acetate percentage by volume is 20% ethyl acetate-stone
Oily ether mixed solvent is eluant, eluent.
6. the preparation method of guainane type sesquiterpenoid according to claim 2, it is characterised in that step (2)
In, thin-layer chromatography condition is：It is expansion by 25% ethyl acetate-light petrol mixed solvent of ethyl acetate percentage by volume
Agent.
7. the preparation method of guainane type sesquiterpenoid according to claim 2, it is characterised in that step (2)
In, sieve chromatography condition is：Molecular sieve is Sephadex LH-20, the chloroform-methanol using chloroform percentage by volume as 40%
Mixed solvent is eluant, eluent.
8. application of the guainane type sesquiterpenoid in resisting rheumatoid disease arthritis drug is prepared described in claim 1.
9. the guainane type sesquiterpenoid described in claim 1 is preparing suppression synoviocytes in rheumatoid arthritis propagation
Application in medicine.
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| CN108299178B (en) * | 2018-02-12 | 2021-02-09 | 新疆维吾尔自治区中药民族药研究所 | Guaiane type sesquiterpene B, preparation method thereof and application of guaiane type sesquiterpene B in preparation of tumor prevention and antitumor drugs |
| CN108503521B (en) * | 2018-02-12 | 2021-02-09 | 新疆维吾尔自治区中药民族药研究所 | Guaiane type sesquiterpene A, its preparation method and application in preparing medicine for preventing tumor and resisting tumor |
| CN111675682A (en) * | 2020-06-30 | 2020-09-18 | 海南师范大学 | Benzopyran compound and preparation method and application thereof |
| CN111704546A (en) * | 2020-06-30 | 2020-09-25 | 海南师范大学 | Oxygen-containing cyclohexane derivative and preparation method and application thereof |
| CN115813894A (en) * | 2021-09-18 | 2023-03-21 | 海南师范大学 | Sesquiterpene compound and preparation method and application thereof |
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| CN102260234A (en) * | 2010-05-26 | 2011-11-30 | 福建医科大学 | Preparation method and application of guaiane-type sesquiterpene |
| CN103088083A (en) * | 2013-02-19 | 2013-05-08 | 齐齐哈尔大学 | Method for preparing anti-inflammation and anti-tumor activity sesquiterpene lactone compounds |
| CN103242275A (en) * | 2013-05-13 | 2013-08-14 | 沈阳药科大学 | Sesquiterpenoids in zedoary as well as preparation method and application of sesquiterpenoids |
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