CN101343265A - Medicament containing cave flower active principle and formulation thereof - Google Patents

Abstract

The invention discloses a medicine prepared by an active ingredient C18H16O7 extracted from stone flower. The invention provides a preparation method which mainly adopts solvents such as petroleum ether, normal hexane and ethyl acetate for extraction, and selects the solvents with large polarity to remove coloring matters, and adopts the solvents with small polarity for recrystallization, so as to prepare the active ingredient of C18H16O7. The medicine and the preparation method have advantages of low active ingredient loss, high crystallization purity, stable product yield, good quality and reasonable process. A quality detection method provided in the invention can properly control the product quality and ensure the related drug effects of products, more specifically, the invention relates to a pharmaceutical preparation made by the active ingredient of stone flower and derivates thereof and having the related regulating actions of anti-inflammation, anticancer, antivirus and prevention and cure of hypoimmunity.

Description

A kind of medicine and preparation thereof that contains cave flower active principle

Technical field

The invention discloses a kind of medicine that the new effective constituent C18H16O7 that extracts makes that relates to from parmelia saxatilis.Preparation method of the present invention mainly adopts oil fan, normal hexane, ethyl acetate equal solvent to extract, and selects for use the big solvent of polarity to remove pigment, adopts the less solvent recrystallization of polarity, makes C18H-16O7 effective constituent.Have advantages such as loss of effective components is little, crystallization purity is high and product yield is stablized, and quality is good, and technology is reasonable.Quality determining method of the present invention can be controlled the quality of product well, and can guarantee the relevant drug effect of product, have anti-inflammatory, anticancer, antiviral, a control hypoimmunity associated adjustment effect more specifically to a kind of, and the pharmaceutical preparation made from effective constituent and derivative.

Background technology

Parmelia saxatilis is called white parmelia saxatilis, stone clothing, skin of South and East Asian rice frog, the plum clothing, its Latin is called Parmelia saxatilis Ach., and thallus is wrinkled more, about 30 centimetres of diameter, above canescence, the edge apportion becomes most irregular lobes, sliver tip circle, the edge the fecula bud, and is level and smooth, little glossy, the close living black of central pin bud, more coarse, the lower edge part is level and smooth, and tarnish has rhizoid.Sub-device is few, cup-shaped, the about 1 centimetre of one-tenth of diameter, short handle is arranged: the oval Room 1 of spore, colourless, be born on rock and the bark, all there is distribution all parts of the country, and it mainly contains modern bibliographical information sekikaic acid, parmelia saxatilis element, removes first ox-hide folic acid, Sa La Qin acid etc., still contain black phenolic acid methyl esters etc. in addition, have clearing heat and detoxicating, cool blood, hemostasis, diuresis, the effect of kidney tonifying, invigorating the spleen and replenishing QI existing is treated diseases such as hysteromyoma, calculus, strengthening immunity with it.The report of the effective constituent related drugs preparation of from existing bibliographical information, also not finding among the present invention to be mentioned.

Inflammatory reaction (comprising short scorching reaction and anti-inflammatory response) is the protectiveness stress reaction that body resists external paathogenic factor invasion and attack.But, then can form systemic inflammatory reaction and combine disease (SIRS) and the comprehensive disease (CARS) of compensatory anti-inflammatory response if undue strong, out of hand.Can cause multiple organ dysfunction syndrome (MODS) and multiple system organ failure (MSOF) subsequently.MODS not only has inflammatory mediator excessively to discharge, and with the not enough of endogenous anti-inflammatory medium or excessively release, both all can increase the weight of inflammatory damage, the body internal organs are produced huge destruction, and major complications has clinical critical illnesses such as Sepsis, serious burn, acute pancreatitis, hemorrhagic shock, wound.(multiple organ dysfunctionsyndrome is MODS) so that dead finally to cause multiple organ dysfunction.

Immunity system is that live organism is to heteroplasmonic defense mechanism external source or endogenous, generation is the myelocyte class of representative with scavenger cell and neutrophil leucocyte, lymphocytes such as T cell and B cell, these cell classes not only play independent action, and by intercellular directly contact or solubility factor work mutually, be referred to as cytokine, thereby keep its homeostasis.Defense mechanism produces so subtly, so that fragile equilibrated collapse can cause serious morbid state.

This collapse triggering for generating autoantibody of immunocyte control mechanism or cause the over-drastic immune response it is believed that it can cause collagen diseases, whole body lupus erythematosus and various anaphylactic disease.In AIDS, the infection of HIV and T cell causes immunity system decline, and this decline can further develop, and in addition, because the forfeiture of immunologic balance, the chronic disease that is caused by the development of diabetes or virus or cancer morbid state also part occurs.

In recent years, cytokine formation inhibitor such as S-Neoral have been used for the treatment of above-mentioned disease.In addition, the pair cell factor generates has the agent that disappears of inhibiting steroid class to be used for autoimmune disease, as irritated, atopy is sick and rheumatosis and bronchial asthma, at present in Chinese medicine, natural drug, polysaccharide and fungi plant all are used to strengthen human immune system's disease, and have result of treatment to a certain degree.

As you know, immunity system is absolutely necessary for the temporary transient anti-defense mechanism of parasitism, and gives under the immune deficiency condition that causes in immunosuppressor or carcinostatic, and the host is easy to be smitten by an epidemic.Now, be used to suppress under the immune deficiency condition that a kind of medicine that various cytokines produce such as immunological reagent or carcinostatic administration cause, the host is easy to be smitten by an epidemic.Now, be used to suppress a kind of medicine that various cytokines produce such as immunological reagent or steroid class antiphlogistic and can not arbitrarily be used for autoimmune disease, it is restricted with administering mode or dosing interval.In order to treat and above-mentioned immunity system diseases associated, therefore need a kind of existing immunization of exploitation to can be used for anti-inflammatory, anticancer preparation again.

Summary of the invention

The objective of the invention is to prepare the pharmaceutical preparation that a kind of existing immunization that height specificity and tight security arranged can be used for anti-inflammatory again, this is by to extensively there being the screening of immunization Chinese medicine and crude drug, from parmelia saxatilis obtain a kind of efficiently, the medicine of the anti-inflammatory of single effective constituent, anticancer, strengthening immunity, it makes in the following manner, and preparation method is as follows:

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, use weak polar solvent (preferred oil fan, normal hexane, hexanaphthene, ethyl acetate), solvent load is 10-20 a times of medicinal material weight, being heated to boiling and stopping heating, extract 2 times, extracted 2 hours at every turn, obtain extracting solution.Extracting solution is concentrated.

Resulting extracting solution is concentrated into 1-2 times of medicinal material volume, filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

The gained primary crystal adds boiling water or low polar solvent (preferred oil fan, normal hexane, hexanaphthene, ethyl acetate, the ethanol) dissolving again that coarse-grain weight 4-8 doubly measures, and removes by filter the chlorophyll constituents.

Filter the lysate of the low polar solvent (preferred solvent oil fan, normal hexane, hexanaphthene, ethyl acetate, ethanol) of back solution,, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal below 10 degrees centigrade.

Obtain crystallization through suction filtration or smart filter, and use the recrystallization solvent for use to clean, and obtaining effective constituent by drying, the physicochemical property of its contained effective constituent are as follows: this product is for talking yellow crystal, be soluble in organic solvents such as chloroform, oil fan, be insoluble in the first alcohol and water.

Resulting crystallization turns out to be single component through high performance liquid phase, further by infrared, and hydrogen spectrum, carbon spectrum,

Nuclear-magnetism Chun, single crystal diffraction altogether turns out to be C ₁₈H ₁₆O ₇Structure (seeing accompanying drawing one～six) related data is as follows.

The related data form

Table 1 The note of x-ray of the Figures single crystal diffraction data

Table 2 ¹H (600MHz) and ¹³C (600MHz) NMR chemical shifts for compounds in DMSO-d6 crystalline hydrogen spectrum carbon spectrum chemical displacement value

Table 1 (table one) The note ofx-ray of the figures

Table 2 (table two) ¹H (600MHz) and ¹³C (600MHz) NMR chemical shifts for compounds inCDCl3

Turn out to be single component (seeing accompanying drawing seven) by high performance liquid phase, its purity reaches more than 99%.

Crystallization prepares implementation column 1

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, uses weak polar solvent oil fan to extract, and solvent load is 5-15 a times of medicinal material weight, being heated to boiling and stopping heating, extracts 3 times, extracts 2 hours at every turn, obtains extracting solution.Extracting solution is dense

Resulting extracting solution is concentrated into 1-2 times of medicinal material volume, filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

Boiling water or low polar solvent 10-70% ethanol that gained primary crystal adding coarse-grain weight 4-8 doubly measures dissolve again, remove by filter the chlorophyll constituents.

Filter back solution and carry out lysate,, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal below 10 degrees centigrade with low polar solvent normal hexane.

Obtain crystallization through suction filtration or smart filter, and use the recrystallization solvent for use to clean, obtain effective constituent by drying, the physicochemical property of its contained effective constituent are as follows

Crystallization prepares implementation column 2

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, uses the weak polar solvent normal hexane, and solvent load is 10-20 a times of medicinal material weight, being heated to boiling and stopping heating, extracts 2 times, extracts 2 hours at every turn, obtains extracting solution.Extracting solution is dense

Resulting extracting solution is concentrated into 1-2 times of medicinal material volume, filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

The gained primary crystal adds boiling water or the low polar solvent acetic acid second fat that coarse-grain weight 4-8 doubly measures: 2: 1 mixed solvent removal of impurities of normal hexane removes by filter the chlorophyll constituents.

Filter post crystallization with low polar solvent n-hexane dissolution,, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal below 10 degrees centigrade.

Crystallization prepares implementation column 3

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, uses the weak polar solvent ethyl acetate, and solvent load is 18-20 a times of medicinal material weight, being heated to boiling and stopping heating, extracts 3 times, extracts 2 hours at every turn, obtains extracting solution.Extracting solution is dense

Resulting extracting solution is concentrated into 1-2 times of medicinal material volume, filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

The gained primary crystal adds coarse-grain weight 4-8 and doubly measures the ethanol removal of impurities, removes by filter the chlorophyll constituents.

Filter the dissolving of post crystallization,, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal below 10 degrees centigrade with low polar solvent chloroform.Obtain crystallization through suction filtration or smart filter, and use the recrystallization solvent for use to clean, obtain effective constituent by drying.

Use the high performance liquid phase detection method: with acetonitrile-0.1%, phosphoric acid (90: 10) is made moving phase, detects wavelength 284, chromatographic column: C18ODS pillar.Verified is single component.

Crystallization prepares implementation column 4

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, uses polar solvent ethanol to extract, and solvent load is 10-20 a times of medicinal material weight, being heated to boiling and stopping heating, extracts 2 times, extracts 2 hours at every turn, obtains extracting solution.Extracting solution is dense

Resulting extracting solution is concentrated into 1-2 times of medicinal material volume, filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

The gained primary crystal adds the methyl alcohol heating removal of impurities that coarse-grain weight 4-10 doubly measures, and removes the chlorophyll constituents after the filtration, obtains crystallization.

Crystallization after the filtration is with ethyl acetate: the dissolving of hexanaphthene (ratio is 3: 1) mixed solvent, filter, filtrate is below 10 degrees centigrade, sufficient standing 24-48 hour, separates out yellow crystal.

The high performance liquid phase detection method: with acetonitrile-0.1%, phosphoric acid (90: 10) solution is moving phase, detects wavelength 284, chromatographic column: the C18ODS pillar, detect and be single component.

Experimental example 1 composition anti-inflammatory pharmacodynamics of the present invention and Study on mechanism experiment

One, LPS is stimulated the influence experiment of the TNF-a of RAW264. cell

Test materials

1, medicine (knot/crystalline substance): the administration group, be this compound, design three concentration, be followed successively by 10ug/ml, 5ug/ml, 1ug/ml, self-control; The negative control medicine is an astragalus polysaccharides, and concentration is 100ug/ml, self-control; Normal group is not administration group; The blank group is an experimental group of having only lipopolysaccharides to stimulate; Positive controls is a dexamethasone administration group.

The RPMI-1640 substratum is available from Gibco company, and MTT is available from AMRESCO company; TRIZOL Reagent is available from U.S. Promega company, and polymerase chain reaction (PCR) reagent is available from Japanese Toyobo company, and primer and fluorescent probe are synthetic by Shanghai Shen You biotech firm.35mm Petri Tissue Culture Dish (Japanese Meridian Instrument company), NAPCO2-5420-1 type carbonic acid gas formula incubator U.S. exact science company, the inverted microscope IMT-2 Japan Olympus LPS of company (E.coli 0127:B8, Difico Lab., the U.S.), dexamethasone is Shijiazhuang first pharmaceutical factory's product; Other reagent is analytical pure.The low-temperature and high-speed whizzer: model is 5415D, originates from German Eppendorf company; The low temperature low speed centrifuge: model is DLBR, originates from the ShangHai City centrifugal Machine Institute; Bechtop: originate from Purifying Equipment Co., Ltd., Suzhou; Ultraviolet spectrophotometer: model is 752 types, originates from Shanghai Precision Scientific Apparatus Co., Ltd; The pcr amplification instrument: model is 9600, originates from U.S. PE company.

2, RAW264.7 cell, RAW264.7 cell (Turnover of Mouse Peritoneal Macrophages tumour cell, the present of immunity teaching and research room of Tongji Medical College, Huazhong Science and Technology Univ.).

Two, experimental technique and result

1, all ingredients preparation

1) preparation of scavenging solution: potassium bichromate 360g is dissolved in the 3L distilled water, is stirred to dissolving fully, slowly adds industrial vitriol oil 600mL, stirs heat radiation, is mixed with time strong acid washing lotion.

2) RPMI 1640 substratum: take by weighing 10.2g RPMI-1640 cell culture medium and be dissolved in the fresh distilled water of 1000ml, use NaHCO ₃Regulate pH to 7.1-7.4, suction filtration degerming, 4 ℃ of preservations are filtered in degerming behind the adding additive.

3) lipopolysaccharides solution: lipopolysaccharides dilutes with sterile saline, and concentration is 1mg/mL, the suction filtration degerming.By once testing the consumption packing, be stored in-20 ℃.During experiment mother liquor is diluted to 100mg/mL with RPMI 1640 substratum that contain 10% calf serum, by the requirement of experiment application of sample, making its final concentration is 10ug/mLo again

4) preparation of PBS liquid: every liter of PBS liquid contains 0.2g KCl, 0.2g KH2PO4,8.0g NaCl and 1.56g Na ₂HPO4HzO, suction filtration degerming, 4 ℃ of preservations.

2, drug cell toxicity test

The relative proliferation rate of cell tetrazolium (MTT) colorimetry: cultivate RAW264.7 cell (Turnover of Mouse Peritoneal Macrophages tumour cell, the present of immunity teaching and research room of Tongji Medical College, Huazhong Science and Technology Univ.) with 96 plates, at 37 ℃, 5%CO ₂Under the condition, with containing 10% calf serum, penicillin (1 * 10 ⁵UL ^-1) and Streptomycin sulphate (100mgL ^-1) the cultivation of going down to posterity of RPMI-1640 nutrient solution, experiment all is in logarithmic phase with cell.According to requirement of experiment with 2 * 10 ⁸Cells/L cultivates 24h in 96 orifice plates, treat cell attachment, abandon supernatant, wash 3 times with Hanks liquid, add different concns parmelia saxatilis crystallization (vat liquor concentration is respectively 1%, 5%, 10%, 15%, 20%, 30%), negative control DMEM nutrient solution is respectively at 24h, 48h and 72h, through 5mgPml MTT

TNF-α, IL-1 β and IL-6 are the present clearer and more definite common inflammatory factors that comes across inflammatory reaction.Therefore we at first observe these several different yellow crystals stimulates the short inflammatory factor that produces behind the RAW264.7 cell whether the regulating effect of reduction is arranged to LPS, adopts euzymelinked immunosorbent assay (ELISA) to measure corresponding content.

The result is from Fig. 2 to Fig. 4, after cell is intervened by LPS and parmelia saxatilis crystallization, the content of TNF-α, IL-1 β and IL-6 is than obviously reducing (p＜0.01or p＜0.05) in the independent stimulated cells of LPS. simultaneously, along with the increase of carrying the parmelia saxatilis crystallization content, its restraining effect also strengthens, and shows that the pharmacological action and the dosage of extract has positive correlation.But these excretory inflammatory mediators and the factor can not return to normal level, show that medicine has the anti-inflammatory action of keeping.(seeing accompanying drawing eight)

Effect experiment two,

After A, the preceding B of intervention, the intervention, (seeing accompanying drawing eight)

The result proves that medicine does not have cytotoxicity under this concentration.

Activity rating

1) cell cultures and incentive condition

There is not to carry out under the Cytotoxic drug level drug intervention effect experiment above-mentioned.

RAW264.7 cell (Turnover of Mouse Peritoneal Macrophages tumour cell, the present of immunity teaching and research room of Tongji Medical College, Huazhong Science and Technology Univ.), at 37 ℃, 5%CO ₂Under the condition, with containing 10% calf serum, penicillin (1 * 10 ⁵UL ^-1) and Streptomycin sulphate (100mgL ^-1) the cultivation of going down to posterity of RPMI-1640 nutrient solution, experiment all is in logarithmic phase with cell.According to requirement of experiment with 2 * 10 ⁸Cells/L cultivates 24h in 24 orifice plates, treat cell attachment, abandon supernatant, wash 3 times, divide into groups: cultured cells is divided into 7 groups (every group of each 3 hole): PBS control group: add phosphate buffered saline buffer (PBS) (not having any processing) in the nutrient solution with different extract experiments with Hanks liquid; LPS control group: the LPS that adds 10ng/ml in the nutrient solution; LPS+ different concns drug treating group (3 groups), LPS+ water treatment group.Adopt the RT-PCR method to detect TNF-α mRNA content after cultivating 24h.

2) total RNA extracts

According to the MTT experimental result, the maximum dose level of Cytotoxic minimum dose as the screening and assessment test appears with every group of medicine, intervene cell, cultivate sample under the specified time then according to requirement of experiment.Extract cell total rna with Trizol (U.S. Gibco company) by operation instructions, with the DNA enzyme I digestion trace amount DNA of no RNA enzyme, diethyl coke acid (DEPC) treating water dissolving RNA.

3)RT-PCR

(Roche Diagnostics, Mannheim Germany) carry out RT-PCR on the amplification instrument at LightCycler instrument.Adopt FastStart DNA Master SYBR Green I reagent to close (also from Roche), the LDCA software system analysis experimental result of using instrument to carry then.(seeing accompanying drawing nine)

Annotate: compare with model group: ^*P＜0.05, ^*P＜0.01

The result shows that medicine can reduce the effect of TNF-a total nucleic acid in 3 different concns.

Show certain anti-inflammatory activity.

Experimental example two, to TNF-α, IL-1 β, IL-6 and IL-10 content influence

(1) test materials

Medicine: the RAW264.7 cell (is bought in Chinese DSMZ,) the RPMI1640 substratum is available from (the Grand Island of Gibico company, NY). lipopolysaccharides (Escherichia coli O111:B4) and methyl thiazolyl tetrazolium (MTT) were obtained available from Sigma company (St.Louis, MO). the sheep anti-mouse antibody HO-1 (R﹠amp of purifying; DSystems, Minneapolis, MN), the sheep anti-mouse antibody COX-2 of purifying, rabbit anti-mouse antibody NF-κ B and I-κ B buy in Santa Cruz Biotechnology (CA, USA).Mouse TNF-α, IL-1 β and IL-6 enzyme linked immunoassay detection kit is bought in Quantikine R﹠amp; D Systems (Minneapolis, MN), Griess reagent NO assay kit is bought in green skies biotechnology companies (Chinese Jiangsu); Mouse source property IL-10ELISA test kit buy in BenderMedsystem (Vienna, Austria).Dexamethasone, dexamethasone (Dexamethasone, antiphlogiston) positive control drug (dexamethasone is Shijiazhuang first pharmaceutical factory's product).

(2) experimental technique

Adopt sandwich ELISA method to measure the content of TNF-α, IL-1 β, IL-6 and IL-10 in the RAW264.7 cell of LPS stimulation.After 24 hours intervention, collect supernatant liquor and read the absorbance at 450nm wavelength place with Microplate reader, calculate with corresponding ELISA test kit specification sheets operation and according to typical curve, the content of TNF-α, IL-1 β, IL-6 and IL-6, draw a diagram, compare between organizing.

(3) experimental result

TNF-α, IL-1 β and IL-6 are the present clearer and more definite common inflammatory factors that comes across inflammatory reaction.So we at first observe these several different extracts and white crystal stimulates the short inflammatory factor that produces behind the RAW264.7 cell whether the regulating effect of reduction is arranged to LPS, adopt euzymelinked immunosorbent assay (ELISA) to measure corresponding content.

After cell is intervened by LPS and parmelia saxatilis crystallization as a result, the content of TNF-α, IL-1 β and IL-6 is than obviously reducing (p＜0.01 or p＜0.05) in the independent stimulated cells of LPS. simultaneously, increase along with extract concentrations, its restraining effect also strengthens, and shows that parmelia saxatilis crystalline pharmacological action and dosage have positive correlation.But these excretory inflammatory mediators and the factor can not return to normal level, show that medicine has the anti-inflammatory action of keeping, and see (accompanying drawing ten, 11)

Experimental example three

Experiment material

The same.

(1) experimental technique

With cell pyrolysis liquid (150mmol/L NaCl, 50mmol/L Tris, pH 7.5,1%NP-40,0.25% sodium deoxycholate, 1mmol/L phenylmethylsulfonyl fluoride (PMSF), 1mmol/L NaVO4,2mmol/L EGTA, 50 μ g/ml leupeptins) hatch lysing cell 20min on ice, with homogenate in 10000r/min, 4 ℃ of centrifugal 5min.After BCA protein quantification test kit (U.S. Pierce company) is quantitative, respectively get 50 μ g albumen in 10% sodium laurylsulfonate-polyacrylamide gel (SDS-PAGE) electrophoresis, change film to pvdf membrane (U.S. Millipore company), contain the 2h that vibrates under PBST (PBS that contains 0.01% tween 20) the confining liquid room temperature of 5% skimmed milk, (1: " KG-*2 " 1000) 3h is hatched to the antibody of anti-carbonic anhydride enzyme II in (U.S. Santa Cruz company), contain horseradish peroxidase mark two anti-(1: 5000) (U.S. Santa Cruz companies) and hatch 1h, under the PBST room temperature behind the rinsing 15min, chemoluminescence method develop (U.S. Santa Cruz company).

(2) experimental result

Adopt western blot method to detect, find that different extracts all can obviously reduce COX-2 protein content (seeing accompanying drawing 12-13).Therefore can infer that the parmelia saxatilis crystallization can suppress the generation of epoxide hydrolase-2 on protein level.Its anti-inflammatory action may be also relevant therewith.

Find out that from Figure 10-11 the nucleic acid LPS of HO-1 stimulates back and normal cell contrast not to have the significance difference, show that it is the cytokine that produces in the inflammation later stage.Different parmelia saxatilis crystallization stimulating group, Dexamethasone group and astragalus polysaccharides (APS) group are all than the nucleic acid and the protein content height (p＜0.05) of independent LPS stimulating group and normal cell group.Simultaneously, when parmelia saxatilis crystallization concentration was high more, the nucleic acid content expression amount of HO-1 was big more, showed that the parmelia saxatilis crystallization can strengthen anti-inflammatory action.Pass through from Figure 12-13 as can be seen simultaneously, different parmelia saxatilis crystallizations are the protein expression amount of energy significance increase HO-1 all.From these results, the enough secretions of these parmelia saxatilis crystallizations from protein level control anti-inflammatory factors.

Test four, the influence of NF-kB and I-kB is tested

(1) test materials

1. the same.

(2) experimental technique

Reference, immunohistochemical method.Elder generation's fixed cell, and poststaining selects any 5 visuals field to observe at microscopically.

(3) experimental result

Document shows that it is to regulate the key factor that LPS stimulates cytokines such as the NOS that produces in the scavenger cell and COX-2 that NF-κ B activates.This paper adopts immunohistochemical method to analyze, and from Figure 10-11 as can be seen, different extracts all can obviously suppress the NF-kB activity., and see that yellow crystal may reach by the activity that suppresses NF-κ B for the influence of IL-1 β, TNF-α, iNOS and COX-2 in conjunction with result of upper experiment according to the regulation process of each cytokine in the inflammatory reaction.

Bibliographical information, I-kB protein content is reduced in the NF-κ B activation expression and has keying action in the LPS stimulated cells.Combine with I-κ B when having confirmed at present that NF-κ B does not activate, but when NF-κ B is activated, I-κ B understands and separate with NF-κ B.Therefore the content of measuring I-κ B in the cell can reflect the state of activation of NF-κ B.Find out that from the result of immunohistochemistry figure different parmelia saxatilis crystallizations all can obviously suppress the generation (p＜0.05) of I-κ B.These results show, after 24 hours, crystallization still has good antiphlogistic effects to the RAW264.7 cell, illustrates that these materials may have secular anti-inflammatory action by LPS and parmelia saxatilis crystalline different stimulated.

Test five, the influence of growth of tumour cell cycle is tested

The normal cell cell be transformed into cancer cells and the cell cycle closely related, tumour cell be in all the time vegetative state can not enter static its, with the petrochemical industry crystallization to ECCS, Hela, the effect of L1210 inhibition of proliferation, and adopt the influence of flow cytometry analysis petrochemical industry crystallization to the above-mentioned cell cycle, result's demonstration is crossed ECCS through the petrochemical industry crystallization treatment, Hela, L1210, cell rests on G ₀-G ₁Phase, S phase cell count obviously reduces.Do not influencing cylina A with the petrochemical industry crystallization, optionally suppressing cylinB 1 albumen under the situation of cylinE, causing the CDC2 kinase activity to suppress fully, making cell rest on G ₁Phase.The effect that proof has anticancer to increase.

Medication preparation implementation column 1

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, uses the weak polar solvent normal hexane, and solvent load is 10-20 a times of medicinal material weight, being heated to boiling and stopping heating, extracts 2 times, extracts 2 hours at every turn, obtains extracting solution.

Resulting extracting solution is concentrated into 1-2 times of medicinal material volume, filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

Low polar solvent oil fan, normal hexane that gained primary crystal adding coarse-grain weight 4-8 doubly measures dissolve again, remove by filter the chlorophyll constituents.

Filter the lysate of the low polar solvent oil fan of back solution, normal hexane,, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal below 10 degrees centigrade.

Crystallization is pulverized the back and is added Microcrystalline Cellulose with 1.6: 1 ratio mixings, adds 50% ethanol, makes softwood, granulates, and adds sodium starch glycolate, magnesium stearate again, compressing tablet, and the bag film-coat promptly gets film coated tablet.

Medication preparation implementation column 2

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, uses the weak polar solvent normal hexane, and solvent load is 10-20 a times of medicinal material weight, being heated to boiling and stopping heating, extracts 2 times, extracts 2 hours at every turn, obtains extracting solution.

Resulting extracting solution is concentrated into 1-2 times of medicinal material volume, filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

The boiling water that gained primary crystal adding coarse-grain weight 4-8 doubly measures dissolves again, removes by filter the chlorophyll constituents.

Filter the low polar solvent n-hexane dissolution liquid of back solution,, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal below 10 degrees centigrade.

Crystallization is pulverized and added starch: crystallization adds 40% ethanol with 2: 1 ratio mixings, makes softwood, granulates, and adds magnesium stearate again, according to the common process filled capsules, is prepared into the capsule of clinical acceptance;

Medication preparation implementation column 3

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, uses the weak polar solvent normal hexane, and solvent load is 10-20 a times of medicinal material weight, being heated to boiling and stopping heating, extracts 2 times, extracts 2 hours at every turn, obtains extracting solution.

Resulting extracting solution is concentrated into 1-2 times of medicinal material volume, filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

Boiling water that gained primary crystal adding coarse-grain weight 4-8 doubly measures or low polar solvent acetic acid second fat, ethanol dissolve again, remove by filter the chlorophyll constituents.

Filter the low polar solvent acetic acid second fat of back solution, alcoholic acid lysate,, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal below 10 degrees centigrade.

Crystallization is pulverized and is added Microcrystalline Cellulose, sodium starch glycolate with the certain proportion mixing, adds 60% ethanol, makes softwood, granulates, and adds micropowder silica gel again, and compressing tablet promptly gets dispersible tablet;

Medication preparation implementation column 4

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, uses the weak polar solvent ethyl acetate, and solvent load is 10-20 a times of medicinal material weight, being heated to boiling and stopping heating, extracts 2 times, extracts 2 hours at every turn, obtains extracting solution.

Resulting extracting solution is concentrated into 1-2 times of medicinal material volume, filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

The boiling water that gained primary crystal adding coarse-grain weight 4-8 doubly measures dissolves again, removes by filter the chlorophyll constituents.

Filter the lysate of the low polar solvent acetic acid second fat of back solution,, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal below 10 degrees centigrade.

Crystallization is pulverized and is added Zulkovsky starch with 1.5: 1 ratio mixings, and adding starch slurry or polyethylene pyrrole lattice alkane ketone is tackiness agent, spraying granulation, and bag slowly-releasing film-coat adds magnesium stearate again, according to the common process filled capsules, is prepared into the slow releasing capsule of clinical acceptance;

Medication preparation implementation column 5

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, uses the weak polar solvent normal hexane, and solvent load is 10-20 a times of medicinal material weight, being heated to boiling and stopping heating, extracts 2 times, extracts 2 hours at every turn, obtains extracting solution.

Resulting extracting solution is concentrated into 1-2 times of medicinal material volume, filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

The gained primary crystal adds the low polar solvent normal hexane that coarse-grain weight 4-8 doubly measures: ethyl acetate (2: 1) dissolves again, removes by filter the chlorophyll constituents.

Filter the lysate of the low polar solvent acetic acid second fat of back solution,, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal below 10 degrees centigrade.Crystallisate adds a certain proportion of dissolution of sodium hydroxide, heats and adds a certain proportion of ethyl acetate, CCL4 by diverse ways prepared in reaction derivative, and obtain the crystallization sodium salt.

Crystallization is pulverized and is added starch with 2: 1 ratio mixings, adds 40% ethanol, makes softwood, granulates, and adds magnesium stearate again, according to the common process filled capsules, is prepared into the capsule of clinical acceptance;

Medication preparation implementation column 6

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, uses the weak polar solvent normal hexane, and solvent load is 10-20 a times of medicinal material weight, being heated to boiling and stopping heating, extracts 2 times, extracts 2 hours at every turn, obtains extracting solution.

The 1-2 that resulting extracting solution is concentrated into the medicinal material volume doubly filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

Boiling water or low polar solvent acetic acid second fat that gained primary crystal adding coarse-grain weight 4-8 doubly measures dissolve again, remove by filter the chlorophyll constituents.

Filter the lysate of the low polar solvent acetic acid second fat of back solution,, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal below 10 degrees centigrade.Crystallisate adds the dissolving of a certain proportion of sodium bicarbonate, heats and adds a certain proportion of ethyl acetate, prepares derivative by the diverse ways reacting by heating, and the gained precipitation is used ethyl alcohol recrystallization, and obtains the crystallization sodium salt.

The crystallization sodium salt is pulverized and is added stearic acid, lanolin, aqueous paraffin wax, glycerine mixing in varing proportions, adds an amount of distilled water, makes ointment,, according to common process, be prepared into the ointment of clinical acceptance;

Medication preparation implementation column 7

Take by weighing the qualified parmelia saxatilis of quality inspection, extractor is put in fragmentation, uses the weak polar solvent normal hexane, and solvent load is 10-20 a times of medicinal material weight, being heated to boiling and stopping heating, extracts 2 times, extracts 2 hours at every turn, obtains extracting solution.The 1-2 that resulting extracting solution is concentrated into the medicinal material volume doubly filters the back concentrated solution, below 10 degrees centigrade, sufficient standing 24-48 hour, separates out coarse crystallization.

The boiling water that gained primary crystal adding coarse-grain weight 4-8 doubly measures dissolves again, removes by filter the chlorophyll constituents.

Filter the lysate of the low polar solvent acetic acid second fat of back solution,, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal below 10 degrees centigrade.Crystallisate adds the dissolving of a certain proportion of sodium bicarbonate, heats and adds a certain proportion of ethyl acetate, prepares derivative by the diverse ways reacting by heating, and the gained precipitation is used ethyl alcohol recrystallization, and obtains the crystallization sodium salt.

The crystallization sodium salt is pulverized the suspensoid that adds, and presses the different ratios mixing, adds an amount of distilled water, makes medicinal fluid, according to common process, is prepared into the liquid preparation that is used for anti-inflammatory of clinical acceptance.

Claims (6)

1, a kind of medicine that contains the plant cave flower active principle is characterized in that described effective constituent is to extract resulting a kind of yellow crystal from parmelia saxatilis.

R ₂-OH

2, the medicine that contains cave flower active principle according to claim 1 is characterized in that described cave flower active principle is C ₁₈H ₁₆O ₇Or the effective constituent by its gained of deriving, (R1=OH R2=OH or R1=Ona R2=ONa) is as sodium salt.

3, the preparation method who contains plant cave flower active principle medicine according to claim 1 is characterized in that preparing described cave flower active principle and makes as follows:

(1), take by weighing the qualified parmelia saxatilis of quality inspection, fragmentation uses weak polar solvent (preferred oil fan, normal hexane, hexanaphthene, ethyl acetate) to extract, and obtains extracting solution.

(2), resulting extracting solution be concentrated into the medicinal material volume 1-2 doubly, concentrated solution is filtered, below 10 degrees centigrade, sufficient standing 24-48 hour, separate out coarse crystallization.

(3), the gained primary crystal joins (preferred oil fan, normal hexane, hexanaphthene, ethyl acetate, ethanol) dissolving again in the boiling water or low polar solvent that crystallization weight 4-8 doubly measures, and removes by filter the chlorophyll constituents.

(4), filter back solution and make lysate with low polar solvent (preferred solvent oil fan, normal hexane, hexanaphthene, ethyl acetate, ethanol), below 10 degrees centigrade, sufficient standing 24-48 hour, separate out faint yellow to yellow crystal.

(5), obtain crystallization, and use the recrystallization solvent for use to clean, get effective constituent by drying through suction filtration or smart filter.

(6), gained effective constituent confirms it is effective constituent C by infrared, nuclear-magnetism, single crystal diffraction ₁₈H ₁₆O ₇

(7), gained effective constituent by structural modification obtain new effective constituent, as sodium salt.

4, the medicine that contains claim 1 effective constituent has anti-inflammatory, antiviral, anticancer effect.

5, the medicine that contains claim 1 effective constituent has the effect that prevents and treats the immunocompromised associated adjustment.

6, contain claim 1 effective constituent and derivative thereof, and at the made pharmaceutical composition of pharmaceutically acceptable carrier, as tablet, capsule, liquid preparation, externally used paste.

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