CN101318946B - Dimeric ainsliaea terpene A, preparation and application thereof - Google Patents

Abstract

The invention belongs to the medicine technical field, in particular to a compound Ainsliaidimer A, a preparation method and an application thereof. The compound Ainsliaidimer A is obtained through extracting and separating Ainsliaiea macrocephala, and is a colorless prismatic crystal with a molecular formula C30H34O7. Experiments show that the compound has obvious functions in inhibiting tumor and inflammations, so the compound can be used for preparing anti-tumor drugs and anti-inflammatory drugs.

Description

Dimeric ainsliaea terpene A and its production and application

Technical field

The invention belongs to medical technical field, be specifically related to a kind of new Dimerized sesquiterpenoids dimerization Tuerfeng terpene A (Ainsliaidimer A) that separation obtains from plant major part Tuerfeng and its production and application.

Background technology

It is that one in composite family (Compositae) the broom chrysanthemum wood family (Mutisieae Cass) belongs to that Tuerfeng belongs to (Ainsliaea DC) plant, kind surplus the whole world 70, and there be 44 kind of 4 mutation in China, accounts for 37.5% of sum.The medicinal kind of Tuerfeng platymiscium is more.Be widely used in treatment flu cough breathe heavily, diseases such as rheumatic arthralgia, wound, urinary system.The Tuerfeng plant resources of China mainly is distributed in the Yangtze valley and each province, southwestern south China." Shanghai City medicinal material standard ", " Jiangxi Province's Chinese medicinal materials standard ", " Yunnan Province's drug standard ", " the Sanitation Ministry medicine standard " all record.

Chemical constitution study to the Tuerfeng platymiscium starts from the eighties in 20th century, mainly concentrates on Frangrant Ainsliaea Herb and the tool leaf Tuerfeng.It is reported that the Tuerfeng platymiscium mainly contains sesquiterpene lactones, sesquiterpene glycosides, steroidal, triterpene compound, studies show that sesquiterpene lactones has good antitumor activity.And modern pharmacology and clinical application show that this platymiscium has good anti-inflammatory action.Be blank to major part Tuerfeng (Ainsliaea macrocephala) The Chemical Constituents at present.

The major part Tuerfeng originates in the northwestern Yunnan Province and the west and south, Sichuan, is born in hillside border, softwood forest down or in the ridge shrubbery, 2600～3100 meters of height above sea level are perennial herb." Chinese Plants will " is described in detail the morphological specificity of major part Tuerfeng.We in the hope of clear and definite its chemical ingredients, are Natural Medicine Chemistry research stockpile to gathering the chemical constitution study that has carried out system from the major part Tuerfeng in Yunnan; Discovery has the natural product of excellent activity, for follow-up deep research and development provide material base.

Summary of the invention

The object of the present invention is to provide a kind of new dimerization sesquiterpenoids natural compounds and preparation method thereof and its application in preparation anti-inflammatory, antitumor Chinese traditional medicine.

The present invention is a kind of new Dimerized sesquiterpenoids that extraction separation obtains from major part Tuerfeng (growing in the Chinese yunnan Lijing), called after dimerization Tuerfeng terpene A (AinsliaidimerA), and its chemical structural formula is as follows:

The preparation method of above-claimed cpd is as follows:

(1) extract: with the dry herb of major part Tuerfeng, pulverize, with 80～95% alcohol reflux 1～3 time, each 1～2 hour; United extraction liquid becomes fluid extract to the extracting solution concentrating under reduced pressure, earlier with petroleum ether extraction, again with ethyl acetate extraction, obtains ethyl acetate extract behind the thin up;

(2) separate: above-mentioned ethyl acetate extract applying silicon plastic column chromatography, it with volume ratio 20: 0～4: 1 chloroform-methanol system gradient elution, thin-layer chromatography detects, collection contains the flow point of dimerization Tuerfeng terpene A, again through the dextrane gel column chromatography, is 1: 1 chloroform-methanol wash-out with volume ratio, after the C18 reversed phase column chromatography, with volume ratio is that 4: 6～6: 4 methanol-water carries out wash-out, and thin-layer chromatography detects, dimerization Tuerfeng terpene A.

Dimerized sesquiterpenoids Ainsliaidimer A of the present invention is colourless prism, by electron spray(ES) ion massspectrum signal m/z505[M-H] ^-, high resolution mass spectrum must the accurate total mass number of this compound is 505.2170[M-H] ^-, calculate that its molecular formula is C ₃₀H ₃₄O ₇This compound structure is further determined by the X-diffraction. ¹H and ¹³The C nuclear magnetic resonance data sees Table 1.

Show that through experimental study compound of the present invention has the obvious suppression effect to tumour.Therefore, can be used for preparing antitumor drug.In addition, this compound also has the obvious suppression effect to inflammation, therefore, can be used for preparing anti-inflammatory medicaments.

Table 1Ainsliaidimer A's ¹H and ¹³The C nuclear magnetic resonance data

J: coupling constant

Embodiment

The invention is further illustrated by the following examples, makes those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.

Embodiment 1: major part Tuerfeng (Ainsliaea macrocephala) herb 5kg, extract 2 times each 2 hours with 80% alcohol reflux, the extracting solution concentrating under reduced pressure becomes fluid extract, earlier with petroleum ether extraction,, obtain ethyl acetate extract 320g behind the thin up again with ethyl acetate extraction; Above-mentioned ethyl acetate extract mixed with 300g silica gel mix sample, the applying silicon plastic column chromatography, it with volume ratio 20: 1～4: 1 chloroform-methanol system gradient elution, thin-layer chromatography detects, collection contains the flow point of dimerization Tuerfeng terpene A, again through the dextrane gel column chromatography, it with volume ratio 1: 1 chloroform-methanol wash-out, after the C18 reversed phase column chromatography, with volume ratio is that 4: 6～6: 4 methanol-water carries out wash-out, thin-layer chromatography detects, and gets the pure product 198mg of dimerization Tuerfeng terpene A (Ainsliaidimer A).

The cytotoxic activity experiment of embodiment 2:Ainsliaidimer A

1, experiment material

1.1, given the test agent

After Ainsliaidimer A dissolves with DMSO (Merck), add solution or uniform suspension that PBS (-) is made into 1000 μ g/ml, then with PBS (-) dilution that contains DMSO.

1.2, cell strain

A549 (human lung carcinoma cell)

LOVO (people's colon-cancer cell)

6T-CEM (human T cell leukemia cell)

HL-60 (human leukemia cell)

HepG-2 (human liver cancer cell)

1.3, nutrient solution

RPMI1640+15%NBS+ is two anti-

1.4, other materials

Full-automatic microplate reader: model: WellscanMK-2, production firm: Labsystems

Import 96 well culture plates etc.

2, test method

Mtt assay: it is 4～6 * 10 that the every hole of 96 orifice plates adds concentration ⁴The cell suspension 100 μ l of individual/ml put 37 ℃, 5%CO ₂In the incubator.Behind the 24h, add sample liquid, two multiple holes are established in 10 μ l/ holes, and 37 ℃, 5%CO ₂Effect 72h.Every hole adds the MTT solution 20 μ l of 5mg/ml, adds lysate behind the effect 4h, and put in the incubator in 100 μ l/ holes, and 570nm OD value is surveyed with the full-automatic microplate reader of MK-2 in the dissolving back.

3, test-results

The results are shown in Table 2, the result shows that Ainsliaidimer A is to human lung carcinoma cell, people's colon-cancer cell, the human T cell leukemia cell, human leukemia cell and human liver cancer cell all have better inhibited activity, Ainsliaidimer A has favorable anti-tumor effect, has the excellent development prospect.

Embodiment 3:AinsliaidimerA is to the efficacy experiment of mouse S180 sarcoma (solid-type)

1, experiment material

1.1 given the test agent

AinsliaidimerA uses the 0.5%CMC wiring solution-forming after with a small amount of tween-80 hydrotropy.

1.2 animal

Strain: Kunming mouse

The source: the The 2nd Army Medical College Experimental Animal Center provides.

Conformity certification number: Shanghai is moving closes the card word No. 107

Body weight: 18-20g

Sex: female.

1.3 transplanted tumor

Mouse S180 sarcoma is gone down to posterity by Shanghai Institute of Pharmaceutical Industry and to keep.

2, experimental technique

Get well-grown mouse S180 sarcoma ascites, dilute with 1: 4 with physiological saline, every mouse armpit subcutaneous vaccination 0.2ml, random packet is divided into control group, endoxan group (CTX group, 20mg/kg, ip * 7), AinsliaidimerA (5mg/kg) group, next day is played administration in the inoculation back, the administration volume is the 0.5ml/20g body weight, continuous irrigation stomach 7 days.Inoculate back 10 days and take off neck execution animal, dissect behind the title the weight of animals and get the knurl piece, claim knurl heavy.The result judges according to following formula:

3, experimental result

Ainsliaidimer A has significant tumor-inhibiting action.Experimental result sees Table 3.

Table 2AinsliaidimerA is to the in-vitro multiplication restraining effect of human body tumour cell

Table 3AinsliaidimerA is to the restraining effect of mouse S180 sarcoma

Compare with control group: ^*P＜0.05, ^*P＜0.01.

Embodiment 4:Ainsliaidimer A anti-inflammatory experiment mice ear swelling model

1 laboratory animal

Swiss kind mouse, male, body weight 20-24g.

2. experimental model and testing method

Get 50 of mouse, be divided into 5 groups at random, press the dosage gastric infusion 3d shown in the table 4,1h after the last administration, dimethylbenzene with 0.05ml causes inflammation with mouse left side ear, the punch tool that with diameter is 7mm is the overlapping punching of ears, and the disk of laying is gone up weighing at torsion(type)balance (sensitivity be ten thousand/), represents its swelling degree with the weight difference of mouse ear two disks.

3 statistical study:

All experimental result is all represented with mean ± standard error.Respectively organize data and the blank significant difference of organizing with student t-check, P＜0.05, expression difference has the significance meaning.

4 test-results:

Table 4.Ainsliaidimer A is to the influence of mice ear test

Compare with the blank group, ^*P＜0.05, ^*P＜0.01

The result shows that positive control drug hydrocortisone, Ainsliaidimer A have significant inhibitory effect to the inflammation that the mice ear model causes.

Embodiment 5:Ainsliaidimer A anti-inflammatory experimental rat carrageenin foot swelling model

1 laboratory animal

The SD rat, male and female dual-purpose, body weight 180-220g.

2. experimental model and testing method

Get 50 of rats, be divided into 5 groups at random, press the dosage gastric infusion 4d shown in the table 5, before the last administration, measure and cause scorching preceding volume; 30min after the last administration, the sufficient subcutaneous injection 1% carrageenin 0.1ml that wastes time causes inflammation in rat right hind leg.Respectively at cause scorching back 2,4,6h measures the following volume of right ankle joint.

3 statistical study:

All experimental result is all represented with mean ± standard error.Respectively organize data and the blank significant difference of organizing with student t-check, P＜0.05, expression difference has the significance meaning.

4 test-results:

Table 5.Ainsliaidimer A is to the influence of rat carrageenan foot swelling test

Compare with the blank group, ^*P＜0.05, ^*P＜0.01

The result shows that positive control drug dexamethasone, Ainsliaidimer A have significant inhibitory effect to the caused inflammation of rat carrageenan foot swelling model.

The capillary permeability test of embodiment 6:AinsliaidimerA anti-inflammatory experiment mice abdominal cavity

1 laboratory animal

NH is a mouse, male and female dual-purpose, body weight 18-22g.

2. experimental model and testing method

Get 50 of rats, be divided into 5 groups at random, press the dosage gastric infusion 1h shown in the table 6, tail vein injection 2% AZO-blue normal saline solution 0.1ml/10g body weight, abdominal injection 0.8% acetic acid normal saline solution 0.20ml/ only takes off cervical vertebra and puts to death mouse behind the 20min immediately, divides with 5ml physiological saline and washs the abdominal cavity for several times, 3000 rev/mins, centrifugal 15min; Getting supernatant liquor uses microplate reader in its optical density(OD) of 570nm colorimetric estimation (OD) value.

3 statistical study:

All experimental result is all represented with mean ± standard error.Respectively organize data and the blank significant difference of organizing with student t-check, P＜0.05, expression difference has the significance meaning.

4 test-results:

Table 6.Ainsliaidimer A is to the influence of mouse peritoneal capillary permeability test

Compare with the blank group, ^*P＜0.05, ^*P＜0.01

The result shows that positive control drug dexamethasone, the Ainsliaidimer A inflammation that test causes to the mouse peritoneal capillary permeability have significant inhibitory effect.

Claims (4)

1. compound, name is called dimerization Tuerfeng terpene A, it is characterized in that chemical structural formula is as follows:

2. the preparation method of dimeric ainsliaea terpene A as claimed in claim 1 is characterized in that concrete steps are as follows:

(1) extract: with the dry herb of major part Tuerfeng, pulverize, with 80～95% alcohol reflux 1～3 time, each 1～2 hour; United extraction liquid becomes fluid extract to the extracting solution concentrating under reduced pressure, earlier with petroleum ether extraction, again with ethyl acetate extraction, obtains ethyl acetate extract behind the thin up;

(2) separate: above-mentioned ethyl acetate extract applying silicon plastic column chromatography, it with volume ratio 20: 0～4: 1 chloroform-methanol system gradient elution, thin-layer chromatography detects, collection contains the flow point of dimerization Tuerfeng terpene A, again through the dextrane gel column chromatography, is 1: 1 chloroform-methanol wash-out with volume ratio, after the C18 reversed phase column chromatography, with volume ratio is that 4: 6～6: 4 methanol-water carries out wash-out, and thin-layer chromatography detects, dimerization Tuerfeng terpene A.

3. the application of dimeric ainsliaea terpene A according to claim 1 in the preparation anti-inflammatory drug.

4. the application of dimeric ainsliaea terpene A according to claim 1 in the preparation antitumor drug.