CN109908133A - A kind of pimarane type diterpene is preparing the application in anti-oxidation stress medicine - Google Patents
A kind of pimarane type diterpene is preparing the application in anti-oxidation stress medicine Download PDFInfo
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Abstract
Present disclose provides a kind of pimarane type diterpene to prepare the application in anti-oxidation stress medicine.The antioxidant system that the compound can activate Nrf2 to regulate and control, mechanism are to increase the stability of Nrf2, inhibit ubiquitination；Use its cytoprotection of the human bronchial epithelial Beas-2B cell oxidative damage model evaluation of AS (III) induction; the compound can raise the content of intracellular GSH as the result is shown, inhibit the Beas-2B cellular damage and apoptosis of AS (III) induction；Experiment in vivo shows that the compound is able to suppress zebra fish vivo oxidation stress reaction, to prevent and treat diabetes, neurodegenerative disease, cancer, cardiovascular disease, chronic obstructive pulmonary disease, chronic kidney disease, respiratory inflammation.
Description
Technical field
The disclosure belongs to anti-oxidation stress medicine field, is related to a kind of pimarane type diterpene Sphaeropsidin A, includes
The composition of the compound is preparing the application in anti-oxidation stress medicine and health care product.
Background technique
The information for disclosing the background technology part is merely intended to increase the understanding to the general background of the disclosure, without certainty
It is considered as recognizing or implying in any form that information composition has become existing skill well known to persons skilled in the art
Art.
The diseases such as diabetes, neurodegenerative disease, cancer, cardiovascular disease, chronic obstructive pulmonary disease seriously threaten people
Class health, causes human death to lead raising.These disease pathology processes have a general character, and oxidative stress is the important induction of above-mentioned disease
Factor, and with its entire pathogenesis.Oxidative stress is by active oxygen, active nitrogen or the electrophilic daughter isoreactivity substance of reactivity
Superoxidant state caused by imbalance with anti-oxidation stress system of defense.Active oxygen caused by oxidative stress increases, and can break
Lipid, protein and nucleic acid in bad cell structure, cause cellular damage, induced Diabetic, neurodegenerative disease, cancer,
A series of diseases such as cardiovascular disease.2 correlation factor of nuclear factor red blood cell, 2 (Nrf2) access is sent out in regulating cell redox
Important function is waved, and Nrf2 access is activated to be resistant to one of major defence mechanism of oxidative stress.Under steady state conditions, a reactor, Nrf2
It is combined in endochylema with Keap1, low-level is maintained by proteasome degradation after ubiquitination；Body is lured by the invasion of extraneous poisonous substance
Oxidative stress is sent out, Nrf2 and Keap1 is can lead to and dissociates, indexing enters core, with the antioxygen for being located at cytoprotection gene promoter region
Change response element (ARE) to combine, start the transcription of anti-oxidant albumen or II phase detoxication enzyme, remove noxious pollutant, reduces activity
Levels of substance.It activates Nrf2 to regulate and control defense reaction, cell can be promoted to restore redox equilibrium, glycosuria can be prevented and treated
Disease, neurodegenerative disease, cancer, cardiovascular disease, chronic obstructive pulmonary disease.
Therefore, exogeneous small molecule Nrf2 agonist be prevention or treatment oxidative stress induce an illness drug it is important come
Source.Small molecule Nrf2 agonist can raise anti-oxidant albumen or II phase is detoxified expression of enzymes, reduces intracellular reactive oxygen level, increases
Add the content of endogenous antioxidant GSH, inhibits to protein in eucaryotic cell structure, the damage of lipid and nucleic acid molecule, thus
Diabetes, neurodegenerative disease, cancer, cardiovascular disease, chronic obstructive pulmonary disease chronic diseases are prevented and treated, in reduction
State the disease incidence of disease.
Natural products has drug discovery to be contributed significantly, compares chemically synthesized antioxidant, and natural products has
The advantage of low toxicity is to have irreplaceable role.Terpenoid has the bioactivity of multiplicity, studies have reported that one
A little effects of the terpenoid in Nrf2/ARE access, for example, andrographolide by the accumulation of Nrf2 in enhancing nucleus and
Promote the expression of the gene target of ARE adjusting to protect lung from damage caused by cigarette, oleanolic acid activates Nrf2 to inhibit
Atherosclerosis.
Summary of the invention
Sphaeropsidin A is found to have certain external melanoma as a kind of fungal metabolite at present
Activity in order to further develop there is prevention or the treatment active pimarane type compound of oxidative stress diseases, inventor to be directed to
The antioxidant activity of Sphaeropsidin A is studied.The disclosure is for pimarane type diterpene Sphaeropsidin A's
Antioxidant activity is studied, research shows that the compound is strong Nrf2 agonist, has the expression for raising anti-oxidant albumen, drop
The effect of low intracellular reactive oxygen level is expected to be applied to diabetes, neurodegenerative disease, cancer, cardiovascular disease, chronic
The prevention and treatment of obstructive lung disease chronic diseases.
In order to realize above-mentioned technical effect, the disclosure the following technical schemes are provided:
The Korean pine alkane type diterpenoid provided in the disclosure has the chemical structure as shown in following formula I, entitled
Sphaeropsidin A。
The disclosure is preparing anti-oxidation stress in a first aspect, providing Korean pine alkane type diterpenoid Sphaeropsidin A
Application in drug, which is characterized in that the structure of the compound is shown below:
Disclosure second aspect, provides a kind of pharmaceutical composition, and described pharmaceutical composition includes pimarane type diterpene chemical combination
Object Sphaeropsidin A and/or its pharmaceutically acceptable salt.
Preferably, the pharmaceutically acceptable salt be sulfate, phosphate, hydrochloride or be compound form.
The disclosure third aspect, provides a kind of drug, and the drug includes pharmaceutical composition described in second aspect and medicine
Acceptable auxiliary material on.
Preferably, the dosage form of the drug is tablet, mixture, granule, tincture, capsule, injection, vina, pill
Or oral solution.
Preferably, the auxiliary material of the tablet includes starch, dextrin, sodium carboxymethylcellulose and magnesium stearate.
Disclosure fourth aspect provides drug described in second aspect described pharmaceutical composition or the third aspect and is preparing antioxygen
Change the application in stress medicine or health care product.
Preferably, the anti-oxidation stress medicine is to prevent or treat diabetes, neurodegenerative disease, cancer, painstaking effort
Pipe disease, chronic obstructive pulmonary disease, chronic kidney disease or respiratory inflammation drug or health care product.
Preferably, the anti-oxidation stress medicine is Nrf2 or NQO1 and γ GCS agonist or endogenous antioxidant
GSH agonist or T-SOD vigor agonist.
Compared with prior art, the beneficial effect of the disclosure is:
Disclosure research provides the antioxidant activity of Sphaeropsidin A, and specifically provides the compound
Anti-oxidation stress mechanism is prepared into corresponding drug for the compound and provides valuable foundation；Compound
Sphaeropsidin A is preferably anti-oxidant compared to the pimarane type compound of similar structures as a kind of strong Nrf2 agonist
Activity, the preparation applied to anti-oxidation stress medicine have a good application prospect.
Detailed description of the invention
The Figure of description for constituting a part of this disclosure is used to provide further understanding of the disclosure, and the disclosure is shown
Meaning property embodiment and its explanation do not constitute the improper restriction to the disclosure for explaining the disclosure.
The NQO1 induced activity that Fig. 1 is Sphaeropsidin A in embodiment 1 tests histogram；
Concentration range used in Sphaeropsidin A is 0.78~25 μM in figure, concentration used in positive control sulforaphen
It is 2.0 μM；The result shows that Sphaeropsidin A can raise the expression of II phase detoxication enzyme NQO1 in Hepa 1c1c7 cell；
Fig. 2 is the immunoblotting assay figure of Sphaeropsidin A dose dependent research experiment in embodiment 1；
Concentration range used in Sphaeropsidin A is 0~6 μM in figure；The result shows that Sphaeropsidin A being capable of agent
Raise Nrf2 and its downstream gene NQO1 and γ GCS protein level to amount dependence；
Fig. 3 is the immunoblotting assay figure of Sphaeropsidin A protein half-life in embodiment 1；
Concentration used in Sphaeropsidin A in figure is 0.75 μM；The result shows that Sphaeropsidin A is able to extend
Nrf2 half-life period；
Fig. 4 is immunofluorescence micrograph；
Concentration used in Sphaeropsidin A is 0.75 μM in figure, and concentration used in positive control sulforaphen is 5.0 μM；
The result shows that Sphaeropsidin A promotes Nrf2 indexing to enter core；
Fig. 5 is the histogram that Sphaeropsidin A influences intracellular GSH content in various concentration embodiment 1；
Concentration range used in Sphaeropsidin A is 0.375~3 μM in figure, concentration used in positive control sulforaphen
It is 5.0 μM；The result shows that Sphaeropsidin A can dose-dependently increase in human bronchial epithelial Beas-2B cell
The content of endogenous antioxidant GSH；
Fig. 6 is the cell survival amount of the protective effect for the cell death that Sphaeropsidin A induces arsenic in embodiment 1
Histogram；
Wherein, Fig. 6 A is that various concentration Sphaeropsidin A (0.095~3 μM) causes cytotoxicity to 10 μM of arsenic
Protective effect；Fig. 6 B is the protective effect that 0.75 μM of Sphaeropsidin A causes cytotoxicity to various concentration arsenic；As a result
Show that Sphaeropsidin A can improve the cytotoxicity of arsenic induction.
Fig. 7 is AO/EB double-staining fluorescence micrograph；
Concentration used in Sphaeropsidin A is 0.75 μM in figure, and the concentration of arsenic is 5.0 μM；The result shows that
Sphaeropsidin A is able to suppress the Apoptosis of arsenic induction.
Fig. 8 is the column that various concentration Sphaeropsidin A influences correlation factor GSH content anti-oxidant in zebra fish body
Shape figure；
Concentration range used in Sphaeropsidin A is 0.5~1.5 μM in figure, and concentration used in LPS is 20 μ g/mL；As a result
Show that Sphaeropsidin A can dose-dependently improve the GSH content reduction of LPS induction.
Fig. 9 is various concentration Sphaeropsidin A to correlation factor T-SOD effect of vigor anti-oxidant in zebra fish body
Histogram；
Concentration range used in Sphaeropsidin A is 0.5~1.5 μM in figure, and concentration used in LPS is 20 μ g/mL；As a result
Show that 1.5 μM of Sphaeropsidin A can improve the T-SOD content reduction of LPS induction.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the disclosure.Unless another
It indicates, all technical and scientific terms used herein has usual with disclosure person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the disclosure.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
As background technique is introduced, the prior art is insufficient to the research of Sphaeropsidin A antioxidant activity, in order to
Technical problem as above is solved, the application proposes pimarane type diterpene Sphaeropsidin A in preparation prevention or treatment oxidation
Application in stress induction disease medicament, health care product.
A kind of exemplary embodiment of the disclosure provides pimarane type diterpene Sphaeropsidin A and prevents in preparation
Or the application in treatment the oxidative stress drug or health care product that induce an illness, the chemical formula of the pimarane type diterpene are as follows:
The disclosure can increase Nrf2 and its downstream gene γ GCS by research discovery Sphaeropsidin A, NQO1's
Protein expression, mechanism are the stability by increasing Nrf2, and ubiquitination is inhibited to realize；The people's branch induced using AS (III)
The cytoprotection of tracheal epithelium Beas-2B cell oxidative damage model evaluation Sphaeropsidin A, the as the result is shown change
The content of intracellular GSH can be raised by closing object, inhibit the Beas-2B cellular damage and apoptosis of AS (III) induction；
Sphaeropsidin A is able to suppress zebra fish vivo oxidation stress reaction, has and prevents or treat diabetes, nervus retrogression
The effect of disease, cancer, cardiovascular disease, chronic obstructive pulmonary disease, chronic kidney disease, respiratory inflammation.
Preferably, for above-mentioned application, the concentration range of Sphaeropsidin A is 0.095~0.75 μM.It is further excellent
Choosing, the concentration of Sphaeropsidin A is 0.75 μM.Cells play best protection is acted under the concentration.
Preferably, above-mentioned oxidative stress induces an illness as diabetes, neurodegenerative disease, cancer, cardiovascular disease, slow
Property obstructive lung disease, chronic kidney disease, respiratory inflammation.
Preferably, the method for separating and preparing of above-mentioned pimarane type diterpene are as follows: by endogenetic fungus Botrysphaeria
The Czapek's medium of laricina filters, and filtrate is extracted with ethyl acetate.It is separated after reduced pressure with silicagel column, using stone
Oily ether-ethyl acetate (100:1 → 1:1) carries out gradient elution, and 10 obtained components are denoted as Fr.1-Fr.10 respectively.
Fr.5 is further separated with silicagel column, gradient elution is carried out with petroleum ether-ethyl acetate (70:1 → 1:1), is obtained
To 8 components (Fr.5.1-Fr.5.8).
Fr.5.3 successively uses gel Sephadex LH-20 column (methylene chloride-methanol 1:1), octadecyl silane column
(50% → 100% methanol) isolated 4 components (Fr.5.3.1-Fr.5.3.4).
Again by Fr.5.3.4 silica gel post separation, petroleum ether-ethyl acetate (50:1 → 1:1) is mobile phase elution, is obtained
Colorless needle crystals, i.e. Sphaeropsidin A.
In order to enable those skilled in the art can clearly understand the technical solution of the disclosure, below with reference to tool
The technical solution of the disclosure is described in detail in the embodiment and comparative example of body.
The separation preparation of embodiment 1:SA and structural identification
The Czapek's medium of endogenetic fungus Botrysphaeria laricina to be filtered, filtrate is extracted with ethyl acetate,
It extracts 4 times altogether, each 20L.Gained extracting solution is concentrated under reduced pressure to give 200.2g red oil.Silicagel column crude separation is used afterwards,
Gradient elution is carried out using petroleum ether-ethyl acetate (100:1 → 1:1), wherein petroleum ether-ethyl acetate ratio is followed successively by
100:1,50:1,20:1,10:1,5:1,2:1,1:1, the eluting agent of each ratio are 9L, obtained 10 components difference
It is denoted as Fr.1-Fr.10.Fr.5 (22.5g) is further separated with silicagel column, is stream with petroleum ether-ethyl acetate (70:1 → 1:1)
It is dynamic mutually to carry out gradient elution, wherein petroleum ether-ethyl acetate ratio is followed successively by 70:1,30:1,10:1,5:1,2:1,1:1, often
The eluting agent of a ratio is 2L, obtain 8 components (Fr.5.1-Fr.5.8).Fr.5.3 successively uses gel column LH-20 (two
Chloromethanes-methanol 1:1), the isolated 4 component (Fr.5.3.1- of octadecyl silane column (50% → 100% methanol)
Fr.5.3.4).Again by Fr.5.3.4 silica gel post separation, petroleum ether-ethyl acetate (50:1 → 1:1) is mobile phase, obtains nothing
Color acicular crystal, i.e. Sphaeropsidin A.
Structural Identification is carried out to gained compound using nuclear magnetic resonance spectroscopy: from13 methyl are observed in H-NMR spectrum
Unimodal signal (δH1.10,3H,s；1.19,3H, s and 1.22,3H, s)；Proton signal (the δ of 1 group end double bondH 5.78,1H,
Dd, J=17.6,10.7Hz；5.07,1H, dd, J=10.7,0.5Hz and 5.04,1H, dd, J=17.6,0.5Hz)；1 conjugation
Proton signal (the δ of double bondH6.83,1H,s).From13C-NMR spectrum observes 3 methyl signals (δC24.4,22.7 and 33.2)；
6 methylene signals (including sp2The carbon atom δ of hydridizationC113.5)；3 methine signals (including sp2The carbon atom δ of hydridizationC
145.0 with 153.4)；6 quaternary carbon signals (including a company oxygen carbon atom δC71.7 and 1 company dioxygen carbon atom δC104.4)；
2 carbonyl signals (including a ketone carbonyl carbon atom δCA 192.6 and ester carbonyl group carbon atom δC176.1).In conclusion
The compound is pimarane type diterpene, and consulting literatures identify that the compound is Sphaeropsidin A.
The nuclear magnetic resonance data of Sphaeropsidin A are as follows:1H-NMR signal is δH: 2.22 (1H, brd, J=8.1Hz, H-
1α)；1.56(1H,m,H-1β)；1.55(1H,m,H-2α)；1.54(1H,m,H-2β)；1.85(1H,m,H-3α)；1.80(1H,
m,H-3β)；2.70(1H,s,H-5)；1.83(1H,m,H-11α)；1.65(1H,m,H-11β)；1.33(1H,m,H-12α)；
1.23(1H,m,H-12β)；6.83(1H,s,H-14)；5.78 (1H, dd, J=17.6,10.7Hz, H-15)；5.04(1H,dd,J
=17.6,0.5Hz, H-16 α)；5.07 (1H, dd, J=10.7,0.5Hz, H-16 β)；1.10(3H,s,H-17)；1.19(3H,
s,H-18)；1.22(3H,s,H-19).13C-NMR signal is δC: 23.3 (C-1)；18.1(C-2)；27.0(C-3)；32.3(C-
4)；52.2(C-5)；104.4(C-6)；192.6(C-7)；133.2(C-8)；71.7(C-9)；58.5(C-10)；41.2(C-
11)；30.7(C-12)；38.4(C-13)；153.4(C-14)；145.0(C-15)；113.5(C-16)；24.4(C-17)；33.2
(C-18)；22.7(C-19)；176.1(C-20).
The NQO1 induced activity of embodiment 2:Sphaeropsidin A is evaluated
(1) culture of murine hepatocarcinoma cell Hepa 1c1c7
Murine hepatocarcinoma cell Hepa 1c1c7 purchase is from American Type Culture collection warehousing (ATCC), using containing 10% tire ox
The DMEM culture medium of serum (FBS), is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
(2) NQO1 induced activity is tested
By in Hepa 1c1c7 cell inoculation to 96 orifice plates, various concentration is added after cell is adherent
Sphaeropsidin A (confirmation of embodiment 1) is incubated for for 24 hours, and with 0.8% digitonin solution lytic cell, detection liquid is added
[1.0mL 0.5M trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl), 15mg bovine serum albumin(BSA), 6mg MTT, 150 μ L are spat
Warm -20,150 μ L 150mM D-Glucose -6- phosphoric acid, 15 μ L 7.5mM flavin adenine dinucleotide (FAD)s, 27 μ L 50mM nicotinoyl
Amine adenine-dinucleotide phosphoric acid, 20 μ L 50mM menadiones are placed 3 minutes, measure luminous intensity in 630nm.
As a result: as shown in Figure 1, Sphaeropsidin A can activate NQO1 activity in Hepa 1c1c7 cell, it is maximum
Induced activity is 1.89 times (3.12 μM) of control group, and positive control drug sulforaphen (2.0 μM) is 1.5 times of blank control group
Left and right.The above results show that Sphaeropsidin A can activate II phase detoxification enzyme NQO1, have protective effect to human body cell.
Embodiment 3:Sphaeropsidin A can dose-dependently raise Nrf2 and its downstream gene γ GCS and
The protein level of NQO1.
(1) culture of people's pulmonary branches tracheal epithelium Beas-2B cell
People's pulmonary branches tracheal epithelium Beas-2B cell is purchased from American Type Culture collection warehousing (ATCC), using containing 10% tire
1640 culture mediums of cow's serum (FBS), are placed in 37 DEG C, 5%CO2It is cultivated in incubator.
(2) immunoblotting assay
By Beas-2B cell inoculation in D35 culture dish, after density reaches 70%-80%, be added various concentration to
It surveying compound and handles 18h, PBS is washed 2 times, addition cell pyrolysis liquid (0.5mM phenylmethylsulfonyl fluoride, 50 μ g/ml Aprotinins,
10mM sodium fluoride, 1mM sodium vanadate, 10mM β-phosphoglycerol), albumen is collected, takes appropriate amount of sample albumen loading, SDS- respectively
PAGE protein isolate component, on electrotransfer to cellulose nitrate film.Film passes through the PBST solution room temperature containing 5% skimmed milk power
After closing 1h, it is incubated overnight respectively with 4 DEG C of test antibodies.The secondary antibody room of horseradish peroxidase is added after PBST is washed
After temperature is incubated for 1h, analysis of protein is carried out with enhanced ECL chemiluminescence.
As a result: as shown in Fig. 2, after the transpulmonary bronchiolar epithelium processing 18h of cell, Nrf2 and its downstream gene NQO1 and γ
GCS protein level dose dependent increases, and maximum effective dose is 3 μM.Keap1 protein level dose dependent reduces, it was demonstrated that
The compound can activate Nrf2 signal path on protein level.
Embodiment 4: pulmonary branches tracheal epithelium extends Nrf2 half-life period
Method: it by Beas-2B cell inoculation in D35 culture dish, after density reaches 70%-80%, is added
50 μM of cycloheximides are then added in Sphaeropsidin A preincubate 8h (blank group is not added), respectively 0,10,20,30,
40min collects albumen, carries out immunoblotting assay detection, is quantified using Image J software.
As a result: as shown in figure 3, Beas-2B cell Nrf2 protein half-life was extended to by 15.81 minutes after agent-feeding treatment
30.11 minutes, shows that Sphaeropsidin A is able to extend Nrf2 protein half-life, increase protein stability.
Embodiment 5:Sphaeropsidin A can promote Nrf2 protein translocation to enter core
Method: cell climbing sheet is placed in D35 culture dish, is inoculated with Beas-2B cell, is added after cell is adherent
Sphaeropsidin A handles 8h, and the PBS of 4 degree of pre-coolings is washed 2 times, and methanol-acetone (1:1) is added and fixes 10 minutes, PBS is washed
It washs 2 times, 4 degree of Nrf2 antibody overnight incubations is added, PBS is washed 3 times, and fluorescence secondary antibody is added and is incubated at room temperature 1h, with DAPI dyeing 10
Minute, using fluorescence microscope and take pictures.
As a result: as shown in figure 4, untreated fish group Nrf2 albumen is predominantly located in cytoplasm, Sphaeropsidin A is added
After (5.0 μM) processing 8h of (0.75 μM) and positive control sulforaphen, Nrf2 is concentrated mainly in nucleus.The result shows that
Sphaeropsidin A can induce Nrf2 indexing to enter core.
Embodiment 6:Sphaeropsidin A can raise Beas-2B glutathione levels
Method: it by Beas-2B cell inoculation in D60 culture dish, after density reaches 70%-80%, is added different dense
For 24 hours, PBS is washed 2 times for Sphaeropsidin A (confirmation of the embodiment 1) processing of degree, collects cell, centrifugation, to cell precipitate
Middle addition 0.5mL 50mM sodium phosphate and 1mM edta buffer liquid lytic cell, ultrasonic 1min, cell suspension and one 1:1 of reagent are mixed
It is even, 3500 revs/min, it is centrifuged 10min, takes supernatant, operated according to glutathione assay kit specification, 405nm measurement is inhaled
Luminosity calculates glutathione content.
As a result: as shown in figure 5, Sphaeropsidin A can dramatically increase glutathione levels, enhancing cell
Oxidation resistance.
Embodiment 7:Sphaeropsidin A can be improved survival rate of the Beas-2B cell under arsenic damaging condition
Method: by Beas-2B cell inoculation in 96 orifice plates, after reaching suitable density, using prescribed concentration
The Sphaeropsidin A of Sphaeropsidin A pretreatment cell 8h, arsenic and prescribed concentration that various concentration is added (are implemented
Example 1 is confirmed) it handles for 24 hours, after MTT 3h is added, 570nm measures absorbance, calculates cells survival rate.
As a result: as shown in Figure 6A, Sphaeropsidin A pretreatment cell 8h can inhibit 10 μM of arsenic to induce significantly
Cytotoxicity improves cells survival rate, wherein 0.75 μM of Sphaeropsidin A protective effect is best.As shown in Figure 6B, it adopts
With 0.75 μM of Sphaeropsidin A preincubate 8h, the cytotoxicity that 5,10,20 μM of arsenic induces is significantly inhibited, cell is improved
Survival rate.The result shows that Sphaeropsidin A is able to suppress the toxicity of carcinogenic substance arsenic induction.
Embodiment 8:Sphaeropsidin A is able to suppress the natural death of cerebral cells of arsenic induction
Method: by Beas-2B cell inoculation in D35 culture dish, 0.75 μM is added after cell is adherent
Sphaeropsidin A and 5 μM of arsenic coprocessing 12h is separately added into acridine orange (AO)/ethidium bromide (EB) and is dyed, and used
Fluorescence microscope cell state.
As a result: as shown in fig. 7, the cell that arsenic and Sphaeropsidin A coprocessing 12h can significantly inhibit arsenic induction withers
It dies.
Embodiment 9:Sphaeropsidin A can raise glutathione level of the zebra fish under LPS existence condition
(1) zebrafish embryo obtains
Male and female zebra fish is separately fed, illumination 14h/ dark 10h alternately, timing feed with artificial grain's shape bait and just
The artemia nauplii hatched.The sexually matured zebra fish of health is taken to be put into mating cylinder when adopting ovum in the ratio of male and female 1:1, next day
Fertilized eggs are obtained when 9-10.(the chlorination containing 5.0mM of zebrafish embryo culture water is moved into after fertilized eggs are carried out disinfection and washed
Sodium, 0.17mM potassium chloride, 0.4mM calcium chloride, 0.16mM magnesium sulfate) in, optical culture is controlled at 28 DEG C.
(2) GSH content detection in zebra fish body
Each processing group juvenile fish is collected, three times with distillation washing, washes away processing medical fluid, then exhaust moisture.To every group of juvenile fish
1.5mL physiological saline is added in sample, and 10 magnetic beads are added, and 6 grades, 2 minutes, homogenate was repeated 2 times, and was sufficiently homogenized.2500 revs/min,
Centrifugation 10 minutes, takes supernatant, operates according to glutathione assay kit specification.With the zeroing of 255uL distilled water, 420nm
Place's measurement light absorption value, while protein concentration in supernatant is detected with BCA determination of protein concentration method.Calculate GSH content in Supernatant samples.
As a result: as shown in figure 8, Sphaeropsidin A can dose-dependently raise zebra fish in LPS existence condition
Under glutathione level, enhance oxidation resistance.
Embodiment 10:Sphaeropsidin A can raise T-SOD vigor of the zebra fish under LPS existence condition
Method: collecting each processing group juvenile fish, three times with distillation washing, washes away processing medical fluid, then exhaust moisture, homogenate.
It 2500 revs/min, is centrifuged 10 minutes, takes supernatant.It is operated according to total number born testing cassete specification.Every group is taken when detection
Supernatant matches liquid in proportion in 1.5mL centrifuge tube, is then mixed well with swirl mixing device, set 37 DEG C of water bath with thermostatic control 40min,
Then color developing agent is added, mixes, is placed at room temperature for 10min, the place Yu Bochang 550nm, distilled water zeroing, measurement light absorption value uses simultaneously
BCA determination of protein concentration method detects protein concentration in supernatant.Calculate total SOD vigor in Supernatant samples.
As a result: as shown in figure 9,1.5 μM of Sphaeropsidin A can raise T-SOD vigor in zebra fish body, enhancing
Oxidation resistance.
Embodiment 11: the preparation of tablet
Sphaeropsidin A (confirmation of embodiment 1) 1g, starch 1g, dextrin 2g are sieved after evenly mixing, and carboxylic first is added
Base sodium cellulosate is pelletized in right amount.Add Magnesium Stearate proper quantity, mix, tabletting to get.
The foregoing is merely preferred embodiment of the present disclosure, are not limited to the disclosure, for the skill of this field
For art personnel, the disclosure can have various modifications and variations.It is all within the spirit and principle of the disclosure, it is made any to repair
Change, equivalent replacement, improvement etc., should be included within the protection scope of the disclosure.
Claims (10)
1. Korean pine alkane type diterpenoid Sphaeropsidin A exists preparing the application in anti-oxidation stress medicine, feature
In the compound has the chemical structure being shown below:
2. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes Korean pine alkane type diterpenoid
Sphaeropsidin A and/or its pharmaceutically acceptable salt.
3. pharmaceutical composition as claimed in claim 2, which is characterized in that the pharmaceutically acceptable salt be sulfate, phosphate,
Hydrochloride or form for compound.
4. a kind of drug, which is characterized in that the drug includes pharmaceutical composition described in claim 2 or 3 and pharmaceutically may be used
The auxiliary material of receiving.
5. drug as claimed in claim 4, which is characterized in that the dosage form of the drug be tablet, mixture, granule, tincture,
Capsule, injection, vina, pill or oral solution.
6. drug as claimed in claim 5, which is characterized in that the auxiliary material of the tablet includes starch, dextrin, carboxymethyl cellulose
Plain sodium and magnesium stearate.
7. drug as claimed in claim 6, which is characterized in that Sphaeropsidin A, starch and dextrin matter in the tablet
Amount is than being 1~2:1~2:2~4.
8. pharmaceutical composition or the described in any item drugs of claim 4-7 that Claims 2 or 3 is stated are preparing anti-oxidation stress
Application in drug.
9. application as claimed in claim 8, which is characterized in that the anti-oxidation stress medicine is Nrf2 or NQO1 and γ GCS
Agonist or endogenous antioxidant GSH agonist or T-SOD vigor agonist.
10. application as claimed in claim 8, which is characterized in that the anti-oxidation stress medicine be prevent or treatment diabetes,
Neurodegenerative disease, cancer, cardiovascular disease, chronic obstructive pulmonary disease, chronic kidney disease or respiratory inflammation drug.
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| CN113082007A (en) * | 2021-05-27 | 2021-07-09 | 天津中医药大学 | Application of dihydropiceatannol in preparing medicine for preventing and/or treating cardiovascular diseases |
| CN114853704A (en) * | 2022-06-14 | 2022-08-05 | 云南省中医中药研究院 | Pimarane lactone, preparation method and application thereof |
| CN115192569A (en) * | 2022-08-10 | 2022-10-18 | 山东大学 | Application of SphaeropsidinA in preparation of medicine for preventing or treating inflammation-induced diseases |
| WO2023070480A1 (en) * | 2021-10-29 | 2023-05-04 | 海南师范大学 | Halimane diterpenoid in leucas zeylanica, and preparation method and application thereof |
| CN116675602A (en) * | 2023-08-02 | 2023-09-01 | 山东省食品药品检验研究院 | Pinane diterpene and preparation method and application thereof |
| CN115192569B (en) * | 2022-08-10 | 2024-05-10 | 山东大学 | Use of Sphaeropsidin A in preparing medicine for preventing or treating inflammation induced diseases |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113082007A (en) * | 2021-05-27 | 2021-07-09 | 天津中医药大学 | Application of dihydropiceatannol in preparing medicine for preventing and/or treating cardiovascular diseases |
| WO2023070480A1 (en) * | 2021-10-29 | 2023-05-04 | 海南师范大学 | Halimane diterpenoid in leucas zeylanica, and preparation method and application thereof |
| CN114853704A (en) * | 2022-06-14 | 2022-08-05 | 云南省中医中药研究院 | Pimarane lactone, preparation method and application thereof |
| CN115192569A (en) * | 2022-08-10 | 2022-10-18 | 山东大学 | Application of SphaeropsidinA in preparation of medicine for preventing or treating inflammation-induced diseases |
| CN115192569B (en) * | 2022-08-10 | 2024-05-10 | 山东大学 | Use of Sphaeropsidin A in preparing medicine for preventing or treating inflammation induced diseases |
| CN116675602A (en) * | 2023-08-02 | 2023-09-01 | 山东省食品药品检验研究院 | Pinane diterpene and preparation method and application thereof |
| CN116675602B (en) * | 2023-08-02 | 2023-10-20 | 山东省食品药品检验研究院 | Pinane diterpene and preparation method and application thereof |
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Application publication date: 20190621 |