CN1329038C - Water soluble nightshade extract, its preparation method and pharmaceutical compositions - Google Patents

Water soluble nightshade extract, its preparation method and pharmaceutical compositions Download PDF

Info

Publication number
CN1329038C
CN1329038C CNB031494404A CN03149440A CN1329038C CN 1329038 C CN1329038 C CN 1329038C CN B031494404 A CNB031494404 A CN B031494404A CN 03149440 A CN03149440 A CN 03149440A CN 1329038 C CN1329038 C CN 1329038C
Authority
CN
China
Prior art keywords
water
soluble extract
eggplant
soluble
nightshade
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
CNB031494404A
Other languages
Chinese (zh)
Other versions
CN1565473A (en
Inventor
郭国华
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
G and E Herbal Biotechnology Co Ltd
Original Assignee
G and E Herbal Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by G and E Herbal Biotechnology Co Ltd filed Critical G and E Herbal Biotechnology Co Ltd
Priority to CNB031494404A priority Critical patent/CN1329038C/en
Publication of CN1565473A publication Critical patent/CN1565473A/en
Priority to HK05102739A priority patent/HK1069995A1/en
Application granted granted Critical
Publication of CN1329038C publication Critical patent/CN1329038C/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Abstract

The present invention provides water-soluble extractant coming from nightshade, which is basically composed of at least 60% to 90% of Australia solasonine and solamargine. The present invention also provides a method for preparing the water-soluble extractant of nightshade. The method comprises the steps of acid hydrolysis, alkali deposition and separation with solvent, such as chloroform, alcohol, water, etc. The water-soluble extractant prepared by the method can be directly dissolved in purified water or water with a neutral pH value under the condition that no other solvent and/or auxiliary solvent is added to form yellow transparent water solution, and the dissolvability can reach more than 2 to 20 mg/ml. The present invention also provides a pharmacy composition containing the water-soluble extractant of nightshade which serves as an active constituent used for inhibiting tumours/cancer cells (particularly hepatoma carcinoma cells, lung cancer cells and mammary cancer cells) from growing.

Description

The water-soluble extract of nightshade, its preparation method and pharmaceutical compositions thereof
Technical field
The present invention relates to water-soluble extract and its preparation method of a kind of nightshade (plant of Solanum genus), and the pharmaceutical compositions that includes this water-soluble extract.
Background technology
Cancer is one of human topmost cause of death in the present whole world, and pulmonary carcinoma, hepatocarcinoma and breast carcinoma then are human topmost cancer.Though the formation mechanism of cancer is not understood yet fully, what it is believed that cancer is attributable to cell division (Chen, P.L. etc., (1990), Science, 250,1576-1580 out of hand; Finlay, C.A. etc., (1989), Cell, 57,1083-1093; And Baker, S.J. etc., (1990) Science, 249,912-915).
Usually, the intravital cell growth of the mankind or animal is controlled with the strictness that differentiation can be subjected to the body inner growth hormone.When genovariation that cellular accumulation endogeny or extrinsic factor cause, this cell will produce wrong message transmission, and cause this cell at merisis down out of hand, and form cancerous cell (Kerr gradually, J.F.R. (1971) J.Pathol., 105,13-20).
The research worker of countries in the world all is devoted to the research of cancer now, but the achievement of being reached in treatment of cancer at present is still desirable not to the utmost, this is except the individual factor of patient own, and the serious side effects of anticarcinogen and the problems such as Drug resistance of cancerous cell are the maximum of the being run into puzzlements on clinical treatment.In view of the Western medicine medicine that uses clinically at present still can't effectively improve treatment for cancer, some research worker sees through the mechanism of research cancer, and attempt from Chinese medicine (traditional Chinese medicines, TCM) or seek the active constituent of the symptom that can be used for treating or eliminate cancer in the herbaceous plant (herbs).
Apoptosis (Apoptosis) is considered to the machine-processed Martin of a kind of natural regulating cell growth of zooblast, S.J.and Green, D.R. (1995), Crit.Rev.Oncol.Hemat., 18,137-153), it plays the part of a key player in the naturality of many cells is dead regulates (for example, animal growth process organize nature atrophy and absorption).In addition, when the human cell came to harm and can't repair, the death of nature cell program also can be activated to avoid producing cancerous cell.
The principal character of apoptosis has: the fragmentation of formation, chromatin agglutination and the DNA of apoptosis corpusculum (apoptotic bodies) (Arends, M.J.and Wyllie, A.H. (1991) Int.Rev.Exp.Pathol., 32,223-254; Dive, C. etc., (1992) Biochim.Biophys.Acta 1133,275-285; And Darzynkiewicz, Z. etc., (1992) Cytometry, 13,795-808).In the apoptosis process, the fragment of dead cell can promptly be absorbed via phagocytosis by peripheral cell and phagocyte, does not produce (Sarraf, F.E.and Bowen and do not have inflammatory response, I.D. (1988) Cell Tissue Res.21,45-49).In addition, when utilizing stream type cell analyzer to measure the variation of cell cycle, Sub-G1 ripple (Alzerreca, A.and Hart, G. (1982) Toxicology Lett.12,151-155 can appear; And Lin, C.N. etc., (1986) J.Taiwan Pharm.Assoc.38,166), so the Sub-G1 ripple is considered to the typical marks that cell carries out apoptosis.
Reported in literature is arranged, and when cell changes and its apoptosis mechanism when out of control, this cell can form cancerous cell (Carson, D.A.and Ribeiro, J.M. (1993) Lancet 341,1251-1254; And Kaufmann, S.H. (1989) Cancer Res.49,5870-5878).Therefore, apoptosis has become the target of oncology studies in recent years.In addition, reported in literature is also arranged, some cancer therapy drug can cause that cancerous cell carries out apoptosis (Wyllie, A.H. etc., (1980) Int.Rev.Cytol.68,251-306; Wyllie, A.H. etc., (1984) JPathol.142,67-77; Barry, M.A. etc., (1990) Biochem.Pharmacol., 40,2353-2362; And Hickman, J.A. (1992) Cancer Metast.Rev., 11,121-139), therefore, apoptosis also become whole world research cancer therapy drug important directions one.
Chinese medicine (TCM) or medical herbs (herbal medicines) are applied to the existing one phase quite long history of treatment of diseases, and also have many research worker to attempt seeking out useful cancer therapy drug in the middle of Chinese medicine and medical herbs at present.But the use of Chinese medicine and medical herbs is mostly still based on traditional experience, and may not have sufficient scientific evidence to support.In addition, Chinese medicine or medical herbs because of extracting process, dosage control and the character surveillance of active ingredient also not by scientific, and the curative effect difficulty that makes the dispensing back be produced reaches unanimity.Moreover it is water-insoluble that the active ingredient of Chinese medicine or medical herbs mostly is greatly, and non-water soluble substance is by oral administration medicine supplying or when being injected in the animal body, may have the difficulty in the absorption and can't show curative effect.These are restriction Chinese medicine and the application of medical herbs and the main cause of development.
At present in the world can be by numerous when the plant that is used for medicinal purpose, known have the many kinds of protein inhibitors that get from the vegetable material extraction to can be used for anticancer therapy.Have in the protein inhibitor of anticancer potentiality at these, found to have potentiality as cancer therapy drug by the resulting steroid alkaloid of plant of Solanaceae body (steroidal alkaloids).
Known yellow fluid eggplant (Latin formal name used at school: Solanum incanum L., alias: Solanum incanum Ruiz.﹠amp; Pav., Solanum coagulans Forsskal; English name: bitter apple) contain steroid glycosyl alkaloid (steroidal glycoalkaloid) (Kuo, K.w. etc., (2000), Biochemical Pharmacology, 60 (12): 1865-73).In addition, according to reports, have the plant that much belongs to Solanaceae also to contain steroid glycosyl alkaloid, this comprises, for example, and India eggplant (Solanum indicum), Herba Solani Nigri (Latin formal name used at school: Solanum nigrum; The Chinese phonetic alphabet: Long kui; 35-8), Jerusalem cherry (Latin formal name used at school: Solanum capsicastrum English name: black nightshade) (Hu, K. etc., (1999), Planta Medica, 65 (1):; False jerusalemcherry), Fructus solani xanthocarpi (Solanum xanthocarpum), melon eggplant (Solanum melongena) (Blankemeyer, J.T. etc., (1998), Food ﹠amp English name:; Chemical Toxicology, 36 (5): 383-9), yellow Solanum aculeatissimum Jacq. (Solanumcoagulans), Rhizoma Solani tuber osi (Solanum tuberosum) (Friedman, M. etc., (1996), Journal of Nutrition, 126 (4): 989-99), Satan eggplant (Solanum sodomeum) (apple of Sodom is otherwise known as in Australia), gyro eggplant (Solanum turburosum), Solanum aculeatissimum Jacq. (Solanum aculeastrum) (Wanyonyi, A.W. etc., (2002), Phytochemistry, 59 (1): 79-84), Lycopodium clavatum eggplant (Solanum lycocarpum) (Peters, V.M. etc., (2001), Contraception, 63 (1): 53-5), Keshen inferior eggplant (Solanum khasianum) (Putalun, W. etc., (2000), Biological ﹠amp; Pharmaceutical Bulletin, 23 (1): 72-5), Radix Solani Torvi (Solanumsuaveolens) (Ripperger, H. etc., (1997), Phytochemistry, 46 (7): 1279-82), three-Flowered nightshade (Solanum uporo) (Ripperger, H. etc., (1997), Phytochemistry, 44, (4): 731-4)), lettuce eggplant (Solanum abutiloides) (Tian, R.H., et al (1997), Phytochemistry, 44 (4): 723-6), the West eggplant (Solanum coccineum) (Lorey, S. etc., (1996), Phytochemistry, 41 (6): 1633-5), hoof shape eggplant (Solanum unguiculatum) (Sarg, T.M. etc., (1995), Pharmacy World ﹠amp; Science, 17 (6): 191-4), tough eggplant (Solanum robustum) (Ripperger, H. (1995), Phytochemistry 39 (6): 1475-7), Serpentis shape eggplant (Solanum anguivi) (Ripperger, H. etc., (1994), Phytochemistry, 37 (6): 1725-7), lobule eggplant (Solanum platanifolium) (Puri, R. etc., (1 994), Journal of NaturalProducts 57 (5): 587-96), America eggplant (Solanum mammosum) (Alzerreca, A. etc., (1982), Toxicology Letters, 12 (2-3): 151-5) or the like.
Known at present, the steroid alkaloid that obtains from these plants of Solanaceae includes, for example, solamargine (solamargine), solasonine (solasonine), Keshen inferior plain (khasianine) and solasodine composition (Chataing such as (solasodine), B. etc., (1998), Planta Medica 64,31-36; And Weissenberg, M. etc., (1998), Phytochemistry 47,203-209).The chemical constitution of solasonine and solamargine is as follows:
In addition, existing discovering, the solamargine that gets from different plant origin extractions has the effect of the growth that suppresses following organism: parasite, as withered Xi Shi trypanosomicide (Trypanosoma cruzi); Insecticide is as red flour beetle worm (Tribolium castaneum, English red flour beetle by name), maduca sexta (Manduca sexta, English tobacco hornworm by name); Mycete is as mould dekamycin (Phoma medicaginis), root bundlin (Rhizoctomia solani); And Mollusca, as Lima spiral shell (Lymnaea cubensis), fresh water Limax (Biomphalaria glabrata) (Chataing, B. etc., (1998), Planta Medica, 64,31-36; Fewell, A.M. etc., (1994), Phytochemistry, 37,1007-1011; Lin, C.N. etc., (1990), J.Nat.Prod., 53,513-516).
In addition, people such as Chun-Nan Lin once reported, can extract solamargine from the fruit of yellow fluid eggplant (Solanum incanum), the chemical constitution through identifying this chemical compound belongs to steroid alkaloid sugar, and this chemical compound is found for CCl through biological activity determination 4-the hepatic injury that brings out can show the liver protective effect, and can suppress the growth (Chun-Nan Lin etc. (1986), J.NaturalProd., 53,513-516) of JTC-26 and human PLC/PRF/5 tumor cell of liver.
People such as Shu-Hui Hsu further study the cytotoxic effect mechanism of solamargine, find the death that solamargine can improve cell (such as human liver cancer cell Hep3B and normal skin fibroblast) by apoptosis.Especially, known cancer mecrosis factor receptors I (TNF receptor I) relates to the process of apoptosis, and solamargine is found and can makes this receptor produce over-drastic gene performance (Shu-Hui Hsu etc., (1996), Biochem.Biophys.Res.Comm., 229,1-5).
Katsuya Fukuhara and Isao Kubo are in Phytochemistry, 30 (2): 685-687, report in 1991: the mature fruit of the yellow fluid eggplant that extracts with methanol at room temperature moves down in decompression then and desolventizes and with the residue lyophilization, and obtains a dark-brown extract; Connect and this extract loose float in the water that contains 1% methanol, after removing water-fast part, distribute separation (partitioning), and obtain the water layer of biologically active with n-hexane, chloroform, ethyl acetate and water.The water layer of this biologically active connects and is rotated chamber type countercurrent chromatography (rotation locular countercurrent chromatography) and droplet type countercurrent chromatography (droplet countercurrent chromatography), and can obtain solamargine, these two kinds of main compound of solasonine.
Ke Hu equals (1999), Planta Medica 65, disclose among the 35-38, use the Herba Solani Nigri (Solanumnigrum) of whole strain, make reflow treatment with 75%EtOH, and remove solvent under vacuum, the brown residue of gained connects and makes degrease with petroleum ether and handle, the extract of gained is loose to float in the water and with the macroresin tubing string and is made chromatography thus, contains reactive compound in the 60%EtOH eluant.This 60%EtOH eluant is distributed separation with water and is extracted with n-BuOH, and gained n-BuOH extract is directed to and uses CHCl 3-MeOH-H 2The silica gel tube column chromatography of O and use MeOH-H 2The Sephadex LH-20 tubing string chromatography of O (60: 40), and obtain β 2-solamargine (β 2-solamargine), solamargine and take off galactose carry the fruit soap (degalactotigonin).This piece paper do not have teaching how from Herba Solani Nigri obtain can be directly water-soluble the bioactive extract that has.
EP 0 020 029 A1 disclose, earlier extract Solanumsodomeum (apple of Sodom is otherwise known as in Australia) vegetable material to obtain one first acid extraction thing (aqueous solution part) with a dilute acid solution (2% or 3% acetic acid), extract solid residue with this dilute acid solution again after this first acid extraction thing separated with solid residue and obtain one second acid extraction thing (aqueous solution part), after merging this first acid extraction thing and this second acid extraction thing, added alkali and obtain precipitate.This precipitate is dissolved in after the ebullient ethanol, ethanol is removed again and obtains being the extract of careful powder, and this extract is called BEC 001.This BEC 001 extract can obtain multiple glycosyl alkaloid (glycoalkaloids) through further separation and purification treatment, wherein comprise the list of solamargine, solasonine and solasodine-with two-sugar.
Though EP 0 020 029 A1 mention, can make the carrier (carrier) of water as BEC 001 extract, in the embodiment of this case, BEC 001 extract mainly is to be made into dimethyl sulfoxide (DMSO) solution, or be assigned in paraffin wax, zinc ointment (zinc ointment), zinc cream (zinc cream) and the west holder Marko's height (cetomacrogol, a kind of interface activating agent).
Moreover, according to US 5,958,770 announcement, solasodine sugar (solasodine glycosides) is used in vitro in the cytotoxicity experiment, and is to be dissolved in earlier in the DMSO, is diluted to a 5%DMSO solution then and uses.In addition, solasodine sugar in the middle of being applied to testing is to be a mixture (BEC, include solamargine (33%), solasonine (33%) together with two-sugar and list-sugar (34%)), or be separation component (mixture of solamargine, solasonine, two-sugar and list-sugar, and the sugared aglucon of solasodine (theaglycones of solasodine)).
The above-mentioned steroid alkaloid of mentioning is water-insoluble; therefore; preceding case of above-mentioned patent or the traditional method that document disclosed are carried out with the pure way of distillation often in the do extraction, and the composition that is extracted is analyzed after can dissolving with the organic solvent dimethyl sulfoxine usually again.Because non-water soluble substance not too is suitable for by direct injection to animal body, when oral administration medicine supplying, also may embarrass digestive tract to absorb and can't show curative effect, and make its medicinal application with develop on be restricted.
The applicant finds in test, the dried powder of solamargine and/or solasonine is if handle earlier without DMSO, can't dissolve extremely, even also can't dissolve fully after handling via DMSO, especially the method extraction that is disclosed with EP 0 020 029 A1 and steroid alkaloid and can't be dissolved in the distilled water.Moreover solamargine or solasonine be in case earlier with after the DMSO dissolving, adds then solubilized of water again, if but the concentration of solamargine or solasonine when too high (surpassing 5mg/ml), solamargine or solasonine still can be separated out in crystallization.In addition, DMSO (more than 1%) itself promptly has very strong toxic action for cell, so the concentration of DMSO must be controlled at below 5%.Demonstrate still restricted existence when wanting to utilize this organic solvent of DMSO to dissolve from steroid alkaloid that plant of Solanaceae extracts these The fact has been fully proveds.
Based on the above, these steroid alkaloid products are in the supply of actual product at present, only limit to the production of a small amount of and single batch of chemicals company, and lack effective ways come on a large scale from the plant of Solanaceae body extract can be directly water-soluble the steroid alkaloid product for commercial exploitation, thereby limit these steroid alkaloids and will be applied to development in the medicament manufacturing.
Summary of the invention
So, for a large amount of generations can supply use and the water solublity steroid alkaloid that stem from nightshade of industry, aspect first, the invention provides the water-soluble extract of a kind of nightshade, it is by solasonine and the solamargine of 60%-90% are constituted at least basically.This water-soluble extract can directly be dissolved under not adding any other solvent and/or cosolvent in the water of pure water or pH neutral and be formed the aqueous solution of a yellow clear, and dissolubility can reach 2～20mg/ml or more than.
Aspect second, the invention provides a kind of pharmaceutical compositions, it includes above-mentioned water-soluble extract with as the active constituent in order to the growth that suppresses lesion/cancer cell (particularly hepatoma carcinoma cell, lung carcinoma cell and breast cancer cell).
Aspect the 3rd, the invention provides a kind of method that is used to prepare the water-soluble extract of a kind of nightshade, it comprises the following step:
(a) making a kind of vegetable material that belongs to a nightshade carry out one, to use a pH value be that the extraction of 3～5 acidic aqueous solution is handled, and obtains an aqueous solution by this;
(b) pH value of aqueous solution that will be obtained from step (a) with an alkali is adjusted to pH 8～10, by this, has a precipitate to be formed;
(c) clean the precipitate that is obtained from step (b) with water, give drying then, obtain a desciccate by this;
(d) desciccate that will derive from step (c) mixes with chloroform, adds one an amount of 100% alcohol then, obtains one chloroform-alcohol mixture by this;
(e) will be obtained from the chloroform of step (d)-alcohol mixture mixes with the predetermined water of a tool one: the water/alcoholic solution of pure ratio, by this, one have one based on the layer of chloroform and one non-based on chloroform layer mixture be formed;
(f) layer based on chloroform in the formed mixture of step (e) is removed, add an an amount of water then; And
(g) obtain a supernatant from the formed mixture of step (f), give drying then, by this, formed desciccate can be by directly soluble in water and form the aqueous solution of a yellow clear.
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 show use from yellow fluid eggplant extraction and water-soluble extract (25mg) carry out the result of high pressure liquid chromatography (HPLC) (HPLC);
Fig. 2 show use from yellow fluid eggplant extraction and the HPLC analysis result of water-soluble extract (25mg) and solasonine (5mg);
Fig. 3 show use from yellow fluid eggplant extraction and the HPLC analysis result of water-soluble extract (25mg) and solamargine (5mg);
Fig. 4 show use from Herba Solani Nigri extraction and water-soluble extract (25mg) carry out the result of HPLC;
Fig. 5 show use from Herba Solani Nigri extraction and the HPLC analysis result of water-soluble extract (25mg) and solasonine (10mg);
Fig. 6 show use from Herba Solani Nigri extraction and the HPLC analysis result of water-soluble extract (25mg) and solamargine (10mg);
Fig. 7 to Figure 12 shows that the yellow fluid eggplant water-soluble extract that uses varying number carries out the result of HPLC;
Figure 13 shows the standard inspection amount line of the yellow fluid eggplant water-soluble extract master composition that estimates according to the result of Fig. 7 to Figure 12;
Figure 14 to Figure 18 shows the result who carries out HPLC according to yellow fluid eggplant water-soluble extract of the present invention under different pH value;
Figure 19 shows the method that is disclosed according to yellow fluid eggplant water-soluble extract of the present invention and according to EP 0 020 029 A1 and the granularmetric analysis comparative result of the yellow fluid eggplant extract that makes;
Figure 20 and Figure 21 show respectively, gets the method that is disclosed according to EP 0 020 029 A1 of equal weight (50mg) and the yellow fluid eggplant extract that makes and according to yellow fluid eggplant water-soluble extract of the present invention, dissolved the result who carries out HPLC afterwards with pure water;
Figure 22 to Figure 24 shows respectively according to yellow fluid eggplant water-soluble extract of the present invention growth inhibited effectiveness for human liver cancer, pulmonary carcinoma and breast cancer cell under various dose;
Figure 25 demonstration is analyzed according to the automatic radiography result of yellow fluid eggplant water-soluble extract of the present invention for the gene regulation mechanism of lung carcinoma cell with gene chip, wherein lung carcinoma cell took out later on RNA in 2 hours being handled with this yellow fluid eggplant water-soluble extract (100 μ g/ml), again with [ 32P]-dCTP carries out reverse transcription and makes the cDNA that is labeled, and do hybridization with gene chip then and detect and make automatic radiography with X-light film;
Figure 26 shows the metamorphosis that human liver cancer cell (A is capable), lung carcinoma cell (B is capable) and breast cancer cell (C is capable) are produced when being handled with foundation yellow fluid eggplant water-soluble extract of the present invention;
Figure 27, Figure 28 and Figure 29 show the nude mice that transplant at the back the Human Lung Cancer cell respectively, will not offer medicine according to yellow fluid eggplant water-soluble extract of the present invention, offer medicine via feeding and to offer medicine according to viewed result under the yellow fluid eggplant water-soluble extract of the present invention according to yellow fluid eggplant water-soluble extract of the present invention and via lumbar injection, wherein each the bar curve shown in each figure is respectively represented the result of a test nude mice; And
Figure 30 shows from the resulting solasonine of yellow fluid eggplant water-soluble extract of the present invention and the solamargine growth inhibited effectiveness for the Human Lung Cancer cell.
The specific embodiment
The invention provides the water-soluble extract of a kind of extraction from nightshade, it contains can be directly soluble in water and form the steroid alkaloid of the aqueous solution of a clear, thereby be suitable for the manufacturing of medicament and preparation.
Especially, the invention provides the water-soluble extract of a kind of nightshade, it is by solasonine and the solamargine of 60%-90% are constituted at least basically.This water-soluble extract can directly be dissolved under not adding any other solvent and/or cosolvent in the water of pure water or pH neutral and be formed the aqueous solution of a yellow clear, and dissolubility can reach 2～20mg/ml or more than.
The present invention also provides a kind of method that is used to prepare this water-soluble extract, and it comprises the following step:
(a) making a kind of vegetable material that belongs to a nightshade carry out one, to use a pH value be that the extraction of 3～5 acidic aqueous solution is handled, and obtains an aqueous solution by this;
(b) pH value of aqueous solution that will be obtained from step (a) with an alkali is adjusted to pH 8～10, by this, has a precipitate to be formed;
(c) clean the precipitate that is obtained from step (b) with water, give drying then, obtain a desciccate by this;
(d) desciccate that will derive from step (c) mixes with chloroform, adds one an amount of 100% alcohol then, obtains one chloroform-alcohol mixture by this;
(e) will be obtained from the chloroform of step (d)-alcohol mixture mixes with the predetermined water of a tool one: the water/alcoholic solution of pure ratio, by this, one have one based on the layer of chloroform and one non-based on chloroform layer mixture be formed;
(f) layer based on chloroform in the formed mixture of step (e) is removed, add an an amount of water then; And
(g) obtain a supernatant from the formed mixture of step (f), give drying then, by this, formed desciccate can be by directly soluble in water and form the aqueous solution of a yellow clear.
Preferably, the vegetable material that is used in Solanum (Solanum) plant in this method step (a) is handled through chopping earlier.
Preferably, be used in the middle of fruit that vegetable material in this method step (a) is this nightshade, root, stem, the leaf at least one.In a preferred embodiment of the present invention, the vegetable material that is used in this method step (a) is the fruit of this nightshade.In another preferred embodiment of the present invention, the vegetable material that is used in this method step (a) is this nightshade of whole strain.
Preferably, this water-soluble extract is to be extracted and got by a nightshade that is selected from the following group that constitutes: yellow fluid eggplant (Solanum incanum L.), India eggplant (Solanum indicum), Herba Solani Nigri (Solanumnigrum), Jerusalem cherry (Solanum capsicastrum), Fructus solani xanthocarpi (Solanum xanthocarpum), melon eggplant (Solanum melongena), yellow Solanum aculeatissimum Jacq. (Solanum coagulans), Rhizoma Solani tuber osi (Solanumtunigrum), Satan eggplant (Solanum sodomeum), gyro eggplant (Solanum turburosum), Solanum aculeatissimum Jacq. (Solanum aculeastrum), Lycopodium clavatum eggplant (Solanum lycocarpum), the inferior eggplant (Solanumkhasianum) in Keshen, Radix Solani Torvi (Solanum suaveolens), three-Flowered nightshade (Solanum uporo), lettuce eggplant (Solanumabutiloides), the West eggplant (Solanum coccineum), hoof shape eggplant (Solanum unguiculatum), tough eggplant (Solanum robustum), Serpentis shape eggplant (Solanum anguivi), lobule eggplant (Solanum platanifolium) and America eggplant (Solanum mammosum).
In a preferred embodiment of the present invention, this water-soluble extract is to be got by the extraction of yellow fluid eggplant.In another preferred embodiment of the present invention, this water-soluble extract is to be got by the Herba Solani Nigri extraction.
Preferably, this aqueous solution that is obtained from the step (a) of this method is by carrying out centrifugal after handling in this extraction or filtration and obtained.
Preferably, used acidic aqueous solution is one to contain the aqueous solution of formic acid, acetic acid or hydrochloric acid in the extraction of the step (a) of this method is handled.
Preferably, used alkali is one to contain the alkaline aqueous solution that is selected from the chemical compound in the following group in the step (b) of this method: alkali-metal hydroxide and ammonium hydroxide.In a preferred embodiment of the present invention, this alkaline aqueous solution contains ammonium hydroxide.In another preferred embodiment of the present invention, this alkaline aqueous solution contains sodium hydroxide.
Preferably, this precipitate that is obtained from the step (b) of this method is by carrying out centrifugal after the pH value adjustment or filtration and obtained.
Preferably, the dried of being carried out in the step (c) of this method is to be selected from one by the following group that constitutes: lyophilization processing, spray drying treatment, evaporation process, heat drying are handled, and these a combination.In a preferred embodiment, the present invention carries out this dried with lyophilization, and this is to desire to improve the stability of active constituent with active in a low temperature mode.
Preferably, in the step (c) of this method, the precipitate that is obtained from step (b) cleans with water and then gives centrifugally, to remove excessive alkali, adds distilled water then, then carries out this dried again.
In the step (d) of this method, to the consumption of chloroform and alcohol and have no special requirements, as long as can will derive from the desciccate dissolving of step (c).In a preferred embodiment of the present invention, in the step (d) of this method, the use amount of the no more than chloroform of use amount of alcohol.
Preferably, being used in this method step (d) is to be selected from one by the following group that constitutes with alcohol (e): methanol, ethanol, propanol and these combination.In a preferred embodiment of the present invention, be used in this method step (d) with (e) in alcohol be methanol.
Preferably, this water/alcoholic solution that is used in this method step (e) has the pure content that a water content is not less than institute's tool.More preferably, the water of this water/alcoholic solution: pure ratio is 1: 1.
Preferably, in the step (f) of this method, the layer based on chloroform that removes in the formed mixture of step (e) is to be undertaken by centrifugal or filtration.
Preferably, in the step (g) of this method, this supernatant is by giving the formed mixture of step (f) centrifugal or filtration and obtained.
Preferably, the dried of being carried out in the step (g) of this method is to be selected from one by the following group that constitutes: lyophilization processing, spray drying treatment, evaporation process, concentrating under reduced pressure, heat drying are handled, and these a combination.In a preferred embodiment, the dried of being carried out in the step (g) of this method comprises concentrating under reduced pressure and lyophilization processing.
If needed, can be with the resulting desciccate of step (g) water-soluble once more in, give then centrifugal after, give drying again.
The resulting extract of the present invention can directly be dissolved in general drinkable water or the sterilized water, and forms the solution of a yellow clear.Therefore, the resulting extract of the present invention is a kind of real water-soluble extract.
By the prepared water-soluble extract of this case method is by solasonine and the solamargine of 60%-90% are constituted at least basically.In addition, the applicant is found to some factor may influence the interior solasonine of the resulting water-soluble extract of this case method and the content and the ratio of solamargine.These factors comprise: the species of the nightshade that is used to extract and use position, used alcohol and used alkali.
For example, the water-soluble extract that the mature fruit of use yellow fluid eggplant makes, when analyzing with HPLC, the content of solamargine is higher than the content of solasonine.In addition, when with 33%NH 4The OH alkaline solution is compared, and when using 10M NaOH alkaline solution to carry out step (b), the content of solamargine can be lower than the content of solasonine in the resulting water-soluble extract.Moreover when comparing with methanol, in the resulting water-soluble extract, the content of solasonine and solamargine can reduce when using ethanol to carry out this case method, 50% during approximately only for use methanol.In addition, when extracting with Herba Solani Nigri, in the resulting extract of its crude fruit (dark green), the content of solasonine and solamargine is higher, and ripe fruit (black purple) and other positions are then less.And in the resulting extract of Herba Solani Nigri, the content ratio of solasonine and solamargine is different from from yellow fluid taker that eggplant is come together.Therefore, have the knack of the visual required species of selecting nightshade for use of this technology personage and use position and other operating conditions to generate after being modified to meet required water-soluble extract.
In a preferred embodiment of the present invention, in step (a), use an aqueous solution that contains acetic acid to extract processing, in step (b), use one contain ammonium hydroxide alkaline aqueous solution so that a precipitate be formed, and step (d) with (e) in employed alcohol be methanol.
Preferably, this water-soluble extract has more than 75% and is made of solasonine and solamargine.
Preferably, it is between 0.3: 1.0～1.0: 0.6 with respect to the ratio of solamargine that this water-soluble extract has a solasonine, and perhaps to have a solasonine be between 0.4: 1.0～0.9: 1.0 with respect to the ratio of solamargine to this water-soluble extract.In a preferred embodiment of the present invention, this water-soluble extract has a solasonine and is about 0.7: 1.0 with respect to the ratio of solamargine.
Preferably, this water-soluble extract is the form that is the water-soluble granular with nanoscale particle diameter.More preferably, this water-soluble extract is to be to have a form less than the water-soluble granular of the particle diameter of 1 μ m.
Had the effectiveness of the growth that suppresses lesion/cancer cell (the particularly cancerous cell of liver tumor, pulmonary carcinoma and breast carcinoma) in confirmation according to water-soluble extract of the present invention, and also be proved the growth that can suppress the lesion/cancer cell equally from resulting solasonine of water-soluble extract of the present invention and solamargine.Therefore, the present invention also expects to also have the application on preparation antitumor or anti-cancer composition by its resulting solasonine and solamargine according to water-soluble extract of the present invention.
Therefore, the present invention also provides a pharmaceutical compositions, and it comprises one according to water-soluble extract of the present invention, or by resulting solasonine of this water-soluble extract and solamargine.Be further purified and the solasonine that obtains and solamargine similarly can be by in directly water-soluble from this water-soluble extract.
Optionally, pharmaceutical compositions of the present invention can comprise a pharmaceutically acceptable carrier that is widely used in medicine producing technology extraly.For example, this pharmaceutically acceptable carrier can comprise one or more following reagent: water, normal saline, buffer solution, distintegrant, adhesive, excipient, lubricant, absorption delay agent etc.
Pharmaceutical compositions of the present invention can be a proper dosage form by parenteral ground or oral the dispensing.The proper dosage form comprises: aseptic aqueous solution or dispersion, aseptic powder, lozenge, capsule, cream, ointment or the like.Preferably, be manufactured into the form that is suitable for injection according to pharmaceutical compositions of the present invention, for example water type injection, powder injection (powder injection), injection freeze-drying prods forms such as (lyophilizationproduct for injection).
Optionally, pharmaceutical compositions of the present invention can be offerd medicine individually, or combination is offerd medicine with an extra antitumor/anticarcinogen (such as mitomycin, amycin, D actinomycin D, dichloro diamidogen platinum (cis-platin) etc.).
The dispensing dosage of foundation pharmaceutical compositions 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 normally 2 to the 6mg/Kg body weight of dosage of offeing medicine every day of pharmaceutical compositions of the present invention, be single dose or be divided into the form of several dosage, and can be offerd medicine by oral ground or parenteral ground.
The present invention is described in more detail with reference to the following examples, and it is the purpose that illustrates for reaching that these embodiment are provided, and is not intended to be used for limiting the scope of the invention.
The preparation of embodiment 1. water-soluble extracts
Take by weighing the yellow fluid eggplant mature fruit of 500 grams, behind the pure water that adds 1000ml, rub, and formed aqueous mixture dropwise added 99.5% acetic acid, and being adjusted to 4.0 with pH value with formed aqueous mixture, shaking out lasts 12 hours under the room temperature then.Afterwards, aqueous mixture is centrifugal and obtain a supernatant, dropwise add 33%NH 4OH alkalescence is water-soluble to be adjusted to 9.0 with the pH value with supernatant, and have precipitate and separate out this moment.In 4, carry out centrifugal under the 500rpm (Beckman Coulter, AvantiJ-25, JA-14 Rotor) and obtain a precipitate, clean and again in 4 with water, centrifugal under the 500rpm, to remove residual alkaline solution.Resulting precipitate is suspended in the distilled water, gives to get after the lyophilization (Virtis, Freezemobile 12ES) dried powder of 5g.Resulting herein dried powder only can be suspended in the aqueous solution, and can not or only can be micro-ground directly water-soluble.
Be the direct water-soluble extract of preparation, will top resulting dried powder take by weighing 2 and restrain, dissolved, add 100% methanol of 40ml then and give jolting to form an even aaerosol solution with the chloroform of 50ml SILVER REAGENT.Then, (4,500rpm) or filter the back and obtain a supernatant, added the methanol of 70ml: water (1: 1) is also mixed jolting by centrifugal.Formed mixture is 12, give under the 000rpm centrifugally lasting 10 minutes, get supernatant and added the distilled water of 120ml again and mix jolting, supernatant will become muddy slightly this moment, again in 12, carry out under the 000rpm centrifugally lasting 10 minutes to remove the precipitate of being separated out.Resulting supernatant carries out concentrating under reduced pressure to remove methanol under 55 ℃, give lyophilization then, and obtain dried powder.
Can be in resulting dried powder of this stage directly with dissolved in distilled water, and form the yellow solution of a clear.If when solution still has a little not consoluet precipitation, can make it again in 12, centrifugally under the 000rpm last 10 minutes to remove precipitate, the supernatant that obtains does not need through concentrating under reduced pressure, and directly lyophilization can obtain the water-soluble dry powder.
Generally speaking, the yellow fluid solanberrys of per 500 grams can extract in fact the water-soluble dry powder of about 800mg.When analyzing with HPLC, contained Main Ingredients and Appearance is solamargine and reaches solasonine in this water-soluble dry powder, and wherein the solamargine proportion is higher than solasonine (referring to following embodiment 2 and Fig. 1).
About above-mentioned preparation process, can select in addition directly place fruit in 3% or 5% the 1000ml acetic acid aqueous solution and shred.In addition, formed aqueous mixture can carry out jolting and last 5 hours under 50 ℃, or carries out jolting and last 2 hours under 80 ℃, to be alternative in the step that this aqueous mixture of jolting is lasted 12 hours under the room temperature.In addition, can use sodium hydrate aqueous solution to substitute above-mentioned used NH in addition with selecting 4The OH aqueous solution.
The composition of embodiment 2. water-soluble extracts is identified
In order to confirm which kind of Main Ingredients and Appearance the water-soluble extract that obtains according to the present invention contains, (HPLC) carries out analysis of components with high pressure liquid chromatography (HPLC).
Experimental technique:
In this embodiment, the high pressure liquid chromatography (HPLC) instrument (HPLC) that uses as Agilent Technologyies (Waldbronn, Germany), 1100 types; The tubing string that uses is LiChroCART 250-4 Lichropher100 RP-18e (5 μ M), length: 250mm * 4mm; Mobile phase: 60% acetonitrile/40% redistilled water (pH=2.8); Flow velocity is controlled as 1ml/min.In addition, the standard substance of used solasonine and solamargine are that Lin Zhongnan professor institute generosity by department of pharmacy of Kaohsiung Medical University provides in experiment, and these two standard substance are according to Gan, K.H., Lin, C.N. and Won, S.J. (1993), Journal of Natural Products56, the purification process that is disclosed among the 15-21 and being made.
Preparation method according to shown in the embodiment 1 makes water-soluble extract respectively with the fruit of yellow fluid eggplant and Herba Solani Nigri, and after getting appropriate amount and being dissolved in pure water, condition is carried out HPLC and analyzed below cooperating:
1. carry out HPLC with yellow fluid eggplant water-soluble extract (25 μ g), yellow fluid eggplant water-soluble extract (25 μ g)+solasonine (5 μ g) and yellow fluid eggplant water-soluble extract (25 μ g)+solamargine (5 μ g);
2. carry out HPLC with Herba Solani Nigri water-soluble extract (25 μ g), Herba Solani Nigri water-soluble extract (25 μ g)+solasonine (10 μ g) and Herba Solani Nigri water-soluble extract (25 μ g)+solamargine (10 μ g); And
3. the yellow fluid eggplant water-soluble extract of getting 50 μ g, 40 μ g, 30 μ g, 20 μ g, 10 μ g and 5 μ g carries out HPLC and analyzes.
The result:
Fig. 1 shows the HPLC result of the water-soluble extract of yellow fluid solanberry reality, and contrast Fig. 2 and Fig. 3 judge between demurrage, and the crest that occurs at first among Fig. 1 should be a solasonine as can be known, and second crest should be solamargine, and the content of solamargine will be higher than the content of solasonine.
Referring to Fig. 4 to Fig. 6, from the HPLC analysis result as seen, two crests corresponding to solasonine and solamargine can appear in the water-soluble extract of Solanum nigrum fruit equally.But, with the water-soluble extract of yellow fluid solanberry reality different be that in the water-soluble extract of Solanum nigrum fruit, the content of solasonine will be higher than the content of solamargine.
In addition, from Fig. 1 to Fig. 3 and Fig. 4 to Fig. 6 as seen, resulting water-soluble extract does not contain solasodine according to the present invention, and obviously is different from the resulting extract of EP 0 020 029 A1.
Fig. 7 to Figure 12 shows that the yellow fluid eggplant water-soluble extract with varying number (75mg, 50mg, 40mg, 30mg, 20mg, 10mg) carries out the result that HPLC analyzes, and can find that the peak value of the central contained two kinds of Main Ingredients and Appearances of yellow fluid eggplant water-soluble extract can present systematicness growth and decline along with the using dosage of water-soluble extract.
Content ratio as for solasonine and solamargine, can be according to resulting HPLC elution figure, do the automatic integration estimation by computer software (Agilent ChemStation Integrator Algorithm), waveform shown in it is if having a little sharp-pointed or during cylinder, may be influenced by the Maturity of fruit and seasonal factor.The applicant once carried out 26 times estimation with yellow fluid eggplant water-soluble extract, and obtain solasonine with respect to the ratio of solamargine between 0.3: 1.0～1.0: 1.0, and most ratio is to drop between 0.4: 1.0～0.9: 1.0.
The applicant finds that from the gained experimental result the resulting water-soluble extract of the present invention has at least 60% to 95% (being preferably more than 75%) to be made of solasonine and these two kinds of Main Ingredients and Appearances of solamargine.
Simultaneously when HPLC analyzes, the applicant also find the content of solasonine and these two kinds of main compositions of solamargine with than regular meeting along with employed dosage just is linear growth and decline relation.Figure 13 shows and to get the yellow fluid eggplant water-soluble extract of 7 various dose, respectively carries out HPLC and analyzes 3 times, and come calculating mean value ± standard deviation with the peak integration area of contained two kinds of Main Ingredients and Appearances, and the inspection amount line of being drawn out with linear regression.Result shown in Figure 13 is clear to be shown, these two kinds of Main Ingredients and Appearances of solasonine and solamargine can be linear growth and decline relation along with the dosage height.Therefore, these two kinds of Main Ingredients and Appearances of solasonine and solamargine should can be used as the quality control index components according to water-soluble extract of the present invention.
In addition, can utilize above-mentioned HPLC condition and isolate from water-soluble extract of the present invention can directly water-soluble solamargine and solasonine through what be further purified.
Embodiment 3.pH value is for the elution influence of water-soluble extract
Experimental technique:
For understanding the elution influence of pH value for water-soluble extract of the present invention, use is same as the HPLC instrument of embodiment 2, and the water-soluble extract (40 μ g) that uses the yellow fluid eggplant that makes according to the preparation method shown in the embodiment 1, and condition is carried out the HPLC analysis below cooperating:
1. the tubing string that uses is LiChroCART 250-4 Lichropher 100 RP-18e (5 μ M), length: 250mm * 4mm; And
2. mobile phase: 60% acetonitrile/40% redistilled water, and with pH value be made as respectively pH 2.3, pH 2.5, pH 2.8, pH 3.0, with pH 3.2.
The result:
Figure 14 to Figure 18 shows that the water-soluble extract of yellow fluid eggplant carries out the result that HPLC analyzes under different pH value.Can find out by these figure, the main composition of these water-soluble extracts can and prolong its holdup time along with the rising of mobile phase pH value, though and the elution figure (elution profiles) of latter two main composition can change a little, still keep the figure of two main compositions basically.
The particle diameter comparative analysis of embodiment 3. water-soluble extracts
Present embodiment is further relatively according to prepared yellow fluid eggplant water-soluble extract of the present invention and the difference of yellow fluid eggplant extract on particle diameter that makes according to the method that embodiment 2 disclosed of EP 0 020 029 A1.
Experimental technique:
The yellow fluid eggplant extract that makes according to the method that embodiment 2 disclosed of EP 0 020 029 A1 according to prepared yellow fluid eggplant water-soluble extract of the present invention and 50mg of 50mg is dissolved in separately in 50 milliliters the distilled water, and utilize a particle size analyzer (Beckman Coulter LS 230, CoulterCorporation, Miami USA) carries out granularmetric analysis.
The result
Referring to Figure 19, the yellow fluid eggplant extract that the method that the applicant is disclosed according to the embodiment 2 of EP 0 020 029 A1 makes, its granule mean size is 238.2 μ m, and distribution of particles is between 1.8 μ m to 1500 μ m, and has many precipitate can come across in the aqueous solution and can't molten with water (referring to the top of Figure 19).Relatively, according to the prepared yellow fluid eggplant water-soluble extract of the present invention, its granule mean size is 0.418 μ m, and distribution of particles is between 0.28 μ m to 0.65 μ m, and can fully be dissolved in the water (referring to the below of Figure 19).
The dissolubility of embodiment 4. water-soluble extracts relatively
Present embodiment further compares, according to the difference of prepared yellow fluid eggplant water-soluble extract of the present invention and the dissolubility of yellow fluid eggplant extract in water that make according to the method that embodiment 2 disclosed of EP 0 020 029 A1.
Experimental technique
With 5mg according to the method that is disclosed according to EP 0,020 029 A1 embodiment 2 of prepared yellow fluid eggplant water-soluble extract of the present invention and 5mg and the yellow fluid eggplant extract that makes be dissolved in separately in the distilled water of 2ml, 12, carry out centrifugally under the 000rpm, respectively take out 20 μ l then and carry out HPLC according to method described in the foregoing description 2 and analyze to obtain supernatant.
The result:
Figure 20 and Figure 21 show respectively, the yellow fluid eggplant extract (Figure 20) that makes according to the method that embodiment 2 disclosed of EP 0 020 029 A1 and according to the prepared dissolubility of yellow fluid eggplant water-soluble extract (Figure 21) in water of the present invention.Can find out by these two figure, in water, show to have obviously preferable dissolubility according to the prepared yellow fluid eggplant water-soluble extract of the present invention.
Above embodiment and graphic result are clear to be shown, according to the water-soluble extract of the nightshade that the present invention obtained, compared to the prepared extract of prior art, really in the water soluble and form the aqueous solution of a clear.This maybe might be because can be have the nanoscale particle diameter form of water-soluble granular of (particularly less than 1 μ m) according to the water-soluble extract of the nightshade that the present invention obtained.Can expect thus, be used to make the medicament that contains steroid alkaloid according to the suitable supply of the water-soluble extract of the nightshade that the present invention obtained, especially for the preparation of treatment of cancer.
Therefore, carry out pharmacological testing in following with foundation embodiment 1 prepared yellow fluid eggplant water-soluble extract according to the biological activity of the water-soluble extract of the nightshade that the present invention obtained for confirming.
In vitro active anticancer analysis of pharmacological testing 1.
For whether the water-soluble extract of measuring according to the nightshade that the present invention obtained has active anticancer, the applicant selects for use human topmost cancer cell as subjects, wherein comprise: hepatoma carcinoma cell (Hep3B), lung carcinoma cell (H441) and breast cancer cell (MCF-7), wherein hepatoma carcinoma cell (Hep3B) and pulmonary carcinoma (H441) cell are available from American type culture collection (ATCC, P.O.Box 1549, Manassas, and breast cancer cell (MCF-7) is available from Foodstuff Industrial and Development Inst. (No. 331,300 Xinzhu City food roads, Taiwan Province) VA 20108 USA).
Experimental technique:
Hepatoma carcinoma cell be incubated at Du's Bei Keshi improvement according to Ge Shi culture medium (Dulbecco ' s ModifiedEagle ' s medium, DMEM) in, and lung carcinoma cell and breast cancer cell are incubated in the RPMI-1640 culture medium, and are added with 10% hyclone and 40mg/L gentamycin in the above-mentioned culture medium.
Toxotest for cancerous cell is to use a kind of tetrazolium salts analytic process (tetrazolium salt assay, MTS) (Mosmann.T., 1983, Immmunol.Meth., 65,55-63), it is operation sequence (the Cell Titer 96TM AQ (Promega according to manufacturer, Madison, USA)) carrying out cell can active colorimetric determination (colomertric determination of cell viability).
With quantitative cell (1 * 10 4Cells/well) cultivate in the culture dish of 96-hole, and at 37 ℃, 5%CO 2At least cultivate more than 16 hours in the incubator.Then, the yellow fluid eggplant water-soluble extract of various dose is dissolved in the aseptic water for injection, is added into respectively again in the cancerous cell culture fluid in each hole.After waiting to act on 12 hours, every hole was added the MTS solution of 20 μ l and homologation reaction 3 hours.After reaction is finished, use the enzyme linked immunological immunity analysis instrument (ELISA reader 312e, Bio-TEK) and measure the light absorption value in each hole down in wavelength 490nm.All test datas are to represent with the meansigma methods ± standard deviation of 3 experiments.
The result:
Figure 22, Figure 23 and Figure 24 show respectively according to the inhibition effectiveness of yellow fluid eggplant water-soluble extract of the present invention for hepatoma carcinoma cell (Hep3B), lung carcinoma cell (H441) and breast carcinoma (MCF-7).The result shows, can suppress the growth of these three kinds of cancerous cell effectively according to yellow fluid eggplant water-soluble extract of the present invention.
Pharmacological testing 2. is analyzed according to the gene regulation mechanism of yellow fluid eggplant water-soluble extract of the present invention for lung carcinoma cell with gene chip
For understanding according to the mechanism of action of yellow fluid eggplant water-soluble extract of the present invention for the cancerous cell gene, so that a useful R﹠D direction in the treatment of cancer to be provided, and can help cancerigenic factor research and develop an effective antitumor medicine, this experiment further utilizes biochip technology to analyze according to the regulation and control situation of water-soluble extract of the present invention for the cancerous cell gene.
In this experiment, the applicant uses a kind of commercial gene array of buying, and (MD USA) analyzes according to the gene regulation situation of water-soluble extract of the present invention for lung carcinoma cell (H441) for SuperArray Inc., Bethesda.
At first, will extract RNA respectively through two hours lung carcinoma cell (experimental group) of yellow fluid eggplant water-soluble extract of the present invention (100 μ g/ml) effect and the matched group of handling without extract, and via use [ 32P]-dCTP carries out reverse transcription and makes the cDNAs that is labeled.The cDNAs through labelling that is obtained can be used as label probe, and be used to and the position this gene array (SuperArray Inc., Bethesda, MD, USA) gene on carries out hybridization.Reacted gene array carries out automatic radiography with X-light film.
The result:
Referring to shown in Figure 25, when lung carcinoma cell with yellow fluid eggplant water-soluble extract of the present invention effect after two hours, show with the gene that promotes " Tumor Necrosis Factor Receptors I (Tumor necrosis factorreceptor I) " that cancer cell death is relevant and " TNF (tumor necrosis factor) receptor associated factor-1 (TNF receptor-associated factor-1; TRAF-1) " and obviously to be increased, and prevent cell death " inhibitor of apoptosis protein matter 2 (Inhibitor of apoptosis protein 2; IAP-2) " and " (Apoptosisinhibitor 4, API-4), and " isogenic performance then is subjected to the effect of this extract and reduces apoptosis inhibitor 4.It seems that by these results yellow fluid eggplant water-soluble extract of the present invention can start the tumor necrosis factor receptor I (TNFR-I) of lung carcinoma cell and the gene performance of TNF (tumor necrosis factor) receptor associated factor-1 (TRAF-1) significantly.
Pharmacological testing 3. yellow fluid eggplant water-soluble extracts of the present invention are for the influence of the cell cycle of cancerous cell
This experiment utilize stream type cell analyzer (Flow cytometry, " FACScan ", Becton DickinsonCorp.) analyze cancerous cell before yellow fluid eggplant water-soluble extract of the present invention effect, cell cycle afterwards changes.
Experimental technique:
At first, with 1 * 10 6Cancerous cell is incubated in the 35mm culture dish and lasts 16 hours, afterwards, is added yellow fluid eggplant water-soluble extract of the present invention (30 μ g/ml), is respectively the action time of each experimental group 0,1,3,5,8 hour.After treating that each experimental group reaches its action time, cell is laid (supernatant and culture fluid must be collected in the lump) with the trypsin of one times of concentration severally again, again the cell of laying is placed the 15ml centrifuge tube, under 1000rpm, carry out centrifugal last 5 minutes after, outwell supernatant.Behind the 1 * PBS mix homogeneously that adds about 300 μ l, get 300 μ l and place in the micro-centrifuge tube, add 700 μ l dehydrated alcohol with cell fixation.Be placed in 4 ℃ of refrigerators by the cell after the alcohol fixation and last more than 30 minutes, afterwards, under 4 ℃, carry out centrifugally lasting 5 minutes with the 1200rpm rotating speed.Behind centrifugal the finishing, draw supernatant and light mix homogeneously.After 1 * PBS that this supernatant is added 445 μ l made mixing with cells evenly, (RNase 10mg/ml) decomposed so that RNA is destroyed to add the ribonuclease of 5 μ l again.The 10%Triton-X100 that then adds 50 μ l burrows cell, afterwards cell is placed 37 ℃ of incubator internal reactions to last 1 hour, gives centrifugal then.Draw supernatant and add the PBS of 495 μ l, behind the mix homogeneously, (propidium iodide 5mg/ml) carries out DNA dyeing to add iodate third ingot of 5 μ l again, and under 4 ℃ lucifuge environment, react and last 15-30 minute, filter and analyze with filter membrane again.
The mensuration of cell cycle:
Use flow cytometer (Beckman-Coulter FACScan), the above-mentioned cell that is attached on the filter membrane evenly is suspended in 1 * PBS solution, and use one to be that the speed of 100 cell/seconds is directed into cell in the flow cytometer, analyze 10,000 cells at every turn.Allow cell be passed the aperture that radius is 75 μ m, and produce and volume current impulse signal in proportion.Cell excites with the thunder laser beam 488nm of argon ion and sends fluorescent.The gained data cooperates the Winmdi software to come analysis of cells interior dna content number and observation of cell cycle.
The result:
Table 1 shows that the cell cycle that hepatoma carcinoma cell (Hep3B), lung carcinoma cell (H441) and breast carcinoma (MCF-7) are taken place after through yellow fluid eggplant water-soluble extract of the present invention effect changes, and wherein on behalf of cancerous cell, the feature that increases of Sub-G1 ripple be subjected to this water-soluble extract effect and carried out apoptosis.
As seen from Table 1, the cell cycle S ub-G1 ripple of various cancerous cell has remarkable increase in 1 hour, and cancerous cell is broken extremely when using the extract of high dose because of after birth, and is stronger along with extract increase action time simultaneously.These results show that yellow fluid eggplant water-soluble extract provided by the present invention can start the apoptosis mechanism of action of these three kinds of cancerous cell really.
Table 1. yellow fluid eggplant of the present invention water-soluble extract is for the influence of the cell cycle of hepatoma carcinoma cell (Hep3B), lung carcinoma cell (H441) and breast carcinoma (MCF-7)
Sub-G1 G0/G1 S G2/M
Time (hr) Meansigma methods ± S.D ％ Meansigma methods ± S.D ％ Meansigma methods ± S.D ％ Meansigma methods ± S.D ％
Hepatoma carcinoma cell
0 1 3 5 8 5.5±0.2 38.3±0.6 69.5±0.4 90.7±0.7 99.1±0.1 100 246 446 581 635 58.8±0.3 31.9±0.9 15.4±0.3 4.3±0.3 0.6±0.1 100 64 31 9 1 17.4±0.3 15.1±0.1 9.9±0.2 3.6±0.7 0.3±0.0 100 87 57 21 2 18.0±0.5 14.6±0.4 5.6±0.2 1.5±0.2 0.1±0.0 100 81 31 8 1
Lung carcinoma cell
0 1 3 5 8 9.4±0.2 46.5±1.7 61.8±0.7 96.6±0.2 97.2±0.6 100 441 660 1030 1037 43.3±0.3 23.6±0.4 13.1±1.4 2.3±0.1 1.9±0.4 100 54 30 5 4 7.1±0.1 11.8±0.1 9.8±0.7 0.8±0.0 0.4±0.4 100 168 139 11 6 40.8±0.1 18.5±1.2 15.0±0.7 0.4±0.0 0.4±0.5 100 45 36 1 1
Breast cancer cell
0 1 3 5 8 2.9±0.6 12.0±0.8 77.1±0.4 92.0±0.3 97.1±0.4 100 441 2633 3141 3313 66.8±1.2 71.2±0.4 18.7±1.0 6.8±0.3 2.5±0.3 100 107 27 10 4 13.1±0.6 6.4±0.6 2.4±0.3 0.8±0.0 0.2±0.0 100 49 18 6 2 1 8.2±0.4 10.7±1.0 1.3±0.2 0.4±0.0 0.2±0.1 100 59 7 2 1
Pharmacological testing 4. yellow fluid eggplant water-soluble extracts of the present invention are for the influence of the cellular morphology of cancerous cell
For the research cancerous cell changes in the cellular morphology that is subjected to being produced after the yellow fluid eggplant water-soluble extract of the present invention effect, this experiment is further observed by the metamorphosis of the cancerous cell of brazilwood extract dyeing with optical microscope.
Experimental technique:
At first, to act on hepatocarcinoma, pulmonary carcinoma and the breast cancer cell that lasts 1,3,5,8 hour through yellow fluid eggplant water-soluble extract of the present invention (30 μ g/ml), take out suitable cell number, use cell section centrifuge Cytospin cell centrifugation to be lasted 10 minutes to make cell section with the rotating speed of 800rpm.Then, last 30 minutes so that cell fixation with 4% paraformaldehyde effect.Cell section carries out nucleus dyeing with hematoxylin, is stripped the dyestuff of surplus again by 70%, 80%, 90%, 95% to 100% dehydrated alcohol with the ethanol of dilution.Then cell section is placed in the dimethylbenzene carrying out processed, and with coverslip and mounting glue with the slide capping, and get off to observe the also variation of film recording cellular morphology in 400 times of enlargement ratios with optical microscope (Olympus CX-40).
The result:
Figure 26 shows, with before yellow fluid eggplant water-soluble extract of the present invention (30g/ml) effect and act on last 1,3,5,8 hour after, the morphological change of three-type-person's quasi-cancer cell, wherein A is capable be hepatoma carcinoma cell (Hep3B), B capable be lung carcinoma cell (H441) and C capable be breast cancer cell (MCF-7).The cell of matched group for dealing with without extract, and on behalf of typical morphocytology, the arrow indication change.
From Figure 26 as seen, three kinds of cancerous cell through yellow fluid eggplant water-soluble extract of the present invention effect, can be observed promptly at 1 hour that nucleus dwindles, the generation of chromatin agglutination and apoptosis corpusculum, simultaneously with the growth of works with the time, the metamorphosis of these cancerous cell more is obviously, last membranolysis.
Because the generation of Sub-G1 ripple, chromatin agglutination and apoptosis corpusculum all is apoptotic typical characteristics, the result of this pharmacological testing and top pharmacological testing 3 is clear to show that can start cancerous cell according to water-soluble extract of the present invention carries out apoptotic mechanism, and makes cancer cell death.
The in vivo animal model test of pharmacological testing 5. yellow fluid eggplant water-soluble extracts of the present invention
For can confirm in vivo producing anticancer effectiveness according to water-soluble extract of the present invention, present embodiment further carries out in vivo animal model test with nude mice.
Experimental technique:
At first, in hypodermic mode with Human Lung Cancer cell H441 (2 * 10 7Individual cell) is implanted in the back of nude mice (BABL/c-nu-nu, 8 weeks are big, the about 20-25g of body weight), after treating tumor formation and beginning growth (needing 10 days approximately), will be formed with the nude mice random packet (each group has 6-7 nude mice approximately) of tumor.
Nude mice for the injection group, the water-soluble extract of the entry needle that uses 0.8mm 220 μ g in water-soluble with lumbar injection once a day, continuously injection 3 days drug withdrawal 4 days again, every two days record nude mice body weight, and measure the tumor size variation at nude mice back with mircrometer gauge (micrometer), proceed the next course of treatment again, and continue to observe more than two months.The account form of tumor size: tumor size=length x width x (highly/2).
For oral group, adopt feeder that every nude mice is given water-soluble extract (600 μ g) in water-soluble with oral throwing, every day feeding once, feeding drug withdrawal 2 days again after 5 days continuously.Adopting the mode that is same as the injection group writes down the nude mice body weight and measures the tumor size.Lasting feeding is also observed more than two months.The account form of tumor size is identical with the injection group.
The result:
Figure 27, Figure 28 and Figure 29 show the nude mice that transplant at the back the Human Lung Cancer cell respectively, extract at the foundation yellow fluid eggplant water solublity of the present invention of will not offeing medicine, offer medicine with feeding and offer medicine according to the viewed result of yellow fluid eggplant water-soluble extract of the present invention according to yellow fluid eggplant water-soluble extract of the present invention and with lumbar injection, wherein on behalf of matched group (6 nude mices), Figure 28, Figure 27 represent oral experimental group (7 nude mices) and Figure 29 to represent the tumor variation situation of injection experimental group (7 nude mices).
Can find out that from Figure 27, Figure 28 and result shown in Figure 29 it is quick according to the nude mice of control group in-vivo tumour growth rate of yellow fluid eggplant water-soluble extract of the present invention to be offerd medicine, about 6 times of can grow into original size in 1 month; By oral throwing give according to the intravital tumor of nude mice of yellow fluid eggplant water-soluble extract of the present invention then major part dwindle or disappear, though there is minority to grow up a little, growth rate is far below matched group; By intraperitoneal injection then to dwindle fully according to the intravital tumor of nude mice of yellow fluid eggplant water-soluble extract of the present invention or to disappear.These results show, offeing medicine all can make effectively by commentaries on classics according to water-soluble extract of the present invention with oral or intraperitoneal injection mode is implanted in that the intravital tumor growth of nude mice slows down, size is dwindled even eliminates, and the effect that intraperitoneal injection is offerd medicine is better than oral result.
Pharmacological testing 6. from yellow fluid eggplant water-soluble extract of the present invention be further purified and solasonine and the in vitro anticancer test of solamargine
This experiment is research further, utilizes HPLC and the purified solasonine that obtains from yellow fluid eggplant water-soluble extract of the present invention and the biological activity of solamargine.
Experimental technique:
With reference to HPLC method described in the foregoing description 2, can from yellow fluid eggplant water-soluble extract according to the present invention, further separation and purification go out solasonine and these two chemical compounds of solamargine, and they similarly can be by directly soluble in water.
With reference to pharmacological testing 1, carry out the cytotoxicity test according to the tetrazolium salts analytic process.Handling human lung carcinoma cell (H441) respectively from the resulting purified solasonine of yellow fluid eggplant water-soluble extract of the present invention and solamargine and last 12 hours with various dose, then according to operation sequence (the CellTiter 96 TM AQ (Promega of manufacturer, Madison, USA)) carrying out cell can active colorimetric determination.Experimental data is to represent with the meansigma methods ± standard deviation of 3 experiments.
The result:
Figure 30 shows from the purified solasonine that goes out of yellow fluid eggplant water-soluble extract of the present invention and the solamargine growth inhibited effectiveness for the Human Lung Cancer cell.From result shown in Figure 30 as seen, has significant anti-cancer activity respectively from purified solasonine and the solamargine that goes out of yellow fluid eggplant water-soluble extract of the present invention.
Comprehensive above experimental result, the applicant successfully obtains the water-soluble extract of nightshade, set up simultaneously this water-soluble extract in extraction process scientific analysis condition and find out Main Ingredients and Appearance, and can accomplish the quality management and control effectively.In addition, the activity that is proved growth and starts the nature cell apoptosis mechanism of cancerous cell according to water-soluble extract of the present invention with anticancer, and fully show according to the development of water-soluble extract of the present invention in the detection of the growth of anticancer and cancerous cell.
All patents and the document quoted from this description are merged in this case as the reference data with its integral body.If to some extent when conflicting, this case describes (comprise be defined in) in detail and will get the upper hand.

Claims (55)

1. the water-soluble extract of a nightshade is characterized in that:
Described water-soluble extract is the form that is the water-soluble granular with a nanoscale particle diameter basically, and is that solasonine and solamargine by 60%-90% constituted basically.
2. water-soluble extract as claimed in claim 1 is characterized in that:
Described water-soluble extract is to be extracted and got by a nightshade that is selected from the following group that constitutes: yellow fluid eggplant, India eggplant, Herba Solani Nigri, Jerusalem cherry, Fructus solani xanthocarpi, melon eggplant, yellow Solanum aculeatissimum Jacq., Rhizoma Solani tuber osi, Satan eggplant, gyro eggplant, Solanum aculeatissimum Jacq., Lycopodium clavatum eggplant, the inferior eggplant in Keshen, Radix Solani Torvi, three-Flowered nightshade, lettuce eggplant, Western eggplant, hoof shape eggplant, tough eggplant, Serpentis shape eggplant, lobule eggplant, America eggplant and their combination.
3. water-soluble extract as claimed in claim 2 is characterized in that:
Described water-soluble extract is to be got by the extraction of yellow fluid eggplant.
4. water-soluble extract as claimed in claim 2 is characterized in that:
Described water-soluble extract is to be got by the Herba Solani Nigri extraction.
5. water-soluble extract as claimed in claim 1 is characterized in that:
Described water-soluble extract is to adopt the method that comprises the following step to make:
(a) using pH value is that 3～5 acidic aqueous solution extracts processing to the vegetable material that belongs to nightshade, obtains an aqueous solution;
(b) pH value of aqueous solution that will be obtained from step (a) with an alkali is adjusted to pH8～10, forms a precipitate;
(c) clean the precipitate that is obtained from step (b) with water, give drying then, obtain a desciccate;
(d) desciccate that will derive from step (c) mixes with chloroform, adds one an amount of 100% alcohol then, obtains one chloroform-alcohol mixture;
(e) chloroform-alcohol mixture and that will be obtained from step (d) has a predetermined water: the water/alcoholic solution of pure ratio mixes, forms one based on chloroform layer and one non-based on chloroform layer;
(f) after removing the formed layer of step (e), add an an amount of water then based on chloroform; And
(g) obtain a supernatant from the formed mixture of step (f), give drying then, by this, formed desciccate can be directly soluble in water and be formed the aqueous solution of a yellow clear.
6. water-soluble extract as claimed in claim 5 is characterized in that:
The vegetable material that is used for described method step (a) is at least one in the middle of the fruit of this nightshade, root, stem, the leaf.
7. water-soluble extract as claimed in claim 6 is characterized in that:
The vegetable material that is used for described method step (a) is the fruit of this nightshade.
8. water-soluble extract as claimed in claim 6 is characterized in that:
The vegetable material that is used for this method step (a) is this nightshade of whole strain.
9. water-soluble extract as claimed in claim 5 is characterized in that:
The vegetable material that is used for the nightshade of this method step (a) is handled through chopping earlier.
10. water-soluble extract as claimed in claim 5 is characterized in that:
This aqueous solution that is obtained from this method step (a) is to obtain by carrying out centrifugal after handling in this extraction.
11. water-soluble extract as claimed in claim 5 is characterized in that:
Used acidic aqueous solution is one to contain the aqueous solution of formic acid, acetic acid or hydrochloric acid in the extraction of this method step (a) is handled.
12. water-soluble extract as claimed in claim 5 is characterized in that:
Used alkali is one to contain the alkaline aqueous solution that is selected from the chemical compound in the following group in the step (b) of this method: alkali-metal hydroxide and ammonium hydroxide.
13. water-soluble extract as claimed in claim 12 is characterized in that:
Used alkali is one to contain the alkaline aqueous solution of ammonium hydroxide in the step (b) of this method.
14. water-soluble extract as claimed in claim 12 is characterized in that:
Used alkali is one to contain the alkaline aqueous solution of sodium hydroxide in the step (b) of this method.
15. water-soluble extract as claimed in claim 5 is characterized in that:
In the step (b) of this method, this precipitate is to obtain by carrying out centrifugal after the pH value adjustment.
16. water-soluble extract as claimed in claim 5 is characterized in that:
The dried of being carried out in the step (c) of this method is to be selected from one by the following group that constitutes: lyophilization processing, spray drying treatment, evaporation process, heat drying are handled and combination.
17. water-soluble extract as claimed in claim 5 is characterized in that:
In the step (c) of this method, the precipitate that is obtained from step (b) is added into water after cleaning with water, connects and is frozen dry to obtain this desciccate.
18. water-soluble extract as claimed in claim 5 is characterized in that:
The alcohol that is used for this method step (d) and step (e) is to be selected from one by the following group that constitutes: methanol, ethanol, propanol and combination thereof.
19. water-soluble extract as claimed in claim 5 is characterized in that:
In the step (f) of this method, the layer based on chloroform that removes in the formed mixture of step (e) is to be undertaken by centrifugal.
20. water-soluble extract as claimed in claim 5 is characterized in that:
The dried of being carried out in the step (g) of this method is to be selected from one by the following group that constitutes: lyophilization processing, spray drying treatment, evaporation process, heat drying are handled and combination.
21. water-soluble extract as claimed in claim 1 is characterized in that:
This water-soluble extract is to be to have a form less than the water-soluble granular of the particle diameter of 1 μ m.
22. water-soluble extract as claimed in claim 1 is characterized in that:
This water-soluble extract is made of solasonine more than 75% and solamargine.
23. water-soluble extract as claimed in claim 1 is characterized in that:
This water-soluble extract have a solasonine with respect to the ratio of solamargine between 0.3: 1.0～1.0: 0.6.
24. water-soluble extract as claimed in claim 1 is characterized in that:
This water-soluble extract have a solasonine with respect to the ratio of solamargine between 0.4: 1.0～0.9: 1.0.
25. water-soluble extract as claimed in claim 1 is characterized in that:
This water-soluble extract has a solasonine and is about 0.7: 1.0 with respect to the ratio of solamargine.
26. water-soluble extract as claimed in claim 1 is characterized in that:
The water solubility of this water-soluble extract be 2～20mg/ml or more than.
27. the purposes of each described water-soluble extract in the medicine of a kind of growth that can suppress the lesion/cancer cell of preparation in the claim 1 to 26.
28. a pharmaceutical compositions that is used to suppress the growth of lesion/cancer cell is characterized in that:
This pharmaceutical compositions includes just like each described water-soluble extract in the claim 1 to 26, and a pharmaceutically acceptable carrier optionally.
29. a method that is used to prepare the water-soluble extract of a kind of nightshade is characterized in that this method comprises the following step:
(a) using pH value is that 3～5 acidic aqueous solution extracts processing to a kind of vegetable material that belongs to nightshade, obtains an aqueous solution;
(b) pH value of aqueous solution that will be obtained from step (a) with an alkali is adjusted to pH8～10, forms precipitate;
(c) clean the precipitate that is obtained from step (b) with water, give drying then, obtain a desciccate;
(d) desciccate that will derive from step (c) mixes with chloroform, adds one an amount of 100% alcohol then, obtains one chloroform-alcohol mixture;
(e) chloroform-alcohol mixture that will be obtained from step (d) water predetermined with a tool one: the water/alcoholic solution of pure ratio mixes, form one have based on chloroform layer and one non-based on chloroform layer;
(f) after removing the formed layer of step (e), add an an amount of water then based on chloroform; And
(g) obtain a supernatant from the formed mixture of step (f), give drying then, by this, formed desciccate can be by directly soluble in water and form the aqueous solution of a yellow clear.
30. method as claimed in claim 29 is characterized in that:
The vegetable material that is used for this nightshade of described step (a) is handled through chopping earlier.
31. method as claimed in claim 29 is characterized in that:
The vegetable material that is used for described step (a) is at least one in the middle of the fruit of this nightshade, root, stem, the leaf.
32. method as claimed in claim 29 is characterized in that:
The vegetable material that is used for described step (a) is the fruit of this nightshade.
33. method as claimed in claim 29 is characterized in that:
The vegetable material that is used for described step (a) is this nightshade of whole strain.
34. method as claimed in claim 29 is characterized in that:
The vegetable material that is used for described step (a) is to belong to one to be selected from by the nightshade in the following group that constitutes: yellow fluid eggplant, India eggplant, Herba Solani Nigri, Jerusalem cherry, Fructus solani xanthocarpi, melon eggplant, yellow Solanum aculeatissimum Jacq., Rhizoma Solani tuber osi, Satan eggplant, gyro eggplant, Solanum aculeatissimum Jacq., Lycopodium clavatum eggplant, the inferior eggplant in Keshen, Radix Solani Torvi, three-Flowered nightshade, lettuce eggplant, Western eggplant, hoof shape eggplant, tough eggplant, Serpentis shape eggplant, lobule eggplant, America eggplant and combination thereof.
35. method as claimed in claim 34 is characterized in that:
The vegetable material that is used for this nightshade of described step (a) is the yellow fluid eggplant.
36. method as claimed in claim 34 is characterized in that:
The vegetable material that is used for this nightshade of described step (a) is a Herba Solani Nigri.
37. method as claimed in claim 29 is characterized in that:
This aqueous solution that is obtained from described step (a) is centrifugal and obtained by carrying out after handling in this extraction.
38. method as claimed in claim 29 is characterized in that:
Used acidic aqueous solution is one to contain the aqueous solution of formic acid, acetic acid or hydrochloric acid in the extraction of step (a) is handled.
39. method as claimed in claim 29 is characterized in that:
Used alkali is one to contain the alkaline aqueous solution that is selected from the chemical compound in the following group in described step (b): alkali-metal hydroxide and ammonium hydroxide.
40. method as claimed in claim 39 is characterized in that:
Used alkali is one to contain the alkaline aqueous solution of ammonium hydroxide in described step (b).
41. method as claimed in claim 39 is characterized in that:
Used alkali is one to contain the alkaline aqueous solution of sodium hydroxide in described step (b).
42. method as claimed in claim 29 is characterized in that:
This precipitate that is obtained from step (b) is centrifugal and obtained by carrying out after the pH value adjustment.
43. method as claimed in claim 29 is characterized in that:
The dried of being carried out in described step (c) is to be selected from one by the following group that constitutes: lyophilization processing, spray drying treatment, evaporation process, heat drying are handled and combination.
44. method as claimed in claim 29 is characterized in that:
In described step (c), the precipitate that is obtained from step (b) is added in the water after cleaning with water, connects and is frozen drying and obtains this desciccate.
45. method as claimed in claim 29 is characterized in that:
The alcohol that is used for step (d) and step (e) is to be selected from the following group that constitutes: methanol, ethanol, propanol and combination thereof.
46. method as claimed in claim 29 is characterized in that:
In described step (f), the layer based on chloroform that removes in the formed mixture of step (e) is to be undertaken by centrifugal.
47. method as claimed in claim 29 is characterized in that:
The dried of being carried out in described step (g) is to be selected from one by the following group that constitutes: lyophilization processing, spray drying treatment, evaporation process, heat drying are handled and combination.
48. method as claimed in claim 29 is characterized in that:
By the resulting water-soluble extract of described method is the form that is the water-soluble granular with a nanoscale particle diameter.
49. method as claimed in claim 48 is characterized in that:
By the resulting water-soluble extract of described method is to be to have a form less than the water-soluble granular of the particle diameter of 1 μ m.
50. method as claimed in claim 29 is characterized in that:
By the resulting water-soluble extract of described method is by solasonine and the solamargine of 60%-90% are constituted at least basically.
51. method as claimed in claim 50 is characterized in that:
Constituted by solasonine more than 75% and solamargine by the resulting water-soluble extract of described method.
52. method as claimed in claim 29 is characterized in that:
By the resulting water-soluble extract of described method have a solasonine with respect to the ratio of solamargine between 0.3: 1.0～1.0: 0.6.
53. method as claimed in claim 29 is characterized in that:
By the resulting water-soluble extract of described method have a solasonine with respect to the ratio of solamargine between 0.4: 1.0～0.9: 1.0.
54. method as claimed in claim 29 is characterized in that:
Have a solasonine by the resulting water-soluble extract of described method and be about 0.7: 1.0 with respect to the ratio of solamargine.
55. method as claimed in claim 29 is characterized in that:
By the water solubility of the resulting water-soluble extract of described method be 2～20mg/ml or more than.
CNB031494404A 2003-06-20 2003-06-20 Water soluble nightshade extract, its preparation method and pharmaceutical compositions Expired - Lifetime CN1329038C (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CNB031494404A CN1329038C (en) 2003-06-20 2003-06-20 Water soluble nightshade extract, its preparation method and pharmaceutical compositions
HK05102739A HK1069995A1 (en) 2003-06-20 2005-04-01 Water soluble extract from plant of solanum genus and the preparation process thereof, and pharmaceutical composition containing the water soluble extract

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNB031494404A CN1329038C (en) 2003-06-20 2003-06-20 Water soluble nightshade extract, its preparation method and pharmaceutical compositions

Publications (2)

Publication Number Publication Date
CN1565473A CN1565473A (en) 2005-01-19
CN1329038C true CN1329038C (en) 2007-08-01

Family

ID=34472540

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB031494404A Expired - Lifetime CN1329038C (en) 2003-06-20 2003-06-20 Water soluble nightshade extract, its preparation method and pharmaceutical compositions

Country Status (2)

Country Link
CN (1) CN1329038C (en)
HK (1) HK1069995A1 (en)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101108872B (en) * 2006-07-21 2012-07-18 江苏中康药物科技有限公司 Plants natural base extract and formulated product and use thereof
CN101032246B (en) * 2007-02-06 2010-05-19 东北师范大学 Application of glycoalkaloids as pesticides for controlling plant diseases
TWI414304B (en) * 2011-05-04 2013-11-11 G & E Herbal Biotechnology Co Ltd Treatment of wart with a water soluble extract from plant of solanum genus
TWI476012B (en) 2011-05-12 2015-03-11 G & E Herbal Biotechnology Co Ltd Treatment and/or prevention of inflammation and cutaneous photodamage and photoprotection of the skin with a water soluble extract from plant of solanum genus
CN102872260A (en) * 2011-06-10 2013-01-16 德英生物科技股份有限公司 Method for treating and/or preventing inflammation and cutaneous photodamage by using water soluble extract from plant of Solanum genus and having photoprotective effect
CN102846837B (en) * 2011-06-27 2016-05-11 德英生物科技股份有限公司 The water-soluble extract of a kind of yellow water eggplant is for the purposes of the pharmaceuticals for the preparation of the pointed condyloma for the treatment of
CN103181994A (en) * 2013-03-03 2013-07-03 济南康众医药科技开发有限公司 Solanum pseudo-capsicumvar diflorum (Vell.) Bitter fruit for treating cancers
CN103181995A (en) * 2013-03-03 2013-07-03 济南康众医药科技开发有限公司 Processing method of twoflower jerusalemcherry
CN103181990A (en) * 2013-03-03 2013-07-03 济南康众医药科技开发有限公司 Fresh solanum pseudo-capsicumvar diflorum (Vell.) Bitter for treating cancers
CN103181996A (en) * 2013-03-03 2013-07-03 济南康众医药科技开发有限公司 Solanum pseudo-capsicumvar diflorum (Vell.) Bitter for treating cancers

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958770A (en) * 1990-01-18 1999-09-28 Cham; Bill Elliot Glycoalkaloids
CN1345728A (en) * 2000-09-29 2002-04-24 刘良 Solasodine hydrochlorate and productive method and application in medicine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958770A (en) * 1990-01-18 1999-09-28 Cham; Bill Elliot Glycoalkaloids
CN1345728A (en) * 2000-09-29 2002-04-24 刘良 Solasodine hydrochlorate and productive method and application in medicine

Also Published As

Publication number Publication date
HK1069995A1 (en) 2005-06-10
CN1565473A (en) 2005-01-19

Similar Documents

Publication Publication Date Title
Bagnis et al. Herbs and the kidney
CA2455425C (en) Water soluble extract from plant of solanum genus and the preparation process thereof, and pharmaceutical composition containing the water soluble extract
Ansori et al. Medicinal properties of Muntingia calabura L.: A Review
US8168238B2 (en) Extracts of Aquilaria hulls and use thereof in the treatment of cancer
CN104013668B (en) Licoflavone class extract is used to prepare to be applied in treatment ulcerative colitis medicine
Wani et al. Phytochemical screening and aphrodisiac property of Tinospora cordifolia
Pitchaipillai et al. In vitro antidiabetic activity of ethanolic leaf extract of bruguiera Cylindrica L.–glucose uptake by yeast cells method
CN1329038C (en) Water soluble nightshade extract, its preparation method and pharmaceutical compositions
CN105050608A (en) Algal extract comprising sulphated and non-sulphated polyanionic polysaccharides and uses thereof
Jodh et al. Pharmacological review on Madhuca longifolia
CN102743401A (en) Application of panaxadiol saponins fraction in preparing medicine for preventing epilepsia
Mukinda Acute and chronic toxicity of the flavonoid-containing plant, Artemisia afra in rodents
EP3441076A1 (en) Pharmaceutical product for treating tumor and combined immune defect, and preparation and application thereof
KR20200081553A (en) Composition for the prevention and improvement of Antitussive and Expectorant
TWI389701B (en) Extracts of aquilaria hulls and use thereof in the treatment of cancer
EP1508334B1 (en) Water soluble extract from plant of solanum genus and the preparation process thereof, and pharmaceutical composition containing the water soluble extract
CN111329871A (en) Preparation method and application of product of cordyceps militaris for preventing and treating liver cancer
CN102641342A (en) Traditional Chinese medicine extract for treating nephropathy and preparation method
CN114031656A (en) Medicine for treating urinary calculus related diseases and preparation method thereof
CN100427099C (en) Juglans mandshurica maxim pdysaccharide preparation capable of effectively treating brain cancer and its preapring method
CN107252444A (en) It is a kind of to be used to treat and/or prevent herbal composite of prostatic disorders and its preparation method and application
AJAGBA et al. EFFECTS OF ETHANOL EXTRACT OF DESMODIUM VELUTINUM STEM ON LIVER MARKER ENZYME IN ALBINO WISTAR RATS FED WITH COW’S BRAINS
Magfirah et al. Acute Toxicity Test of Ekor Naga (Rhaphidophora pinnata (Lf) Schott) Leaf Extract in Mice and Kidney Histological Examination
JP2003252780A (en) Apoptosis inducer
CN111393497A (en) Preparation method and application of natural product for preventing and treating lung cancer

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1069995

Country of ref document: HK

C14 Grant of patent or utility model
GR01 Patent grant
CX01 Expiry of patent term
CX01 Expiry of patent term

Granted publication date: 20070801