CN105348095A - Novel diterpenoid as well as preparation method and medical application thereof - Google Patents
Novel diterpenoid as well as preparation method and medical application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/58—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/612—Esters of carboxylic acids having a carboxyl group bound to an acyclic carbon atom and having a six-membered aromatic ring in the acid moiety
- C07C69/618—Esters of carboxylic acids having a carboxyl group bound to an acyclic carbon atom and having a six-membered aromatic ring in the acid moiety having unsaturation outside the six-membered aromatic ring
Abstract
The invention discloses a novel diterpenoid as well as a preparation method and medical application thereof, and belongs to the technical field of medicines. The novel diterpenoid is reported for the first time, is novel in structure and can be obtained by extraction, separation and purification of dry buds of lilac daphne flower. In-vitro tests show that the novel diterpenoid has the osteoclast differentiation and proliferation inhibition function, and the inhibition function is concentration-dependent. Applying the novel diterpenoid (I) to treatment of bone destructive diseases mainly featured by osteolast viability increase can probably become a promising therapeutic method, and the novel diterpenoid can be further used for developing medicines for treating bone destructive diseases.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to from the dry flower of lilac daphne, be separated obtain a kind of and there is diterpene-kind compound for the treatment of osteoclasia disease effect and preparation method thereof.
Background technology
Lilac daphne is also known as purple lilac daphne, southern lilac daphne, headache flower, medicine water shield etc., and begin to be loaded in Shennong's Herbal, being classified as low-grades, is traditional drastically purgating water drug.Compendium of Material Medica lists poisonous weeds class in, and " life in February leafiness, thicken then black, Hua Youzi, red, Bai Zhe, March is honest most, Ye Naisheng to call lilac daphne by " WU Pu Bencao "." " shortcut has it everywhere to meaning to Han Bao Noboru " another name for Sichuan Province book on Chinese herbal medicine ", height of seedling two or three chi.Leaf is like Cynanchum glaucescens and willow leaf, and root Beijing opera is like mulberry root, and February in the first month of the lunar year, flower was sent out, and purple green look, receives during Ye Weisheng." combine " Bencao Tujing " " Anhui Chuzhou's lilac daphne " and " Jinzhou, Sichuan lilac daphne " accompanying drawing and see, unanimously, its former plant is Daphnegenkwasieb.Etzucc. undoubtedly to medicinal lilac daphne kind at all times." Chinese Pharmacopoeia " 2010 editions records its dry flower being thymelaeceae (Thymelaeaceae) Daphne (Daphne) plant lilac daphne (Daphnegenkwasiebetzucc).Xie Zongwan had once carried out Herbal Textuals textual criticism to lilac daphne, and confirming lilac daphne described in history tree should be the dry flower of purple lilac daphne Daphnegenkwasieb.Etzucc.This plant is the clinical kind commonly used in national most of area, and it is wild mainly originates in the provinces such as Henan, Shandong, Jiangsu, Anhui, Sichuan, and Henan Province is mainly distributed in the mountain area on the ground such as Xinyang, Nanyang.
Up to the present, from the flower of lilac daphne, bud, leaf, branch, root, extraction and isolation goes out compound kind more than 70, wherein containing Multiple components such as flavones ingredient, diterpene ortho-ester composition, coumarins composition, chlorogenic acid composition, lignan component, phenolic glycoside classes.
Lilac daphne is traditional drastically purgating water drug, poisonous, function removing fluid-retention by purgation, removing toxic substances desinsection, and modern study shows that it has following pharmacological action: 1. diuretic properties; 2. Antitussive and Expectorant Effect; 3. desinsection, parasiticide effect; 4. induced labor effect; 5. anti-inflammatory, antitumor action; 6. immunoregulation effect.
Summary of the invention
The object of this invention is to provide and a kind ofly from the dry flower of lilac daphne, be separated obtain a kind of there is diterpene-kind compound for the treatment of osteoclasia disease effect and preparation method thereof.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
There is the compound (I) of following structural formula,
The preparation method of described compound (I), comprise following operation steps: the dry flower of lilac daphne is pulverized by (a), extract with 70 ~ 80% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, then use 80% ethanol elution, 10 column volumes, collect 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract; C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,25:1,15:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,15:1 and 8:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 10 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
Further, described macroporous resin is D101 macroporous adsorbent resin.
Further, described alcohol heat reflux extracts the alcohol concn adopted is 75%.
A kind of pharmaceutical composition, the described compound (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
The application of described compound (I) in the medicine of preparation treatment osteoclasia disease.
The application of described pharmaceutical composition in the medicine of preparation treatment osteoclasia disease.
When the compounds of this invention is used as medicine, directly can uses, or use with the form of pharmaceutical composition.
This pharmaceutical composition contains the compounds of this invention (I) for the treatment of significant quantity, and all the other are acceptable on pharmacology, nontoxic to humans and animals and pharmaceutically acceptable carrier of inertia and/or vehicle.
Described pharmaceutically acceptable carrier or vehicle are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, dripping pill, micropill, suspensoid, emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into water-based or oily solution, aseptic powder injection, liposome or the emulsion etc. of sterilizing.
Accompanying drawing explanation
Fig. 1 is compound (I) structural formula;
Fig. 2 is that the theoretical ECD value of compound (I) compares with experiment ECD value.
Embodiment
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
Embodiment 1: compound (I) is separated preparation and structural identification
Reagent source: ethanol, sherwood oil, ethyl acetate, propyl carbinol, methylene dichloride are analytical pure, purchased from Shanghai Ling Feng chemical reagent company limited, methyl alcohol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Preparation method: the dry flower (10kg) of lilac daphne is pulverized by (a), (25L × 3 time) are extracted with 75% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste (3L), use sherwood oil (3L × 3 time), ethyl acetate (3L × 3 time) and water saturated propyl carbinol (3L × 3 time) to extract successively, obtain petroleum ether extract, acetic acid ethyl ester extract (431g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract D101 macroporous resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, use 80% ethanol elution, 10 column volumes again, collect 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract (161g); C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, successively with volume ratio be 75:1 (8 column volumes), the methylene chloride-methanol gradient elution of 45:1 (8 column volumes), 25:1 (8 column volumes), 15:1 (10 column volumes) and 1:1 (5 column volumes) obtains 5 components; D component 4 (52g) is separated further by purification on normal-phase silica gel in () step (c), successively with volume ratio be 20:1 (8 column volumes), the methylene chloride-methanol gradient elution of 15:1 (10 column volumes) and 8:1 (8 column volumes) obtains 3 components; E in () step (d), component 2 (26g) reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 10-12 column volume elutriant, elutriant concentrating under reduced pressure obtains pure compound (I) (237mg).
Structural identification: white amorphous powder; HR-ESIMS shows [M+Na]
+for m/z537.2502, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
29h
38o
8, degree of unsaturation is 11.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 500MHz): H-1 (2.51, t, J=13.5), H-1 (1.59, br, d, J=13.5), H-2 (2.50, m), H-3 (5.51, t, J=4.0), H-4 (3.16, d, J=4.0), H-7 (1.79, t, J=15.5), H-7 (1.25, dd, J=15.5, 4.0), H-8 (1.97, m), H-8 (1.48, t, J=13.5), H-9 (3.40, dd, J=10.0, 5.5), H-11 (3.14, d, J=1.5), H-12 (4.11, d, J=1.5), H-16 (1.01, d, J=7.5), H-17 (1.11, s), H-18 (0.91, s), H-19 (0.89, s), H-20 (1.17, s), H-2 ' (7.57, br, d, J=7.0), H-3 ' (7.33, br, t, J=7.0), H-4 ' (7.35, br, t, J=7.0), H-5 ' (7.34, br, t, J=7.0), H-6 ' (7.58, br, d, J=7.0), H-7 ' (7.69, d, J=16.5), H-8 ' (6.62, d, J=16.5), 9-OH (4.60, d, J=5.5), 11-OH (5.38, s), 12-OH (5.37, s), 15-OH (6.55, s), carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 125MHz): 41.7 (CH
2, 1-C), 35.4 (CH, 2-C), 78.0 (CH, 3-C), 54.1 (CH, 4-C), 211.7 (C, 5-C), 49.1 (C, 6-C), 31.8 (CH
2, 7-C), 28.6 (CH
2, 8-C), 79.7 (CH, 9-C), 38.4 (C, 10-C), 81.1 (CH, 11-C), 71.6 (CH, 12-C), 56.3 (C, 13-C), 207.3 (C, 14-C), 85.7 (C, 15-C), 15.9 (CH
3, 16-C), 18.3 (CH
3, 17-C), 25.2 (CH
3, 18-C), 11.7 (CH
3, 19-C), 10.1 (CH
3, 20-C), 134.8 (C, 1 '-C), 128.3 (CH, 2 '-C), 127.2 (CH, 3 '-C), 130.5 (CH, 4 '-C), 129.2 (CH, 5 '-C), 128.3 (CH, 6 '-C), 145.1 (CH, 7 '-C), 118.7 (CH, 8 '-C), 167.1 (C, 9 '-C), carbon atom mark is see Fig. 1.Infrared spectra shows that this compound contains hydroxyl (3587,3502 and 3376cm
-1), carbonyl (1708 and 1686cm
-1) and aromatic ring (1605 and 1498cm
-1) group.
1this compound of HNMR spectrum display contains a trans-cinnamic acid ester group [δ H7.57 (br, d, J=7.0Hz, H-2 '), 7.33 (br, t, J=7.0Hz, H-3 '), 7.35 (br, t, J=7.0Hz, H-4 '), 7.34 (br, t, J=7.0Hz, H-5 '), 7.58 (br, d, J=7.0Hz, H-6 '), 7.69 (d, J=16.5Hz, H-7 '), 6.62 (d, J=16.5Hz, H-8 '), δ C134.8 (1 '-C), 128.3 (2 '-C), 127.2 (3 '-C), 130.5 (4 '-C), 129.2 (5 '-C), 128.3 (6 '-C), 145.1 (7 '-C), 118.7 (8 '-C), 167.1 (9 '-C)], four containing oxygen methyne [δ H5.51 (t, J=4.0Hz, H-3), 3.40 (dd, J=10.0Hz and 5.5Hz, H-9), 3.14 (d, J=1.5Hz, H-11) and 4.11 (d, J=1.5Hz, H-12)], three methylene radical, and five methyl [δ H1.01 (d, J=7.5Hz, H
3-16), 1.11 (s, H
3-17), 0.91 (s, H
3-18), 0.89 (s, H
3-19) and 1.17 (s, H
3-20)].In HMBC spectrum, the dependency of H-3 and C-9 ' confirms that this styracin ester group is positioned on C-3 position.Except trans-cinnamate acid ester moiety,
13cNMR and DEPT composes display 20 carbon signals, also comprises six quaternary carbons (two carbonyl carbon δ C211.7 and 207.3, and containing oxygen quaternary carbon δ C85.7) except the functional unit that correspondence is above-mentioned.Above-mentioned nuclear magnetic data shows that this compound is a diterpene alcohol laurate compounds.In HMBC spectrum, H
2-1 and C-2, C-3, C-4, C-15 and C-16; H-3 and C-1, C-15 and C-9 '; H
3-16 and C-1, C-2 and C-3; The dependency of OH-15 and C-4 and C-14, and in conjunction with the chemical shift of these protons with resonance carbon, show that this Compound C-2, C-3 and C-15 position are connected with methyl, laurate fragment and oh group respectively.H-4 and C-5 and C-6 in being composed by HMBC; H
3-17 and C-5, C-6 and C-13; H
3-20 and C-6, C-13 and C-14; The dependency of OH-15 and C-14, known Isosorbide-5-Nitrae-cyclohexanedione ring is connected with five-ring by C-4 with C-15, and C-6 and C-13 position is connected with a methyl respectively.In addition, H-9 and H-11 and C-18 and C-19; H-11 and C-12 and C-13; H-12 and C-13 and C-20; H
3-17 and C-7; H
3-18 and H
3-19 and C-9, C-10 and C-11; H
3-20 and C-12; The dependency of OH-9 and C-9 and C-10 shows that this compound contains an octatomic ring, and be connected with six-ring by C-6 with C-13, C-9, C-11 and C-12 position is respectively connected with a hydroxyl, and C-10 position is connected with two methyl.In NOESY spectrum, H-3 and H-2 and H-4; H-4 and H-2, H-3 and H
3-17; H-9 and H-11 and H
3the dependency of-18 shows that these protons are α configuration.Comprehensive hydrogen spectrum, carbon spectrum, HMBC spectrum and NOESY spectrum, and document is about correlation type nuclear magnetic data, can substantially determine this compound as shown in Figure 1, steric configuration is determined further by ECD test, theoretical value and experimental value basically identical (Fig. 2).
Embodiment 2: compound (I) pharmacological action is tested
One, material and instrument
SD male rat (4 week age) is purchased from Hebei Medical University's animal experimental center, cleaning grade, body weight 150g.Compound (I) is made by oneself, and HPLC normalization method purity is greater than 98%.1 α, 25 (OH)
2d
3be purchased from Plymouth, Britain biological research laboratories.α-MEM substratum is purchased from U.S. GIBCOBRL.SolubleRANKL is purchased from Britain PeprotecScience.Tartrate resistant acid phosphatase (TRAP) test kit is purchased from Sigma Co., USA.Dextrane gel is purchased from Shanghai Ru Ji development in science and technology company limited.Standard foetal calf serum is purchased from the Shandong biological company limited of the fragrant great achievement of silver.Benzylpenicillin sodium for injection is purchased from North China pharmaceutical Co. Ltd.Streptomycin sulphate for injection is purchased from Shenzhen China medicine south pharmaceutical Co. Ltd.Acetone, formaldehyde solution are purchased from Shijiazhuang City Organic Chemical Plant.Isoflurane is purchased from Lunan Beite Pharmaceutical Co., Ltd..Dimethyl sulfoxide (DMSO) is purchased from Tianjin recovery fine chemistry industry institute.
BB5060/BB16 CO2gas incubator (Shanghai Heraeus company), the VD-650 type table ultra-clean operator's console of above formula cell cultures (Purifying Equipment Co., Ltd., Suzhou), BFX5-320 type low speed centrifuge (Chinese Shanghai Lu Nan scientific instrument related factory), XD-1019810 inverted microscope (south of the River company), CX-21 opticmicroscope (Japanese OLYMPUS), micro sample adding appliance (French GILSON), 2510 type oscillation inverter instrument (Xinbo Biological Technology Co., Ltd., Shanghai), GF-300 type electronics sky (JapanA & DCompany, limited), 725 type Ultralow Temperature Freezers (FormaScientific.Inc).The material do not mentioned and instrument are conventional instrument.The solution preparation do not mentioned all adopts this area conventional soln compound method to prepare.
The preparation of drug solution preparing and sephadex column:
1, the preparation of α-MEM nutrient solution:
(1) α-MEM powder one bag+ultrapure water 800ml, dissolves completely;
(2) add sodium bicarbonate 3.02g add until completely dissolved hydrochloric acid survey PH reach 7.2;
(3) ultra-pure water is again added to 1000ml;
(4) added with antibiotic (penicillin and Streptomycin sulphate);
(5) sterilizing (0.22 micrometer Millipore membrane filtration) is filtered
(6) 500ml sterilizing bottle packing, 4 DEG C are in store for.
2,1 α, 25 (OH)
2d
3the packing of storage liquid
(1) 1 α is got, 25 (OH)
2d
3stoste 50 microlitre (50 microlitre one), adds dehydrated alcohol 1150 microlitre, makes total amount to 1200 microlitre (i.e. 24 times of dilutions)
(2) with 100 microlitres/be only sub-packed in 12 sterilizing test tubes, store for future use in-30 DEG C of refrigerators;
(3) again dilute 1000 times before application, make ultimate density be 1 × 10
-8m.
3, SolubleRANKL storage liquid packing
(1) RANKL lyophilized powder one (10 microgram) is got;
(2) 0.1Mtris-HClbuffer50 microlitre dissolves RANKL;
(3) with 5 microlitres/be only sub-packed in 10 sterilizing freeze pipes, store for future use in-30 DEG C of refrigerators;
(4) one is taken out before using, first with dissolving containing 10%FBS α-MEM nutrient solution 495 microlitre (namely diluting 100 times); Again dilute 100 times before interpolation, make final concentration be 20ng/ml.
4, the preparation of 0.1Mtris-HClbuffer
(1)tris-HCl12.11g(0.1M)；
(2) ultrapure water 800ml (add after dissolving, regulate pH to 8.2);
(3) ultrapure water is added for subsequent use to 1000ml.
5, the preparation of compound (I) storage liquid
(1) concentration of storage liquid: 1mg/ml and 100ug/ml.
(2) preparation of storage liquid: Weigh Compound (I) 1mg is put in sterilizing freeze pipe, adds dimethyl sulfoxide (DMSO) 1ml and dissolves completely, is made into the storage liquid of 1mg/ml.From the storage liquid of 1mg/ml, take out 100 microlitres be put in another sterilizing freeze pipe, add the storage liquid that namely dimethyl sulfoxide (DMSO) 900 microlitre is made into 100 mcg/ml, be stored in 4 DEG C of constant temperature refrigerators, be no more than two weeks duration of service.The storage liquid freezen protective of 1mg/ml.
6, the preparation of TRAP stationary liquid
Before dyeing, prepare by product description.First get one, aseptic 20ml test tube, add reagent successively in the following order: acetone 6.5ml, Citric Acid 2.5ml, formaldehyde 0.8ml, fully mix, meter 9.8ml, now with the current.
7, the preparation of TRAP staining fluid
Prepare before dyeing equally, by description of product preparation, now with the current.
8, the preparation of sephadex column
(1) 50ml syringe one after sterilization, removing inner core;
(2) injection tube and injection head join and dispose the rayon balls one after sterilizing;
(3) anchor fixes syringe, and syringe needle place connects the threeway of closing shape;
(4) dextrane gel of sterilizing is mixed, extract 30ml and inject in syringe, open threeway, slow filtering-depositing, until gel precipitation liquid level to during 15ml; Close threeway, put into 4 DEG C of constant temperature refrigerators and spend the night;
(5) test and slowly inject in gel column with the α-MEM nutrient solution 50ml containing 15% foetal calf serum for first 1 hour, open threeway, stand-by after natural filtration detergent gel post.
Two, test method
1, full medullary microeirculation
1.1 full medullary cells extract
Experiment starts standard foetal calf serum to be put into 37 DEG C of water baths in first 30 minutes and thaws; Getting 50mL sterile centrifugation tube one, load 40mL not containing the α-MEM nutrient solution of foetal calf serum, using in order to rinsing full medullary cell.
(1) select SD male rat 1 in 4 week age, after 75% ethanol disinfection, adopt isoflurane inhalation anesthesia; (2) after anesthesia onset, under aseptic condition, get rat both sides shin bone and femur, cut bone dirt end and expose medullary space; (3) extract not containing the α-MEM nutrient solution of serum with 10mL syringe, in medullary space, go out medullary cell after using No. 25 syringe needles instead in 50mL sterile centrifugation tube; (4) extracted cell suspension is is fully blown and beaten, after there is no agglomerate, the centrifuge tube filling cell is put into whizzer together with equilibration tube centrifugal, adjustment rotating speed (2000 revs/min), centrifugal 5 minutes; In centrifugal process, the α-MEM nutrient solution 50mL (7.5mL foetal calf serum add in 42.5mL α-MEM nutrient solution) of preparation containing 15% foetal calf serum is for subsequent use; (5) abandoning supernatant after centrifugal end, adds the α-MEM nutrient solution 20mL containing 15% foetal calf serum, fully blows and beats mixing, and form cell suspension, this liquid is referred to as cell stoste.
The calculating (original liquid concentration and extension rate) of 1.2 cell count
(1) the cell suspension 25 μ L getting above extraction puts in vitro, then adds 5% acetic acid 475 μ L, namely forms the cell suspension of 20 times of dilutions; Put by test tube on the oscillator, vibrated for 20 seconds, object removes red corpuscle; (2) cell count is calculated: after getting vibration, cell suspension instills cell counting dish, does not exceed edge, and cover glass covers, and calculate cell count under inverted microscope, in counting dish 4 large lattice, all cells all counts.This experimental calculation cell count is 537, according to formula (cell count/4) × 20 × 10
4cells/mL calculates the concentration of cell stoste, following (537/4) × 20 × 10 of this experimental calculation
4=26.85 × 10
6cells/mL i.e. this experimental cell original liquid concentration is 26.85 × 10
6cells/mL, and the object concentration that we need is 2 × 10
6cells/mL, so cell stoste needs dilution.(3) extension rate is calculated as follows: extension rate=original liquid concentration/object concentration; This experiment extension rate=26.85 × 10
6cells/mL/2 × 10
6cells/mL=13.425, namely stoste needs dilution 13.425 doubly just can reach the object concentration of needs.This experiment needs 2 × 10
6the cell suspension 40mL of cells/mL, referred to as object amount.(4) amount of the stoste needed for concentration that achieves the goal, is calculated as follows: the amount=object amount/extension rate of the stoste needed for concentration that achieves the goal.This experimental calculation is as follows: the amount=40mL/13.425 ≌ 2.98mL of the stoste needed for concentration that achieves the goal.Namely 26.85 × 10 are got
6the cell stoste 2.98mL of cells/mL puts into the 50mL centrifuge tube of sterilizing, then adds 37.02mL (40-2.98) and can be made into 2 × 10 containing the α-MEM nutrient solution of 15% foetal calf serum
6the cell suspension 40mL of cells/mL, then adds 1 α, 25 (OH)
2d
3storage liquid 40 μ L, lucifuge is answered in operating process, i.e. 1 α, 25 (OH)
2d
3final concentration is 1 × 10
-8m; Add 4 μ LsolubleRANKL storage liquid again, namely solubleRANKL final concentration is 20ng/mL; Liquid feeding process operates on Bechtop, so far, and required 2 × 10
6the cell suspension object amount of cells/mL has configured.
1.3 culturing cells, dosing
(1) cell inoculation: get 4 row 6 row 24 well culture plate one piece, every hole adds 2 × 10
6the cell suspension 0.5mL (1 × 10 of cells/mL
6cells).(2) compound (I) is added: get 100 μ g/mL compound (I) packing liquid one in the 2nd row of culture plate and add 2.5 μ L respectively, often adding a hole all needs to change a suction nozzle; The 3rd every hole of row adds 5 μ L; The 4th every hole of row adds 10 μ L.After adding the 4th hole, this liquid is put into 4 DEG C of constant temperature refrigerators and preserve, get 1mg/mL compound (I) storage liquid one.The 5th every hole of row adds 1mg/mL compound (I) storage liquid 2.5 μ L; The 6th every hole of row adds 5 μ L.Add rear residue liquid and put into 4 DEG C of constant temperature refrigerators preservations.First row does not add medicine, as a control group.So far in 24 well culture plates, there are 6 concentration the every provisional capital of compound (I), and be respectively 0,0.5,1,2,5,10 μ g/mL, each concentration is row 4 hole (n=4) often, and first is classified as control group.(3) culturing cell: after adding medicine, puts into CO by culture plate
2in incubator, at 37 DEG C, 5%CO
2and under saturated humidity condition, cultivate 7 days.Nutrient solution is changed once when being cultured to the 4th day, α-MEM nutrient solution the 40m (l6mL foetal calf serum add in 34mL α-MEM nutrient solution) of preparation containing 15% foetal calf serum as stated above, include 10 μ L penicillin 8 μ L Streptomycin sulphate (800,000 u penicillin, 2mL aseptic distillation water dissolution used respectively by 1000000 Streptomycin sulphates), add 4 μ LsolubleRANKL storage liquid, then 1 α is got, 25 (OH)
2d
3storage liquid 40 μ L adds.Take out culture plate in incubator after, observation of cell upgrowth situation under inverted microscope, every hole sucking-off old nutrient solution 300 μ L, add new nutrient solution 400 μ L, every sucking-off one arranges replacing suction nozzle, often adds a hole and changes a suction nozzle.Change after liquid completes, culture plate is continued to put into CO
2in incubator, at 37 DEG C, 5%CO
2and under saturated humidity condition, then cultivate 3 days.
2, broken bone precursor cell (POC) is cultivated
2.1 the extraction of full medullary cell: as the above method, prepare full bone marrow cell suspension 1.5mL.
The extraction of 2.2 non-attachment medullary cells
(1) with the flushed gel column of nutrient solution, be fixed on anchor; (2) slowly in gel column, inject full medullary cell 1.5mL, open threeway, slowly filter; Infiltrate after in gel column until the full medullary cell of 1.5mL, more slowly inject the α-MEM nutrient solution 10mL containing 15% foetal calf serum.(3) nutrient solution collected containing non-attachment medullary cell amounts to 12-15mL, and this liquid is called non-attachment medullary cell stoste.
The calculating (original liquid concentration and extension rate) of 2.3 cell count and the amount of the stoste needed for concentration that achieves the goal
As the above method, calculate cell count, extension rate, the amount of the stoste needed for concentration that achieves the goal.This experimental calculation cell count is 337, calculates that the concentration of cell stoste is 16.85 × 10
6cells/mL; Extension rate is 8.425, and namely cell stoste need dilute the object dense (2 × 10 that 8.425 times just can reach needs
6cells/mL); The amount 40mL/8.425 ≌ 4.75mL of the stoste needed for concentration that achieves the goal, namely gets the 50mL centrifuge tube that 4.75mL cell stoste puts into sterilizing, then adds 35.25mL (40-4.75) and can be made into 2 × 10 containing the α-MEM nutrient solution of 15% foetal calf serum
6the object cell suspension 40mL of cells/mL, then adds 1 α, 25 (OH)
2d
3storage liquid 40ul, lucifuge is answered in operating process, i.e. 1 α, 25 (OH)
2d
3final concentration is 1 × 10
-8m; Add solubleRANKL storage liquid 4uL, namely solubleRANKL final concentration is 20ng/mL; Liquid feeding process all operates on Bechtop, so far, does not adhere to bone marrow cell suspension object amount and has configured.
2.4 culturing cells and dosing are as aforementioned.
3, observe under inverted microscope
The adjustment of 3.1 inverted microscopes
(1) by microscope sympodium;
(2) visual field aperture is opened greatly to consistent with condenser diaphragm edge;
(3) phase-plate consistent with object lens magnification is put into condensing apparatus;
(4) eyepiece in tune is changed in eyepiece stalk, the focusing ring in turn tune on eyepiece is to the clear picture of phase-plate phase ring;
(5) knob in regulating the phase ring on condensing apparatus to adjust, makes two doughnut pictures be overlapped into concentric(al) circles state;
(6) eyepiece in tune is changed with common eyepiece.
3.2TRAP dyeing
(1) cultivate after 7 days, culture plate is taken out in incubator, discards nutrient solution; (2) add stationary liquid 0.4mL/ hole, fix 1 minute; (3) stationary liquid is discarded, distilled water flushing four times; (4) add staining fluid 3-4 and drip/hole, wrap up culture plate with tinfoil; (5) at 37 DEG C, 5%CO
2and after hatching 30 minutes under saturated humidity condition, take out culture plate, discard staining fluid, distilled water flushing four times, seasoning.
3.3 observe
Cultivate and after 7 days, culture plate is taken out in incubator, before discarding nutrient solution, under inverted microscope, observe upgrowth situation and the form of cell under different pharmaceutical concentration, determine whether dye.Color is observed under band staining fluid inverted microscope after dyeing.
4, om observation
After observing under band staining fluid inverted microscope, discard staining fluid, distillation washing 4 times, seasoning, ordinary light Microscopic observation calculates osteoclast number.
5, statistical analysis
SPSS16.0 statistics software is used to analyze.Data processing adopts mean ± SD to represent, to the different pharmaceutical concentration same treatment time, each group is compared with between control group, compound (I) is on the impact of osteoclast formation and differentiation, the data analysis drawn adopts variance analysis, thus draws the variation relation of drug effect and drug level.
Three, result and conclusion
In full medullary microeirculation system, 0.5,1,2 μ g/mL groups compare with control group, TRAP staining positive cells (more than 3 cores) number is only 49.04%, 28.85%, 5.77% of control group, through statistical analysis, dosing group is formed with restraining effect to osteoclast, when drug level is 1 μ g/mL compared with control group, there is remarkable restraining effect (P ﹤ 0.01) to osteoclast formation.In table 1.
In broken bone precursor cell training system, 0.5,1,2,5 μ g/mL groups are compared with control group, TRAP staining positive cells (monokaryon or 2 cores) number is only 85.11%, 65.79%, 37.83%, 2.21% of control group, through statistical analysis, dosing group is formed with restraining effect to broken bone precursor cell, when drug level is 2 μ g/mL compared with control group, remarkable restraining effect (P ﹤ 0.01) is formed with to broken bone precursor cell.In table 2.
Conclusion, this result of study shows that compound (I) is to the differentiation of osteoclast and breed inhibited, and there is concentration-dependent relation.Application compound (I) treatment likely becomes a kind of promising methods for the treatment of based on the osteoclasia disease of osteoclast activity enhancing.
TRAP staining positive cells (more than 3 cores) number in the full medullary microeirculation system of table 1
| 0 (control group) | 0.5μg/mL | 1μg/mL | 2μg/mL | 5μg/mL | 10μg/mL | |
| Parallel running 1 | 83 | 56 | 26 | 5 | 2 | 0 |
| Parallel running 2 | 81 | 44 | 28 | 4 | 0 | 0 |
| Parallel running 3 | 81 | 22 | 19 | 4 | 0 | 0 |
| Parallel running 4 | 67 | 31 | 17 | 5 | 0 | 2 |
Table 2 breaks TRAP staining positive cells in bone precursor cell training system (monokaryon or 2 cores) number
| 0 (control group) | 0.5μg/mL | 1μg/mL | 2μg/mL | 5μg/mL | 10μg/mL | |
| Parallel running 1 | 132 | 120 | 71 | 48 | 1 | 0 |
| Parallel running 2 | 135 | 102 | 91 | 56 | 5 | 0 |
| Parallel running 3 | 108 | 90 | 86 | 46 | 3 | 0 |
| Parallel running 4 | 122 | 111 | 79 | 38 | 2 | 0 |
Embodiment 3
The preparation of tablet: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, vehicle is added, pelletizing press sheet than the ratio for 1:7 in itself and excipient weight.
Embodiment 4
Prepared by oral liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, oral liquid method for making makes oral liquid routinely.
Embodiment 5
The preparation of capsule or granule: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, add vehicle in itself and excipient weight than the ratio for 1:7, make capsule or granule.
Embodiment 6
The preparation of injection liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, inject with water routinely, essence filter, injection liquid is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, be dissolved in sterile water for injection, stirring makes molten, filter with aseptic suction funnel, aseptic essence filter again, be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains powder injection.
The effect of above-described embodiment is essentiality content of the present invention is described, but does not limit protection scope of the present invention with this.Those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the protection domain of technical solution of the present invention.
Claims (7)
1. there is the compound (I) of following structural formula,
2. the preparation method of compound according to claim 1 (I), it is characterized in that comprising following operation steps: the dry flower of lilac daphne is pulverized by (a), extract with 70 ~ 80% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), first use 10% ethanol elution, 8 column volumes, then use 80% ethanol elution, 10 column volumes, collect 80% ethanol eluate, concentrating under reduced pressure obtains 80% ethanol elution thing medicinal extract; C in () step (b), 80% ethanol elution medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 75:1,45:1,25:1,15:1 and 1:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 20:1,15:1 and 8:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 10 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
3. the preparation method of compound according to claim 2 (I), is characterized in that: described macroporous resin is D101 macroporous adsorbent resin.
4. the preparation method of compound according to claim 2 (I), is characterized in that: it is 75% that described alcohol heat reflux extracts the alcohol concn adopted.
5. a pharmaceutical composition, is characterized in that: the compound according to claim 1 (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
6. the application of compound according to claim 1 (I) in the medicine of preparation treatment osteoclasia disease.
7. the application of pharmaceutical composition according to claim 5 in the medicine of preparation treatment osteoclasia disease.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106083766A (en) * | 2016-06-03 | 2016-11-09 | 武晓丹 | A kind of miscellaneous note compounds and preparation method thereof |
| JP2019534338A (en) * | 2016-11-14 | 2019-11-28 | コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジーＫｏｒｅａ Ｒｅｓｅａｒｃｈ Ｉｎｓｔｉｔｕｔｅ Ｏｆ Ｂｉｏｓｃｉｅｎｃｅ Ａｎｄ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ | Pharmaceutical composition for prevention or treatment of neurodegenerative diseases, comprising Fujimodoki flower extract or a fraction thereof as an active ingredient |
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| EP1296697A1 (en) * | 2000-06-07 | 2003-04-02 | Peplin Research Pty Ltd | Therapeutic agents - i |
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2015
- 2015-12-07 CN CN201510887582.4A patent/CN105348095A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1296697A1 (en) * | 2000-06-07 | 2003-04-02 | Peplin Research Pty Ltd | Therapeutic agents - i |
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| TIAN, YE等: "Diterpenoids with Diverse Skeletons from the Roots of Euphorbia micractina", 《JOURNAL OF NATURAL PRODUCTS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106083766A (en) * | 2016-06-03 | 2016-11-09 | 武晓丹 | A kind of miscellaneous note compounds and preparation method thereof |
| JP2019534338A (en) * | 2016-11-14 | 2019-11-28 | コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジーＫｏｒｅａ Ｒｅｓｅａｒｃｈ Ｉｎｓｔｉｔｕｔｅ Ｏｆ Ｂｉｏｓｃｉｅｎｃｅ Ａｎｄ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ | Pharmaceutical composition for prevention or treatment of neurodegenerative diseases, comprising Fujimodoki flower extract or a fraction thereof as an active ingredient |
| JP7221207B2 (en) | 2016-11-14 | 2023-02-13 | コリア リサーチ インスティチュート オブ バイオサイエンス アンド バイオテクノロジー | A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, containing a flower extract of Fujimodoki or a fraction thereof as an active ingredient |
| US11738063B2 (en) | 2016-11-14 | 2023-08-29 | Korea Research Institute Of Bioscience And Biotechnology | Pharmaceutical composition for preventing or treating neurodegenerative diseases which includes flower extract of Daphne genkwa or fractions thereof as active ingredient |
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