Contain the product of effective ingredient of anti-inflammatory, anti-platelet aggregation or antibacterial activity and the medical component of method and product fully from processed with Rhizoma Zingiberis Recens
The invention relates to a kind of method that from Rhizoma Zingiberis Recens, prepares the product of the effective ingredient that contains anti-inflammatory, anti-platelet aggregation or antibacterial activity.
In China or Japanese, Rhizoma Zingiberis Recens edible or medicinal aspect all be very general material.Rhizoma Zingiberis Recens is a seasoning condiment indispensable in general common people's daily life, clinical the be used for dispersing wind-cold, arresting vomiting by invigorating the stomach of the traditional Chinese medical science more, detoxifcation, Sucus Zingberis can be used as external application, has effect (the clinical practice science of color Chinese medicine grand ceremony of anti-inflammatory analgetic, first of international Chinese edition, 76 pages, Li De publishing house).In Saudi Arabia, Jiang Ze be used for the treatment of vomiting, facilitating digestion or as carminative (Ageel et al, 1987, Plants Usedin Saudi Folk Medicine, King Saud University Press, Riyadh, p.400).
In recent years, many scientific researches demonstrate Rhizoma Zingiberis Recens and have physiologically active widely.For example: the compound recipe that contains Rhizoma Zingiberis Recens can suppress cancer metastasis (Japanese patent publication 07258104), antiviral (Japanese patent publication 0711861), the generation of auxiliary nervous trophic factors, improves brain diseasess such as Alzheimer disease and Parkinson's disease (Japanese patent publication 07025777), arthritis (Japanese patent publication 06293653, No. the 5494668th, United States Patent (USP), No. the 5683698th, United States Patent (USP)), antimicrobial (Japanese patent publication 06227931), analgesia (Japanese patent publication 06107556) etc.In fact, Rhizoma Zingiberis Recens contains 1~4% volatile oil, wherein has 200 kinds of materials to be separated approximately.Mention in No. the 5804603rd, United States Patent (USP), Jiang Suohan zingiberone (zingerone) is the main source of acid, and zingiberol (gingerol) also has hot fragrance with shogaol (shogaol).In addition, zingiberol has heart tonifying, suppresses the effects such as contraction that the stripped vein contraction of mouse, regulation and control mouse and rabbit blood vessel cause because of eicosanoid, and shogaol then can suppress vasoconstriction.Show that in the research of Srivastava and Mustafa Rhizoma Zingiberis Recens can suppress epoxidation ferment (cyclooxygenase) and generate with lipid oxidation ferment (lipoxygenase) product, synthetic (the Medical Hypotheses that suppresses eicosanoid simultaneously, 1992,39:342-348).MascoloN. wait the people then to find, after oral dose administration with 100mg/kg, 80% alcoholic extract of Rhizoma Zingiberis Recens and acetylsalicylic acid (acetylsalicylic acid) are suitable to the inhibition effect of the mouse sole edema that antler glue (carageenan) brings out, and suppression ratio is respectively 39% and 38%.Find also that in same research 80% alcoholic extract of Rhizoma Zingiberis Recens can suppress the caused heating of yeast, and can suppress gram-negative bacteria and gram-positive bacteria growth (Journal of Ethnopharmocology, 1989,27,129-140).Srivastava and Mustafa are with 56 clinical patients of Rhizoma Zingiberis powder end treatment, the result shows that 3/4ths arthritis patient eases the pain or swelling, and the patient of all muscle discomforts all feels pain alleviate, and at three months during two-and-a-half years clinical, do not have patient table be shown with ill effect produce (1992, ibid).
Dermatophytes (Dermatophytes), especially trichophyton purpureatum (Trichophyton rubrum) reaches and sends out skin ulcer budlet spore tinea bacterium (Trichophyton mentagrophytes), common cause of disease [Roberts DT. for mycosis ungualis (onychomycosis) and tinea pedis (tinea pedis), British Journal of Dermatology.141 Supple 56:1-4,1999 Nov.; Roldan YB.et al., Mycoses, 43 (5): 181-3,2000].Pityrosporum ovale [Pityrosporum ovale (Malasseziafurfur)] is the cause of disease that tinea versicolor (pityriasis vesicolor), skin brood cell bacterium filter born of the same parents' inflammation (Pityrosporum folliculitis) and the peppery plug of horse Salmonella are wiped rash (Malassezia intertrigo).Existing several studies show that pityrosporum ovale (Pityrosporum ovale) relevant with the seborrheic dermatitis (seborrheic dermatitis) and the dandruff (dandruff belongs to a kind of seborrheic dermatitis of light symptom) [Nenoff P.et al., Dermatology.191 (4): 311-4,1995; Bulmer AC.et al., Mycopathologia, 147 (2) 63-5,1999].
The present invention utilizes organic solvent (ether, acetone, methanol, ethanol) or supercritical carbon dioxide extraction, or prepare a rough liquid from Rhizoma Zingiberis Recens (Zingiber officinale) rhizome with vapor distillation, refining and obtain containing the product of shogaol (shogaols), zingiberol (gingerols) and/or dehydrogenation Rhizoma Zingiberis Recens diketone (dehydrogingerdione) through the reverse-phase chromatography method again.These products confirm to have certain activity through external anti-platelet aggregation test, and then confirm to have the swollen effect of tangible anti-floating, and has tangible antibacterial activity by the activity assessment confirmation that suppresses to send out skin ulcer budlet spore tinea bacterium (Trichophytonmentagrophytes) and pityrosporum ovale (Pityrosporum ovale) through the animal experiment of antler glue (Carrageenan) method.
As described in background, Sucus Zingberis has the record of antiinflammatory, pain relieving effectiveness.
The present invention aims to provide a kind of effective method is provided the effective ingredient of anti-inflammatory, anti-platelet aggregation and antibacterial activity from the preparation of Rhizoma Zingiberis Recens rhizome product, and prepared product has effective component contents such as fixed in fact, thereby has clear and definite pharmacological action.
A kind ofly contain the method for product of the effective ingredient of anti-inflammatory, anti-platelet aggregation or antibacterial activity fully according to what content of the present invention was finished, comprise the following step from processed with Rhizoma Zingiberis Recens:
A) prepare a rough liquid from Rhizoma Zingiberis Recens (Zingiber officinale) rhizome;
B) should import a reverse-phase chromatography tubing string by rough liquid, and wash with water, first-class washing liquid and the second flowing lotion stream in regular turn, molten molten from the thing and second flowing lotion of collecting wherein first-class washing liquid from thing, wherein the polarity of this first-class washing liquid is greater than this second flowing lotion, and the polarity of this second flowing lotion is greater than chloroform; And
C) volatilization remove this first-class washing liquid molten in thing first-class washing liquid and one first concentrate moltenly from thing, this first concentrates and moltenly can be used as this product from thing and be removed; And
D) volatilization remove this second flowing lotion molten in thing second flowing lotion and one second concentrate moltenly from thing, this second concentrates and moltenly can be used as this product from thing and be removed,
Wherein this step a) comprises the following step i) extremely iv), step I), step I ') or step I "), this step I) to iv) being:
I) the fresh rhizome of Rhizoma Zingiberis Recens (Zingiber officinale) is pulverized and filtered, and obtain filtrate and residue part;
Ii) with this filtrate with one first organic solvent extraction, take out the first organic solvent extraction liquid obtained, and volatilization removes first organic solvent wherein and obtains spissated first an organic solvent extraction liquid;
Iii) with this residue partly with one second organic solvent extraction, take out the second organic solvent extraction liquid that is obtained, and volatilization removes second organic solvent wherein and obtains spissated second an organic solvent extraction liquid; And
Iv) merge this spissated first organic solvent extraction liquid and this spissated second organic solvent extraction liquid and obtain this rough liquid;
This step I) be:
I) with the dry rhizome of the pulverized Rhizoma Zingiberis Recens of this second organic solvent extraction (Zingiber officinale), take out the second organic solvent extraction liquid that is obtained, and volatilization removes second organic solvent wherein and obtains this rough liquid;
This step I ') be:
I ') with the dry rhizome of the pulverized Rhizoma Zingiberis Recens of vapor distillation (Zingiber officinale), the distillation that concentrating under reduced pressure obtained and obtain this rough liquid; And
This step I ") be:
I ") with the dry rhizome of the pulverized Rhizoma Zingiberis Recens of supercritical carbon dioxide extraction (Zingiber officinale), and volatilization removes carbon dioxide wherein and obtains this rough liquid.
Comprise the shogaol (shogaol) of 0-10mg/g, the 6-dehydrogenation Rhizoma Zingiberis Recens diketone (6-dehydrogingerdione) of the zingiberol of 1-150mg/g (gingerol) and 0-40mg/g according to this prepared product that contains the effective ingredient of anti-inflammatory, anti-platelet aggregation or antibacterial activity of the inventive method.
The present invention also provides a kind of medical component with anti-inflammatory activity, anti-platelet aggregation activity or antibacterial activity, comprise the prepared rough liquid of the step a) according to aforementioned the inventive method of anti-inflammatory treatment effective dose as effective ingredient, and pharmaceutically acceptable carrier or diluent.
The present invention also provides a kind of medical component with anti-inflammatory activity, anti-platelet aggregation activity or antibacterial activity, comprises to comply with the prepared product of aforementioned the inventive method as effective ingredient, and pharmaceutically acceptable carrier or diluent.Preferably, this product be this first concentrate molten from thing.Optionally, this product be this second concentrate molten from thing.
Preferably, this first-class washing liquid is that methanol and this second flowing lotion are acetone.
Preferably, this step a) comprises step I) extremely iv).
Preferably, this first organic solvent is an ether.
Preferably, this second organic solvent is acetone, methanol, ethanol or their mixture.More preferably, this second organic solvent is an acetone.
Preferably, this step a) comprises step I).
Optionally, this step a) comprises step I ').
Optionally, this step a) comprises step I ").
Be applicable to that the reverse-phase chromatography tubing string of the inventive method includes, but is not limited to be filled with the resin of porous polymer, for example Diaion HP-20 (Mitsubishi Mitsubishi Co.), SephadexLH-20 (Pharmicia Co.) or RP-18 reverse-phase chromatography resins such as (Nacalai tesque Co.).
Medical component with antibacterial activity of the present invention preferably is employed partly, and for example the form with shampoo, bath oil, soap, body lotion, health cream and abluent is used.Preferable, be used to treatment and send out skin ulcer budlet spore tinea bacterium (Trichophyton mentagrophytes) and pityrosporum ovale (Pityrosporum ovale) diseases associated, include but not limited to tinea pedis (tineapedis), tineatonsurans (tinea capitis), epidermophytosis inguinale (tinea cruris), sliding tinea (tinea glabrosa), mycosis ungualis (onychomycosis), tinea capitis (pityriasis capitis), tinea versicolor (pityriasisvesicolor), skin brood cell bacterium filter born of the same parents' inflammation (Pityrosporum folliculitis), seborrheic dermatitis (seborrheic dermatitis), and the dandruff (dandruff).Especially the medical component with antibacterial activity of the present invention is one to be used for the treatment of the shampoo form of the dandruff.
Below in conjunction with embodiment technical scheme of the present invention is further described, but illustrated embodiment only for explanation but not be used to limit the scope of the invention.
The mensuration of effective ingredient:
In following examples, use high performance liquid chroma-tography (high performance liquidchromatography is called for short HPLC) to measure the effective ingredient of the prepared product of the inventive method.HPLC spectrum is to be recorded in a high-performance liquid chromatograph (HPLC Shimadzu LC-10AT, Japan), wherein an amount of sample is with mobile phase solution [formonitrile HCN and water (65: 35, V/V) mixed liquor] quantitatively to 25ml, behind 0.25 μ m membrane filtration, filtrate injection one is filled with tubing string (long 25cm, the internal diameter 4.6mm of Cosmosil 5C-18, particle size 5 μ m), carry out stream with this mobile phase solution and wash (elution).(Shimadzu SPD-6AV Japan), detects the absorption of eluat (eluate) at wavelength 230nm to adopt UV-detector.
Embodiment 1
Rhizoma Zingiberis Recens 2100g is smashed after-filtration get filtrate and Rhizoma Zingiberis Recens residue.Get 500ml filtrate with 500ml extracted with diethyl ether three times, the water layer that extraction is at every turn obtained separates with ether layer, merges separated ether layer, volatilization remove wherein ether and must ether layer concentrate (I-OE).In addition with the Rhizoma Zingiberis Recens residue with 3000ml acetone extract three times, after the filtration acetone extract liquid, will extract at every turn and obtain that acetone extract liquid merges and concentrating under reduced pressure gets acetone extract (I-O) 14.5g.To get 7g after ether layer concentrate (I-OE) and acetone extract (I-O) merging, injection one is filled with anti-phase chromatograph chromatography tubing string (the length 30cm of 180g Diaion HP-20 resin, internal diameter 3cm, particle diameter 500 μ m-800 μ m), in regular turn with 1500ml water, 2500ml methanol, 2000ml acetone and 2000ml chloroform carry out stream and wash (elution), collect water-soluble from thing, methanol is molten from thing, acetone is molten molten from thing from thing and chloroform, must concentrate water-soluble from thing (I-OW) 0.27g behind the concentrating under reduced pressure, it is molten from thing (I-OM) 1.45g to concentrate methanol, it is molten molten from thing (I-OC) 0.83g from thing (I-OA) 2.68g and concentrated chloroform to concentrate acetone.Wherein, the content of 6-shogaol, 6-zingiberol and 6-dehydrogenation Rhizoma Zingiberis Recens diketone is as shown in Table 1 among I-O, I-OM and the I-OA.
Table one
Content (mg/g) | I-O | I-OM | I-OA |
6-shogaol 6-zingiberol 6-dehydrogenation Rhizoma Zingiberis Recens diketone | 1.10±0.14 59.98±0.99 7.68±0.42 | 1.15±0.0 103.37±8.57 8.94±0.41 | - 2.51±0.89 - |
Embodiment 2
Directly divide three extractions (each 10L) after Rhizoma Zingiberis 500g smashed with 30L acetone, after the filtration acetone filtrate, merging filtrate and concentrating under reduced pressure get acetone extract (II-O) 24g.Get the 20g acetone extract, by filling the anti-phase chromatograph chromatography tubing string of 600g DiaionHP-20 resin, wash with 4L water, 6.5L methanol, 15L acetone and 5L chloroform stream in regular turn, collect water-soluble molten molten molten from thing from thing and chloroform from thing, acetone from thing, methanol, must concentrate behind the concentrating under reduced pressure respectively water-soluble from thing (II-OW) 2.5g, concentrate methanol molten from thing (II-OM) 7.1g, to concentrate acetone molten molten from thing (II-OC) 3.5g from thing (II-OA) 6.9g and concentrated chloroform.Wherein, the content of 6-shogaol, 6-zingiberol and 6-dehydrogenation Rhizoma Zingiberis Recens diketone is as shown in Table 2 among II-O, II-OM and the II-OA.
Table two
Content (mg/g) | U-O | II-OM | II-OA |
6-shogaol 6-zingiberol 6-dehydrogenation Rhizoma Zingiberis Recens diketone | 1.98±0.00 43.06±0.84 9.33±0.85 | 4.96±0.00 70.87±1.85 19.15±4.57 | - 2.54±0.00 2.35±0.28 |
Embodiment 3
Rhizoma Zingiberis 10kg is smashed the back with vapor distillation 5 hours, the distillation concentrating under reduced pressure is got distillation (III-O) 410g.Get the 20g distillation by filling the anti-phase chromatograph chromatography tubing string of 600g Diaion HP-20 resin, wash with 4.5L water, 4.5L methanol, 3L acetone and 5L chloroform stream in regular turn, collect water-soluble molten molten molten from thing from thing and chloroform from thing, acetone from thing, methanol, must concentrate behind the concentrating under reduced pressure water-soluble from thing (III-OW) 0.03g, concentrate methanol molten from thing (III-OM) 14.5g, to concentrate acetone molten molten from thing (III-OC) 0.2g from thing (III-OA) 0.85g and concentrated chloroform.The vapor distillation thing (III-O) of Rhizoma Zingiberis is analyzed through HPLC and is not contained 6-shogaol, 6-zingiberol and 6-dehydrogenation Rhizoma Zingiberis Recens diketone.
Embodiment 4
Get 10g pulverize Rhizoma Zingiberis with 1000ml acetone in 50 ℃ of extractions 2 hours, remove by filter plant residue, behind the concentrating under reduced pressure with vacuum drying (40 ℃, 75mmHg vacuum) acetone extract thing IV-O.The color and luster and the viscosity of productive rate and product see Table three.
Embodiment 5
Get 10g and pulverize Rhizoma Zingiberis, with its oil-soluble and the lyophilization of water solublity extraction part, get oil-soluble extract V-O and water-soluble extract V-W respectively with vapor distillation.The color and luster and the viscosity of productive rate and product see Table three.
Embodiment 6
Get 10g and pulverize the extraction tank that Rhizoma Zingiberis places volume 250ml, with high pressure group Pu (EK-1 of LEWA company type, the U.S.) control supercritical CO
2Pressure, setting pressure are 2500 to 4000psia, and temperature cooperates the control of extraction tank external circulation line by heat exchanger (H-2410 of HOTEC company type, Taiwan), keeps operative temperature at 35-60 ℃, and flow speed control is worked as CO at 45L/min
2When volume reaches 300L, stop extraction step, get extract VI-O.The color and luster and the viscosity of productive rate and product see Table three, and the pungent component content of product sees Table four.
Table three
| IV-O | V-O | VI-O |
L
* A
* B
* | 87.6 -9.1 31.1 | 80.4 -0.1 9.6 | 96.3 -9.6 22.0 |
Viscosity (cPs) | 15.6 | 11.8 | 12.1 |
Productive rate (%) | 3.8 | 2.2 | 3.9 |
*: the value of L, A, B be with colour difference meter (∑ 90 color measuring system, Nippon DenshokuInc, co., LTD Japan) measures, lightness represents that with L colourity is represented with A and B.
Table four
Content (mg/g) | VI-O |
6-shogaol 6-zingiberol 6-dehydrogenation Rhizoma Zingiberis Recens diketone | 17.30±0.00 26.29±0.00 19.20±1.19 |
The pharmacologically active assessment of embodiment 7 anti-platelet aggregations
With EDTA (100mM) is anticoagulant, by rabbit ear vein blood drawing (1: 14), with blood after under 90 * g centrifugal 10 minutes, get the upper strata and be rich in platelet blood plasma under 500 * g centrifugal 10 minutes, after removing upper plasma, with platelet suspension in calcic not but contain Tyrode ' the s solution of 2mM EDTA, again under 500 * g centrifugal 10 minutes subsequently, after removing Tyrode ' s solution, platelet is suspended in again Tyrode ' the s solution that does not contain EDTA, and centrifugal through above-mentioned condition, at last with platelet suspension (mM): NaCl (138.6) in the buffer of following composition, KCl (2.8), NaHCO
3(11.9), MgCl
2(1.1), NaH
2PO
4(0.33), CaCl
2(1.0), glycerol (glucose) (11.2) and bovine serum albumin (0.35%), PC then is adjusted in every ml buffer and contains 4.5 * 10
8Individual platelet.The results are shown in table five.
Table five, Rhizoma Zingiberis Recens extract cause the inhibitory action of platelet aggregation to arachic acid and collagen protein
A) The Rhizoma Zingiberis Recens extract
b) | Suppress the required concentration of 50% agglutination (μ g/ml) |
Arachic acid | Collagen protein |
I-O I-OM II-O II-OA II-OC II-OM | 3.8±0.8 1.7±0.3 3.1±0.5 10.9±3.2 6.9±0.7 2.0±0.2 | 5.5±0.4 2.7±0.4 6.5±1.2 21.8±2.2 16.6±4.3 6.9±2.4 |
A)With platelet and solvent or Rhizoma Zingiberis Recens extract 37 ℃ warm 3 minutes down, add arachic acid (100 μ M) subsequently or collagen protein (10 μ g/ml) causes platelet aggregation, after 6 minutes when reaction when reaching maximum, record platelet aggregation degree, extract calculates in the following manner to hematoblastic inhibition percentage ratio:
The n value of data is 3 to 5 in [(control group coagulation degree-experimental group coagulation degree)/(control group coagulation degree)] * 100% table.
B)Preparation among the embodiment 1 and 2
The bullate pharmacologically active assessment of embodiment 8 anti-floatings
The swollen activity of anti-floating is according to Winter, and people's such as C.A. method is assessed (Winter C.A.etal., Proc.Soc.Exper.Biol.Med.111:544-547,1962.), and it thes contents are as follows:
After the public Wistar rat fasting overnight with 150 ± 20g, squeeze into antler glue (Carrageenan) (o.1ml at the left foot palm, 1% suspension (suspension)), immediately solvent or testing sample (10mg/paw) are evenly spread upon on the left back sole, after three hours, the hind paw volume is with plethysmograph (Cat.#7150, UGO Basil, Italy) measured, and deducted the pointer of right crus of diaphragm palm volume as the edema degree with left foot palm volume.
The anti-floating of table six, Rhizoma Zingiberis Recens extract is swollen active
A) Handle
b) | Dosage (mg/paw) | Edema suppresses (%) (Inhibition on carrageenan edema) |
I-O I-OE I-OM I-OA | 10 10 10 10 | 18 19 29 25 |
II-O II-OW II-OM II-OA II-OC | 10 10 10 10 10 | 18 0 26 25 8 |
III-O III-OM III-OA | 10 10 10 | 0 11 15 |
[6]-dehydrogingedione | 5 | 26 |
A). the percentage ratio that suppresses edema calculates in the following manner:
[(control group mouse edema degree)-(experimental group mouse edema degree)/(control group mouse edema degree)] * 100%
Data are the meansigma methods of 3 to 6 mouse in the table.
B)Preparation among the embodiment 1,2 and 3
Embodiment 9 suppresses to send out the activity assessment of skin ulcer budlet spore tinea bacterium (Trichophyton mentagrophytes) and pityrosporum ovale (Pityrosporum ovale)
To sending out the inhibiting assessment of skin ulcer budlet spore tinea bacterium
The Rhizoma Zingiberis Recens extract is to sending out skin ulcer budlet spore tinea bacterium (Trichophyton mentagrophytes; ATCC9533) inhibitory action is according to Edwards, J.R. wait people's method to be assessed (Edwards, J.R.etal., Antimicrobial Agents Chemotherapy 33:215-222,1989), and with minimal inhibitory concentration represent (minimum inhibitory concentration; MIC), the Rhizoma Zingiberis Recens extract is become the basic solution (stock solution) of various desired concns with dimethyl sulfoxide (dimethyl sulfoxide is called for short DMSO) dissolving back redilution.0.01 milliliter of basic solution added contain 0.99 milliliter of potato glucose nutritional solution [Potato Dextrose Broth (DIFCO, U.S.A.)] with 10
3To 10
4CFU/ml sends out in the 48 hole culture plates of skin ulcer budlet spore tinea bacterium, culture plate is inspected the decision extract with naked eyes after 28 ℃ down temperature is bathed 72 hours inhibitory action, and as the blank group, each test is all carried out in two multiple modes in the experiment and with solvent.The result as shown in Table 7.
Inhibitory action to pityrosporum ovale
The Rhizoma Zingiberis Recens extract is to pityrosporum ovale (Pityrosporum ovale; ATCC 38593) inhibitory action also according to above-mentioned Edwards, people's such as J.R. method is assessed, and represents (minimum inhibitory concentration with minimal inhibitory concentration; MIC), the Rhizoma Zingiberis Recens extract is become the basic solution of desired concn with DMSO dissolving back redilution.0.01 milliliter of basic solution added contain in 0.99 milliliter of flow-like Sa Pu Rockwell medium [Fluid Sabouraud Medium (DIFCO, U.S.A.)] with 10
3-10
4In the 48 hole culture plates of CFU/ml pityrosporum ovale, after 37 ℃ down temperature was bathed 48 hours, with the inhibitory action of macroscopy decision extract, as the blank group, each test was all carried out in two multiple modes in the experiment and with solvent DMSO.The result as shown in Table 7.
Table seven Rhizoma Zingiberis Recens extract is to the inhibitory action of a pityrosporum ovale (Pityrosporum ovale) and a skin ulcer budlet spore tinea bacterium (Trichophyton mentagrophytes)
The Rhizoma Zingiberis Recens extract
a) | MIC(μg/ml) |
Pityrosporum ovale | Send out skin ulcer budlet spore tinea bacterium |
I-O | 100 | 30 |
I-OM | 100 | 30 |
II-O | 500 | 30 |
II-OC | 100 | 30 |
II-OM | 100 | 30 |
III-O | 500 | 100 |
The blank group | --
b) | --
b) |
A)Prepared at embodiment 1,2 and 3
B)The unrestraint effect