CN109293729A - Two kinds of steroid compounds and a kind of triterpene compound and its extracting method and application - Google Patents

Two kinds of steroid compounds and a kind of triterpene compound and its extracting method and application Download PDF

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CN109293729A
CN109293729A CN201811224405.8A CN201811224405A CN109293729A CN 109293729 A CN109293729 A CN 109293729A CN 201811224405 A CN201811224405 A CN 201811224405A CN 109293729 A CN109293729 A CN 109293729A
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compound
polar solvent
pharmaceutically acceptable
highly polar
steroid
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CN109293729B (en
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何祥久
王宜海
严飞
黄雨颖
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Guangdong Pharmaceutical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • C07J71/001—Oxiranes
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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Abstract

The invention discloses two kinds of steroid compounds and a kind of triterpene compound and its extracting method and applications, belong to field of medicaments.The compound is novel steroid compound and triterpene compound, and it is identified by physicochemical constant and Modern spectroscopy, it specifies its physicochemical property and chemical structure, provides strong reference using the value of triterpenes in banaba and steroid chemical component for further research and development；Separation and purification method of the invention is simple, efficiently and mild；Simultaneously, pharmacodynamics test shows: two kinds of novel steroid compounds provided by the invention have preferable extracorporeal anti-inflammatory activity, there is anti-inflammatory activity to BV-2 cell, show that there is the further exploitation as the great potential for preparing novel anti-inflammatory disease or drug for treating Alzheimer's disease, for anti-inflammatory or drug for treating Alzheimer's disease to lay a good foundation for the compound, its tautomer and its pharmaceutically acceptable salt.

Description

Two kinds of steroid compounds and a kind of triterpene compound and its extracting method and application
Technical field
The invention belongs to field of medicaments, and in particular to two kinds of steroid compounds and a kind of triterpene compound and its extraction Method and application.
Background technique
Inflammation is that have the living tissue of vascular system to defense reaction caused by damage factor, most of diseases all companions There are the generation of inflammation, the occurrence and development that inflammation can aggravate disease, some chronic inflammations will lead to the generation of tumour, therefore, right The control and treatment of inflammation have very important significance.Tumour refer to body the various tumorigenesis factors effect under, local organization Hyperplasia is formed by neoformation, it is current one of the primary killers for threatening human health, there is no effectively cure at present Drug, the treatment for tumour are always the content that scientific research is constantly studied.
The drug that natural drug especially derives from plant has the spy of chemical structure diversity and diverse biological activities Point is always the main source that the mankind prevent and treat disease.The many drugs clinically applied all directly or indirectly derive from Natural products, natural products act not only as the semi-synthetic precursor of drug, are also used as the template of chemical synthetic drug, New approaches are provided for new drug design, currently, natural products has become discovery one of novel drugs or the main source of lead compound.
Banaba (Lagerstroemia speciosa (L.) Pers), also known as Lagerstroemia speciosa (Lagerstroemia Flos-reginae Retz), it is commonly called as crape myrtle, Lythraceae (Lythraceae) Lagerstroemia (Lagerstroemia) plant, It falls leaves high megaphanerophyte.Tropical Asian is originated in, Sri Lanka is distributed in, India is Malaysian, Philippine, Vietnam, and China is wide There is cultivation in east, Guangxi, Fujian.It is mild-natured, it is mildly bitter flavor, puckery, return liver, stomach, large intestine channel.Bark and leaf can make cathartic；Seed has fiber crops Liquor-saturated property；Root contains tannin, can make astringent；Leaf has hypoglycemic, anti-oxidant and antimycotic activity.
Modern pharmacology experimental study shows: banaba has hypoglycemic, lipid-loweringing, anti-oxidant, antimycotic isoreactivity.Its master Wanting active constituent is triterpene and Analysis of Steroids, but existing research is still not thorough enough to the chemical constitution study of banaba, because And the triterpene and steroid chemical component in banaba are still worth further research and development to utilize.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide two kinds of new steroidals of separation and Extraction in banaba Class compound and a kind of new triterpene compound and its extracting method and application.
The technical solution used in the present invention is:
The present invention has carried out system separation to the triterpene and Analysis of Steroids of banaba, obtains two kinds of novel steroids Compound and a kind of novel tetraterpene class compound, and its chemical structure and anti-inflammatory anti-tumor activity are researched and analysed.
Two kinds of steroid compounds and a kind of triterpene compound, tautomer and its pharmaceutically acceptable salt, should The structural formula of compound is as follows:
Preferably, above two steroid compound and a kind of triterpene compound pharmaceutically acceptable salt be its sodium, Potassium, calcium, magnesium, iron, ferrous iron, lead, barium, copper, ammonium or zinc salt.
Above two steroid compound and a kind of extracting method of triterpene compound, include the following steps:
(1) fruit of banaba crushed into drying, alcohol extracting, obtain extracting solution；
(2) extracting solution is successively extracted with low polar solvent, middle polar solvent and highly polar solvent, obtains middle polarity Layer；
(3) middle polar layer is subjected to silica gel column chromatography, the middle highly polar solvent of polarity-carries out gradient elution, obtains volume ratio The elution fraction of 80:1 is denoted as sample A (drying sample)；
(4) silica gel column chromatography is carried out to sample A, after the middle highly polar solvent gradient elution of polarity-, passes through reversed phase thin-layer point Analysis, centering polarity and highly polar solvent volume are than the elution fraction for 3:1-carry out ODS column chromatography, lower alcohol or its aqueous solution ladder It after degree elution, is analyzed by silica gel and reversed phase thin-layer, half preparation reverse phase is carried out to its 40% aqueous lower alcoholic solutions elution fraction HPLC obtains compound 1；
(5) silica gel column chromatography is carried out to sample A, after the middle highly polar solvent gradient elution of polarity-, centering polarity and highly polar Solvent volume after lower alcohol or its aqueous solution gradient elution, passes through thin layer point than carrying out ODS column chromatography for the elution fraction of 6:1 Analysis carries out silica gel column chromatography to its 50% lower alcohol aqueous solvent elution fraction, after the middle highly polar solvent gradient elution of polarity-, leads to Cross reverse phase and silica gel thin-layer chromatography spectrum analysis, the elution fraction that the volume ratio of centering polarity and highly polar solvent is 15:1 carries out half and makes Standby reversed-phase HPLC obtains compound 2；
(6) silica gel column chromatography is carried out to sample A, after the middle highly polar solvent gradient elution of polarity-, centering polarity and highly polar Solvent volume is than carrying out silica gel column chromatography for the elution fraction of 8:1, in after the highly polar solvent gradient elution of polarity-, by anti- Phase tlc analysis, centering polarity and highly polar solvent volume carry out mesolow ODS column chromatography than the elution fraction for 5:1, rudimentary After alcohol or its aqueous solution gradient elution, by reverse phase and silica gel thin-layer chromatography spectrum analysis, to its 40% aqueous lower alcoholic solutions elution portion Point partly being prepared reversed-phase HPLC obtains compound 3；
Wherein, lower alcohol refers to the alkylol of C1-6.
Preferably, it is 35 μm~64 μm that banaba fruit, which is crushed to partial size, in step (1).
Preferably, step (1) is extracted using 60~90% alcohol.
It is highly preferred that step (1) is extracted using 70% alcohol.
Preferably, alcohol used in step (1) alcohol extracting is ethyl alcohol.
In the case where non-special declaration, the alcohol of certain concentration of the invention refers to the concentration in its aqueous solution.
Preferably, step (1) alcohol extracting 1 time or more.
Preferably, step (1) alcohol extracting 1~5 time.
It is highly preferred that step (1) alcohol extracting 4 times.
Preferably, the volume of alcohol and the weight ratio of banaba fruit are (3~5) L:1Kg in step (1).
It is highly preferred that the volume of alcohol and the weight ratio of banaba fruit are 4.8L:1Kg in step (1).
Preferably, step (2), (3), (4), (5), the low polar solvent in (6) are selected from hexamethylene, petroleum ether, hexane, different At least one of hydrocarbon solvents such as octane, trimethylpentane, pentamethylene, heptane；Middle polar solvent is selected from ethyl acetate, chlorine At least one of imitative, methylene chloride, ether, methyl formate, nitromethane, butyl acetate, isopropyl ether；Highly polar solvent is selected from At least one of n-butanol, methanol, the tert-butyl alcohol, propyl alcohol, isopropanol, ethyl alcohol, acetone, tetrahydrofuran, pyridine.
Preferably, low polar solvent is selected from hexamethylene in step (2)；Middle polar solvent is selected from ethyl acetate；It is highly polar molten Agent is selected from n-butanol.
Preferably, the highly polar solvent gradient elution sequence of middle polarity-in step (3) are as follows: 100:1,80:1,50:1,30: 1,20:1,15:1,10:1,5:1,3:1,1:1,0:1.Wherein, the middle polar solvent is chloroform, and highly polar solvent is methanol.
Preferably, step (4), the middle polar solvent in (6) are selected from chloroform；Highly polar solvent is selected from acetone.
Preferably, step (5) carries out silica gel column chromatography to sample A, the middle pole in the middle highly polar solvent gradient elution of polarity- Property solvent be selected from chloroform；Highly polar solvent is selected from acetone.In second in the highly polar solvent gradient elution of polarity-, middle polarity is molten Agent is selected from hexamethylene, and highly polar solvent is selected from ethyl acetate.
Preferably, step (4), (5), the lower alcohol in (6) are selected from least one of methanol, ethyl alcohol, propyl alcohol.Wherein, Concentration in the aqueous lower alcoholic solutions refers to the percentage by volume of lower alcohol in solution.
It is highly preferred that step (4), (5), the lower alcohol in (6) are selected from methanol.
Preferably, when the middle polar solvent in step (4) is selected from chloroform, highly polar solvent is selected from acetone, and lower alcohol is selected from When methanol, chloroform-acetone gradient elution sequence is the chloroform-acetone solution of 100:1~0:1 (volume ratio), and methanol-water gradient is washed De- sequence is 10% methanol aqueous solution~100% methanol aqueous solution.
Preferably, when step (5) carries out silica gel column chromatography, the middle pole of the middle highly polar solvent gradient elution of polarity-to sample A Property solvent be selected from chloroform, highly polar solvent is selected from acetone, and when lower alcohol is selected from methanol, methanol-water gradient elution sequence is 10% first Alcohol solution~100% methanol aqueous solution, chloroform-acetone gradient elution sequence are the chloroform-the third of 100:1~0:1 (volume ratio) Ketone solution.In second in the highly polar solvent gradient elution of polarity-, middle polar solvent is selected from hexamethylene, and highly polar solvent is selected from Ethyl acetate, cyclohexane-ethyl acetate gradient elution sequence is 20:1~0:1 (volume ratio).
Preferably, when the middle polar solvent in step (6) is selected from chloroform, highly polar solvent is selected from acetone, and lower alcohol is selected from When methanol, chloroform-acetone gradient elution sequence is 100:1~0:1 (volume ratio), and methanol-water gradient elution sequence is 10% methanol Aqueous solution~100% methanol aqueous solution.
The present invention also provides above two steroid compound and a kind of triterpene compound pharmaceutically acceptable salts The preparation method comprises the following steps: steroid compound is dissolved in a solvent with corresponding basic salt, steroid chemical combination is settled out from solution Object pharmaceutically acceptable salt；Triterpene compound is dissolved in a solvent with corresponding basic salt, three are settled out from solution Terpenoid pharmaceutically acceptable salt.
Preferably, above two steroid compound and a kind of preparation side of triterpene compound pharmaceutically acceptable salt Method are as follows: any one in the compound 1,2,3 that extracts of the present invention is mixed in a solvent with basic salt, stirring and dissolving, standing, Sediment is isolated to get pharmaceutically acceptable salt.
Preferably, above-mentioned basic salt is selected from basic lead acetate, alkali formula calcium acetate, alkali formula magnesium acetate, basic ferric acetate, alkali formula Ferrous acetate, zinc acetate basic, barium hydroxide, sodium hydroxide, any one in potassium hydroxide.
Preferably, above-mentioned solvent is selected from least one of water, ethyl alcohol, methanol, butanol, amylalcohol.
Preferably, mixing time is 0.1~30min, and whipping temp is 40~80 DEG C.
It is highly preferred that mixing time is 8~12min, whipping temp is 45~60 DEG C.
Preferably, it is placed in 0.1~60min of standing in 2~6 DEG C.
It is highly preferred that being placed in 20~40min of standing in 3~5 DEG C.
Preferably, the molar ratio of compound 1 and basic salt is 1:1~6, the molar ratio of compound 2 and basic salt be 1:1~ 2, the molar ratio of compound 3 and basic salt is 1:1~4.
The study shows that: the steroid compound of said extracted is more significant to the inhibiting effect of inflammatory mediator NO, and it is imitated Fruit is substantially better than anti-inflammatory medicaments rice Lip river ring element, illustrates the steroid compound, its tautomer and its pharmaceutically acceptable Salt has as the great potential for preparing novel anti-inflammatory disease drug.
In addition, Alzheimer's disease (AD) is the nervous centralis degeneration of a kind of progressive cognitive disorder and memory injury Disease seriously affects the ability to work and life quality of patient with the change of emotion and personality.Nowadays A Erzhihaimo disease Disease incidence increase year by year.The major pathologic features of AD are that extracellular β-amyloid proteins deposit the albumen patch to be formed and intracellular The neurofibrillary tangles that microcosmic relevant protein hyperphosphorylation is formed, eventually leads to inflammation, oxidative stress, neuronal death Etc. causing one to be class AD illness.Microglia (BV-2) is central immune cell, can inhibit beta amyloid egg under state of activation White deposition and aggregation.A large number of studies show that many factors cause BV-2 to activate the Neuroinflammation of initiation in turn in the hair of AD Play the part of important role in exhibition, test proves: the steroid compound of extraction has anti-inflammatory activity, thus the steroid to BV-2 cell Body class compound, its tautomer and its pharmaceutically acceptable salt, which have to be used as, prepares novel therapeutic Alzheimer's disease medicine The great potential of object.
It is prepared by above-mentioned steroid compound and triterpene compound, tautomer and its pharmaceutically acceptable salt Application in anti-inflammatory medicaments or treatment Alzheimer's disease or anti-tumor drug.
Preferably, above-mentioned inflammation is neuroinflamation, pneumonia, hepatitis, mazoitis, gastritis, bursal synovitis, thrombosis-obstructive vessel Any one inflammation scorching, in myocarditis.
A kind of anti-inflammatory or treatment Alzheimer's disease or anti-tumor drug, active constituent include above-mentioned steroid Close object, tautomer and its pharmaceutically acceptable salt further include pharmaceutically acceptable carrier, diluent, excipient, steady Determine agent, antioxidant.
Preferably, carrier be selected from starch, chitosan, alginic acid, agar, fibrin, collagen, polyphosphoric acid esters, At least one of polyurethanes, polyacids anhydride, liposome, polyethylene glycol, mannose, galactolipin, povidone.
Preferably, diluent is selected from least one of microcrystalline cellulose, lactose, mannitol, starch, saccharin.
Preferably, excipient is selected from least one of mannose, glycine, lactose, sodium chloride, glucose.
Preferably, stabilizer is selected from albumin, collagen, cyclodextrin and its derivative, polyethylene glycol, tween, sapn, dextrorotation At least one of glucosides, mannitol.
Preferably, antioxidant is selected from VC, VE, benzoic acid, citric acid and its salt, sorbic acid, sodium sulfite, bisulfite At least one of sodium, sodium pyrosulfite, sodium thiosulfate.
Preferably, above-mentioned anti-inflammatory or drug for treating Alzheimer's disease be selected from oral agents, injection, powder, granule, Capsule, pill, tablet, bolt i.e., film, aerosol, spray, powder spray, sustained release and controlled release agent, targeting preparation, in pulvis Any one dosage form.
Above-mentioned steroid compound and triterpene compound, tautomer and its pharmaceutically acceptable salt are being made Application in standby health care product.
A kind of health care product includes above-mentioned steroid compound and/or triterpene compound in the health care product, mutually makes a variation Structure body and its pharmaceutically acceptable salt.
The present invention is experiments have shown that the anti-proliferative capacity of above-mentioned three kinds of compounds cancer cell different types of for three kinds is more positive Property medicine cis-platinum it is bad it, but also have certain antiproliferative effect.And the compound is extracted from banaba fruit Out, so having the prospect for being added to and using in health care product as routine healthcare.
The beneficial effects of the present invention are:
1, the present invention extracts two kinds of steroid compounds and a kind of triterpene compound from banaba fruit, passes through Physicochemical constant and Modern spectroscopy are identified, specify its physicochemical property and chemical structure, for further research and development benefit Strong reference is provided with the value of steroid in banaba fruit and triterpenes chemical component.
2, separation and purification method of the invention is simple, efficient, and mild, can fully save steroid and triterpenes The component of object is closed, and its structure is clear, it is quality controllable.
3, pharmacodynamics test shows: steroid compound and triterpene compound provided by the invention have preferable external Anti-inflammatory activity has anti-inflammatory activity to BV-2 cell, shows that the steroid compound, its tautomer and its pharmacy can connect The salt received have be used as the great potential for preparing novel anti-inflammatory disease or drug for treating Alzheimer's disease, be anti-inflammatory or treat Ah The further exploitation of the silent medicine in Wurz sea is laid a good foundation.
4, the present invention experiments have shown that above-mentioned three kinds of compounds cancer cell different types of for three kinds anti-proliferative capacity compared with Positive drug cis-platinum it is bad it, but also have certain antiproliferative effect.And the compound is mentioned from banaba fruit It takes out, so having the prospect for being added to and using in health care product as routine healthcare.
Detailed description of the invention
Fig. 1 is compound 11H-NMR spectrum；
Fig. 2 is compound 113C-NMR spectrogram；
Fig. 3 is 135 spectrogram of DEPT of compound 1；
Fig. 4 is the HR-ESI-MS spectrogram of compound 1；
Fig. 5 is the hsqc spectrum figure of compound 1；
Fig. 6 is the HMBC spectrogram of compound 1；
Fig. 7 is compound 21H-NMR spectrum；
Fig. 8 is compound 213C-NMR spectrogram；
Fig. 9 is 135 spectrogram of DEPT of compound 2；
Figure 10 is the HR-ESI-MS spectrogram of compound 2；
Figure 11 is the hsqc spectrum figure of compound 2；
Figure 12 is the HMBC spectrogram of compound 2；
Figure 13 is the NOESY spectrogram of compound 2；
Figure 14 is compound 31H-NMR spectrum；
Figure 15 is compound 313C-NMR spectrogram；
Figure 16 is 135 spectrogram of DEPT of compound 3；
Figure 17 is the HR-ESI-MS spectrogram of compound 3；
Figure 18 is the hsqc spectrum figure of compound 3；
Figure 19 is the HMBC spectrogram of compound 3；
(A) is protein expression of the compound to LPS BV-2 the cell COX-2 and INOS induced of various concentration in Figure 20 Figure；(B) quantify figure for the expressing quantity to (A).
Specific embodiment
Enumerate embodiment further below with the present invention will be described in detail.It will similarly be understood that following embodiment is served only for this Invention is further described, and should not be understood as limiting the scope of the invention, those skilled in the art are according to the present invention Some nonessential modifications and adaptations that the principle of elaboration is made all belong to the scope of protection of the present invention.Following specific works of example Skill parameter etc. is also only an example in OK range, i.e., those skilled in the art can explanation through the invention do properly Selection in range, and do not really want to be defined in the exemplary specific data of description of the invention.
Embodiment 1
One, the extraction of steroid compound and triterpene compound
(1) dry banaba fruit 20kg is taken, the two batches for being divided into equivalent are extracted, and every batch of is with 60 liter of 70% ethyl alcohol at 60 DEG C Lower refluxing extraction four times, merging all extracting solutions and being concentrated under reduced pressure into extracting liquid volume is 15L；
(2) extracting solution in step 1) is successively extracted with isometric hexamethylene, chloroform, ethyl acetate and n-butanol It takes, obtains 109.5g chloroform layer；
(3) by chloroform layer (114.9g) carry out silica gel column chromatography, using chloroform methanol gradient elution (100:1,80:1,50: 1,30:1,20:1,15:1,10:1,5:1,3:1,1:1,0:1；V/v), the sample A (26.7863g) of 80:1 elution fraction is obtained；
(4) A is divided into 10 parts through silica gel column chromatography with chloroform-acetone gradient elution (100:1~0:1:v/v), It is analyzed by reversed phase thin-layer, to its 4th part A-8, (volume ratio is the elution fraction of the chloroform-acetone solution of 3:1, it may be assumed that 3:1 chlorine Imitative-acetone elution fraction) mesolow ODS column chromatography is carried out, after carrying out gradient elution with methanol aqueous solution (20%~100%), It is analyzed by silica gel and reversed phase thin-layer, to wherein the 3rd fraction (40% methanol-water elution fraction), then carries out HPLC preparation, obtain Compound 1；
(5) by A-5 (elution fraction that sample A is eluted through chloroform-acetone solvent that volume ratio is 6:1) through mesolow ODS Column chromatography carries out gradient elution with methanol-water (30%~100%), by silica gel thin-layer chromatography spectrum analysis, to its 4th fraction (50% methanol-water elution fraction) carries out silica gel column chromatography, with cyclohexane-ethyl acetate gradient elution (20:1~0:1；V/v after) It is divided into 5 parts, HPLC preparation is carried out again to get chemical combination to its 3rd fraction (15:1 cyclohexane-ethyl acetate elution fraction) Object 2；
(6) elution fraction for eluting A-4 sample A through chloroform-acetone solvent that volume ratio is 8:1, it may be assumed that 8:1 chloroform-the third Ketone elution fraction) through silica gel column chromatography, with chloroform-acetone gradient elution (30:1~0:1；V/v), it is divided into 10 part parts, leads to After crossing reversed phase thin-layer analysis, the 6th part (5:1 chloroform-acetone elution fraction) is subjected to mesolow ODS column chromatography, uses methanol-water (20%~100%) after carrying out gradient elution, after reverse phase and silica gel thin-layer chromatography spectrum analysis, to wherein the 2nd fraction (40% Methanol-water elution fraction) carry out HPLC preparation again to get compound 3.
Two, the identification of steroid compound
1, the identification of compound 1:
The compound 1 isolated and purified is white amorphous powder, the aobvious red of 10% sulfuric acid-ethyl alcohol.
Further compound 1 is carried out1H-NMR、13C-NMR, DEPT135, HR-ESI-MS, HSQC, HMBC spectrum analysis, knot Fruit sees Fig. 1~6:
By Fig. 1's1Known to H-NMR map: in high field region, there are two methyl singlets signal δH0.63 (3H, s) and 1.16 (3H, s), the bimodal signal δ of four methylH0.81 (3H, d, J=6.9Hz), 0.83 (3H, d, J=6.6Hz), 0.90 (3H, d, J =6.9Hz), 0.99 (3H, d, J=6.5Hz).In δHAt 3.13 (1H, s) and 3.87 (1H, m), thus it is speculated that there may be two companies The proton signal of oxygen, in addition in δH5.13 (1H, dd, J=15.2,7.8Hz), 5.21 (1H, dd, J=15.2,7.8Hz), 5.44 (1H, m) go out prompt, and there are three alkene hydrogen signals, and two of them alkene hydrogen signal intercouples, and are one group of alkene hydrogen signal.Tool Volume data is shown in Table 1 column in 1.
By Fig. 2's13Known to C-NMR map: the compound shares 28 carbon signals, δCIt 62.6,68.2,68.6 is three The even carbon signal of oxygen, δC121.0,132.4,135.4,136.9 be four olefinic carbon signals, and δC132.4,135.4 being ergot steroid The double bond characteristic signal that alcohol compound is 22,23, δC208.2 be a carbonyl carbon signals.It again can by the DEPT135 of Fig. 3 , wherein primary carbon has 6, and secondary carbon has 6, and tertiary carbon 11, quaternary carbon has 5.Specific data are shown in Table 1 column in 1.
The HR-ESI-MS map of Fig. 4 shows quasi-molecular ion peak m/z 449.3024 [M+Na]+(Calcd for C28H42O3Na, 449.3026), prompting its molecular weight is 426, in conjunction with1H-NMR,13C-NMR, DEPT135 can determine its molecular formula For C28H42O3。
From the HSQC map of Fig. 5: first from δH3.87 (H-3) set out, with δC68.6 (C-3) are directly related；Again from δC 62.6 (C-6) set out, with δH3.13 (H-6) are directly related；And δC68.2 (C-5) determine C-5, C-6 without directly related hydrogen signal Between exist even oxygen ring structure；By δH5.13 (H-22), δH5.21 (H-23) and δC135.4 (H-22), 132.4 (H-23) are direct Correlation determines that there are the double bond signals of ergosterol.
By δ can be observed in the HMBC map of Fig. 6H5.44 (1H, m) and δC 38.7(C-10),41.4(C-12),48.1 (C-14) there are long-range correlation, δC136.9,121 and δHThere are long-range correlation, δ by 2.21 (H-12)H1.16 (H-19) and δC 136.9 existing long-range related.Finally determine position of double bond at C9 and C11.
The above analysis can determine that compound 1 is 5 α, 6 α-epoxy-3 β-hydroxy- (22E, 24R)- Ergosta-9 (11), 22-diene-7-one, structural formula are as follows:
2, the identification of compound 2:
The compound 2 isolated and purified is green amorphous powder, the aobvious red of 10% sulfuric acid-ethyl alcohol.
Further compound 2 is carried out1H-NMR、13C-NMR, DEPT135, HR-ESI-MS, HSQC, HMBC and NOESY spectrum Analysis, as a result see Fig. 7~13:
By Fig. 7's1Known to H-NMR map: six methyl hydrogen signals occur in high field region, be δ respectivelyH 0.54(3H, S), 0.81 (3H, d, J=6.8Hz), 0.84 (3H, d, J=6.8Hz), 0.91 (3H, d, J=6.8Hz), 1.00 (3H, s), 1.01 (3H, s), the hydrogen signal on two company's oxygen carbon, is δ respectivelyH4.21 (1H, br s) and 4.38 (1H, br s), in addition, also There are four the signals of alkene hydrogen, are δ respectivelyH5.16 (1H, dd, J=15.2,7.8Hz), 5.21 (1H, br s), 5.23 (1H, dd, J=15.2,7.8Hz), 5.63 (1H, br d, J=6.5Hz), two of them alkene hydrogen signal intercouples, and is sentenced by coupling constant Disconnected, compound 2 is also ergosterol class compound.Specific data are shown in Table 2 column in 1.
By Fig. 8's13Known to C-NMR map: occurring 28 carbon signals in map altogether, wherein δC68.3,71.9,77.7 it is The carbon signal of three company's oxygen, δC120.8,122.6,132.3,135.5,137.3,139.0 be six double bond carbon signals, and its Middle δCIt 132.3,135.5 is the double bond carbon signal of feature on ergosterol class compound branch.Again by the DEPT135 map of Fig. 9 It can obtain, in the compound, primary carbon has 6, and secondary carbon has 6, and tertiary carbon has 11, and quaternary carbon has 5.Specific data are shown in Table 2 in 1 Column.
The HR-ESI-MS map of Figure 10 shows quasi-molecular ion peak m/z 451.3181 [M+Na]+(Calcd for C28H44O3Na, 451.3182), prompting the molecular weight of compound 2 is 428, binding compounds 21H-NMR,13C-NMR, DEPT135 can determine that its molecular formula is C28H44O3。
From the HSQC map of Figure 11: from δH4.21 (H-3) set out, and can find δC68.6 (C-3) directly phase It closes, from δH4.38 (H-6) set out, and can find δC62.6 (C-6) are directly related, finally by δH 5.16(H-22), 5.23 (H-23) set out, and can find δC135.5 (C-22) and 132.2 (C-23) are directly related therewith respectively.
From the HMBC map of Figure 12: from δH4.38 (H-6) set out, and can find δC77.7 (C-5) and δC 120.8 (C-7) long-range related to it, from δH1.01 (H-19) set out, and can find δC122.6 (C-11) are long-range related to it.To sentence Disconnected other two groups of double bonds out are located at the position of C-7,8 and C-9,11.
It is composed by the NOESY of Figure 13, it can be observed that δH4.35 (6-OH) and H-12 (α), judge 6-OH for α configuration, by δH4.21 (H-3), W1/2≈ 7.0Hz, can obtain 5-OH is beta comfiguration.
The above analysis, can determine compound 2 be (22E, 24R)-ergosta-7,9 (11), 22-triene-3 β, 5 β, 6 α-triol, structural formula are as follows:
Three, the identification of triterpene compound
1, the identification of compound 3:
The compound 3 isolated and purified is white amorphous powder, and it is to vanillic aldehyde-sulfuric acid and 10% sulfuric acid- Ethyl alcohol shows bluish violet, thus it is speculated that the compound is triterpene compound.
Further compound 3 is carried out for above-mentioned supposition1H-NMR、13C-NMR、DEPT135、HR-ESI-MS、HSQC、 The analysis of HMBC map, the result is shown in Figure 1 4~19:
By Figure 14's1Known to H-NMR map: there are 6 feature methyl signals of triterpene compound in high field region, respectively It is δH0.83 (3H, d, J=6.2Hz), δH0.94 (6H, overlapped), δH1.07 (3H, s), 1.28 (3H, s), 1.99 (3H, s), in δHAt 5.32 (1H, br s), 6.30 (1H, s), it is understood that there may be there are two the signals of alkene hydrogen, and 6.43 (1H, Br s) at then there may be the hydrogen signal of a phenolic hydroxyl group, specific data be shown in Table in 13 column.
By Figure 15's13Known to C-NMR map: there are 29 carbon signals altogether in map, wherein δC 10.8,17.0,17.1, 21.2,21.7,23.1 be the signal of methyl, δC 125.2,125.5,126.1,138.6,144.4,167.3,181.6,182.5 For unsaturated carbon signal, and δC125.5 and 138.6 be the feature carbon signal of Ursane triterpene structure.Again by Figure 16's DEPT135 map can obtain, wherein primary carbon has 6, and secondary carbon has 7, and tertiary carbon has 6, and quaternary carbon has 10.Specific data are shown in Table in 1 3 column.
The HR-ESI-MS map of Figure 17 shows quasi-molecular ion peak m/z 475.2818 [M+Na]+(Calcd for C29H40O4Na, 475.2818), prompting the molecular weight of compound 3 is 452, in conjunction with1H-NMR,13C-NMR, DEPT135 can determine it Molecular formula is C29H40O4。
From the HSQC map of Figure 18: from δH6.30 (H-1) set out, δC125.2 (C-1) are directly related, from δH 5.32 (H-12) set out, δC125.5 (C-12) are directly related.
From the HMBC map of Figure 19: from δH 1.28(CH3- 25) it sets out, δ can be foundC125.2 (C-1) are remote therewith Cheng Xiangguan, then from δH 1.99(CH3- 23) it sets out, δ can be foundC126.1 (C-4), δC167.3 (C-5) long-range phase therewith It closes, therefore, it is determined that there is two groups of double bonds to be located at C-1,2 and C-4,5 position.Then, from δH6.30 (H-1) set out, can be with Find δC144.4 (C-2), δC181.6 (C-3), δC167.3 (C-5) are long-range related therewith, from δH 1.99(CH3- 23) go out Hair, can find δC 181.6(C-3),δC 126.1(C-4),δC 167.3(C-5),δC43.6 (C-10) are associated, from And it can determine carbonyl and be located at the position C-3.
The above analysis, can determine compound 3 be 2-hydroxy-3-oxo-24-norursa-1,4,12- Triene-28-oic acid, structural formula are as follows:
Table 1
Note:aSample uses deuterated chloroform as solvent, and hydrogen spectrum is tested under conditions of 600MHz, and carbon is composed in 151MHz Under conditions of test.
bAll signals assignments are all dependent on DEPT, HSQC, HMBC and NOESY map.
Four, the anti-inflammatory activity of steroid and triterpene compound is tested
External inflammatory model is established using the BV-2 (mouse microglia) that LPS (lipopolysaccharides) induces, using MTT and Influence of the compounds of this invention to the BV-2 inflammatory mediator NO after LPS is induced, anti-inflammatory agent are investigated in Griess experiment Minocycline (meter Luo Huan element) is used as positive control.
1, MTT is tested
By BV-2 cell inoculation in 96 orifice plates, after culture 24 hours, test sample to be measured is added, is further cultured for using MTT afterwards for 24 hours Method measures inhibiting rate of the sample to tumor cell proliferation, cell proliferation inhibition rate=(negative control group OD value average value-sample sets OD value average value) ÷ (negative control group OD value average value-blank control group OD value average value) × 100%, and use CalcuSyn Software calculates the half-inhibitory concentration (IC50) of tested sample.
2, Griess is tested
By BV-2 cell inoculation in 96 orifice plates, after culture 24 hours, test sample to be measured is added, after being further cultured for for 24 hours, draws Each 50 μ L of hole culture solution, is added 50 μ L Griess A reagents and 50 μ L Griess B reagents mix, with microplate reader at 546nm Measurement OD value, the inhibiting rate that calculating generates NO, NO inhibiting rate=(model control group OD value average value-sample sets OD value is average Value) ÷ (model control group OD value average value-negative control group OD value average value) × 100%, and calculated with CalcuSyn software The half-inhibitory concentration (IC50) of tested sample.
Above-mentioned test result see the table below 2:
Table 2
3, Western Blot is tested
By in exponential phase of growth cell kind in 96 orifice plates, be separately added into various concentration compound 1~2 (5,10, 20μmol·L-1), (15,30, the 60 μm of olL of compound 3-1), and each group total protein is extracted in corresponding time point, using 10% Polyacrylamide gel carry out electrophoresis, routinely operating method carry out Western blot experiment, finally use ECL kit Develop and is imaged.Every group of experiment is repeated 3 times, and experimental result is shown in Figure 20, and wherein " * " in figure indicates LPS relative to the significant of CTL Property, conspicuousness of the " # " expression triterpene saponin componds relative to actin (actin),#P < 0.05,##P < 0.01,###p < 0.001；*P < 0.05,***P < 0.01,***P < 0.001).
To sum up, by the IC in 2 experimental result of table50It is worth it is found that the anti-inflammatory effect of 1,2 pairs of BV-2 cells of compound is compared with positive drug Indomethacin is eager to excel, and compound 1,2 is all steroid compound.The anti-inflammatory effect of compound 3 is weaker.This is the result shows that above-mentioned The steroid compound of extraction is more significant to the inhibiting effect of inflammatory mediator NO, and its effect is substantially better than anti-inflammatory medicaments rice Lip river Ring element.As shown in Figure 20: when concentration is 5 μm of olL-1, compound 1 can significantly lower LPS induction BV-2 cell iNOS egg White expression, when concentration is 15 μm of olL-1, compound 3 can significantly lower LPS induction BV-2 cell COX-2 and iNOS albumen Expression, and the significant difference of concentration and protein expression is then not present in compound 2.Prompt: compound 1 can be by inhibiting INOS's Expression, so that the biosynthesis of NO is lowered, and compound 3 then can be by inhibiting the expression of two kinds of albumen of COX-2 and iNOS come under The biosynthesis of NO is adjusted, this may be one of compound 1,3 anti-inflammatory mechanisms, and the anti-inflammatory mechanisms of compound 2 then need further to grind Study carefully.
Alzheimer's disease (AD) is the nervous centralis degenerative disease of a kind of progressive cognitive disorder and memory injury, With the changes of emotion and personality, the ability to work and life quality of patient are seriously affected, 70 years old or more the elderly is mainly in, Nowadays disease incidence increases year by year.The major pathologic features of AD are that extracellular β-amyloid proteins deposit the albumen patch to be formed and thin The neurofibrillary tangles that microcosmic relevant protein hyperphosphorylation intracellular is formed, eventually leads to inflammation, oxidative stress, neuron It is class AD illness that death etc., which causes one,.Microglia (BV-2) is central immune cell, can inhibit β-starch under state of activation Sample proteinosis and aggregation.A large number of studies show that many factors cause, BV-2 is activated and then the Neuroinflammation of initiation is in AD Development in play the part of important role, experiments have shown that: the steroid and triterpene compound of extraction to BV-2 cell have difference The anti-inflammatory activity of degree, thus the steroid and triterpene compound, its tautomer and its pharmaceutically acceptable salt have As the great potential for preparing novel therapeutic Alzheimer medicine.
Five, the anticancer activity of steroid and triterpene compound is tested
Isolated three noval chemical compounds from banaba fruit are tested to Hela (human cervical carcinoma using mtt assay Cell), HepG2 (human liver cancer cell), the cytotoxicity of three kinds of human body tumour cells of SGC-7901 (gastric carcinoma cells) and just The toxicity of normal cell L-929 (l cell).Anticarcinogen cis-dichlorodiamineplatinum (II) (cis-platinum) As positive control.
1, the culture of cell and the configuration of test sample
The human tumor cells such as Hela and normal cell L-929 are incubated in RPMI-1640 culture solution, 10% tire ox is added Serum, 1% antibiotic, in 5%CO2, cultivate under the conditions of 37 DEG C.Every two days squamous subcultures, keep a cell in logarithmic growth phase. Each monomeric compound is configured to stock solution with DMSO and is stored at 4 DEG C, is diluted to respective concentration with complete medium when use. MTT is made into 5mg/mL with PBS (phosphate buffer solution) solution, is kept in dark place in 4 DEG C.It is basic when use Culture medium is diluted to 0.5mg/mL.
2, active test (mtt assay)
Tetrazolium salts colorimetric test is a kind of method for detecting cell survival and growth.Color developing agent tetrazolium salts chemical name used For 3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide, trade name Thiazolyl blue, abbreviation MTT.Living cells line grain Succinate dehydrogenase in body can make exogenous MTT be reduced to the crystal violet (first a ceremonial jade-ladle, used in libation) of slightly solubility and be deposited in cell, and Dead cell is without this function.Dodecyl sodium sulfate (SDS) can dissolve the first a ceremonial jade-ladle, used in libation in cell.It is measured at 546nm using microplate reader Absorption value can reflect the quantity of living cells indirectly.
The 100 well-grown tumour cells of μ L (containing about 5000 cells) is added in each hole of 96 well culture plates, 5%CO2, cultivate 12h under the conditions of 37 DEG C after test sample is added, every group sets 3 multiple holes in parallel, while setting blank control (without thin The cell culture fluid of born of the same parents and test sample) and negative control (cell liquid that no test sample is added).In 5%CO2, train under the conditions of 37 DEG C After supporting 48h, centrifugation discards culture solution, and the MTT solution (0.5mg/mL) that 100 μ L are added in every hole continues to cultivate 4h, and 100 μ L are added OD value is measured at 546nm with microplate reader after SDS, 6h, cell proliferation inhibition rate is calculated as follows, with pharmacology software meter Calculate half-inhibitory concentration IC50Value.
Cell proliferation inhibition rate=(negative control group OD value average value-sample sets OD value average value) ÷ (negative control group OD value average value-blank control group OD value average value) × 100%
Above-mentioned test result see the table below 3:
Table 3
It can be obtained by 3 data of table, on antiproliferative activity, compound 1 has centainly Hela cell and SGC-7901 cell Antiproliferative effect, compound 2 also has certain antiproliferative effect to SGC-7901, but compared with cis-platinum, and effect is not very By force.
Cancer, is also malignant tumour, and opposite has benign tumour.Tumour refer to body various tumorigenesis factors effect under, The cell paraplasm of local organization and the local lump formed.Benign tumour is easy to remove completely, does not shift generally, is not multiple Hair, to organ, tissue only extruding and blocking action.But malignant tumour can also destroy the structure and function of tissue, organ, draw Necrotic hemorrhage concurrent infection is played, patient may finally be dead due to organ failure.The basic unit of cancerous lesion is cancer Cell.Neonatal cell is had after human body cell aging is dead and replaces it, to maintain body function.As it can be seen that the human body overwhelming majority is thin Born of the same parents can hyperplasia, but this hyperplasia be it is limited, and the hyperplasia of cancer cell be then it is endless, this makes the battalion of patient's body Substance is supported largely to be consumed.It is demonstrated experimentally that by inhibiting one kind of cancer cell multiplication and treating cancer to have means.On although State three kinds of compounds cancer cell different types of for three kinds anti-proliferative capacity it is bad compared with positive drug cis-platinum it, but also have one Fixed antiproliferative effect.Therefore, these two types of compounds are proliferated inhibiting tumor cell, it may have certain directive significance and practice base Plinth.

Claims (10)

1. two kinds of steroid compounds and a kind of triterpene compound, tautomer and its pharmaceutically acceptable salt, Be characterized in that: the structural formula of the compound is as follows:
2. two kinds of steroid compounds according to claim 1 and a kind of triterpene compound, tautomer and its Pharmaceutically acceptable salt, it is characterised in that: the pharmaceutically acceptable salt be its sodium, potassium, calcium, magnesium, iron, ferrous iron, lead, Barium, copper, ammonium or zinc salt.
3. two kinds of steroid compounds described in claim 1 and a kind of extracting method of triterpene compound, it is characterised in that: Include the following steps:
(1) fruit of banaba crushed into drying, alcohol extracting, obtain extracting solution；
(2) extracting solution is successively extracted with low polar solvent, middle polar solvent and highly polar solvent, obtains middle polar layer；
(3) middle polar layer is subjected to silica gel column chromatography, the highly polar solvent of polarity-carries out gradient elution in, obtains volume ratio For the elution fraction of 80:1, it is denoted as sample A；
(4) silica gel column chromatography, after the middle highly polar solvent gradient elution of polarity-, centering polarity and highly polar solvent are carried out to sample A The elution fraction that volume ratio is 3:1 carries out ODS column chromatography, after lower alcohol or its aqueous solution gradient elution, passes through silica gel and reverse phase Tlc analysis is partly prepared reversed-phase HPLC to its 40% aqueous lower alcoholic solutions elution fraction and obtains compound 1；
(5) silica gel column chromatography, after the middle highly polar solvent gradient elution of polarity-, centering polarity and highly polar solvent are carried out to sample A The elution fraction that volume ratio is 6:1 carries out ODS column chromatography, right by tlc analysis after lower alcohol or its aqueous solution gradient elution Its 50% aqueous lower alcoholic solutions elution fraction carries out silica gel column chromatography, after the middle highly polar solvent gradient elution of polarity-, passes through reverse phase With silica gel thin-layer chromatography spectrum analysis, the elution fraction that the volume ratio of centering polarity and highly polar solvent is 15:1 carries out half preparation reverse phase HPLC obtains compound 2；
(6) silica gel column chromatography, after the middle highly polar solvent gradient elution of polarity-, centering polarity and highly polar solvent are carried out to sample A The elution fraction that volume ratio is 8:1 carries out silica gel column chromatography, thin by reverse phase in after the highly polar solvent gradient elution of polarity- Layer analysis, centering polarity and highly polar solvent volume than for 5:1 elution fraction carry out mesolow ODS column chromatography, lower alcohol or After its aqueous solution gradient elution, by reverse phase and silica gel thin-layer chromatography spectrum analysis, to its 40% aqueous lower alcoholic solutions elution fraction into Row partly prepares reversed-phase HPLC and obtains compound 3；
Wherein, lower alcohol refers to the alkylol of C1-6.
4. extracting method according to claim 3, it is characterised in that: step (2), (3), (4), (5), the low pole in (6) Property solvent in the hydrocarbon solvents such as hexamethylene, petroleum ether, hexane, isooctane, trimethylpentane, pentamethylene, heptane at least It is a kind of；Middle polar solvent is selected from ethyl acetate, chloroform, methylene chloride, ether, methyl formate, nitromethane, butyl acetate, different At least one of propyl ether；Highly polar solvent is selected from n-butanol, methanol, the tert-butyl alcohol, propyl alcohol, isopropanol, ethyl alcohol, acetone, tetrahydro At least one of furans, pyridine.
5. two kinds of steroid compounds of any of claims 1 or 2 and a kind of triterpene compound pharmaceutically acceptable salt Preparation method, it is characterised in that: steroid compound is dissolved in a solvent with corresponding basic salt, steroid is settled out from solution Body class compound pharmaceutically acceptable salt；Triterpene compound is dissolved in a solvent, from solution with corresponding basic salt It is settled out triterpene compound pharmaceutically acceptable salt.
6. two kinds of steroid compounds of any of claims 1 or 2 and a kind of triterpene compound, tautomer and its Pharmaceutically acceptable salt is preparing anti-inflammatory medicaments or is treating the application in Alzheimer disease drugs or anti-tumor drug.
7. application according to claim 6, it is characterised in that: the inflammation is selected from neuroinflamation, pneumonia, hepatitis, mammary gland Inflammation, gastritis, bursal synovitis, Buerger's disease, any one inflammation in myocarditis.
8. a kind of anti-inflammatory or treatment Alzheimer disease or anti-tumor drug, it is characterised in that: the active constituent of the drug In include steroid compound of any of claims 1 or 2 and/or triterpene compound, tautomer and its pharmaceutically Acceptable salt further includes pharmaceutically acceptable carrier, diluent, excipient, stabilizer, antioxidant.
9. two kinds of steroid compounds of any of claims 1 or 2 and a kind of triterpene compound, tautomer and its Pharmaceutically acceptable salt is preparing the application in health care product.
10. a kind of health care product, it is characterised in that: include steroid compound of any of claims 1 or 2 in the health care product And/or triterpene compound, tautomer and its pharmaceutically acceptable salt.
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