CN106187984B - A kind of mouth xanthones compounds and its preparation method and application - Google Patents
A kind of mouth xanthones compounds and its preparation method and application Download PDFInfo
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- CN106187984B CN106187984B CN201610536316.1A CN201610536316A CN106187984B CN 106187984 B CN106187984 B CN 106187984B CN 201610536316 A CN201610536316 A CN 201610536316A CN 106187984 B CN106187984 B CN 106187984B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
- C07D311/82—Xanthenes
- C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
- C07D311/86—Oxygen atoms, e.g. xanthones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/51—Gentianaceae (Gentian family)
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Abstract
The invention discloses a kind of mouth xanthones compounds for suppressing 5 alpha-reductases（1）, the mouth xanthones compounds are from black purple Swertia patens（Swertia atroviolacea）In it is isolated, its molecular formula is C18H18O5, the Compound nomenclature is：The methyl mouth mountain ketone of 6 (2 ethoxy) 1,7 dimethoxy 3, the entitled methyl xanthone of 6 (2 hydroxyethyl) 1,7 dimethoxy 3 of English, has following structural formula：（1）The preparation method of the mouth xanthones compounds, be using black purple Swertia patens as raw material, extracted through medicinal extract, MCI decolourings, silica gel column chromatography, high pressure liquid chromatography separating step and obtain.The mouth xanthones compounds have good inhibiting effect through 5 alpha-reductase active testings to 5 alpha-reductases.The compounds of this invention is simple in construction, and has preferable 5 alpha-reductase inhibitory action, can be as the lead compound for preparing 5 alpha reductase inhibitors.
Description
Technical field
The invention belongs to technical field of phytochemistry, and in particular to a kind of mouth for extracting to obtain first from black purple Swertia patens
Xanthones compounds.Meanwhile the invention further relates to the preparation method of the compound and its 5α-reductase inhibitory activity.
Background technology
Swertia（Swertia）It is a category under Gentianaceae, about 170 kinds of the whole world, China has more than 80 to plant, main distribution
In Yunnan, Sichuan and other places.In China, the platymiscium is used as medicine with a long history, medicinal to have 35 kinds, has clearing heat secreting bile, removing dampness and detoxicating etc.
Effect, it is usually used in treating the diseases such as acute Jaundice Jaundice, osteomyelitis.The chemical composition of the platymiscium is enriched, and mainly has a mouthful mountain
The compounds such as ketone, flavones, iridoid, triterpene and alkaloid.Black purple Swertia patens（Swertia atroviolacea）For river deer tooth
Lepidium herbaceos perennial, it is born in the m of height above sea level 3400~4575 patana, more tor tops or rock seam, is Yunnan Province
The peculiar kind in Diqingzangzu area.
Mouth mountain ketone also known as dibenzopyrans ketone, its derivative are widely present in nature, are the important of medicinal plant
One of active ingredient.Because mouth mountain ketones derivant is that have Fen Xing functional groups on three rings of linear array, mostly with rich
Rich bioactivity and medical value；Xanthone derivative has extensive physiology and pharmacological activity, has antitumor, guarantor
Shield heart blood vessel, the pharmacological actions such as blood glucose, anti-oxidant, antibacterial are reduced, but many pharmacological mechanisms are still in the research and probe stage,
So that its pharmacological activity and its being received much attention with the research of configuration relationship.The present invention is isolated one from black purple Swertia patens
Relevant report is not yet seen in mouth xanthones compounds of the kind with 5α-reductase inhibitory activity, the compound.
The content of the invention
The first object of the present invention is to provide a kind of new mouth xanthones compounds；Second purpose is to provide the mouth
The preparation method of xanthones compounds；3rd purpose is to provide the mouth xanthones compounds as 5α-reductase inhibitor
Application.
The first object of the present invention is achieved in that described mouth xanthones compounds are complete from dry black purple Swertia patens
Isolated in grass, its molecular formula is C18H18O5, it is named as：6- (2- ethoxys) -1,7- dimethoxys -3- methyl-mouth mountain ketone,
Entitled 6- (2-hydroxyethyl) -1, the 7-dimethoxy-3-methyl- xanthone of English, have following structural formula：
The compound is light yellow gum thing.
The second object of the present invention is achieved in that the preparation method of the mouth xanthones compounds described in one, is with dry
Dry black purple Swertia patens herb is raw material, extracted through medicinal extract, MCI decolourings, silica gel column chromatography, high pressure liquid chromatography step, specifically
For：
A, medicinal extract extracts：It is raw material to take dry black purple Swertia patens herb, is crushed, 3 are extracted at room temperature with 95% ethanol
~ 5 times, 3 days every time, merge extract solution, filtering, be concentrated under reduced pressure extract solution, stands, filters out sediment, be condensed into medicinal extract a；
B, MCI decolourizes：Medicinal extract a MCI posts decolourize, and are eluted with 80%-95% methanol aqueous solutions, merge organic phase, and decompression is dense
Shorten medicinal extract b into；
C, silica gel column chromatography：Medicinal extract b carries out silica gel column layer with 80 ~ 100 mesh silica gel dry column-packings of the weight than 8 ~ 10 times of amounts
Analysis；Using volume proportion as 1:0、9:1、8:2、7:3、6:4、1:1 and 0:1 chloroform/acetone solution carries out gradient elution, collects ladder
Eluent, concentration are spent, merges identical part；
D, high pressure liquid chromatography separates：The 7 of step C eluent:The inverted C18 medium pressure liquid chromatographies in 3 parts isolate and purify,
Eluent corresponding to the min chromatographic peaks of gained 20 ~ 35, is further isolated and purified through high pressure liquid chromatography, produces described mouth mountain ketone
Class compound.
The structure of mouth xanthones compounds prepared by method described above is to identify to come by the following method：
The compounds of this invention is light yellow gum thing；High resolution mass spectrum (HRESIMS) provides quasi-molecular ion peakm/z
337.1059 [M+Na]+(calculated value 337.1052).With reference to1H and13 C H NMR spectroscopies provide a molecular formula C18H18O5, insatiable hunger
It is 10 with degree.Ultraviolet spectra has absorption maximum at 338,256 and 210 nm, it was demonstrated that aromatic ring structure in compound be present；It is red
External spectrum data, which are also confirmed that in compound, has hydroxyl (3420 cm-1), carbonyl (1654 cm-1) and aromatic ring 1606,1529,
1468 cm-1) functional group.Compound1H and13C NMR data (table -1), which are shown in compound, 18 carbon and 18 hydrogen letters
Number, including the quaternary mouth mountain ketone skeletons of a 1,3,6,7- [Ind. Crop. Prod., 58, 125 (2014)](C-1
~ C-9, C-4a, C-8a ~ C10a；H-2, H-4, H-5 and H-8), ethoxy (C-1 ' and a C-2 '；H2- 1 ' and H2- 2 '), one
The individual nuclear substituted methyl of virtue (C-3 ' and H3- 3 '), 2 methoxyl groups (δ H55.9 q and 56.3 q；3.80 s and 3.83
s).H in compound be present2-1′ (δH2.62) and C-5 (δ C 119.8)、C-6 (δ C 141.3)、C-7 (δ C158.5),
H2-2′ (δH3.64) and C-6 (δ C141.3), and H-5 (δ H7.02) and C-1 ' (δ C 35.2) HMBC is related
(Fig. 3), it can be verified that ethoxy is substituted in the C-6 positions of compound.According to H3-3′ (δH2.38) and C-2 (δ C 112.2)、C-3
(δ C 147.2)、C-4 (δ C108.6) HMBC is related, it can be verified that methyl is substituted in the C-3 positions of compound.Two methoxyl groups take
In generation, can be by two methoxyl group hydrogen (δ in C-1 and C-7H 3.80 and 3.83) respectively with C-1 (δ C160.2) and C-7 (δ C
158.5) HMBC correlations are confirmed.Typical proton signal in compound [δ H 7.08 d (2.2)、6.87 d (2.2)、
7.02 s, 7.43 s] also support that mouth mountain ketone B rings of the invention are the 6,7- bis- substitutes, and C rings are that 1,3- bis- substitutes.So far
The structure of the compounds of this invention is determined that Compound nomenclature is：6- (2- ethoxys) -1,7- dimethoxys -3- methyl-mouth mountain
Ketone.
Infrared, the ultraviolet and mass spectrometric data of compound：UV (methanol),λ max (log ε) 210 (4.06)、256
(3.64), 338 (3.47), IR (pressing potassium bromide troche)ν max 3420、3085、2928、1654、1606、1529、1468、
1412、1360、1273、1208、1158、1064、869 cm-1；1H NMR and13C NMR data (CDCl3, 500 and 125
MH), it is shown in Table -1；ESI-MS (positive ion mode)m/z 337 [M+Na]+;HR-ESI-MS (positive ion mode)m/z [M
+ Na]+337.1059 (calculated value 337.1052, C18H18NaO5)。
The compound of table -1.1H NMR and13C NMR data (solvent C5D5N)
| No. | d C | d H (m, J, Hz) | No. | d C | d H (m, J, Hz) |
| 1 | 160.2 s | 4a | 154.4 s | ||
| 2 | 112.2 d | 7.08 d (2.2) | 8a | 113.9 s | |
| 3 | 147.2 s | 9a | 110.2 s | ||
| 4 | 108.6 d | 6.87 d (2.2) | 10a | 148.2 s | |
| 5 | 119.8 d | 7.02 s | 1′ | 35.2 t | 2.62 t (7.2) |
| 6 | 141.3 s | 2′ | 63.2 t | 3.64 t (7.2) | |
| 7 | 158.5 s | 3′ | 24.2 q | 2.38 s | |
| 8 | 111.5 d | 7.43 s | 1-OMe | 55.9 q | 3.80 s |
| 9 | 176.7 s | 7-OMe | 56.3 q | 3.83 s |
The third object of the present invention is achieved in that described mouth xanthones compounds as 5α-reductase inhibitor
Application.
The compounds of this invention is separated from black purple Swertia patens first, passes through nuclear magnetic resonance and measuring method of mass spectrum
It is defined as a mouthful xanthones compounds, and characterizes its concrete structure.The compound is tested through 5α-reductase inhibitory activity, as a result
Show, it is 68.4 ± 4.3% to 5α-reductase inhibiting rate, shows that the compound has prominent 5α-reductase inhibitory activity.
The compounds of this invention structure novel active is preferable, can have good answer as the guiding compound of 5α-reductase inhibitor
Use prospect.
Brief description of the drawings
Fig. 1 is the proton nmr spectra of mouth xanthones compounds of the present invention；
Fig. 2 is the carbon-13 nmr spectra of mouth xanthones compounds of the present invention；
Fig. 3 be mouth xanthones compounds of the present invention main HMBC and1H-1H COSY relevant indicators.
Embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples, but not in any way to the present invention
It is any limitation as, based on present invention teach that any conversion or improvement made, each fall within protection scope of the present invention.
Unless otherwise indicated, the percentage employed in the present invention is percentage by volume.
Mouth xanthones compounds of the present invention, it is isolated, its molecule from dry black purple Swertia patens herb
Formula is C18H18O5, there is following structural formula：
The compound is light yellow gum thing, is named as：6- (2- ethoxys) -1,7- dimethoxys -3- methyl-mouth mountain
Ketone, entitled 6- (2-hydroxyethyl) -1, the 7-dimethoxy-3-methyl- xanthone of English.
The preparation method of mouth xanthones compounds of the present invention, be using dry black purple Swertia patens herb as raw material,
Extracted through medicinal extract, MCI decolourings, silica gel column chromatography, high pressure liquid chromatography step, be specially：
A, medicinal extract extracts：It is raw material to take dry black purple Swertia patens herb, is crushed, 3 are extracted at room temperature with 95% ethanol
~ 5 times, 3 days every time, merge extract solution, filtering, be concentrated under reduced pressure extract solution, stands, filters out sediment, be condensed into medicinal extract a；
B, MCI decolourizes：Medicinal extract a MCI posts decolourize, and with 80%-95% methanol water elutions, merge organic phase, are concentrated under reduced pressure into
Medicinal extract b；
C, silica gel column chromatography：Medicinal extract b carries out silica gel column layer with 80 ~ 100 mesh silica gel dry column-packings of the weight than 8 ~ 10 times of amounts
Analysis；Using volume proportion as 1:0、9:1、8:2、7:3、6:4、1:1 and 0:1 chloroform/acetone solution carries out gradient elution, collects ladder
Eluent, concentration are spent, merges identical part；
D, high pressure liquid chromatography separates：The 7 of step C eluent:The inverted C18 medium pressure liquid chromatographies in 3 parts isolate and purify,
Eluent corresponding to the min chromatographic peaks of gained 20 ~ 35, is further isolated and purified through high pressure liquid chromatography, produces described mouth mountain ketone
Class compound.
The medicinal extract b of the step C is more molten than the chloroform/methanol mixing of 1.5~3 times of amounts with weight before through silica gel column chromatography
Agent is dissolved, and 1 ~ 1.5 times of 80~100 mesh silica gel mixed samples are then weighed with medicinal extract.
It is with the mm of 21.2 mm × 250,5 that the anti-phase C18 medium pressure liquid chromatographies of the D steps, which isolate and purify,μM C18
Chromatographic column is stationary phase, and using 45% acetonitrile as mobile phase, flow velocity is 20 mL/min, and UV-detector Detection wavelength is 338
Nm, each mL of sample introduction 2, collect 20 ~ 35 min chromatographic peak.
Eluent first takes off before being isolated and purified through high pressure liquid chromatography corresponding to 20 ~ 35min chromatographic peaks of the D steps
Except solvent again with methanol dissolves.
It is with the mm of 9.4 mm × 250,5 that the high pressure liquid chromatography of the D steps, which isolates and purifies,μM C18Chromatographic column is
Stationary phase, using 52% methanol as mobile phase, flow rate of mobile phase is 3 mL/min, and UV-detector Detection wavelength is 338 nm, often
Secondary sample introduction 50μL, chromatographic peak during 25.2 min is collected, is evaporated after repeatedly adding up.
The application of the present invention is application of the described mouth xanthones compounds in 5α-reductase inhibitor is prepared.
Black purple Swertia patens raw material used in the present invention can not realized by territorial restrictions, the black purple Swertia patens in any source place
The present invention, to pick up from the black purple Swertia patens raw material in Yunnan Province enlightening celebrating area, the present invention will be further described below.
In the examples where no specific technique or condition is specified, according to the technology described by document in the art or condition or
Person is carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can be by buying the normal of acquisition
Advise product.If the solution in the present invention only gives solute, solvent is not disclosed, then those skilled in the art should know molten
Agent is water.Pure chloroform refers to 100% chloroform in the present invention, and pure acetone refers to 100% acetone, and pure methanol refers to 100% methanol.
Embodiment 1
To pick up from the black purple Swertia patens in Yunnan Province enlightening celebrating area as raw material.Black purple Swertia patens herb is sampled into 2.5 kg to dry
Crush, extracted at room temperature 4 times with 95% ethanol, every time extraction 3 days, each 3.5L of extract solution, extract solution merges, filtering, and decompression is dense
Shorten medicinal extract into, obtain medicinal extract 121g.Medicinal extract MCI posts decolourize, and elution requirement is methanol/water 90:10, medicinal extract after decolouring is with 240
The dissolving of gram chloroform/methanol mixed solvent, then adds 120 grams of 100 mesh silica gel and mixes sample.It is dry with 600 grams of 100 mesh silica gel after mixing sample
Method fills post；1 is followed successively by with volume ratio:0、9:1、8:2、7:3、6:4、1:1 and 0:1 chloroform/acetone solution carries out gradient elution,
Gradient eluent, concentration are collected, merges identical part, wherein volume proportion is 7:3 elution fractions are passed through in anti-phase C18 and pressed
Liquid chromatogram, the medium pressure liquid chromatography is using the mm of 21.2 mm × 250,5μM C18Chromatographic column, flow rate of mobile phase 20
ML/min, mobile phase are 45% acetonitrile, and UV-detector Detection wavelength be 210,230 nm, each mL of sample introduction 2, collection 20 ~
Eluent corresponding to 35 min chromatographic peaks, dissolved with methanol after eluent desolvation, lysate is passed through high pressure liquid phase color again
Spectrum is isolated and purified, and the high pressure liquid chromatography is using the mm of 9.4 mm × 250,5μM C18Chromatographic column, flow rate of mobile phase are
3 mL/min, mobile phase are 52% methanol, and UV-detector Detection wavelength is 338 nm, each sample introduction 50μL, collect chromatogram
Peak retention time eluent corresponding when being 25.2 min, it is evaporated after repeatedly adding up, produces the mouth xanthones compounds.
Embodiment 2
To pick up from the black purple Swertia patens in Yunnan Province enlightening celebrating area as raw material.Black purple Swertia patens herb sampling 2.0kg is dried
Crush, extracted at room temperature 3 times with 95% ethanol, every time extraction 3 days, each 3.0L of extract solution, extract solution merges, filtering, and decompression is dense
Shorten medicinal extract into, obtain medicinal extract 105g.Medicinal extract MCI posts decolourize, and elution requirement is methanol/water 90:10, medicinal extract after decolouring is with 200
The dissolving of gram chloroform/methanol mixed solvent, then adds 100 grams of 80 mesh silica gel and mixes sample.After mixing sample, with 80 mesh silica gel, 500 grams of dry method
Fill post；1 is followed successively by with volume ratio:0、9:1、8:2、7:3、6:4、1:1 and 0:1 chloroform/acetone solution carries out gradient elution, receives
Collect gradient eluent, concentration, merge identical part, wherein volume proportion is 7:3 elution fractions are passed through hydraulic fluid in anti-phase C18
Phase chromatogram, the medium pressure liquid chromatography is using the mm of 21.2 mm × 250,5μM C18Chromatographic column, flow rate of mobile phase are 20 mL/
Min, mobile phase are 45% acetonitrile, and UV-detector Detection wavelength is 210,230 nm, each mL of sample introduction 2, collects 20 ~ 35
Eluent corresponding to min chromatographic peaks, dissolved with methanol after eluent desolvation, lysate is passed through high pressure liquid chromatography and entered again
Row isolates and purifies, and the high pressure liquid chromatography is using the mm of 9.4 mm × 250,5μM C18Chromatographic column, flow rate of mobile phase 3
ML/min, mobile phase are 52% methanol, and UV-detector Detection wavelength is 338 nm, each sample introduction 50μL, collect chromatographic peak
Retention time eluent corresponding when being 25.2 min, it is evaporated after repeatedly adding up, produces the mouth xanthones compounds.
Embodiment 3
Compound prepared by Example 1, is light yellow gum thing；
Assay method is：With nuclear magnetic resonance, structure is identified with reference to other spectroscopic techniques.
High resolution mass spectrum (HRESIMS) provides quasi-molecular ion peakm/z 337.1059 [M+Na]+(calculated value
337.1052).With reference to1H and13 C H NMR spectroscopies provide a molecular formula C18H18O5, degree of unsaturation 10.Ultraviolet spectra 338,
There is absorption maximum at 256 and 210 nm, it was demonstrated that aromatic ring structure in compound be present；Ir data is also confirmed that in compound
Hydroxyl ((3420 cm be present-1)), carbonyl (1654 cm-1) and the cm of aromatic ring 1606,1529,1468-1) functional group.Compound1H and13C NMR data (table -1), which are shown in compound, 18 carbon and 18 hydrogen signals, including a 1,3,6,7- tetra- takes
Generation mouth mountain ketone skeleton [Ind. Crop. Prod, 58,125 (2014)] (C-1 ~ C-9, C-4a, C-8a ~ C10a；H-2,
H-4, H-5 and H-8), ethoxy (C-1 ' and a C-2 '；H2- 1 ' and H2- 2 '), the nuclear substituted methyl (C-3 ' of a virtue
And H3- 3 '), 2 methoxyl groups (δ H55.9 q and 56.3 q；3.80 s and 3.83 are s).H in compound be present2-1′ (δH
2.62) and C-5 (δ C 119.8)、C-6 (δ C 141.3)、C-7 (δ C158.5), H2-2′ (δH3.64) and C-6 (δ C
141.3), and H-5 (δ H7.02) and C-1 ' (δ C 35.2) HMBC is related (Fig. 3), it can be verified that being substituted in of ethoxy
The C-6 positions of compound.According to H3-3′ (δH2.38) and C-2 (δ C 112.2)、C-3 (δ C 147.2)、C-4 (δ C 108.6)
HMBC it is related, it can be verified that methyl is substituted in the C-3 positions of compound.Two methoxy substitutions can be by two first in C-1 and C-7
Epoxide hydrogen (δH 3.80 and 3.83) respectively with C-1 (δ C160.2) and C-7 (δ C158.5) HMBC correlations are demonstrate,proved
It is real.The typical proton signal of compound [δ H7.08 d (2.2), 6.87 d (2.2), 7.02 s, 7.43 s] also support this
The mouth mountain ketone B rings of invention are the 6, and 7- bis- substitutes, and C rings are that 1,3- bis- substitutes.So far the structure of the compounds of this invention is able to really
Fixed, Compound nomenclature is：6- (2- ethoxys) -1,7- dimethoxys -3- methyl-mouth mountain ketone.
Infrared, the ultraviolet and mass spectrometric data of compound：UV (methanol),λ max (log ε) 210 (4.06)、256
(3.64), 338 (3.47), IR (pressing potassium bromide troche)ν max 3420、3085、2928、1654、1606、1529、1468、
1412、1360、1273、1208、1158、1064、869 cm-1；1H NMR and13C NMR data (CDCl3, 500 and 125
MH), it is shown in Table -1；ESI-MS (positive ion mode)m/z 337 [M+Na]+;HR-ESI-MS (positive ion mode)m/z [M
+ Na]+337.1059 (calculated value 337.1052, C18H18NaO5)。
The compound of table -1.1H NMR and13C NMR data (solvent C5D5N)
| No. | d C | d H (m, J, Hz) | No. | d C | d H (m, J, Hz) |
| 1 | 160.2 s | 4a | 154.4 s | ||
| 2 | 112.2 d | 7.08 d (2.2) | 8a | 113.9 s | |
| 3 | 147.2 s | 9a | 110.2 s | ||
| 4 | 108.6 d | 6.87 d (2.2) | 10a | 148.2 s | |
| 5 | 119.8 d | 7.02 s | 1′ | 35.2 t | 2.62 t (7.2) |
| 6 | 141.3 s | 2′ | 63.2 t | 3.64 t (7.2) | |
| 7 | 158.5 s | 3′ | 24.2 q | 2.38 s | |
| 8 | 111.5 d | 7.43 s | 1-OMe | 55.9 q | 3.80 s |
| 9 | 176.7 s | 7-OMe | 56.3 q | 3.83 s |
Embodiment 4
The mouth xanthones compounds prepared in Example 1 carry out 5α-reductase inhibitory activity test, and test case is such as
Under：
The method that active testing is used in conjunction using dispersive liquid-liquid microextraction-makings is tested, and concrete operations are：By 50μg
Prostate microsomal protein, 500nM testosterones（T）, 1mM dithiothreitol (DTT)s（DTT）, 25μL DMSO are dissolved in 40mM sodium phosphates
Cushioning liquid（pH 6.5）Reaction solution is configured to, and is dissolved to 0.5 ml, 250 are added in reaction solutionμM nicotinamide adenine dinucleotide phosphates
（NADPH）Start enzymatic reaction, and cultivated 60 minutes in 37 DEG C of vibration water-baths.Afterwards 500 are added in ice bath to reaction solutionμL MeCN stop enzymatic reaction.The mode of the mouth xanthones compounds of preparation, testosterone and dihydrotestosterone dispersive liquid-liquid microextraction
Extracted.Reaction solution continuously adds 50μL internal standard compounds T-d3（175 nM）And DHT-13C3（175 nM）And 50μL trichlorines
Ethene, and be transferred in the conical pipe equipped with 3 ml water.Conical pipe is closed, manually after concussion, is centrifuged 3 minutes with 5000 × g,
After the completion of centrifugation, by 30 including the mouth xanthones compounds comprising preparationμL lower floors material is transferred in new bottle, is used
Gentle nitrogen stream is dried at room temperature for.30 are added after the completion of dryingμl MSTFA+NH4I+DTE（500:4:2 vol/wt/wt）
In domestic microwave（600 w）Lower reaction makes compound carry out silylation reactive for 5 minutes.After the completion of reaction, 1 is extractedμL is anti-
Thing is answered to carry out GC/MS analyses.T/ T-d3With DHT/ DHT-13C3Between ratio can to testosterone and by 5α-reductase it is anti-
The dihydrotestosterone that should be generated is quantified, and test compound is determined by contrasting the amount of the dihydrotestosterone generated after enzymatic reaction
5α-reductase inhibitory activity.
It is the positive control for testing the mouth xanthones compounds 5α-reductase inhibitory activity prepared using finasteride,
Finasteride is dissolved in DMSO, and with 40mM buffer solution of sodium phosphate（pH 6.5）It is diluted, extracts 1μM dilution
Liquid carries out enzymatic reaction.To determine ICs of the finasteride to 5α-reductase inhibitory activity50Value, using various concentrations
finasteride （0.01~1μM）Tested, the equal parallel determination of each concentration 3 times.
By above method of testing, it is determined that the 5α-reductase inhibiting rate of the mouth xanthones compounds prepared for 68.4 ±
4.3%, display compound has prominent 5α-reductase inhibitory activity.
Claims (7)
1. a kind of mouthful xanthones compounds, it is characterised in that the mouth xanthones compounds are from dry black purple Swertia patens herb
In it is isolated, its molecular formula is C18H18O5, it is named as：6- (2- ethoxys) -1,7- dimethoxys -3- methyl-mouth mountain ketone, tool
There is following structural formula：
。
2. a kind of preparation method of mouth xanthones compounds according to claim 1, it is characterised in that with dry black purple
Swertia patens herb is raw material, extracted through medicinal extract, MCI decolourings, silica gel column chromatography, high pressure liquid chromatography step, be specially：
A, medicinal extract extracts：It is raw material to take dry black purple Swertia patens herb, is crushed, 3 ~ 5 are extracted at room temperature with 95% ethanol
It is secondary, 3 days every time, merge extract solution, filtering, be concentrated under reduced pressure extract solution, stands, filters out sediment, be condensed into medicinal extract a；
B, MCI decolourizes：Medicinal extract a MCI posts decolourize, and are eluted with 80 ~ 95% methanol aqueous solutions, merge organic phase, are concentrated under reduced pressure into leaching
Cream b；
C, silica gel column chromatography：Medicinal extract b carries out silica gel column chromatography with 80 ~ 100 mesh silica gel dry column-packings of the weight than 8 ~ 10 times of amounts；With
Volume proportion is 1:0、9:1、8:2、7:3、6:4、1:1 and 0:1 chloroform/acetone solution carries out gradient elution, collects gradient and washes
De- liquid, concentration, merge identical part；
D, high pressure liquid chromatography separates：The 7 of step C eluent:The inverted C18 medium pressure liquid chromatographies in 3 parts isolate and purify, institute
Eluent corresponding to 20 ~ 35min chromatographic peaks is obtained, is further isolated and purified through high pressure liquid chromatography, produces described mouth mountain ketone
Compound.
3. the preparation method of according to claim 2 mouthful of xanthones compounds, it is characterised in that the medicinal extract b of the step C
Before through silica gel column chromatography, dissolved with chloroform/methanol mixed solvent of the weight than 1.5 ~ 3 times of amounts, then weigh 1 ~ 1.5 times with medicinal extract
80 ~ 100 mesh silica gel mixed samples.
4. the preparation method of according to claim 2 mouthful of xanthones compounds, it is characterised in that the D steps it is anti-phase
It is with 21.2mm × 250mm, 5 that C18 medium pressure liquid chromatographies, which isolate and purify,μM C18Chromatographic column is stationary phase, using 45% acetonitrile as
Mobile phase, flow velocity 20mL/min, UV-detector Detection wavelength are 338nm, each sample introduction 2mL, collect 20 ~ 35min color
Spectral peak.
5. the preparation method of according to claim 2 mouthful of xanthones compounds, it is characterised in that the 20 of the D steps ~
Eluent corresponding to 35min chromatographic peaks through high pressure liquid chromatography before isolating and purifying, first desolvation again with methanol dissolving.
6. the preparation method of according to claim 2 mouthful of xanthones compounds, it is characterised in that the high pressure liquid of the D steps
Phase chromatographic separation and purification is with 9.4mm × 250mm, 5μM C18Chromatographic column is stationary phase, using 52% methanol as mobile phase, flowing
Phase flow velocity is 3mL/min, and UV-detector Detection wavelength is 338nm, each sample introduction 50μL, chromatographic peak during 25.2min is collected,
It is evaporated after repeatedly adding up.
A kind of 7. application of the mouth xanthones compounds in 5α-reductase inhibitor is prepared described in claim 1.
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