CN1287814C - Extractive originated from cedrela sinensis, its preparation process and usage - Google Patents
Extractive originated from cedrela sinensis, its preparation process and usage Download PDFInfo
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- CN1287814C CN1287814C CN 200310124477 CN200310124477A CN1287814C CN 1287814 C CN1287814 C CN 1287814C CN 200310124477 CN200310124477 CN 200310124477 CN 200310124477 A CN200310124477 A CN 200310124477A CN 1287814 C CN1287814 C CN 1287814C
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- extract
- folium toonae
- toonae sinensis
- sinensis
- water
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Abstract
The present invention reveals a cedrela sinensis leaf extract which is prepared by orderly extracting cedrela sinensis leaves by water and by alcohol. The present invention also reveals a method for preparing the cedrela sinensis leaf extract, and an application using the extract to prepare medicines used for curing ovarian cancer and/or bladder cancer.
Description
Technical field
The present invention relates to stem from the extract and its preparation method of Tonnae Sinensis (Toona sinensis Roem.) leaf, and this extract supply is used for the purposes of ovarian cancer and/or bladder cancer treatment.
Background technology
(the Latin formal name used at school is Toona sinensis Roem or Cedrela sinensis to Tonnae Sinensis, English Chinesemahogany cedar by name or Chinese Toona) be a kind of tall this plant of perennial fallen leaves that belongs to Meliaceae (Meliaceae), bark is reddish brown brown, and tender leaf can edible and annual adopting.Tonnae Sinensis originates in Southeast China, southwest to the North China, and plantation (Jennifer M.Edmonds and MartinStaniforth, TOONA SINENSIS (Meliaceae) have nowadays all been arranged in the whole world, Curtis ' s Botanical magazine, 15 (3), 186-193,1998; Xiao-Dong Luo et al., Fitoterapia, 71,492-496,2000; Jong-Cheol Park et al., Kor.J.Pharmacogn, 27 (3), 219-223,1996).
Tonnae Sinensis is worth high seeds because the Herb plant all can be utilized and become once Ji.According to reported in literature, each plant parts of Tonnae Sinensis (seed, root bark, bark, rachis, leaves) all has function (the EdmondsJM ﹠amp of health care or treatment; Staniforth M, 1998, (supra) same as above; Park JC et al., 1996, same as above).
Aspect edible, Tonnae Sinensis seed oil is suitable as food with oil very much because of colourless and fragrant odour.Tender shoots of Tonnae Sinensis and leaf contain nutrients such as abundant carotene, amino acid and vitamin, and are loved by the people.In addition, the Lao Ye of Tonnae Sinensis can be used as the high nutritive value feedstuff of domestic animal.
Aspect medicinal, according to document record (Edmonds JM ﹠amp; Staniforth M, 1998, same as above; Xiao-Dong Luo et al., same as above; Park JC et al., 1996, same as above), the bark of Tonnae Sinensis, root bark and seed are at the treatment neuralgia, stop blooding, dispel cold, pain relieving, control stomach, duodenal ulcer, gonorrhea, menoxenia, ascarid, inhibition Bacillus typhi, anti-ameba, rheumatic arthritis and anticancer (Yu Siming and Zhang Ze work as, Jounal of Anhui University Natural Science Edition, No.4,91-94,1990; Liu Yuezhen and Li Yuping, Hebei Forestry Science and Technology, the 4th phase, 51-52, in December, 1997) on have curative effect.
And according to document record, Folium toonae sinensis has antiinflammatory, detoxifcation, parasite killing, controls enteritis, dysentery, cellulitis, treatment, dermatitis rhus, scabies, white bald and improve the medicinal function of body constitution.In addition, pass on from one to another among the people, drink after the Folium toonae sinensis water extract, discovery can improve hypertension and diabetic symptom.Recently, permitted to win the aqueous extract (aqueous extract) that people such as light study Folium toonae sinensis, and the aqueous extract of finding Folium toonae sinensis can reduce blood glucose (the Wang PH et al. of the diabetic mice of being brought out by Alloxan, Toona Sinensis increase GLUT4 glucosetransporter protein in adipose tissue from Alloxan-induced diabetic rats, Annual Conference of Biomedical Sciencr, p.198,2001), and the aqueous extract of finding Folium toonae sinensis in another research has the human lungs adenocarcinoma cell A549 (human lungadenocarcinoma cells A549) of inhibition outgrowth effectiveness (Hui-Chiu Chang et al. (2002), AmericanJournal of Chinese Medicine, Vol.30, Nos.2 ﹠amp; 3,307-314).
In other respects, Folium toonae sinensis can be used as stain or glaze agent.
In addition, the timber of Tonnae Sinensis is solid, careful, do not stick up, do not split, moisture-proof, so and the timber of Tonnae Sinensis often is used to be used as materials such as noble furniture, shipbuilding, bridge, also be applied to making racket, table tennis bat, tennis racket and musical instrument.
Tonnae Sinensis also is a kind of reproducting tree species, when being applied to afforesting, can prevent that earth flow from taking place.In addition, can extract the Chinese toon wood oil from the timber of Tonnae Sinensis, this oil can be used as the odorant of cigar.
Known to the applicant, so far still not having case before any documents and materials or the patent once expressed or hint " Folium toonae sinensis or the extract that stems from Folium toonae sinensis have the activity that suppresses ovarian cancer and/or growth of bladder cancer cells, and so can be applied to preparing the medicine that is used for treating ovarian cancer and/or bladder cancer ".
Summary of the invention
Therefore, aspect first, the invention provides a kind of extract from the Folium toonae sinensis gained, it is to comprise the method for the following step and be made by one:
(1) under heating, extract Folium toonae sinensis and obtain first extract once water extraction with water, and
(2) come resulting second extract that is somebody's turn to do first extract that extracts through water and obtains extracting of extraction step (1) with an alcohol once alcohol.
Aspect second, the invention provides a kind ofly in order to prepare the method for extraction Folium toonae sinensis extract, it comprises the following steps:
(a) under heating, extract Folium toonae sinensis, to obtain an aqueous extraction solution (aqueous extractsolution) with water;
(b) will be obtained from the aqueous extraction solution drying of step (a), to obtain first extract that is dried;
(c) first extract that will be obtained from step (b) is dissolved in the alcoholic solvent, to form a pure extraction solution; And
(d) alcoholic solvent that will be obtained from the pure extraction solution of step (c) removes to doing, to obtain second extract that is dried.
Be proved the growth that can suppress ovarian cancer cell (particularly SKOV3 and PA-1) effectively according to Folium toonae sinensis extract of the present invention.Therefore, aspect the 3rd, the invention provides a kind of pharmaceutical composition, it includes and is an aforesaid Folium toonae sinensis extract that can effectively suppress the quantity of ovarian cancer cell growth.
Above-mentioned and other purposes, feature and advantage of the present invention can become obviously as can be known after examining attached accompanying drawing with reference to following detailed description with preferred embodiment with literary composition.
Description of drawings
The present invention is described in detail below in conjunction with drawings and Examples, in the accompanying drawing:
Fig. 1 is a HPLC elution figure (HPLC elution profile) of a product through water extraction that is derived from Folium toonae sinensis (promptly hereinafter embodiment 1 in resulting " Tonnae Sinensis 5-4 ");
Fig. 2 is a HPLC elution figure of the Folium toonae sinensis extract through alcohol extraction that obtains according to a preferred embodiment of this case method (promptly hereinafter embodiment 1 in resulting " Tonnae Sinensis 5-2 ");
Fig. 3 shows with the cytotoxicity effectiveness (cytotoxicity effect) of the resulting 5 kinds of Folium toonae sinensis extracts of different purification process for bladder cancer cell line T24 (bladder cancer cell line T24), wherein is used to be used as the H of the extractant of these Folium toonae sinensis extracts
2O, 50%EtOH and 99.5%EtOH are used as matched group;
Fig. 4 shows the Folium toonae sinensis extract (i.e. " Tonnae Sinensis 5-2 ") that obtains according to a preferred embodiment of this case method growth inhibited effectiveness for different JEG-3, and wherein matched group is not for adding the experimental group of Folium toonae sinensis extract;
Fig. 5 shows that Folium toonae sinensis extract (i.e. " Tonnae Sinensis the 5-2 ") cellular morphology for ovarian cancer cell strain SKOV3 under variable concentrations (1mg/ml, 100 μ g/ml) that obtains according to a preferred embodiment of this case method influences, and wherein matched group is not for adding the experimental group of Folium toonae sinensis extract;
Fig. 6 shows that Folium toonae sinensis extract (i.e. " Tonnae Sinensis the 5-2 ") cellular morphology for ovarian cancer cell strain PA-1 under variable concentrations (1mg/ml, 10 μ g/ml) that obtains according to a preferred embodiment of this case method influences, and wherein matched group is not for adding the experimental group of Folium toonae sinensis extract; And
Fig. 7 shows that the nude mice have the tumor that ovarian cancer cell grows up to is being changed with the gross tumor volume that the Folium toonae sinensis extract (i.e. " Tonnae Sinensis 5-2 ") that obtains according to a preferred embodiment of this case method back of low dosage and high dose is taken place by dispensing, and wherein tumor size is measured once 7 weeks altogether weekly.
The specific embodiment
At previous research (Hui-Chiu Chang et al. (2002), American Journal of Chinese Medicine, Vol.30, the Nos.2 that doctor Xu Shengguang dominated; 3,307-314), an aqueous extract of Folium toonae sinensis is according to Hui-Chiu Chang et al. (1998), Am.J.Chin.Med., the method that is disclosed among the Vol.30:307-314 and being produced out, wherein the Folium toonae sinensis of 100g be injected towards in the water of 1000mL and the heat treated of being seethed with excitement until the solution of remaining 100mL, come centrifugally to last 20 minutes with 1000x g then, come to use and obtain upper clear liquid for the experiment of this research.
Above-mentioned Folium toonae sinensis aqueous extract is found the cell cycle progress (cell cycle progression) that can block this Human Lung Cancer cell A549 by the plain D1 of cycle (cyclin D1) that suppresses Human Lung Cancer cell A549 with the performance of plain E of cycle (cyclin E) effectively through experiment.
The applicant further attempts extracting Folium toonae sinensis with various processing modes, and obtains a kind of Folium toonae sinensis extract, and it is to be made by a method that comprises the following steps:
(1) under heating, extract Folium toonae sinensis and obtain first extract once water extraction with water, and
(2) come resulting second extract that is somebody's turn to do first extract that extracts through water and obtains extracting of extraction step (1) with an alcohol once alcohol.
So, the invention provides a kind ofly in order to prepare the method for Folium toonae sinensis extract, it comprises the following steps:
(a) under heating, extract Folium toonae sinensis to obtain an aqueous extraction solution (aqueous extractsolution) with water;
(b) will be obtained from the aqueous extraction solution drying of step (a) to obtain first extract that is dried;
(c) first extract that will be obtained from step (b) is dissolved in the alcoholic solvent to form a pure extraction solution; And
(d) alcoholic solvent that will be obtained from the pure extraction solution of step (c) removes to doing to obtain second extract that is dried.
Preferably, in the step (a) of this case method, the acquisition of this aqueous extraction solution is boiled by the water that adding is had Folium toonae sinensis to be concentrated into one in right amount, is filtered then.This filtration treatment can use the filter screen of the selected slot size of gauze, Cotton Gossypii or tool to carry out.In a preferred embodiment of the present invention, this filtration treatment is to carry out with gauze and Cotton Gossypii.In another preferred embodiment of the present invention, this filtration treatment is that the filter screen with 70 sieve apertures (mesh) carries out.
Preferably, the step of this case method (b) to step (d) is to be carried out under cryogenic conditions, in order to do making the destruction to contained active constituent in the Folium toonae sinensis can be reduced to minimum level.
Preferably, the dried of being carried out in the step (b) of this case method is to be selected from the following group that constitutes: (lyophilization) handled in lyophilization, low temperature spray drying is handled (spray-drying), low-temperature evaporation processing (evaporation) and these arbitrary combination.In a preferred embodiment of the present invention, the dried of being carried out in the step (b) is that lyophilization is handled.
The step of preferably, this case method (b) can comprise following treatment step:
(i) the aqueous extraction solution centrifugal of step (a) be will be obtained from and a upper clear liquid and a precipitate obtained; And
(ii) resulting this upper clear liquid in the step (i) is carried out a dried.
Preferably, step (i) is to come centrifugal this to be obtained from the aqueous extraction solution of step (a) with 3000rpm under (for example 4 ℃) at low temperatures and be carried out.
Preferably, being used for step dried (ii) is to be selected from one by the following group that constitutes: lyophilization handles that (lyophilization), low temperature spray drying are handled (spray-drying), low-temperature evaporation is handled (evaporation) and these arbitrary combination.In a preferred embodiment of the present invention, the dried of being carried out in the step (i) is that lyophilization is handled.
In addition, resulting this precipitate of step (i) also can give drying in a similar fashion and generate a desciccate.
Preferably, it is to be undertaken by a processing that is selected from the following group that the alcoholic solvent that is carried out in the step (d) removes: (lyophilization), spray drying treatment (spray-drying) are handled in evaporation process (evaporation), lyophilization, and these arbitrary combination.In a preferred embodiment of the present invention, step (d) is to handle and be carried out by lyophilization.
Preferably, in the step (d) of this case method, carrying out before alcoholic solvent removes, this pure extraction solution that is obtained from step (c) can be filtered or be centrifugal, to remove the insoluble substance that the inside may be contained.More preferably, in step (d), this pure extraction solution that is obtained from step (c) gives centrifugally lasting 12 minutes with 3000rpm earlier under 4 ℃, then carries out alcoholic solvent and removes.
Be applicable to that the alcohol in the step (c) of this case method can be, for example, ethanol, methanol, propanol, isopropyl alcohol, n-butyl alcohol, isobutanol or these a kind of combination.In a preferred embodiment of the present invention, the alcohol that is used in the step (c) of this case method is ethanol.
As shown in following embodiment 1, analyze by resulting this first extract of the step (b) of this case method and by the sampled HPLC that does of resulting this second extract of the step (d) of this case method, and confirm that the main compound crest of these two kinds of extracts truly has difference.
The applicant attempts that also resulting this second extract of the step of this case method (d) is done further extraction and handles, and comprising:
(i) this second extract is dissolved in 50% the ethanol, centrifugal then, and obtain a upper clear liquid part and a precipitate part; Connect and this upper clear liquid part and this precipitate part are given lyophilization respectively, and obtain being derived from this precipitate the 4th extract partly once exsiccant the 3rd extract and that is derived from this upper clear liquid part;
(ii) resulting the 4th extract of above-mentioned steps (i) is dissolved in 25% the ethanol, centrifugal then, and obtain a upper clear liquid part and a precipitate part; Connect and this upper clear liquid part and this precipitate part are given lyophilization respectively, and obtain being derived from this precipitate the 6th extract partly once exsiccant the 5th extract and that is derived from this upper clear liquid part;
(iii) with above-mentioned steps (ii) in resulting the 6th extract be dissolved in the reverse osmosis water (RO water), centrifugal then, and obtain a upper clear liquid part and a precipitate part; Connect and this upper clear liquid part and this precipitate part are given lyophilization respectively, and obtain the first reverse osmosis water extract and the second reverse osmosis water extract that is derived from this precipitate part that are derived from this upper clear liquid part once exsiccant; And
(iv) with above-mentioned steps (iii) in the second reverse osmosis water extract of gained be dissolved in reverse osmosis (RO) water, centrifugal then, and obtain a upper clear liquid part and a precipitate part; Connect and this upper clear liquid part and this precipitate part given lyophilization respectively, and obtain being derived from this precipitate cold limbs infiltration water extract partly once exsiccant the 3rd reverse osmosis water extract and that is derived from this upper clear liquid part,
Wherein in above-mentioned steps (i), (ii), (iii) with (iv) in, upper clear liquid part be separating of precipitate part by under 4 ℃, carrying out centrifugally lasting 12 minutes and being reached with 3000rpm.
For probing into the biological activity of above resultant various different Folium toonae sinensis extracts, the applicant handles the cancerous cell that several stem from women's urinary system (urogynecological system) with selected Folium toonae sinensis extract, whether has cytotoxicity to observe these Folium toonae sinensis extracts.
At the beginning, the applicant uses bladder cancer cell line T24 (bladder cancer cell line T24) to carry out preliminary anticancer cytoactive screening, and find to have the activity that best anticancer is grown according to resulting this second extract of this case method, and take second place by resulting this first extract of the step (b) of this case method.
So, the applicant further handles two kinds of ovarian cancer cell strain SKOV3 and PA-1, two kinds of cervical cancer (cervical cancer) cell strain HeLa and HeLaS3 and a kind of carcinoma of endometrium (endometrial cancer) cell strain RL95-2 with resulting this second extract of foundation this case method, and finds that this second extract has selecting cell toxicity (selective cytotoxicity) for ovarian cancer cell.Therefore, the present invention anticipates that also this Folium toonae sinensis extract is preparing for the application on the pharmaceutical composition that is used for the treatment of ovarian cancer.
So, the invention provides a kind of pharmaceutical composition, it comprises:
(a) this second extract that makes by the aforesaid method of this case of a treatment effective dose; And
(b) a pharmaceutically acceptable supporting agent.
The pharmaceutical composition that includes according to the prepared Folium toonae sinensis extract of this case method can be applied to treating an ovarian cancer or bladder cancer.Therefore, for an individuality of suffering from ovarian cancer or bladder cancer, can will offer medicine to this individuality according to the prepared Folium toonae sinensis extract of this case method.
The present invention also provides a kind of pharmaceutical composition that can be used for treating bladder cancer, and it comprises:
(a) a kind of of treatment effective dose is selected from following Folium toonae sinensis extract:
(i) one extract Folium toonae sinensis and the extract that obtains via water, and
(ii) one extract Folium toonae sinensis via water and an alcohol in order and the extract that obtains; And
(b) a pharmaceutically acceptable supporting agent.
As used herein, " pharmaceutically acceptable supporting agent " this term is meant the supporting agent that medicine is made that is applicable to that this skill is known clearly and known, this comprises, but be not limited to water, normal saline, glycerol, organic solvent, tranquilizer, chelating agen, antiseptic, emulsifying agent, suspending agent, diluent, gel, liposome or the like.
Can utilize according to pharmaceutical composition of the present invention and to have the knack of this skill person and know clearly the technology known and be manufactured into the form that is suitable for parenteral, oral or topical administration, this comprises, but be not limited to injection product (injection), solution (solution), capsule (capsule), dispersion (dispersion), suspension (suspension) or the like.
In order to make oral administration solid shape goods, an excipient and, if need, an adhesive, a disintegrating agent, a lubricant, a coloring agent, a flavoring agent and/or similarly thing can with mix mutually according to Folium toonae sinensis extract of the present invention.Formed mixture connects and can be formed lozenge, lozenge, granule, powder, capsule or similar thing through coating as the method known to this skill by one.These additives can be those and generally are used in person in the middle of now the technical field, comprise excipient: lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, Kaolin (kaolin), microcrystalline Cellulose (micro-crystalline cellulose) and silicic acid (silicic acid); Adhesive: water, ethanol, propanol, sucrose solution, glucose solution, starch solution, gelatin solution, carboxy methyl cellulose, hydroxy propyl cellulose, hydroxypropyl starch, methylcellulose, ethyl cellulose, Lac (shellac), calcium phosphate and poly-second
Cough up ketone (polyvinylpyrrolidone); Disintegrating agent: dried starch, sodium alginate (sodium alginate), by the agar of powdered, sodium bicarbonate, calcium carbonate, sulphuric acid laurel tallow sodium (sodium lauryl sulfate), glycerol monostearate (monoglycerol stearate) and lactose; Lubricant: purified Talcum, stearates (stearate salts), Borax (borax) and Polyethylene Glycol (polyethylene glycol); And correctives (corrigents): sucrose, Pericarpium Citri junoris (bitterorange peel), citric acid (citric acid) and tartaric acid (tartaric acid).
In order to make oral liquid goods, a flavoring agent, a buffer agent, a tranquilizer and similar thing can mix mutually with foundation Folium toonae sinensis extract of the present invention.Formed mixture connect and can by one be formed as the method known to this skill one in the solution, a syrup, an elixir (elixir) taken or similar thing.In this example, this flavoring agent can be same as that in the top person of mentioning.The illustration thing of this buffer agent is a sodium citrate, and the illustration thing of this tranquilizer is Tragacanth (tragacanth), arabic gum (gum arabic) and gelatin.
In order to make injection product, a pH value adjusting control agent, a buffer agent, a tranquilizer, an isotonicity agent (isotonicity) and similar thing can mix mutually with foundation Folium toonae sinensis extract of the present invention.Formed mixture connects and can be by one as the method known to this skill and be formed subcutaneous, intramuscular or intravenous injection product.The example of this pH value adjusting control agent and buffer agent comprises sodium citrate, sodium acetate and sodium sulfate.The illustration thing of this tranquilizer comprises sodium pyrosulfite (sodium pyrosulfite), ethylenediaminetetraacetic acid (EDTA), ethyl mercaptan acid (thioglycollic acid) and 2-mercaptopropionic acid (thiolactic acid).The example of this isotonicity agent (isotonicity) comprises sodium chloride and glucose.
Preferably, be manufactured into the form that is suitable for injection according to pharmaceutical composition of the present invention, for example powder injection (powder injection), injection are frozen brilliant product (lyophilization product for injection), emulsifying injection (emulsion injection), oiliness injection (oily injection), little fat body acupuncture agent forms such as (liposome injection).
As used herein, " treatment effective dose " this term be meant when a kind of contain according to the pharmaceutical composition of Folium toonae sinensis extract of the present invention by throwing give one need that this constituent treats individual the time, one be enough to provide desire to reach the therapeutic efficiency that causes and can not produce the consumption of un-desired serious harm nonstandard tissue or organ.This treatment effective dose can be looked different factor and change, and these factors comprise, for example, and the kind of disease, body weight, age, health and the reaction of the individuality that be treated, the dosing way of medicine etc.This treatment effective dose can be to be had the knack of this skill personage and decides.
The dispensing dosage of foundation pharmaceutical composition of the present invention can be looked following factors with the dispensing number of times and change: the severity of disease that be treated, dosing way, and body weight, age, health and the reaction of the individuality that will be treated.Generally speaking, according to offer medicine every day of pharmaceutical composition of the present invention dosage normally the 0.67mg/Kg body weight be single dose or be divided into the form of several dosage, and can or offer medicine partly to the 6.7mg/Kg body weight by parenteral ground, oral ground.Preferably, can be offerd medicine via intraperitoneal injection (intraperitoneal injection), intravenous injection (continuous intravenousinjection) continuously, the single injection of local intra-arterial (topical arterial single injection), local tumor direct injection modes such as (topical tumor direct injection) according to pharmaceutical composition of the present invention.In a preferred embodiment, can offer medicine by intraperitoneal injection or local tumor direct injection mode according to pharmaceutical composition of the present invention.
Can be offerd medicine by every day according to pharmaceutical composition of the present invention, preferably, can offer medicine continuously weekly 5 days, have a rest then 2 days, carry out altogether one be 43 days during or till occurring alleviating (remission).
Can be offerd medicine individually according to pharmaceutical composition of the present invention, or be used in combination with other Therapeutic Method or medicines that are used for the treatment of ovarian cancer and/or bladder cancer.These Therapeutic Method comprise chemotherapy and external beam X-ray therapy (external beam radiation therapy), and these medicines comprise, but be not limited to, Paclitaxel (paclitaxel), cisplatin (cisplatin), carboplatin (carboplatin), ring phosphorus vinegar amine (cyclophosphamide) and many ropes are as must hot (doxorubicin).
The present invention puts up with following embodiment and is described further, but will be appreciated that, these embodiment are only for illustrating use, and should not be interpreted as the restriction of enforcement of the present invention.
Embodiment
The preparation of embodiment 1. Folium toonae sinensis extracts
Employed Folium toonae sinensis is the Tonnae Sinensis tree of picking up from being planted in Republic of China Yunlin County, Taiwan Province Tu Ku town in the present embodiment.
Extraction procedures A
Take Tonnae Sinensis young tender leaf and water to clean, then use the consumption of the reverse osmosis water (ROwater) of 4L, an amount of reverse osmosis water is mixed mutually with Folium toonae sinensis, and boiled and last 30 minutes with the per kilogram Folium toonae sinensis.Afterwards, take out Folium toonae sinensis, and extract is concentrated, filtered with filter screen (70mesh) then with slow fire.Resulting filtrate can directly use the Virtis machine to carry out lyophilization, and obtains the thick extract of a Folium toonae sinensis (crudeextract), and it is called as " Tonnae Sinensis 5-4 " (claiming " TSL-CE " in addition again).Generally speaking, the mode Folium toonae sinensis of handling 100g on average can obtain the freeze-dried powder of about 5～6g according to this.
In addition, above-mentioned process filtration treatment and the filtrate that obtains can be carried out a centrifugal treating before lyophilization: under 4 ℃ with 3000rpm (Beckman Avanti
TMJ-30I) give centrifugally lasting 12 minutes, and obtain the precipitate part that upper clear liquid part and contains insoluble matter.This upper clear liquid partly connects and uses the Virtis machine to carry out lyophilization, and obtains a water extract (water extract) that is freeze-dried powder, and it is called as " TSL-1 ".
Above-mentioned resulting " Tonnae Sinensis 5-4 " and " TSL-1 " are alleged among the present invention " through first extracts of water extraction ", and for making things convenient for illustration, the applicant uses " TSL-1 " to carry out following further extraction procedures.
Extraction procedures B
Above-mentioned extraction procedures A resulting " TSL-1 " extract (50g) is dissolved in 99.5% ethanol (400ml) to carry out the alcohol extraction.Formed alcoholic solution under 4 ℃ with 3000rpm (Beckman Avanti
TMJ-30I) give centrifugally lasting 12 minutes, and obtain a upper clear liquid part and a precipitate part.This upper clear liquid partly connects and uses the Virtis machine to carry out lyophilization, and obtaining a pure extract through being further purified (thefurther purified alcohol extract) that is freeze-dried powder, it is called as " Tonnae Sinensis 5-2 " (claiming " TSL-2 " in addition again).
Mode is handled " TSL-1 " extract of 100g according to this, can obtain " Tonnae Sinensis 5-2 " freeze-dried powder of about 1g.
Extraction procedures C
The resulting precipitate part of above-mentioned extraction procedures B gives lyophilization with the Virtis machine, is dissolved in then in 50% ethanol.Formed 50% alcoholic solution under 4 ℃ with 3000rpm (Beckman Avanti
TMJ-30I) give centrifugally lasting 12 minutes, and obtain a upper clear liquid part and a precipitate part.This upper clear liquid partly connects and uses the Virtis machine to carry out lyophilization, and obtains an extract that is freeze-dried powder, and it is called as " Tonnae Sinensis 5-5 " (claiming " TSL-3 " in addition again).
Extraction procedures D
The resulting precipitate part of above-mentioned extraction procedures C gives lyophilization with the Virtis machine, is dissolved in then in 25% ethanol.Formed 25% alcoholic solution under 4 ℃ with 3000rpm (Beckman Avanti
TMJ-30I) give centrifugally lasting 12 minutes, and obtain a upper clear liquid part and a precipitate part.Connect and use the Virtis machine to come lyophilization respectively this upper clear liquid part and this precipitate part, and obtain two extraction products that are being called as respectively of freeze-dried powder " TSL-4 " and " TSL-4P ".
Extraction procedures E
The resulting extraction product of above-mentioned extraction procedures D " TSL-4P " is dissolved in the RO water, and the aqueous solution that forms under 4 ℃ with 3000rpm (Beckman Avanti
TMJ-30I) give centrifugally lasting 12 minutes, and obtain a upper clear liquid part and a precipitate part.Connect and use the Virtis machine to come lyophilization respectively this upper clear liquid part and this precipitate part, resultant two are freeze-dried powder and then are called as " TS-H respectively
2O-1 " extraction product of (in addition claim again " TSL-5 ") and " TSL-5P ".
Extraction procedures F
The resulting extraction product of above-mentioned extraction procedures E " TSL-5P " is dissolved in the RO water, and the aqueous solution that forms, under 4 ℃ with 3000rpm (Beckman Avanti
TMJ-30I) give centrifugally lasting 12 minutes, and obtain a upper clear liquid part and a precipitate part.Connect and use the Virtis machine to come lyophilization respectively this upper clear liquid part and this precipitate part, be freeze-dried powder, be called as " Tonnae Sinensis 5-4R " respectively and (claim " TS-H in addition again and obtain two
2O-2 " or " TSL-6 ") with the extraction product of " TSL-7 ".
The HPLC of embodiment 2. Folium toonae sinensis extracts analyzes
For the Main Ingredients and Appearance of understanding the Folium toonae sinensis extract that is obtained according to this case method distributes, " Tonnae Sinensis 5-4 " and " Tonnae Sinensis 5-2 " that extraction procedures A and the extraction procedures B of embodiment 1 obtained respectively brings and analyzes high pressure liquid chromatography (HPLC) (HPLC).
The experimental implementation method:
In present embodiment, the HPLC analytical tool is Hitachi L-7100pump, L-7420 uv/vis detector, D-2410 degaser; Analyzing software is D-7000 HPLC System Manager; Analyzing tubing string is MightysilRP-18GP 250-4.6 (5m); Analysis condition: gradient solvent A: methanol; B: water; 0 ~ 10 minute 20% ~ 50%A; 10 ~ 20 minutes 50% ~ 70%A; 20 ~ 40 minutes 70%A; Flow velocity is 1ml/min; Wavelength 254nm.
The result:
Fig. 1 and Fig. 2 show the HPLC elution figure of " Tonnae Sinensis 5-4 " and " Tonnae Sinensis 5-2 " respectively.Comparison diagram 1 can know with Fig. 2 and find out that under identical separation condition, " Tonnae Sinensis 5-4 " is with " main compound that Tonnae Sinensis 5-2 contains separately is not quite alike." Tonnae Sinensis 5-4 " and " Tonnae Sinensis 5-2 " have same chemical compound crest at 5,16 and 18 minutes holdup time point place, and in " Tonnae Sinensis 5-2 " chemical compound of being contained major part is arranged is to appear in 3 ~ 4 crests of running out previously, but " Tonnae Sinensis-4 " 25 and 26 minutes holdup time the point place more than in addition 2 ~ 3 crest signals.
The XTT analysis of cell proliferation of embodiment 3. Folium toonae sinensis extracts (XTT cell proliferation assay)
The Folium toonae sinensis extract that has the anticancer growth activity for screening, present embodiment uses XTT analysis of cell proliferation (XTT cell proliferation analysis), and to buy from American-type cultivation collection center [American Type Culture Collection (ATCC), P.O.Box 1549, Manassas, VA 20108USA] bladder cancer cell line T24 (bladder cancer cell line T24) come prepared 5 kinds of Folium toonae sinensis extracts in the test implementation example 1, comprise " Tonnae Sinensis 5-4 ", " Tonnae Sinensis 5-2 ", " Tonnae Sinensis 5-5 ", " Tonnae Sinensis 5-5R " and " Tonnae Sinensis 5-4R ".
The experimental implementation method:
Prepared 5 kinds of Folium toonae sinensis extraction products " Tonnae Sinensis 5-4 ", " Tonnae Sinensis 5-2 ", " Tonnae Sinensis-5 ", " Tonnae Sinensis 5-5R " are brought with " Tonnae Sinensis 5-4R " and are carried out XTT analysis of cell proliferation (Roche MolecularBiochemicals) in embodiment 1.The test concentrations of each Folium toonae sinensis extract is respectively 1,10 and 100 μ g/mL, and with H
2O, 50%EtOH and 99.5%EtOH organize in contrast.
Transitional cell bladder carcinoma cell line T24 is incubated in the DMEM-F12 culture medium of amphotericin B (Amphotericin B) of streptomycin (streptomycin), 0.025mg/mL of the penicillin (penicillin) that is added with 10% hyclone (FCS), 10000 units/mL, 10mg/mL.
XTT analysis of cell proliferation (Roche) is undertaken by following manner: with cell with 5 * 10
3The concentration of individual cell/100 μ l/ wells is inoculated in each well of 96 well culture plates, and places one to contain 5%CO culture plate
2Incubator in cultivate and to last 24 hours.Afterwards, each Tonnae Sinensis extract (being dissolved among the PBS) of 1 μ l being added to T24 cell to a ultimate density of being cultivated is 100 μ g/mL, 10 μ g/mL or 1 μ g/mL.Water, 50% ethanol, 99.5% ethanol that other gets 1 μ l are added in the T24 cell to organize in contrast.After cultivation lasts 72 hours, remove culture medium and add XTT mix reagent (the XTT reagent: electronic coupling reagent=50: 1) of the 50 μ l that the fresh culture and of 100 μ l prepares in advance, and culture plate placed last 4 hours in the incubator, afterwards, under wavelength 492nm and 690nm, measure light absorption value (OD=OD with the ELISA reading machine
492-OD
690), and calculate the concentration (IC of inhibition 50% growth of cancer cells of medicine
50), just the light absorption value of experimental group is the tool person's of matched group institute two/for the moment cell concentrations.
The result:
Fig. 3 shows the XTT analysis of cell proliferation result of prepared 5 kinds of Folium toonae sinensis extraction products " Tonnae Sinensis 5-4 " among the embodiment 1, " Tonnae Sinensis 5-2 ", " Tonnae Sinensis 5-5 ", " Tonnae Sinensis 5-5R " and " Tonnae Sinensis 5-4R ", and wherein the low more survival cells of then representing of light absorption value is few more.Clearly visible from Fig. 3, " Tonnae Sinensis 5-2 " is the strongest for the cytotoxicity of bladder cancer cell line T24, and " Tonnae Sinensis 5-4 " is inferior.In addition, " Tonnae Sinensis-2 " has dosage-dependency for the cytotoxicity of bladder cancer cell line T24, and the IC that is calculated
50Be 71.3 μ g/mL.
Embodiment 4. Folium toonae sinensis extracts for bladder cancer cell line T24 the influence of cell cycle
Present embodiment is further probed into " Tonnae Sinensis 5-2 " influence for the cell cycle of bladder cancer cell line T24.The bladder cancer cell line T24 that handles unsynchronized (unsynchronized) with " the Tonnae Sinensis 5-2 " of various dose lasts 24 hours, afterwards, choose other machine analysis [Fluorescence-activated cellsorter (FACS) analysis] with the fluorescent activating cell and assess the cell cycle variation.
The experimental implementation method:
With cancerous cell with 2 * 10
5The concentration of individual cell/dish is inoculated in the 6cm culture plate, and is cultivated and last 24 hours, and culture medium is replaced by the culture medium of " the Tonnae Sinensis 5-2 " that contain 1,10 or 100 μ g/mL afterwards, and continues to cultivate and last 24 hours.Afterwards, (trypsin) handles culture plate with Trypsin, and with the culture medium collected cell that neutralizes, lasts 5 minutes so that 1500rpm is centrifugal, the PBS that removes supernatant and add 300 μ l evenly breaks up cell, and 99.5% ethanol that slowly adds 700 μ l is with cell fixation.Effect is after 30 minutes down through being placed on 4 ℃, and recentrifuge is with the removal supernatant, and adding contains the PBS of 0.1%triton and 0.05%RNase.Carry out after the effect 1hr down centrifugally removing supernatant in 37 ℃, and adding contains the PBS of bromination third ingot (propidium iodide) of 40 μ g/ml.After carrying out the lucifuge effect under 4 ℃ and lasting 30 minutes, break up with 60 μ mMesh Filter and to sieve, analyze with flow cytometer again.
The result:
Referring to following table 1, the applicant finds that after handling with " Tonnae Sinensis 5-2 ", the transitional cell bladder carcinoma cell line T24 that is in the G2 stage increases, particularly when handling with 100 μ g/mL.Because using meeting targetization to the combined therapy of the medicine of the cell that is in different cell cycle phases is standard mode as treatment of cancer, the result shown in the table 1 provides according to the useful data of Folium toonae sinensis extract of the present invention in clinical practice.
Table 1
| Dosage | Be in the percentage ratio of the cell of each cell cycle phase after 24 hours with " Tonnae Sinensis 5-2 " processing | ||
| G1 | S | G2 | |
| Matched group | 45 | 14.1 | 40.9 |
| 100μg/mL | 8.6 | 27.9 | 63.5 |
| 10μg/mL | 33.7 | 13.6 | 52.8 |
| 1μg/mL | 43.9 | 13.2 | 42.9 |
The in vitro anticancer test cell line of embodiment 5. Folium toonae sinensis extracts (in vitro anti-cancer celltest)
Present embodiment is further handled the cancerous cell that several stem from women's urinary system (urogynecological system) with " Tonnae Sinensis 5-2 ", comprise two kinds of ovarian cancers (ovarian cancer) cell strain SKOV3 and PA-1, two kinds of cervical cancer (cervical cancer) cell strain HeLa and HeLaS3 and a kind of carcinoma of endometrium (endometrial cancer) cell strain RL95-2, whether these cancerous cell are had cytotoxicity to observe " Tonnae Sinensis 5-2 ".
The source of JEG-3 and cultivation:
In present embodiment, employed SKOV3, PA-1, HeLa, HeLaS3 and RL95-2 all buy from ATCC, and these JEG-3 all are incubated in the DMEM-F12 culture medium of amphotericin B of streptomycin, 0.025mg/mL of the penicillin that is added with 10%FCS, 10000 units/mL, 10mg/mL.
The experimental implementation method:
With cancerous cell with 15 * 10
5The concentration of individual cell/dish is inoculated in the 10cm culture plate and gives overnight incubation.Afterwards, change culture medium into contain 1mg/mL " Tonnae Sinensis 5-2 " (" Tonnae Sinensis-2 " have be dissolved in earlier in the PBS) culture medium, and continue to cultivate 24 or 48 hours for the concentration allotment.Afterwards, handle culture plate with Trypsin (trypsin) (0.05% Trypsin/0.02%EDTA is assigned in the PBS), and collected cell is placed in little test tube (microtube).Little test tube is placed on the ice bath, make cell suspension in suspension (0.05% Trypsin/0.02%EDTA, be assigned in the PBS) in, and and trypan blue (trypan blue) come uniform mixing with 1: 1 ratio, then under microscope (Nikon TS 100), count the survival cells order with blood cell calculator (hemacytometer).
The result:
For confirming further whether " Tonnae Sinensis 5-2 " has cytotoxicity (cytotoxicity) for the cancerous cell that is derived from women's urinary system (urogynecological system), be that " the Tonnae Sinensis 5-2 " of 1mg/mL handles ovarian cancer cell strain SKOV3 and PA-1, cervical cancer cell strain HeLa and HeLaS3 and endometrial carcinoma cell strain RL95-2 and last 24 hours or 48 hours with concentration, and make comparisons with the matched group that does not add " Tonnae Sinensis 5-2 ", resulting result is shown among Fig. 4, and wherein ordinate is represented the cell survival rate (%) of comparing with matched group.
Clearly visible from Fig. 4, under the activity of 1mg/mL, for ovarian cancer cell strain SKOV3 and PA-1, " Tonnae Sinensis 5-2 " promptly shown powerful growth inhibited effectiveness (almost can suppress the growth of these two kinds of ovarian cancer cells fully) under 24 hours processing time.And for endometrial carcinoma cell strain RL95-2 and cervical cancer HeLaS3, it is not high that the growth of cancer cells that " Tonnae Sinensis 5-2 " shown under 48 hours processing time suppresses effectiveness.For cervical cancer cell strain HeLa, " Tonnae Sinensis 5-2 " shows 60% growth of cancer cells inhibition effectiveness under 48 hours processing time.
Below table 2 show " Tonnae Sinensis 5-2 " IC out of the ordinary for 5 kinds of JEG-3 of being tested
50Value, wherein IC
50The calculating of value be during according to dosing 48 after, have the 50% cell survival drug level of (the survivaling cell sum of matched group is used as 100%).As shown in table 2, " Tonnae Sinensis 5-2 " is for the IC of ovarian cancer cell strain SKOV3 and PA-1
50Value is respectively 28 μ g/mL and 10 μ g/mL.These data show " Tonnae Sinensis 5-2 " have the potentiality that are manufactured into an effective ovarian cancer resistance medicament.
Table 2
| JEG-3 | IC 50 |
| SKOV3 | 28μg/mL |
| PA-1 | 10μg/mL |
| HeLa | 1mg/mL |
| HeLa S3 | ＞1mg/mL |
| RL95-2 | ＞1mg/mL |
Embodiment 6. Folium toonae sinensis extracts are for the cellular morphology of ovarian cancer cell and the influence of cell cycle
Present embodiment is further studied ovarian cancer cell and is changed in cellular morphology that is subjected to being produced after the Folium toonae sinensis extract of the present invention effect and cell cycle.
The experimental implementation method:
With reference to the mode described in the experimental implementation method of embodiment 5, ovarian cancer cell strain SKOV3 and PA-1 are incubated in the culture plate, and after being handled with " the Tonnae Sinensis 5-2 " of 1mg/mL, 100 μ g/mL and 10 μ g/mL respectively, in the variation of microscopically observation of cell form.
In addition, with reference to the mode described in the experimental implementation method of embodiment 4, SKOV3 is incubated in the culture plate with the ovarian cancer cell strain, and after being handled with " the Tonnae Sinensis 5-2 " of 100 μ g/mL and 10 μ g/mL respectively, carries out facs analysis.
The result:
Fig. 5 and Fig. 6 show the form influence that " Tonnae Sinensis 5-2 " caused for ovarian cancer cell strain SKOV3 and PA-1 respectively under employed dosage.Shown in Fig. 5 and Fig. 6 (under the enlargement ratio of 100X), after handling 24 hours under the using dosage of 1mg/mL, " Tonnae Sinensis 5-2 " causes a large amount of apoptosis (apoptosis) and cell floating (cell floating).And after handling 24 hours under the using dosage of 100 μ g/mL and 10 μ g/mL, " Tonnae Sinensis 5-2 " still can cause the remarkable cell death of ovarian cancer cell strain SKOV3 and PA-1 severally.
In addition, ovarian cancer cell strain SKOV3 observes the cell cycle distribution (cell cycledistribution) of ovarian cancer cell strain SKOV3 by facs analysis after " Tonnae Sinensis 5-2 " with various dose handled 24 hours.The result is shown in the following table 3.
Table 3
| Dosage | Handle the percentage ratio that is in the ovarian cancer cell of each cell cycle phase after 24 hours (meansigma methods ± SD) with " Tonnae Sinensis 5-2 " | ||
| G1 | S | G2/M | |
| Matched group | 59.0±0.2 | 27.3±0.9 | 13.8±0.7 |
| 10μg/mL | 53.7±1.3 | 32.1±1.4 | 14.2±1.1 |
| 100μg/mL | 54.2±1.6 | 20.9±0.9 | 25.0±2.5 |
Ovarian cancer cell strain SKOV3 is after being handled 4 hours with " Tonnae Sinensis 5-2 ", (cell survival assay) assesses by the cell survival analysis, and finds that " Tonnae Sinensis 5-2 " has a cytotoxicity (table 4) that raises for the ovarian cancer cell strain SKOV3 that is positioned at the M stage.
Table 4
| SKOV3 | (number) of living | Dead (number) | Apoptotic cell (%) |
| 8h does not have Tx to last 4 hours | 83 | 1 | 1.2 |
| 8h TS 1mg/ml Tx lasts 4 hours | 157 | 12 | 7.1 |
| 12h does not have Tx to last 4 hours | 77 | 1 | 1.3 |
| 12h TS 1mg/ml Tx lasts 4 hours | 72 | 9 | 11.1 |
| 16h does not have Tx to last 4 hours | 113 | 3 | 2.6 |
| 16h TS 1mg/ml Tx lasts 4 hours | 59 | 3 | 4.8 |
| 20h does not have Tx to last 4 hours | 80 | 2 | 2.4 |
| 20h TS 1mg/ml Tx lasts 4 hours | 82 | 6 | 6.8 |
| 24h does not have Tx to last 4 hours | 104 | 8 | 7.1 |
| 24h TS 1mg/ml Tx lasts 4 hours | 88 | 14 | 13.7 |
| The M stage does not have Tx to last 4 hours | 91 | 9 | 9.0 |
| M stage TS 1mg/ml Tx lasts 4 hours | 78 | 36 | 31.6 |
Annotate: abbreviation TS represents " Tonnae Sinensis 5-2 " extract, and abbreviation Tx represents to handle.
Table 3 shows that with the fruit of table 4 " Tonnae Sinensis 5-2 " improves ovarian cancer born of the same parents strain SKOV3 and be parked in G2/M section (G2/M phase), and has more toxicity in the ovarian cancer born of the same parents' strain SKOV3 in the M section
Execute the live body object model of example 7. Tonnae Sinensis extracts
The extract of Folium toonae sinensis is in the test that suppresses tumor cell
For can confirm in vivo producing anticancer effectiveness according to Folium toonae sinensis extract of the present invention, present embodiment further carries out in vivo animal model test with nude mice.
The experimental implementation method:
With 2 * 10
5SKOV3 cell/0.1mL PBS is inoculated in male nude mouse (Foxnlnu/Foxnlnu) (24-29 g of weight, 5-6 week is big, n=5) back is subcutaneous, waits to develop (needing a week approximately) after the tumor that 3mm at least, gives intraperitoneal injection Tonnae Sinensis 5-2 again to observe therapeutic effect.
The preparation of Tonnae Sinensis 5-2 is as follows: Tonnae Sinensis 5-2 is dissolved in the PBS is filtered, and be mixed with 67.25mg/mL and 672.5mg/mL, the body weight according to every nude mice gives Tonnae Sinensis 5-2 (dosage: 0.6725 μ g/g body weight or 6.725 μ g/g body weight) then.
The result:
Have in the body by ovarian cancer cell strain SKOV3 (2 * 10
5Cell) subcutaneous injection develops the male nude mouse of the obvious tumor agglomerate that by " the Tonnae Sinensis 5-2 " of intraperitoneal injection with 0.67 μ g/g body weight (low dosage) or 6.7 μ g/g body weight (high dose), is injected in each week and carries out for 7 weeks altogether in 5 days.
Check discovery from dissecting, being demonstrated naked intravital tumor by dispensing with the test group of the Folium toonae sinensis extract of high dose (6.7 μ g/g body weight) has and obviously is contracted to the degree that disappears that is close to, and has and dwindles and also demonstrated the intravital tumor of nude mice by dispensing with the test group of the Folium toonae sinensis extract of low dosage (0.67 μ g/g body weight).As for the matched group of being injected with phosphate buffered saline (PBS), the intravital tumor of nude mice shows and continues to grow up.Compared to the matched group of handling with PBS, the intraperitoneal injection of " Tonnae Sinensis 5-2 " is handled significantly and is closed the growth that suppresses the tumor agglomerate with a dosage dependency.
Fig. 7 demonstration, under observation through 7 weeks, nude mice tumor agglomerate change in volume on one's body.Especially, injected the nude mice with " the Tonnae Sinensis 5-2 " of high dose, original tumor agglomerate is almost vanished from sight in its body.In addition, referring to table 5, after the intraperitoneal injection of " Tonnae Sinensis 5-2 " lasted for 7 weeks, bone marrow, kidney or the liver to the test nude mice do not cause any significant toxicity.
Table 5
| Meansigma methods+standard deviation | Matched group | Low dosage | High dose |
| WBC(10*3/μl) | 3.32±0.88 | 3.143±0.67 | 4.562±1.14 |
| RBC(10*6/μl) | 9.34±0.17 | 9.21±0.41 | 9.755±0.19 |
| HGB(g/dl) | 12.45±0.20 | 12.5±0.42 | 13.525±0.32 |
| HCT(％) | 41.5±0.76 | 41.266±1.83 | 44.85±0.91 |
| MCV(fl) | 44.45±0.65 | 44.833±0.50 | 46±0.79 |
| MCH(pg) | 13.33±0.14 | 13.6±0.15 | 13.875±0.13 |
| MCHC(％) | 30.05±0.61 | 30.333±0.39 | 30.15±0.33 |
| BUN(mg/dL) | 29.3±2.3 | 28.0±2.0 | 25.0±4.0 |
| Kreatinin (Creatinine) (mg/dL) | 0.43±0.11 | 0.47±0.04 | 0.47±0.16 |
| AST(IU/L) | 101.3±23.3 | 111.7±24.3 | 150.0±30.7 |
| ALT(IU/L) | 29.8±17.7 | 48.0±20.0 | 60.7±51.4 |
Above experimental result is clear to show to have the high potentiality that develop into an ovarian cancer resistance medicament according to Folium toonae sinensis extract of the present invention.
All documents and materials of being quoted from this description are merged in this case as the reference data with its integral body.If to some extent when conflicting, explanation of the present invention (comprise be defined in) will be got the upper hand.
Though the present invention is described with reference to above-mentioned specific concrete example, is not deviating under the scope and spirit of the present invention significantly, can make a lot of modifications and variations.What therefore be intended to is that the present invention only is subjected to as the restriction with the attached claim those shown of literary composition inspection.
Claims (18)
1, a kind of Folium toonae sinensis extract that can be used for treating ovarian cancer or bladder cancer is characterized in that this Folium toonae sinensis extract is to comprise the method for the following step and be made by one:
(i) under heating, extract Folium toonae sinensis and obtain water-soluble first extract once water extraction with water, and
(ii) come resulting second extract that is somebody's turn to do first extract that extracts through water and obtains extracting of extraction step (i) once alcohol with an alcohol.
2, a kind of Folium toonae sinensis extract that can be used for treating ovarian cancer or bladder cancer is characterized in that this Folium toonae sinensis extract is to comprise the method for the following step and be made by one:
(a) under heating, extract Folium toonae sinensis, to obtain an aqueous extraction solution with water;
(b) with resulting aqueous extraction solution drying in the step (a), to obtain first extract that is dried;
(c) resulting first extract in the step (b) is dissolved in the alcoholic solvent, to form a pure extraction solution; And
(d) alcoholic solvent that will be obtained from the pure extraction solution of step (c) removes to doing, to obtain second extract that is dried.
3, the purposes of Folium toonae sinensis extract as claimed in claim 1 or 2 is characterized in that, this Folium toonae sinensis extract is used to prepare a medicine that is used for the treatment of ovarian cancer or bladder cancer.
4, a kind of in order to prepare the method for a Folium toonae sinensis extract, it is characterized in that this method comprises the following step:
(a) under heating, extract Folium toonae sinensis, to obtain an aqueous extraction solution with water;
(b) will be obtained from the aqueous extraction solution drying of step (a), to obtain first extract that is dried;
(c) first extract that will be obtained from step (b) is dissolved in the alcoholic solvent, to form a pure extraction solution; And
(d) alcoholic solvent that will be obtained from the pure extraction solution of step (c) removes to doing, to obtain second extract that is dried.
5, method as claimed in claim 4 is characterized in that:
Wherein in step (a), the acquisition of this aqueous extraction solution is boiled by the water that adding is had Folium toonae sinensis to be concentrated into one in right amount, is filtered then.
6, method as claimed in claim 5 is characterized in that:
Wherein this filtration treatment is to be selected from following utensil by use one to carry out: the filter screen of gauze, Cotton Gossypii, the selected slot size of a tool, and these a kind of combination.
7, method as claimed in claim 4 is characterized in that:
Wherein the dried of being carried out in the step (b) is to be selected from the following group that constitutes: lyophilization processing, low temperature spray drying are handled, low-temperature evaporation is handled, and these a kind of combination.
8, method as claimed in claim 7 is characterized in that:
Wherein the dried of being carried out in the step (b) is that lyophilization is handled.
9, method as claimed in claim 4 is characterized in that:
Wherein step (b) comprises following treatment step:
(i) the aqueous extraction solution centrifugal of step (a) be will be obtained from and a upper clear liquid and a precipitate obtained; And
(ii) resulting this upper clear liquid in the step (i) is carried out a dried.
10, method as claimed in claim 9 is characterized in that:
Wherein the step dried of (ii) being carried out is to be selected from the following group that constitutes: lyophilization processing, low temperature spray drying are handled, low-temperature evaporation is handled and these a combination.
11, method as claimed in claim 9 is characterized in that:
The alcohol that wherein is used in the step (c) is to be selected from the following group that constitutes: ethanol, methanol, propanol, isopropyl alcohol, n-butyl alcohol, isobutanol, and these a kind of combination.
12, method as claimed in claim 11 is characterized in that:
The alcohol that wherein is used in the step (c) is ethanol.
13, method as claimed in claim 4 is characterized in that:
Wherein the alcoholic solvent that is carried out in the step (d) removes, and is to be undertaken by a processing that is selected from the following group: lyophilization processing, evaporation process, spray drying treatment, and these a kind of combination.
14, method as claimed in claim 13 is characterized in that:
Wherein step (d) is to handle and be carried out by lyophilization.
15, a kind of pharmaceutical composition that can be used for treating ovarian cancer or bladder cancer is characterized in that this pharmaceutical composition comprises:
(a) a Folium toonae sinensis extract that is made by the method for claim 4 of a treatment effective dose; And
(b) a pharmaceutically acceptable supporting agent.
16, the purposes of pharmaceutical composition as claimed in claim 15 is characterized in that, is used to prepare the medicine that treatment one is selected from the cancer of ovarian cancer and bladder cancer.
17, a kind of purposes of pharmaceutical composition, this pharmaceutical composition comprises:
(a) a Folium toonae sinensis extract that is made by the method for claim 4 of a treatment effective dose; And
(b) a pharmaceutically acceptable supporting agent,
It is characterized in that described pharmaceutical composition is used to prepare and suppresses a medicine of growth that is selected from the cancerous cell of ovarian cancer cell and transitional cell bladder carcinoma cell line.
18, a kind of pharmaceutical composition that is used for the treatment of bladder cancer or ovarian cancer is characterized in that this pharmaceutical composition comprises:
(a) one of a treatment effective dose be selected from following Folium toonae sinensis extract:
(i) one extract Folium toonae sinensis and water-soluble Folium toonae sinensis extract of obtaining via water;
A (ii) Folium toonae sinensis extract as claimed in claim 1;
(b) a pharmaceutically acceptable supporting agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310124477 CN1287814C (en) | 2003-12-24 | 2003-12-24 | Extractive originated from cedrela sinensis, its preparation process and usage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310124477 CN1287814C (en) | 2003-12-24 | 2003-12-24 | Extractive originated from cedrela sinensis, its preparation process and usage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1631409A CN1631409A (en) | 2005-06-29 |
| CN1287814C true CN1287814C (en) | 2006-12-06 |
Family
ID=34845016
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200310124477 Expired - Fee Related CN1287814C (en) | 2003-12-24 | 2003-12-24 | Extractive originated from cedrela sinensis, its preparation process and usage |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1287814C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100998651B (en) * | 2007-01-06 | 2011-06-15 | 三峡大学 | Medicine for treating thromboembolia type disease and its preparing method |
| CN103356745B (en) * | 2013-06-19 | 2015-07-01 | 华东理工大学 | Chinese medicinal mixed preparation for tumors |
| CN105481876A (en) * | 2015-12-30 | 2016-04-13 | 吴金凤 | Diterpene compound for treating ovarian cancer |
-
2003
- 2003-12-24 CN CN 200310124477 patent/CN1287814C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1631409A (en) | 2005-06-29 |
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