CN109651484A - A kind of new triterpenoid and preparation method thereof and medical usage - Google Patents

Abstract

The invention discloses a kind of new triterpenoid and preparation method thereof and medical usages.The compound is to report for the first time, is a kind of triterpenoid of structure novel, extracting and developing can purify to obtain from the drying branch of shiny-leaved yellowhorn.In vitro test proves A β_1‑40Toxicity damage nerve cell, cause a large amount of apoptosis of PC12, cell activity decline, apoptosis situation is improved after compound (I) is intervened, and can be used to develop into nerve protection medicine.

Description

A kind of new triterpenoid and preparation method thereof and medical usage

Technical field

The invention belongs to technical field of pharmaceuticals, and in particular to isolated one kind has mind from the drying branch of shiny-leaved yellowhorn Triterpenoid and preparation method thereof through protective effect.

Background technique

Shiny-leaved yellowhorn Xanthoceras sorbifolia Bunge is that Sapindaceae (Sapindaceae) shiny-leaved yellowhorn belongs to plant Object, it is single to belong to single, alias Wendeng City pavilion, the road Seng Dengmao, precipice pawpaw, mountain papaw etc..It is distributed in China northeast, North China and Shaanxi, Also often there are a cultivation in the hills hillside on the ground such as Gansu, Ningxia, Anhui, Henan etc., various regions, shiny-leaved yellowhorn first recorded in herbal for Relief of Famines, with Wen Guanhua runs after fame, also on the books in Compendium of Material Medica.

Shiny-leaved yellowhorn is the distinctive rare traditional oil tree in China, there is the title of northern oil tea, because its kernel is rich in fat oil, Its mass fraction is up to 52%, there is very high economic value.In addition to as oil tree, there are also good medicinal, foods for the plant With, the value such as ornamental.Stem, the branch (wood of shiny-leaved yellowhorn) of shiny-leaved yellowhorn be sweet in flavor, slight bitter, cool in nature, has swelling and pain relieving, wind-damp dispelling, holds back dry Huang The effect of water, is usually used in treating the diseases such as hot " Xieri Wusu Symptom ", scrofula, rheumatic arthritis in Mongolian medicine.Its civil kind Benevolence treats infantile enuresis, and Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences is developed into the preparation for the treatment of infantile enuresis, treats Effect is significant.

Isolated chemical component is mostly with triterpenes (You Yiyu stamen alcohol type from the positions such as shiny-leaved yellowhorn husk, carpopodium Triterpene compound is in the majority), based on flavonoids, in addition there are Phenylpropanoid Glycosides, steroid, phenolic acid, alkaloid, monoterpene and fatty acids Close object.Triterpene compound is that most element of the first species is reported in shiny-leaved yellowhorn, and structure parent nucleus is mainly oleanane skeleton knot The beautiful stamen alcohol type (A, B) of structure, in addition there are lupinane type (C), root of gansui alkane type (D), cycloartane type, lanolin alkane types etc..

Shiny-leaved yellowhorn has various pharmacological activity, such as anti-oxidant, anti-inflammatory, antitumor, antibacterial, inhibition hiv protease, improvement Learning and memory function etc..Wherein shiny-leaved yellowhorn kernel Linoleic acid, linolenic acid amount are abundant, often edible to prevent cardiovascular and cerebrovascular disease Disease；Fraxin in calyx piece can be used for antipyretic, sleeping, anti-convulsion；Myricetrin in leaf can be used for sterilizing, norcholesterol etc..Text Be preced with that fruit kernel, the aqueous extract of carpopodium and leaf are significant in efficacy for infantile enuresis, and can the mouse of antagonism resulted from chemical medicine learn Practise memory disorders.

Summary of the invention

The object of the present invention is to provide one kind isolated in a kind of drying branch from shiny-leaved yellowhorn to make with neuroprotection Triterpenoid and preparation method thereof.

Above-mentioned purpose of the invention is achieved by following technical solution:

Compound (I) is had the following structural formula,

The preparation method of the compound (I) includes following operating procedure: (a) crushing the drying branch of shiny-leaved yellowhorn, uses 75~85% alcohol heat reflux extract, and combined extract is concentrated into no alcohol taste, successively use petroleum ether, ethyl acetate and water saturation Extracting n-butyl alcohol, respectively obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract；(b) second in step (a) Acetoacetic ester extract is cleaned with macroreticular resin, first with 10% ethanol elution, 8 column volumes, then with 75% ethanol elution, 10 cylinders Product collects 75% ethanol eluate, 75% ethanol elution object medicinal extract is concentrated under reduced pressure to obtain；(c) 75% ethanol elution soaks in step (b) Cream is separated with purification on normal-phase silica gel, is successively washed with the methylene chloride-methanol gradient that volume ratio is 80:1,45:1,25:1,10:1 and 1:1 It is de- to obtain 5 components；(d) component 4 is further separated with purification on normal-phase silica gel in step (c), successively with volume ratio be 15:1,10:1 and The methylene chloride-methanol gradient elution of 5:1 obtains 3 components；(e) in step (d) octadecylsilane of component 2 be bonded it is anti- The separation of phase silica gel, the methanol aqueous solution isocratic elution for being 75% with concentration expressed in percentage by volume collect 8~12 column volume eluents, Eluent is concentrated under reduced pressure to give pure compound (I).

Further, the macroreticular resin is D101 macroporous absorbent resin.

Further, described to extract the concentration of alcohol that uses with alcohol heat reflux as 80%.

A kind of pharmaceutical composition, wherein the compound (I) and pharmaceutically acceptable load containing therapeutically effective amount Body.

The compound (I) is preparing the application in nerve protection medicine.

The pharmaceutical composition is preparing the application in nerve protection medicine.

It when the compounds of this invention is used as drug, can directly use, or be used in the form of pharmaceutical composition.

The pharmaceutical composition contains the compounds of this invention (I) of therapeutically effective amount, remaining is pharmaceutically acceptable, right The nontoxic and inert pharmaceutical acceptable carrier of humans and animals and/or excipient.

Described pharmaceutical acceptable carrier or excipient is one or more selected from solid, semisolid and liquid diluent, filler And pharmaceutical preparation adjuvant.Pharmaceutical composition of the invention is used in the form of per weight dose.Drug of the present invention can Patient in need for the treatment of is applied to by way of oral or injection.When for taking orally, tablet, sustained release tablets, control can be made into Release piece, capsule, dripping pill, pellet, suspension, emulsion, powder or granule, oral solution etc.；When for injecting, sterilizing can be made into Aqueous or oily solution, aseptic powder injection, liposome or emulsion etc..

Detailed description of the invention

Fig. 1 is compound (I) structural formula；

Fig. 2 is compound (I) theory ECD value compared with testing ECD value.

Specific embodiment

Essentiality content of the invention is further illustrated below with reference to embodiment, but present invention protection model is not limited with this It encloses.Although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should understand that, it can be right Technical solution of the present invention is modified or replaced equivalently, without departing from the spirit and scope of technical solution of the present invention.

Embodiment 1: compound (I) separation preparation and structural identification

Reagent source: ethyl alcohol, petroleum ether, ethyl acetate, n-butanol, methylene chloride are that analysis is pure, insult peaking purchased from Shanghai Reagent Co., Ltd is learned, methanol, analysis is pure, is purchased from Jiangsu Han Bang chemical reagent Co., Ltd.

Preparation method: (a) crushing the drying branch (10kg) of shiny-leaved yellowhorn, extracts (25L × 3 with 80% alcohol heat reflux It is secondary), combined extract is concentrated into no alcohol taste (3L), successively full with petroleum ether (3L × 3 time), ethyl acetate (3L × 3 time) and water The n-butanol (3L × 3 time) of sum extracts, and respectively obtains petroleum ether extract, acetic acid ethyl ester extract (431g) and extracting n-butyl alcohol Object；(b) acetic acid ethyl ester extract is cleaned with D101 macroreticular resin in step (a), first with 10% ethanol elution, 8 column volumes, then With 75% ethanol elution, 10 column volumes, 75% ethanol eluate is collected, 75% ethanol elution object medicinal extract is concentrated under reduced pressure to obtain (172g)；(c) 75% ethanol elution medicinal extract is separated with purification on normal-phase silica gel in step (b), is successively 80:1 (8 cylinders with volume ratio Product), the dichloromethane of 45:1 (8 column volumes), 25:1 (8 column volumes), 10:1 (10 column volumes) and 1:1 (5 column volumes) Alkane-methanol elution gradient obtains 5 components；(d) component 4 (65g) is further separated with purification on normal-phase silica gel in step (c), is successively used Volume ratio is that the methylene chloride-methanol gradient of 15:1 (8 column volumes), 10:1 (10 column volumes) and 5:1 (8 column volumes) is washed It is de- to obtain 3 components；(e) the reverse phase silica gel separation that component 2 (37g) octadecylsilane is bonded in step (d), with volume hundred Dividing concentration is 75% methanol aqueous solution isocratic elution, collects 8-12 column volume eluent, eluent is concentrated under reduced pressure to give pure Compound (I) (45mg).

Structural identification: HR-ESIMS shows [M+Na]⁺For m/z 567.3302, can obtain molecular formula in conjunction with nuclear-magnetism feature is C₃₂H₄₈O₇, degree of unsaturation 9.Hydrogen nuclear magnetic resonance modal data δ_H(ppm, DMSO-d₆, 500MHz): H-1 (2.23, m), H-1 (2.07, m), H-3 (5.02, s), H-5 (1.37, m), H-6 (1.84, m), H-6 (1.67, t, J=14.6), H-7 (2.98, d, J =6.4), (1.83, m) H-11, H-11 (1.92, m), H-12 (1.51, m), H-12 (1.92, m), H-15 (1.47, m), H-15 (1.29, ddd, J=12.5,12.5,5.3), H-16 (1.28, m), H-16 (2.03, m), H-17 (1.60, m), H-18 (0.99, S), (0.93, s) H-19, H-20 (1.31, m), H-21 (0.91, d, J=6.6), H-22 (1.11, m), H-22 (1.48, m), H- 23 (1.78, m), H-23 (1.95, m), H-24 (4.96, tt, J=7.1,1.4), H-26 (1.68, s), H-27 (1.79, s), H- 28 (0.91, s), H-29 (0.91, s), 3-OAc (2.12, s)；Carbon-13 nmr spectra data δ_C(ppm, DMSO-d₆, 125MHz): 42.9(CH₂, 1-C), 201.2 (C, 2-C), 81.3 (CH, 3-C), 40.1 (C, 4-C), 41.5 (CH, 5-C), 24.2 (CH₂, 6- C), 71.1 (CH, 7-C), 82.0 (C, 8-C), 86.3 (C, 9-C), 37.7 (C, 10-C), 22.1 (CH₂, 11-C), 33.2 (CH₂, 12-C), 46.1 (C, 13-C), 59.5 (C, 14-C), 20.9 (CH₂, 15-C), 27.8 (CH₂, 16-C), 51.4 (CH, 17-C), 13.8(CH₃, 18-C), 16.5 (CH₃, 19-C), 34.7 (CH, 20-C), 18.3 (CH₃, 21-C), 35.9 (CH₂, 22-C), 24.7 (CH₂, 23-C), 124.9 (CH, 24-C), 131.1 (C, 25-C), 18.1 (CH₃, 26-C), 25.2 (CH₃, 27-C), 25.1 (CH₃, 28-C), 25.1 (CH₃, 29-C), 177.3 (C, 30-C), 170.3 (C, 3-OAc), 20.8 (CH₃, 3-OAc)；Carbon atom Label is referring to Fig. 1.IR spectrum shows that the compound contains gamma lactone (1765cm^-1) and carbonyl (1731cm^-1) group.¹H NMR Spectrum display contains seven unimodal methyl signals δ H0.99 (H₃- 18), 0.93 (H₃- 19), 1.68 (H₃- 26), 1.79 (H₃- 27), 0.91(H₃- 28), 0.91 (H₃- 29) and 2.12 (3-OAc) and bimodal signal δ H0.91 (H₃- 21, d, J=6.6Hz), Show that the compound is triterpene compound.¹³C NMR and DEPT spectrum 32 carbon signals of display, including eight methyl, six first Base (two oxygen-containing methines and an alkene carbon), eight methylene and ten quaternary carbons (two lactone carbonyl carbon, a ketone Carbonyl carbon, two oxygen-containing carbon and an alkene carbon).In addition, side chain has 24 (25)-double bond (δ C131.1,124.9 and δ H4.96, tt, J=7.1,1.4Hz) signal；In HMBC spectrum, H₃- 26 and H₃- 27 with C-24 and C-25, H₃- 21 and C-17, C-20 And C-22 and H-24 and C-22, C-23, H₃- 26 and H₃The above-mentioned inference of -27 relevance verification.H-3 (δ in HMBC spectrum H5.02, s) and corresponding ester carbonyl group (δ C170.3；δ H2.12, s) correlation show that C-3 are connected with an acetoxyl group.In addition, One ester carbonyl group signal [δ C177.3 (C-30)] and two carbon signals [δ C86.3 (C-9) and 59.5 (C-14)] illustrate the chemical combination There are a gamma lactone structures for object.In HMBC spectrum, H₃- 18 and C-14, H₂- 15 and C-30, H₂- 11 and C-9, H-11 α (δ H1.83, m) and C-8 and H₃- 19 with the relevance verification of the C-9 connection relationship of this functional group of 30,9-.Two oxygen-containing carbon The chemical shift of [C-7 (δ C71.1) and C-8 (δ C82.0)] shows that they are respectively connected with a hydroxyl.Comprehensive hydrogen spectrum, carbon spectrum, HMBC Spectrum and NOESY spectrum and document can determine substantially the compound as shown in Figure 1, spatial configuration about correlation type nuclear magnetic data It is further tested and is determined by ECD, theoretical value and experiment value are almost the same (Fig. 2).

Embodiment 2: compound (I) pharmacological action test

One, material and instrument

PC12 cell strain, Anhui Chinese Medicine College experimental center are gifted.Aβ_1-40Albumen is purchased from Sigma company.Compound (I) For self-control, HPLC normalizes purity and is greater than 98%.(import packing is purchased from the silent winged generation that bioid of Beijing match to DMEM/F12 culture medium Learn Products Co., Ltd.Newbom Calf Serum is purchased from Beijing Solarbio Science&Technology Co., Ltd.MTT is purchased from Amresco company, the U.S..Triumphant base AnnexinV-FITC cell apoptosis detection kit is purchased from Nanjing Kai Jisheng Object Technology Co., Ltd..Paraformaldehyde is purchased from chemical reagent Co., Ltd, Chinese Medicine group.Bcl-2 polyclonal antibody is purchased from force Han Boshide Bioisystech Co., Ltd.Bax polyclonal antibody is purchased from Beijing Bo Aosen Bioisystech Co., Ltd.Cyt-c is more Clonal antibody is purchased from Wuhan doctor's moral Bioisystech Co., Ltd.Goat anti-rabbit igg/FITC, which is purchased from Beijing Bo Aosen biotechnology, to be had Limit company.Sheep anti-mouse igg/(H+L) is purchased from the green skies biotech firm in Jiangsu.Normal Goat Serum is purchased from Wuhan doctor's moral biology skill Art Co., Ltd.SABC immunohistochemical kit is purchased from Wuhan Boster Biological Technology Co., Ltd..DAB colour reagent box is purchased from Wuhan doctor's moral Bioisystech Co., Ltd.

Electronic analytical balance (FA2004 type, Shanghai Lei Ci precision scientific instrument company), water-bath (HHSI1-4 type, north Capital medical apparatus and instruments factory), microplate reader (ELx-800 type, U.S. Bio-Tek), flow cytometer (EPIC XL-MCL type, the U.S. Beckman Couiter company), high speed freezing centrifuge (3K30 type, Sigma Co., USA, carbon dioxide incubator (CO- 150 types, NBS Co., Ltd, the U.S.), (BX41 type, band DP70 camera system, Japanese Olympus are public for OLYMPUS fluorescence microscope Take charge of product).

Two, test method

1、Aβ_1-40It is incubated for

Aβ_1-401000 μm of ol/L storing liquids are configured to deionized water and are dispensed, and are placed in -20 DEG C of refrigerator storages, are faced use It is taken out with the last week and is incubated for 7d in 37 DEG C of incubators, be allowed to assemble aging.

2, the culture of cell

DMEM/F12 culture of the PC12 cell strain containing 10% fetal calf serum, 100U/ and penicillin, 100U/mL streptomysin Base is cultivated in glass culture bottle, CO₂The condition of culture of cell incubator is 37 DEG C, 5%CO₂Concentration, saturated humidity, to cell It is long to using after 80% abundance 0.25% pancreatin had digestive transfer culture for 1 time (about 2-3d), inverted microscope observes cell growth condition, reality Logarithmic growth phase cell is tested when testing.96 well culture plate free serum cultures are seeded cells into before carrying out MTT experiment Base culture；Cell inoculation to 6 well culture plates had both been cultivated with free serum culture before flow cytomery；Immunohistochemical experiment is equal With 24 well culture plate creep plate method culture cells, culture medium is serum free medium.

3, cell administration processing and grouping

Cell is divided into five groups, and serum free medium inoculation carries out pharmaceutical intervention afterwards for 24 hours: 1. Normal group: normal PC12 cell；2. model group: 25 μm of ol/LA β_1-40；Compound 3. (I) low dose group: 25 μm of ol/LA β_1-40+ 50mg/L compound (Ⅰ)；Compound 4. (I) middle dose group: 25 μm of ol/LA β_1-40+ 100mg/L compound (I)；Compound 5. (I) high dose group: 25 μmol/LAβ_1-40+ 200mg/L compound (I).Drug-treated carries out detection indices afterwards for 24 hours.

4, MTT detects group of cells vigor

The cell of logarithmic growth phase, with 1 × 10 after pancreatin digestion⁵The cell density of a/mL, every 100 μ L of hole are inoculated in In 96 orifice plates free serum culture for 24 hours, then according to above-mentioned group technology carry out pharmaceutical intervention, 6 multiple holes of every group of setting, for 24 hours after, The 20 μ L of MTT solution that 5mg/mL is added in every hole continues in CO₂It is incubated for 4h in cell incubator, then takes out 96 well culture plates, abandons Supernatant is removed, every hole is added the DMSO of 150 μ L, is placed on shaking table and vibrates 10min, use microplate reader after purple crystal is completely dissolved The light absorption value in each hole is detected at 490nm wavelength.Only to add the hole of culture solution to be zeroing reference, cell viability percentage table It reaches, regards the cell activity of control group as 100%.As a result it calculates: cell survival rate=(experimental group OD value-blank control group OD Value)/(control group OD value-blank control group OD value) × 100%.

5, it statisticallys analyze

Statistical result is handled using SPSS17.0 analysis software, data use one-way analysis of variance, use mean Scholar's standard deviationIt indicates, is compared between group using LSD, P < 0.05 is with significant difference, and P < 0.01 is extremely conspicuousness Difference, chart are drawn by Excel 2003.

Three, result and conclusion

MTT test result shows that compared with normally group cell, model group cell activity is substantially reduced (P < 0.01), chemical combination Object (I) intervene after cell activity increase, wherein high, middle dose group cell activity raising have statistical significance (P < 0.01, P<0.05), low dose group cell activity changes less (P>0.05).Be shown in Table 1 (compared with model group * P < 0.05, * * P < 0.01)。

Conclusion, this study demonstrates that A β_1-40Toxicity damage nerve cell, cause a large amount of apoptosis of PC12, cell activity decline, Apoptosis situation is improved after compound (I) is intervened.

1 mtt assay compound (I) of table is to A β_1-40Induce PC12 cell activity influence (N=6)

Group	Processing method	Cell activity (%)
			Normal group	–	100±0.0**
Model group	25μmol/LAβ1-40	50.08±6.67
			Compound (I) low dose group	25μmol/LAβ1-40+ 50mg/L compound (I)	58.78±12.64
Compound (I) middle dose group	25μmol/LAβ1-40+ 100mg/L compound (I)	63.77±6.81*
			Compound (I) high dose group	25μmol/LAβ1-40+ 200mg/L compound (I)	83.95±10.58**

Embodiment 3

The preparation of tablet: compound (I) first is made by 1 method of embodiment, and utilizes organic acid such as tartaric acid or lemon Acid or formic acid or ethanedioic acid etc., inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, by its with excipient weight than for 1:7's Excipient, pelletizing press sheet is added in ratio.

Embodiment 4

Oral solution preparation: compound (I) first is made by 1 method of embodiment, and utilizes organic acid such as tartaric acid or lemon Acid or formic acid or ethanedioic acid etc., inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, routinely oral solution preparation method is made oral Liquid.

Embodiment 5

The preparation of capsule or granule: compound (I) first is made by 1 method of embodiment, and utilizes organic acid such as wine Stone acid or citric acid or formic acid or ethanedioic acid etc., inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, by itself and excipient weight Excipient is added than the ratio for being 1:7 in amount, and capsule or granule is made.

Embodiment 6

The preparation of injection: compound (I) first is made by 1 method of embodiment, and utilizes organic acid such as tartaric acid or lemon Lemon acid or formic acid or ethanedioic acid etc., inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, routinely plus water for injection, refined filtration, Injection is made in encapsulating sterilizing.

Embodiment 7

The preparation of aseptic powder injection: by 1 method of embodiment first be made compound (I), and using organic acid for example tartaric acid or Citric acid or formic acid or ethanedioic acid etc., inorganic acid salt as made of hydrochloric acid or sulfuric acid or phosphoric acid, are dissolved in sterile water for injection In, stirring makes molten, is filtered with sterile suction funnel, then sterile refined filtration is sub-packed in ampoule, sterile sealing after frozen drying Obtain powder-injection.

The effect of above-described embodiment indicates that essentiality content of the invention, but protection of the invention is not limited with this Range.Those skilled in the art should understand that can with modification or equivalent replacement of the technical solution of the present invention are made, Without departing from the essence and protection scope of technical solution of the present invention.

Claims (7)

1. compound (I) is had the following structural formula,

2. the preparation method of compound (I) described in claim 1, it is characterised in that include following operating procedure: (a) by Wen Guan The drying branch of fruit crushes, and is extracted with 75~85% alcohol heat reflux, and combined extract is concentrated into no alcohol taste, successively with petroleum ether, Ethyl acetate and water saturated extracting n-butyl alcohol, respectively obtain petroleum ether extract, acetic acid ethyl ester extract and extracting n-butyl alcohol Object；(b) acetic acid ethyl ester extract is cleaned with macroreticular resin in step (a), first with 10% ethanol elution, 8 column volumes, then is used 75% column volume of ethanol elution 10 collects 75% ethanol eluate, 75% ethanol elution object medicinal extract is concentrated under reduced pressure to obtain；(c) step Suddenly 75% ethanol elution medicinal extract is separated with purification on normal-phase silica gel in (b), is successively 80:1,45:1,25:1,10:1 and 1:1 with volume ratio Methylene chloride-methanol gradient elution obtain 5 components；(d) component 4 is further separated with purification on normal-phase silica gel in step (c), successively 3 components are obtained with the methylene chloride-methanol gradient elution that volume ratio is 15:1,10:1 and 5:1；(e) component 2 in step (d) The reverse phase silica gel separation being bonded with octadecylsilane, the methanol aqueous solution isocratic elution for being 75% with concentration expressed in percentage by volume are received Collect 8~12 column volume eluents, eluent is concentrated under reduced pressure to give pure compound (I).

3. the preparation method of compound (I) according to claim 2, it is characterised in that: the macroreticular resin is that D101 is big Macroporous adsorbent resin.

4. the preparation method of compound (I) according to claim 2, it is characterised in that: described to be extracted with alcohol heat reflux The concentration of alcohol used is 80%.

5. a kind of pharmaceutical composition, it is characterised in that: the wherein compound described in claim 1 (I) containing therapeutically effective amount And pharmaceutically acceptable carrier.

6. compound (I) described in claim 1 is preparing the application in nerve protection medicine.

7. pharmaceutical composition described in claim 5 is preparing the application in nerve protection medicine.