CN1329038C - Water-soluble extract of nightshade plant, its preparation method and its pharmaceutical composition - Google Patents

Abstract

The invention provides a water-soluble extract from solanum plants, which is basically composed of at least 60% -90% of solasonine and solamargine. The present invention also provides a method for preparing a water-soluble extract of Solanum genus plant, wherein the method comprises acid hydrolysis, alkali precipitation and separation steps using chloroform, alcohol and water, etc., and the water-soluble extract prepared by the method can be directly dissolved in pure water or water of neutral pH value without adding any other solvent and/or cosolvent to form a yellow clear and transparent aqueous solution, and the solubility can reach 2-20 mg/ml or more. The present invention also provides a pharmaceutical composition comprising the above water-soluble extract of Solanum genus plant as an active ingredient for inhibiting the growth of tumor/cancer cells (especially liver cancer cells, lung cancer cells and breast cancer cells).

Description

Water-soluble extract of nightshade plant, its preparation method and its pharmaceutical composition

technical field

The invention relates to a water-soluble extract of a plant of the genus Solanum (plant of Solanum genus) and a preparation method thereof, and a pharmaceutical composition containing the water-soluble extract.

Background technique

Cancer is currently one of the leading causes of death for humans all over the world, while lung cancer, liver cancer, and breast cancer are the most important cancers in humans. Although the mechanism of cancer formation is still not fully understood, it is believed that the occurrence of cancer can be attributed to uncontrolled cell division (Chen, P.L. et al., (1990), Science, 250, 1576-1580; Finlay, C.A. et al., (1989 ), Cell, 57, 1083-1093; and Baker, S.J. et al., (1990) Science, 249, 912-915).

Normally, cell growth and differentiation in humans or animals are strictly controlled by growth hormones in the body. When a cell accumulates gene mutations caused by endogenous or exogenous factors, the cell will produce wrong message transmission, which will cause the cell to divide and grow out of control, and gradually form cancer cells (Kerr, J.F.R.(1971) J. Pathol., 105, 13-20).

Nowadays, researchers from all over the world are devoted to cancer research, but the results achieved in cancer treatment are still not satisfactory. In addition to the patient's own personal factors, the serious side effects of anticancer drugs and the drug resistance of cancer cells and other issues It is the biggest problem encountered in clinical treatment. In view of the fact that the current clinically used western medicines are still unable to effectively improve the treatment of cancer, some researchers try to find useful medicines from traditional Chinese medicines (TCM) or herbal plants (herbs) by studying the mechanism of cancer occurrence. Active ingredient to treat or eliminate the symptoms of cancer.

Apoptosis is considered to be a mechanism of natural regulation of cell growth of animal cells Martin, S.J.and Green, D.R. (1995), Crit.Rev.Oncol.Hemat., 18, 137-153], it is in many It plays an important role in the regulation of natural cell death (for example, the natural shrinkage and resorption of tissues during animal growth). In addition, when human cells are damaged beyond repair, natural programmed cell death is also activated to avoid the generation of cancer cells.

The main features of programmed cell death are: the formation of apoptotic bodies (apoptotic bodies), chromatin condensation and fragmentation of DNA [Arends, M.J. and Wyllie, A.H. (1991) Int. Rev. Exp. Pathol., 32, 223-254; Dive, C. et al., (1992) Biochim. Biophys. Acta 1133, 275-285; and Darzynkiewicz, Z. et al., (1992) Cytometry, 13, 795-808]. In the process of programmed cell death, the fragments of dead cells will be quickly absorbed by surrounding cells and phagocytic cells through phagocytosis, without inflammatory response [Sarraf, F.E. and Bowen, I.D. (1988) Cell Tissue Res.21, 45-49]. In addition, when a flow cytometer is used to measure changes in the cell cycle, Sub-G1 waves will appear [Alzerreca, A. and Hart, G. (1982) Toxicology Lett.12, 151-155; and Lin, C.N., etc., (1986) J.Taiwan Pharm.Assoc.38, 166], so the Sub-G1 wave is considered to be a typical marker of cell apoptosis.

It has been reported that when a cell undergoes changes such that its apoptosis mechanism goes out of control, the cell forms a cancer cell [Carson, D.A. and Ribeiro, J.M. (1993) Lancet 341, 1251-1254; and Kaufmann, S.H. (1989) Cancer Res .49, 5870-5878]. Consequently, apoptosis has become a target of oncology research in recent years. In addition, there are also reports in the literature that some anticancer drugs can cause apoptosis of cancer cells [Wyllie, A.H. et al., (1980) Int. Rev. Cytol.68, 251-306; Wyllie, A.H. et al., (1984) JPathol. 142, 67-77; Barry, M.A. et al., (1990) Biochem.Pharmacol., 40, 2353-2362; and Hickman, J.A. (1992) Cancer Metast. Rev., 11, 121-139], therefore, programmed cell death It has also become one of the important directions of global research on anticancer drugs.

Traditional Chinese medicine (TCM) or herbal medicines (herbal medicines) have been used in the treatment of diseases for a long time, and many researchers are currently trying to find useful anticancer drugs from traditional Chinese medicines and herbal medicines. However, the use of traditional Chinese medicine and herbal medicine is still mostly based on traditional experience, and there may not be sufficient scientific evidence to support it. In addition, the extraction methods, dose control and quality control of traditional Chinese medicines or herbal medicines have not yet been scientificized, making it difficult to achieve consistent curative effects after administration. Furthermore, most of the active ingredients of traditional Chinese medicines or herbal medicines are water-insoluble, and when insoluble substances are orally administered or injected into animals, they may have difficulties in absorption and fail to show curative effect. These are the main reasons that limit the application and development of traditional Chinese medicine and herbal medicine.

At present, there are many plants that can be used as medicine in the world, and it is known that many kinds of protein inhibitors extracted from plant materials can be used for anticancer treatment. Among these protein inhibitors with anticancer potential, steroidal alkaloids obtained from Solanaceae plants have been found to have potential as anticancer drugs.

It is known that eggplant [Latin name: Solanum incanum L., synonym: Solanum incanum Ruiz. & Pav., Solanum coagulans Forsskal; English name: bitter apple] contains steroidal glycoalkaloid (steroidal glycoalkaloid) [Kuo, K.w. et al., (2000), Biochemical Pharmacology, 60(12):1865-73]. In addition, it has been reported that many plants belonging to the family Solanaceae also contain steroidal glycoalkaloids, including, for example, Indian nightshade (Solanum indicum), nightshade (Latin name: Solanum nigrum; Chinese pinyin: Long kui; English name: black nightshade) 〔Hu, K. etc., (1999), Planta Medica, 65 (1): 35-8〕, winter coral (Latin scientific name: Solanum capsicastrum; English name: false jerusalemcherry), yellow fruit eggplant ( Solanum xanthocarpum), melon eggplant (Solanum melongena) [Blankemeyer, J.T. et al., (1998), Food & Chemical Toxicology, 36(5): 383-9), yellow thorn eggplant (Solanum coagulans), potato (Solanum tuberosum) [Friedman, M. et al., (1996), Journal of Nutrition, 126 (4): 989-99], Solanum sodomeum (also known as apple of Sodom in Australia), Solanum turburosum, Solanum thorns ( Solanum aculeastrum) [Wanyonyi, A.W. et al., (2002), Phytochemistry, 59(1): 79-84], Lycocarpum (Solanum lycocarpum) [Peters, V.M. et al., (2001), Contraception, 63(1): 53- 5〕, Solanum khasianum (Solanum khasianum) 〔Putalun, W. et al., (2000), Biological & Pharmaceutical Bulletin, 23(1):72-5〕, Solanum suaveolens 〔Ripperger, H. et al., (1997 ), Phytochemistry, 46(7): 1279-82], Solanum uporo (Solanum uporo) [Ripperger, H. et al., (1997), Phytochemistry, 44, (4): 731-4)], Lettuce (Solanum uporo) abutiloides) [Tian, R.H., et al (1997), Phytochemistry, 44 (4): 723-6], tomato (Solanum coccineum) [Lorey, S., etc., (1996), Phytochemistry, 41 (6): 1633 -5〕, Solanum unguiculatum 〔Sarg, T.M. et al., (1995), Pharmacy World & Science, 17(6):191-4〕, Solanum robustum 〔Ripperger, H. (1995) , Phytochemistry 39 (6): 1475-7], snake-shaped eggplant (Solanum anguivi) [Ripperger, H. et al., (1994), Phytochemistry, 37 (6): 1725-7], leafy nightshade (Solanum platanifolium) [Puri , R. et al., (1 994), Journal of Natural Products 57(5):587-96], Solanum mammosum (Solanum mammosum) [Alzerreca, A. et al., (1982), Toxicology Letters, 12(2-3): 151-5] and so on.

It is currently known that the steroid alkaloids obtained from these Solanaceae plants contain, for example, components such as solamargine, solasonine, khasianine and solasodine [Chataing , B. et al., (1998), Planta Medica 64, 31-36; and Weissenberg, M. et al., (1998), Phytochemistry 47, 203-209]. The chemical structures of solanine and solanine are as follows:

In addition, studies have found that solanine extracted from different plant sources has the effect of inhibiting the growth of the following organisms: parasites, such as Trypanosoma cruzi (Trypanosoma cruzi); (Tribolium castaneum (red flour beetle), Manduca sexta (tobacco hornworm); molds such as Phoma medicaginis, Rhizoctomia solani; and software Animals, such as Lima snail (Lymnaea cubensis), freshwater snail (Biomphalaria glabrata) [Chataing, B. et al., (1998), Planta Medica, 64, 31-36; Fewell, A.M. et al., (1994), Phytochemistry, 37, 1007-1011; Lin, C.N. et al., (1990), J. Nat. Prod., 53, 513-516].

In addition, Chun-Nan Lin et al. have reported that solanine can be extracted from the fruit of yellow water eggplant (Solanum incanum). The chemical structure of this compound is identified as a steroidal alkaloid sugar, and the biological activity of this compound has been determined It was found that CCl ₄ -induced liver damage will show hepatoprotective effect, and will inhibit the growth of JTC-26 and human PLC/PRF/5 liver tumor cells [Chun-Nan Lin et al. (1986), J.NaturalProd. , 53, 513-516].

Shu-Hui Hsu et al. further studied the cytotoxic mechanism of solanine and found that solanine can increase the death of cells (such as human liver cancer cells Hep3B and normal skin fibroblasts) through programmed cell death. In particular, tumor necrosis factor receptor I (TNF receptor I) is known to be involved in the process of apoptosis, and solanine was found to cause excessive gene expression of this receptor (Shu-Hui Hsu et al., (1996) , Biochem. Biophys. Res. Comm., 229, 1-5).

Katsuya Fukuhara and Isao Kubo in Phytochemistry, 30(2):685-687, 1991 reported: Ripe fruits of Solanum solanum extracted with methanol at room temperature, followed by removal of solvent under reduced pressure and freeze-drying of the residue , to obtain a dark brown extract; then the extract was suspended in water containing 1% methanol, after removing the water-insoluble fraction, carried out with n-hexane, chloroform, ethyl acetate and water Partitioning results in a biologically active aqueous layer. The biologically active water layers are then subjected to rotation locular countercurrent chromatography and droplet countercurrent chromatography to obtain the two main types of solanine and solanine. compound.

Ke Hu et al. (1999), Planta Medica 65, 35-38 disclosed that using whole Solanum nigrum, reflux treatment with 75% EtOH, and removal of solvent under vacuum, the resulting brown residue was While degreasing with petroleum ether, the resulting extract was suspended in water and chromatographed on a macroresin column. The 60% EtOH eluate contained the active compound. The 60% EtOH extract was partitioned with water and extracted with n-BuOH, the obtained n-BuOH extract was introduced to silica gel column chromatography using CHCl ₃ -MeOH-H ₂ O and using MeOH-H _{2 O (60:40) Sephadex LH-20 column chromatography to obtain β 2 -solamargine} , solanine and degalactotigonin. This paper does not teach how to obtain a biologically active extract from Solanum nigrum that is directly soluble in water.

EP 0 020 029 A1 discloses that Solanumsodomeum (also known as apple of Sodom in Australia) plant material is first extracted with a dilute aqueous acid solution (2% or 3% acetic acid) to obtain a first acid extract (aqueous solution part), The first acid extract is separated from the solid residue and the solid residue is extracted with the dilute aqueous acid solution to obtain a second acid extract (aqueous solution portion), after combining the first acid extract and the second acid The extract is followed by the addition of base to give a precipitate. After the precipitate was dissolved in boiling ethanol, the ethanol was removed to obtain an extract as a fine powder, which was called BEC 001. The BEC 001 extract can be further separated and purified to obtain a variety of glycoalkaloids, including mono- and di-saccharides of solanine, solanine and solanine.

Although EP 0 020 029 A1 mentions that water can be used as the carrier (carrier) of the BEC 001 extract, in the examples of the case, the BEC 001 extract is mainly prepared as a dimethyl sulfoxide (DMSO) solution, Or in paraffin wax, zinc ointment, zinc cream and cetomacrogol (a surfactant).

Furthermore, according to the disclosure of US 5,958,770, solasodine glycosides were used in in vitro cytotoxicity experiments, and were first dissolved in DMSO and then diluted into a 5% DMSO solution for use. In addition, the solanamine sugar used in the experiment was a mixture (BEC, containing solanine (33%), solanine (33%) together with di-saccharides and mono-saccharides (34%)], Or as separate components (solanine, solanine, a mixture of di- and mono-saccharides, and theaglycones of solasodine).

The steroid alkaloids mentioned above are water-insoluble. Therefore, the traditional methods disclosed in the above-mentioned patents or literatures are usually extracted by alcohol distillation, and the extracted components are usually extracted with organic solvent dimethyl alcohol. sulfoxide to dissolve and then analyzed. Since water-insoluble substances are not suitable for direct injection into animals, they may also be difficult to be absorbed by the digestive tract during oral administration and fail to show curative effect, which limits their application and development in medicine.

The applicant found in experiments that the dry powder of solanine and/or solanine could never be dissolved without first treatment with DMSO, even after being treated with DMSO, it could not be completely dissolved, especially by the method disclosed in EP 0 020 029 A1 Extracted steroid alkaloids are not soluble in distilled water. Furthermore, once solanine or Australian solanine is dissolved in DMSO first, it can be dissolved by adding water, but if the concentration of solanine or Australian solanine is too high (over 5mg/ml), solanine or Australian solanine The base will still crystallize out. In addition, DMSO (more than 1%) itself has a strong poisonous effect on cells, so the concentration of DMSO must be controlled below 5%. These facts clearly show that there are still limitations when trying to use DMSO as an organic solvent to dissolve steroidal alkaloids extracted from Solanaceae plants.

Based on the above, the current supply of these steroidal alkaloid products is limited to a small amount of single-batch production by chemical preparation companies, and there is no effective method to extract them from Solanaceae plants on a large scale. The production of steroid alkaloids of water is not available for commercial use, thus limiting the development of these steroid alkaloids to be used in the manufacture of pharmaceuticals.

Contents of the invention

Therefore, in order to generate a large amount of water-soluble steroidal alkaloids derived from plants of the genus Solanum which are available for industrial use, in a first aspect, the present invention provides a water-soluble extract of plants of the genus Solanum, which is basically composed of at least 60%-90% Australian solanine and solanine composition. The water-soluble extract can be directly dissolved in pure water or water with a neutral pH value without adding any other solvents and/or co-solvents to form a yellow, clear and transparent aqueous solution, and the solubility can reach 2-20 mg/ml or above.

In a second aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned water-soluble extract as an active ingredient for inhibiting the growth of tumor/cancer cells (especially liver cancer cells, lung cancer cells and breast cancer cells) .

In a third aspect, the present invention provides a method for preparing a water-soluble extract of a plant of the genus Solanum comprising the steps of:

(a) subjecting a plant material belonging to the genus Solanum to an extraction treatment using an acidic aqueous solution having a pH of 3 to 5, thereby obtaining an aqueous solution;

(b) adjusting the pH value of the aqueous solution obtained from step (a) to pH 8-10 with a base, whereby a precipitate is formed;

(c) washing the precipitate obtained from step (b) with water, followed by drying, thereby obtaining a dried product;

(d) mixing the dry product obtained from step (c) with chloroform, followed by adding an appropriate amount of 100% alcohol, thereby obtaining a chloroform-alcohol mixture;

(e) mixing the chloroform-alcohol mixture obtained from step (d) with a water/alcohol solution having a predetermined water:alcohol ratio, whereby a layer having a chloroform-based layer and a non-chloroform-based layer A mixture of predominant layers is formed;

(f) removing the chloroform-based layer in the mixture formed in step (e), and then adding an appropriate amount of water; and

(g) Obtaining a supernatant liquid from the mixture formed in step (f) and then drying it, whereby the dried product formed can be directly dissolved in water to form a yellow clear and transparent aqueous solution.

The above and other objects, features and advantages of the present invention will become apparent with reference to the following detailed description, preferred embodiments and accompanying drawings.

Description of drawings

The present invention is described in detail below in conjunction with accompanying drawing and embodiment, in accompanying drawing:

Figure 1 shows the results of high pressure liquid chromatography (HPLC) using a water-soluble extract (25 mg) extracted from Solanum japonica;

Figure 2 shows the HPLC analysis results using the water-soluble extract (25 mg) obtained from the extraction of Solanum solanum and Solanine (5 mg);

Figure 3 shows the HPLC analysis results using the water-soluble extract (25 mg) and solanine (5 mg) obtained from the extraction of Solanum solanum;

Figure 4 shows the results of HPLC using a water-soluble extract (25 mg) extracted from Solanum nigrum;

Figure 5 shows the HPLC analysis results using the water-soluble extract (25 mg) obtained from Solanum nigrum and solanine (10 mg);

Figure 6 shows the HPLC analysis results using the water-soluble extract (25 mg) and solanine (10 mg) extracted from Solanum nigrum;

Figures 7 to 12 show the results of HPLC using different amounts of water-soluble extracts of Solanum japonica;

Fig. 13 shows the standard calibration line of the main component of the eggplant water-soluble extract estimated according to the results of Fig. 7 to Fig. 12;

Figures 14 to 18 show the results of HPLC at different pH values according to the water-soluble extract of eggplant yellow water according to the present invention;

Figure 19 shows the comparison results of the particle size analysis of the eggplant water-soluble extract according to the present invention and the eggplant extract prepared according to the method disclosed in EP 0 020 029 A1;

Fig. 20 and Fig. 21 show respectively, take the yellow water eggplant extract obtained according to the method disclosed in EP 0 020 029 A1 and the yellow water eggplant water-soluble extract according to the present invention with equal weight (50mg), and use pure water After being dissolved, carry out the result of HPLC;

Figures 22 to 24 respectively show the growth inhibitory effect of the water-soluble extract of Eggplant japonica according to the present invention on human liver cancer, lung cancer and breast cancer cells at different doses;

Figure 25 shows the autoradiographic results of analyzing the gene regulation mechanism of the eggplant water-soluble extract according to the present invention for lung cancer cells by using a gene chip, wherein the lung cancer cells are treated with the eggplant water-soluble extract (100 μg/ml) After being treated for 2 hours, the RNA was taken out, and then reverse-transcribed with [ ³² P]-dCTP to obtain labeled cDNA, and then hybridization detection was performed with a gene chip and automatic radiographic imaging was performed with X-ray film;

Figure 26 shows the morphological changes of human liver cancer cells (row A), lung cancer cells (row B) and breast cancer cells (row C) treated with the water-soluble extract of eggplant according to the present invention;

Figure 27, Figure 28 and Figure 29 respectively show nude mice transplanted with human lung cancer cells on the back, without administering the water-soluble extract of eggplant according to the present invention, and administering the water-soluble extract of eggplant according to the present invention through feeding and the results observed under administration of the water-soluble extract of Solanum japonica according to the present invention through intraperitoneal injection, wherein each curve shown in each figure represents the result of a test nude mouse; and

Figure 30 shows the growth inhibitory effects of solanine and solanine obtained from the water-soluble extract of Solanum solanum of the present invention on human lung cancer cells.

Detailed ways

The present invention provides a water-soluble extract extracted from plants of the genus Solanum, which contains steroidal alkaloids that can be directly dissolved in water to form a clear and transparent aqueous solution, thus being suitable for the manufacture of medicaments and preparations.

In particular, the present invention provides a water-soluble extract of plants belonging to the genus Solanum, which basically consists of at least 60%-90% of solanine and solanine. The water-soluble extract can be directly dissolved in pure water or water with a neutral pH value without adding any other solvents and/or co-solvents to form a yellow, clear and transparent aqueous solution, and the solubility can reach 2-20 mg/ml or above.

The present invention also provides a method for preparing the water-soluble extract, which comprises the following steps:

(a) subjecting a plant material belonging to the genus Solanum to an extraction treatment using an acidic aqueous solution having a pH of 3 to 5, thereby obtaining an aqueous solution;

(b) adjusting the pH value of the aqueous solution obtained from step (a) to pH 8-10 with a base, whereby a precipitate is formed;

(c) washing the precipitate obtained from step (b) with water, followed by drying, thereby obtaining a dry product;

(d) mixing the dry product obtained from step (c) with chloroform, followed by adding an appropriate amount of 100% alcohol, thereby obtaining a chloroform-alcohol mixture;

(e) mixing the chloroform-alcohol mixture obtained from step (d) with a water/alcohol solution having a predetermined water:alcohol ratio, whereby a layer having a chloroform-based layer and a non-chloroform-based layer A mixture of predominant layers is formed;

(f) removing the chloroform-based layer in the mixture formed in step (e), and then adding an appropriate amount of water; and

(g) Obtaining a supernatant liquid from the mixture formed in step (f) and then drying it, whereby the dried product formed can be directly dissolved in water to form a yellow clear and transparent aqueous solution.

Preferably, the plant material of the plant of the genus Solanum (Solanum) used in step (a) of the method is firstly chopped.

Preferably, the plant material used in step (a) of the method is at least one of the fruits, roots, stems and leaves of the Solanum plant. In a preferred embodiment of the present invention, the plant material used in step (a) of the method is the fruit of the plant of the genus Solanum. In another preferred embodiment of the present invention, the plant material used in step (a) of the method is the whole plant of the Solanum plant.

Preferably, the water-soluble extract is extracted from a plant of the genus Solanum selected from the group consisting of Solanum incanum L., Solanum indicum, Nightshade ( Solanum nigrum), Solanum capsicastrum, Solanum xanthocarpum, Solanum melongena, Solanum coagulans, Solanum tunigrum, Solanum sodomeum, Solanum turburosum ), Solanum aculeastrum, Solanum lycocarpum, Solanum khasianum, Solanum suaveolens, Solanum uporo, Solanum abutiloides, Solanum coccineum , Solanum unguiculatum, Solanum robustum, Solanum anguivi, Solanum platanifolium, and Solanum mammosum.

In a preferred embodiment of the present invention, the water-soluble extract is extracted from Solanum solanum. In another preferred embodiment of the present invention, the water-soluble extract is extracted from Solanum nigrum.

Preferably, the aqueous solution obtained from step (a) of the method is obtained by centrifugation or filtration after the extraction treatment.

Preferably, the acidic aqueous solution used in the extraction treatment of step (a) of the method is an aqueous solution containing formic acid, acetic acid or hydrochloric acid.

Preferably, the base used in step (b) of the method is an aqueous alkaline solution containing a compound selected from the group consisting of alkali metal hydroxides and ammonium hydroxide. In a preferred embodiment of the present invention, the alkaline aqueous solution contains ammonium hydroxide. In another preferred embodiment of the present invention, the alkaline aqueous solution contains sodium hydroxide.

Preferably, the precipitate obtained from step (b) of the method is obtained by centrifugation or filtration after pH adjustment.

Preferably, the drying performed in step (c) of the method is selected from the group consisting of freeze drying, spray drying, evaporation, heat drying, and the like a combination. In a preferred embodiment, the present invention uses freeze-drying to carry out the drying treatment, which intends to improve the stability and activity of the active ingredient in a low-temperature manner.

Preferably, in step (c) of the method, the precipitate obtained from step (b) is washed with water and then centrifuged to remove excess alkali, followed by addition of distilled water before the drying process.

In step (d) of the method, there is no particular requirement on the amount of chloroform and alcohol used, as long as the dried product obtained from step (c) can be dissolved. In a preferred embodiment of the present invention, in step (d) of the method, the amount of alcohol used is not more than the amount of chloroform used.

Preferably, the alcohol used in steps (d) and (e) of the method is selected from a group consisting of methanol, ethanol, propanol and combinations thereof. In a preferred embodiment of the present invention, the alcohol used in steps (d) and (e) of the method is methanol.

Preferably, the water/alcoholic solution used in step (e) of the method has a water content not lower than the alcohol content. More preferably, the water/alcohol solution has a water:alcohol ratio of 1:1.

Preferably, in step (f) of the method, removing the chloroform-based layer in the mixture formed in step (e) is performed by centrifugation or filtration.

Preferably, in step (g) of the method, the supernatant is obtained by centrifuging or filtering the mixture formed in step (f).

Preferably, the drying treatment carried out in step (g) of the method is selected from a group consisting of freeze drying treatment, spray drying treatment, evaporation treatment, vacuum concentration, heat drying treatment, and combinations thereof. In a preferred embodiment, the drying treatment in step (g) of the method includes concentration under reduced pressure and freeze-drying treatment.

If desired, the dried product obtained in step (g) can be redissolved in water, centrifuged and then dried.

The extract obtained by the present invention can be directly dissolved in common edible water or sterile water to form a yellow clear and transparent solution. Therefore, the obtained extract of the present invention is a real water-soluble extract.

The water-soluble extract prepared by the method of the present case is basically composed of at least 60%-90% of solanine and solanine. In addition, the applicant found that some factors may affect the content and ratio of solanine and solanine in the water-soluble extract obtained by the method of this case. These factors include: the nightshade species and parts used for the extraction, the alcohol used and the base used.

For example, a water-soluble extract prepared using the ripe fruit of Solanum solani, when analyzed by HPLC, contained more solanine than solanine. In addition, when 10M NaOH alkaline solution is used to carry out step (b) compared with 33% NH ₄ OH alkaline solution, the content of solanine in the obtained water-soluble extract will be lower than that of solanine . Furthermore, when compared with methanol, the content of solanine and solanine in the water-soluble extract obtained when ethanol is used for the method of this case will be reduced, which is only about 50% of that when methanol is used. In addition, when Solanum nigrum is used for extraction, the content of solanine and solanine in the extract obtained from the immature fruit (green) is higher, while the content of mature fruit (black purple) and other parts is less . Moreover, the content ratio of solanine and solanine in the extract obtained from Solanum nigrum is different from that extracted from Solanum solanum. Therefore, those skilled in the art can select the species and parts of Solanum plant and modify other operating conditions to generate the desired water-soluble extract.

In a preferred embodiment of the present invention, an aqueous solution containing acetic acid is used for extraction in step (a), and an alkaline aqueous solution containing ammonium hydroxide is used in step (b) to make a precipitate is formed, and the alcohol used in steps (d) and (e) is methanol.

Preferably, more than 75% of the water-soluble extract is composed of solanine and solanine.

Preferably, the water-soluble extract has a ratio of solanine to solanine of 0.3:1.0 to 1.0:0.6, or the water-soluble extract has a ratio of solanine to solanine The ratio is between 0.4:1.0～0.9:1.0. In a preferred embodiment of the present invention, the water-soluble extract has a ratio of solanine to solanine of about 0.7:1.0.

Preferably, the water-soluble extract is in the form of water-soluble particles having a nanoscale particle size. More preferably, the water-soluble extract is in the form of water-soluble particles having a particle size of less than 1 μm.

According to the water-soluble extract of the present invention, it has been proved to have the effect of inhibiting the growth of tumor/cancer cells (especially cancer cells of liver tumor, lung cancer and breast cancer), and the Australian nightshade obtained from the water-soluble extract of the present invention Alkaline and solanine have also been proven to inhibit the growth of tumor/cancer cells. Therefore, the present invention also anticipates the application of the water-soluble extract and the obtained solanine and solanine in the preparation of anti-tumor or anti-cancer compositions according to the present invention.

Therefore, the present invention also provides a pharmaceutical composition comprising a water-soluble extract according to the present invention, or solanine and solanine obtained from the water-soluble extract. Solanine and solanine, which are further purified from the water-soluble extract, can likewise be directly dissolved in water.

Optionally, the pharmaceutical composition of the present invention may additionally contain a pharmaceutically acceptable carrier, which is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may contain one or more of the following agents: water, physiological saline, buffer solution, disintegrating agent, binder, excipient, lubricant, absorption delaying agent and the like.

The pharmaceutical composition of the present invention can be administered parenterally or orally in a suitable dosage form. Suitable dosage forms include: sterile aqueous solutions or dispersions, sterile powders, lozenges, capsules, creams, ointments and the like. Preferably, the pharmaceutical composition according to the present invention is manufactured in a form suitable for injection, such as aqueous injection, powder injection (powder injection), lyophilization product for injection and other forms.

Alternatively, the pharmaceutical composition of the present invention may be administered alone, or in combination with an additional antineoplastic/anticancer agent [such as mitomycin, doxorubicin, actinomycin, (cis-platin) etc.] to administer the drug.

The dosage and frequency of administration of the pharmaceutical composition according to the present invention will vary depending on the severity of the disease to be treated, the route of administration, and the weight, age, physical condition and response of the individual to be treated. In general, the daily dosage of the pharmaceutical composition according to the present invention is usually 2 to 6 mg/Kg body weight, in the form of a single dose or divided into several doses, and can be administered orally or parenterally.

The present invention will be described in more detail with reference to the following examples, which are provided for the purpose of illustration and are not intended to limit the scope of the invention.

Embodiment 1. Preparation of water-soluble extract

Take by weighing 500 grams of ripe fruit of Solanum solanum, grind after adding 1000ml of pure water, and add 99.5% acetic acid dropwise to the formed aqueous mixture, to adjust the pH value of the formed aqueous mixture to 4.0, and then extracted with shaking at room temperature for 12 hours. Afterwards, the aqueous mixture was centrifuged to obtain a supernatant, which was added dropwise with 33% NH ₄ OH alkaline water solution to adjust the pH of the supernatant to 9.0, at which point a precipitate would precipitate out. Centrifuge at 4,500 rpm (Beckman Coulter, AvantiJ-25, JA-14 Rotor) to obtain a precipitate, wash with water and centrifuge again at 4,500 rpm to remove residual alkaline solution. The obtained precipitate was suspended in distilled water and freeze-dried (Virtis, Freezemobile 12ES) to obtain 5 g of dry powder. The dry powders obtained here can only be suspended in aqueous solutions, but not directly soluble in water, or only in small amounts.

To prepare an extract directly soluble in water, weigh 2 g of the dry powder obtained above, dissolve it in 50 ml of reagent grade chloroform, then add 40 ml of 100% methanol and shake to form a homogeneous suspension. Then, a supernatant obtained by centrifugation (4,500 rpm) or filtration was added to 70 ml of methanol:water (1:1) and mixed and shaken. The resulting mixture was centrifuged at 12,000rpm for 10 minutes, and the supernatant was added to 120ml of distilled water, mixed and shaken. At this time, the supernatant would become slightly turbid, and then centrifuged at 12,000rpm for 10 minutes. Remove the precipitated precipitate. The resulting supernatant was concentrated under reduced pressure at 55° C. to remove methanol, and then freeze-dried to obtain a dry powder.

The dry powder obtained at this stage can be directly dissolved in distilled water to form a clear transparent light yellow solution. If the solution still has a little incompletely dissolved precipitate, it can be centrifuged at 12,000rpm for 10 minutes to remove the precipitate. The obtained supernatant can be directly freeze-dried to obtain water-soluble Dry powder.

Generally speaking, about 800mg of water-soluble dry powder can be extracted from every 500g of Solanum japonica fruit. When analyzed by HPLC, the main components contained in the water-soluble dry powder are solanine and solanine, wherein the proportion of solanine is higher than that of solanine (see Example 2 and Figure 1 below).

Regarding the above-mentioned preparation process, the fruit can alternatively be directly placed in 1000 ml of 3% or 5% aqueous acetic acid solution and chopped. Alternatively, the resulting aqueous mixture may be shaken at 50° C. for 5 hours, or at 80° C. for 2 hours, instead of shaking the aqueous mixture at room temperature for 12 hours. Furthermore, an aqueous sodium hydroxide solution may alternatively be used instead of the aqueous NH ₄ OH solution used above.

Embodiment 2. Composition identification of water-soluble extract

In order to confirm which main components are contained in the water-soluble extract obtained according to the present invention, high pressure liquid chromatography (HPLC) is used for component analysis.

experimental method:

In this embodiment, the high pressure liquid chromatography (HPLC) used is Agilent Technologies (Waldbronn, Germany), 1100 models; The column used is LiChroCART 250-4 Lichropher100 RP-18e (5 μ M), length: 250mm ×4mm; mobile phase: 60% acetonitrile/40% double distilled water (pH=2.8); the flow rate was controlled at 1 ml/min. In addition, the standard substances of solanine and solanine used in the experiment were generously provided by Professor Lin Zhongnan of the Department of Pharmacy, Kaohsiung Medical University, and the two standard substances were based on Gan, K.H., Lin, C.N. and Won, S.J. (1993), the purification method disclosed in Journal of Natural Products56, 15-21 is prepared.

According to the preparation method shown in Example 1, the water-soluble extracts were respectively prepared from the fruits of Solanum solanum and Solanum nigrum, and an appropriate amount was dissolved in pure water, followed by HPLC analysis with the following conditions:

1. With water-soluble extract of eggplant (25μg), water-soluble extract of eggplant (25μg) + solanine (5μg) and water-soluble extract of eggplant (25μg) + solanine (5μg) perform HPLC;

2. Perform HPLC with Solanum nigrum water-soluble extract (25 μg), Solanum solanum extract (25 μg) + Solanine (10 μg) and Solanum solanum extract (25 μg) + Solanine (10 μg); as well as

3. Take 50μg, 40μg, 30μg, 20μg, 10μg and 5μg of water-soluble extracts of Eggplant japonica for HPLC analysis.

result:

Fig. 1 shows the HPLC result of the water-soluble extract of Solanum solanum fruit, and comparing Fig. 2 and Fig. 3, judging from the retention period, it can be seen that the first peak in Fig. 1 should be solanine, and the second peak It should be solanine, and the content of solanine is higher than that of Australian solanine.

Referring to Figures 4 to 6, it can be seen from the HPLC analysis results that the water-soluble extract of Solanum nigrum fruit also has two peaks corresponding to solanine and solanine. However, different from the water-soluble extract of nightshade fruit, the content of solanine in the water-soluble extract of nightshade fruit is higher than that of solanine.

Furthermore, as can be seen from Figures 1 to 3 and Figures 4 to 6, the water-soluble extract obtained according to the present invention does not contain austhalamin, which is clearly different from the extract obtained in EP 0 020 029 A1.

Figures 7 to 12 show the results of HPLC analysis with different amounts (75mg, 50mg, 40mg, 30mg, 20mg, 10mg) of eggplant water-soluble extracts, and it can be found that two The peak value of the main component will show a regular ebb and flow with the dosage of the water-soluble extract.

As for the content ratio of solanine and solanine, it can be calculated automatically by computer software (Agilent ChemStation Integrator Algorithm) based on the obtained HPLC elution graph. If the waveform shown in it is slightly sharp or cylindrical, It may be affected by the maturity of plant fruits and seasonal factors. The applicant has used the water-soluble extract of Solanum solanum to carry out 26 estimates, and obtained the ratio of solanine to solanine from about 0.3:1.0 to 1.0:1.0, and most of the ratios fall in the 0.4:1.0～0.9:1.0.

The applicant found from the obtained experimental results that at least 60% to 95% (preferably more than 75%) of the water-soluble extract obtained in the present invention is composed of two main components, solanine and solanine.

At the same time, during the HPLC analysis, the applicant also found that the content and ratio of the two main components, solanine and solanine, will show a linear relationship of growth and decline with the dosage used. Figure 13 shows that the water-soluble extracts of eggplant yellow water of 7 different doses were taken, each of which was analyzed by HPLC 3 times, and the mean ± standard deviation was calculated by the peak integral areas of the two main components contained, and drawn by linear regression out of the calibration line. The results shown in Figure 13 clearly show that the two main components, solanine and solanine, show a linear relationship of increase and decrease with the dose. Therefore, the two main components of solanine and solanine should be used as quality control index components of the water-soluble extract according to the present invention.

In addition, the further purified solanine and solanine that are directly soluble in water can be separated from the water-soluble extract of the present invention by using the above-mentioned HPLC conditions.

Embodiment 3.pH value is for the elution influence of water-soluble extract

experimental method:

In order to understand the influence of the pH value on the elution of the water-soluble extract of the present invention, use the same HPLC instrument as in Example 2, and use the water-soluble extract of the yellow eggplant prepared according to the preparation method shown in Example 1 (40μg), and carry out HPLC analysis with the following conditions:

1. The column used is LiChroCART 250-4 Lichropher 100 RP-18e (5μM), length: 250mm×4mm; and

2. Mobile phase: 60% acetonitrile/40% double distilled water, and set the pH values to pH 2.3, pH 2.5, pH 2.8, pH 3.0, and pH 3.2 respectively.

result:

Figures 14 to 18 show the results of HPLC analysis of water-soluble extracts of Solanum solani at different pH values. It can be seen from these figures that the main components of these water-soluble extracts will prolong their residence time as the pH value of the mobile phase increases, and although the elution profiles of the last two main components will change slightly, But basically the graphics of the two principal components are maintained.

Embodiment 3. The comparative analysis of the particle size of water-soluble extract

This example further compares the difference in particle size between the eggplant water-soluble extract prepared according to the present invention and the eggplant extract prepared according to the method disclosed in Example 2 of EP 0 020 029 A1.

experimental method:

Dissolve 50 mg of the water-soluble extract of eggplant yellow water prepared according to the present invention and 50 mg of eggplant yellow eggplant extract prepared according to the method disclosed in Example 2 of EP 0 020 029 A1 in 50 ml of distilled water respectively , and utilize a particle size analyzer (Beckman Coulter LS 230, Coulter Corporation, Miami, USA) to carry out particle size analysis.

result

Referring to Fig. 19, the applicant obtained the eggplant extract according to the method disclosed in Example 2 of EP 0 020 029 A1. The average particle size of the eggplant extract is 238.2 μm, and the particle distribution is between 1.8 μm and 1500 μm. And there are many precipitates that will appear in the aqueous solution and cannot be miscible with water (see the top of Figure 19). In contrast, the water-soluble extract of eggplant yellow water prepared according to the present invention has an average particle size of 0.418 μm, and a particle distribution between 0.28 μm and 0.65 μm, and can be completely dissolved in water (see Figure 19 below).

Embodiment 4. The solubility comparison of water-soluble extract

This example further compares the difference in solubility in water between the eggplant water-soluble extract prepared according to the present invention and the eggplant extract prepared according to the method disclosed in Example 2 of EP 0 020 029 A1.

experimental method

5 mg of the water-soluble extract of eggplant yellow water prepared according to the present invention and 5 mg of the eggplant yellow eggplant extract prepared according to the method disclosed in Example 2 of EP 0020 029 A1 were respectively dissolved in 2 ml of distilled water, at 12,000 Centrifugation was performed at rpm to obtain supernatants, and then 20 μl of each was taken out and analyzed by HPLC according to the method described in Example 2 above.

result:

Figure 20 and Figure 21 respectively show, according to the method disclosed in Example 2 of EP 0 020 029 A1, the yellow eggplant extract (Figure 20) and the yellow water eggplant water-soluble extract prepared according to the present invention ( Figure 21) Solubility in water. It can be seen from these two figures that the water-soluble extract of eggplant yellow water prepared according to the present invention has obviously better solubility in water.

The results of the above examples and drawings clearly show that the water-soluble extract of Solanum plant obtained according to the present invention is indeed soluble in water and forms a clear and transparent aqueous solution compared with the extract obtained by the prior art . This may be because the water-soluble extract of Solanum plant obtained according to the present invention may be in the form of water-soluble particles having a nano-scale particle size (especially less than 1 μm). It can thus be expected that the water-soluble extracts of plants of the genus Solanum obtained according to the present invention are suitable for supply for the manufacture of medicaments containing steroid alkaloids, especially for the treatment of cancer.

Therefore, in order to confirm the biological activity of the water-soluble extract of Solanum plant obtained according to the present invention, the following pharmacological test was carried out with the water-soluble extract of Solanaceae prepared according to Example 1.

Pharmacological test 1. Analysis of anticancer activity in vitro

In order to determine whether the water-soluble extract of the Solanum plant obtained according to the present invention has anticancer activity, the applicant selected the most important human cancer cells as test objects, including: liver cancer cells (Hep3B), lung cancer cells (H441) and Breast cancer cells (MCF-7), liver cancer cells (Hep3B) and lung cancer cells (H441) were purchased from the American Type Culture Collection [ATCC, P.O. Box 1549, Manassas, VA 20108 USA], while breast cancer cells (MCF- 7) It was purchased from the Institute of Food Industry Development (No. 331, Food Road, Hsinchu City, Taiwan Province, 300).

experimental method:

Liver cancer cells were cultured in Dulbecco's Modified Eagle's medium (DMEM), while lung cancer cells and breast cancer cells were cultured in RPMI-1640 medium, and the above medium Add 10% fetal bovine serum and 40mg/L gentamicin.

Toxicity testing for cancer cells was performed using a tetrazolium salt assay (MTS) (Mosmann.T., 1983, Immunol.Meth., 65, 55-63) according to the manufacturer's procedure (Cell Titer 96TM AQ (Promega, Madison, USA)) to carry out the colorimetric determination of cell viability (colomertric determination of cell viability).

Quantitative cells (1×10 ⁴ cells/well) were cultured in a 96-well culture dish, and cultured in a 37°C, 5% CO ₂ incubator for at least 16 hours. Next, different doses of water-soluble extracts of Eggplant japonica were dissolved in sterile water for injection, and then added to the cancer cell culture solution in each well respectively. After being treated for 12 hours, 20 μl of MTS solution was added to each well and allowed to react for 3 hours. After the reaction was completed, the absorbance value of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent analyzer (ELISA reader 312e, Bio-TEK). All experimental data are expressed as the mean ± standard deviation of three experiments.

result:

Fig. 22, Fig. 23 and Fig. 24 respectively show the inhibitory effect of the water-soluble extract of Solanum japonica according to the present invention on liver cancer cells (Hep3B), lung cancer cells (H441) and breast cancer cells (MCF-7). The results show that the water-soluble extract of eggplant yellow water according to the present invention can effectively inhibit the growth of these three cancer cells.

Pharmacological test 2. Analyze the gene regulation mechanism of the water-soluble extract of yellow water eggplant according to the present invention for lung cancer cells with a gene chip

In order to understand the mechanism of action of the water-soluble extract of yellow water eggplant according to the present invention on cancer cell genes, to provide a useful research and development direction for cancer treatment, and to contribute to the research of carcinogenic factors and the development of an effective anticancer drug, this experiment further The gene chip technology is used to analyze the regulation of cancer cell genes by the water-soluble extract according to the present invention.

In this experiment, applicants used a commercially available gene array (SuperArray Inc., Bethesda, MD, USA) to analyze the gene regulation of lung cancer cells (H441) by the water-soluble extract according to the present invention.

Firstly, RNA was extracted from lung cancer cells (experimental group) treated with the water-soluble extract of eggplant (100 μg/ml) of the present invention for two hours and a control group without extract treatment, and were extracted by using [ ³² p ]-dCTP for reverse transcription to produce labeled cDNAs. The obtained labeled cDNAs can be used as labeled probes and used for hybridization reaction with the genes located on the gene array (SuperArray Inc., Bethesda, MD, USA). Autoradiography of the reacted gene array was performed on X-ray film.

result:

As shown in Figure 25, when the lung cancer cells were treated with the water-soluble extract of yellow water eggplant of the present invention for two hours, the "tumor necrosis factor receptor I (Tumor necrosis factor receptor I)" and "tumor necrosis factor receptor I" related to the promotion of cancer cell death The gene expression of necrosis factor receptor-associated factor-1 (TNF receptor-associated factor-1, TRAF-1) was significantly increased, while the inhibitor of apoptosis protein 2 (Inhibitor of apoptosis protein 2, The expressions of genes such as IAP-2)" and "apoptosis inhibitor 4 (API-4)" were reduced by the effect of the extract. Seen from these results, the eggplant water-soluble extract of the present invention can significantly activate tumor necrosis factor receptor I (TNFR-I) and tumor necrosis factor receptor-associated factor-1 (TRAF-1) of lung cancer cells. gene expression.

Pharmacological test 3. The effect of the water-soluble extract of yellow water eggplant of the present invention on the cell cycle of cancer cells

In this experiment, a flow cytometry analyzer (Flow cytometry, "FACScan", Becton Dickinson Corp.) was used to analyze the cell cycle changes of cancer cells before and after the action of the yellow water eggplant water-soluble extract of the present invention.

experimental method:

First, 1×10 ⁶ cancer cells were cultured in a 35mm petri dish for 16 hours, after that, the water-soluble extract of yellow water eggplant (30 μg/ml) of the present invention was added, and the action time of each experimental group was 0 and 1 respectively. , 3, 5, 8 hours. After each experimental group has reached its action time, the cells are respectively knocked down with one-fold concentration of trypsin (the supernatant and the culture medium must be collected together), and then the beaten cells are placed in a 15ml centrifuge tube and placed in a 15ml centrifuge tube. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded. After adding about 300 μl of 1×PBS and mixing evenly, take 300 μl and place it in a microcentrifuge tube, and add 700 μl of absolute ethanol to fix the cells. The alcohol-fixed cells were placed in a refrigerator at 4°C for more than 30 minutes, and then centrifuged at 1200 rpm for 5 minutes at 4°C. After centrifugation, aspirate the supernatant and mix gently. 445 μl of 1×PBS was added to the supernatant to mix the cells evenly, and then 5 μl of ribonuclease (RNase, 10 mg/ml) was added to destroy and decompose the RNA. Next, 50 μl of 10% Triton-X100 was added to make holes for the cells, and then the cells were placed in a 37° C. incubator for 1 hour to react, and then centrifuged. Aspirate the supernatant and add 495 μl of PBS, mix well, then add 5 μl of propidium iodide (5 mg/ml) to stain DNA, and carry out the reaction at 4°C in a dark environment for 15-30 minutes, then filtered through a filter membrane and analyzed.

Cell Cycle Assays:

Using a flow cytometer (Beckman-Coulter FACScan), the above-mentioned cells attached to the filter membrane were evenly suspended in 1×PBS solution, and the cells were introduced into the flow cytometer at a rate of 100 cells/second , 10,000 cells per analysis. The cells are passed through a small hole with a radius of 75 μm, and a current pulse signal proportional to the volume is generated. Cells are excited by a laser beam of argon ions at 488nm to fluoresce. The obtained data is combined with Winmdi software to analyze the amount of DNA in the cells and observe the cell cycle.

result:

Table 1 shows the cell cycle changes of liver cancer cells (Hep3B), lung cancer cells (H441) and breast cancer (MCF-7) after the water-soluble extract of yellow water eggplant of the present invention, wherein the characteristics of Sub-G1 wave increase It represents that cancer cells undergo apoptosis under the action of the water-soluble extract.

It can be seen from Table 1 that the Sub-G1 wave of the cell cycle of various cancer cells increased significantly within 1 hour, and when high doses of extracts were used, the cancer cells would die due to cell membrane rupture. The duration of action increases and becomes stronger. These results show that the water-soluble extract of Solanum japonica provided by the present invention can indeed initiate the apoptosis mechanism of these three cancer cells.

Table 1. The effect of Eggplant water-soluble extract of the present invention on the cell cycle of liver cancer cells (Hep3B), lung cancer cells (H441) and breast cancer (MCF-7)

Sub-G1 G0/G1 S G2/M Time (hr) Mean ± S.D % Mean ± S.D % Mean ± S.D % Mean ± S.D % liver cancer cells 01358 5.5±0.238.3±0.669.5±0.490.7±0.799.1±0.1 100246446581635 58.8±0.331.9±0.915.4±0.34.3±0.30.6±0.1 100643191 17.4±0.315.1±0.19.9±0.23.6±0.70.3±0.0 1008757212 18.0±0.514.6±0.45.6±0.21.5±0.20.1±0.0 100813181 lung cancer cells 01358 9.4±0.246.5±1.761.8±0.796.6±0.297.2±0.6 10044166010301037 43.3±0.323.6±0.413.1±1.42.3±0.11.9±0.4 100543054 7.1±0.111.8±0.19.8±0.70.8±0.00.4±0.4 100168139116 40.8±0.118.5±1.215.0±0.70.4±0.00.4±0.5 100453611 breast cancer cells 01358 2.9±0.612.0±0.877.1±0.492.0±0.397.1±0.4 100441263331413313 66.8±1.271.2±0.418.7±1.06.8±0.32.5±0.3 10010727104 13.1±0.66.4±0.62.4±0.30.8±0.00.2±0.0 100491862 1 8.2±0.410.7±1.01.3±0.20.4±0.00.2±0.1 10059721

Pharmacological test 4. The effect of the water-soluble extract of yellow water eggplant of the present invention on the cell morphology of cancer cells

In order to study the morphological changes of the cancer cells after being subjected to the water-soluble extract of the eggplant of the present invention, this experiment further observed the morphological changes of the cancer cells stained by hematoxylin with an optical microscope.

experimental method:

First, take out the appropriate number of cells from the liver cancer, lung cancer and breast cancer cells that have been treated with the eggplant water-soluble extract (30 μg/ml) of the present invention for 1, 3, 5, and 8 hours, and use a cell slice centrifuge Cytospin to The cells were centrifuged at 800 rpm for 10 minutes to slice the cells. Next, cells were fixed with 4% paraformaldehyde for 30 minutes. The cell sections were stained with hematoxylin for nuclei, and then the excess dye was removed with dilute ethanol from 70%, 80%, 90%, 95% to 100% absolute ethanol. Then the cell slices were placed in xylene for dehydration, and the slides were covered with coverslip and mounting glue, and the cells were observed and photographed with an optical microscope (Olympus CX-40) at a magnification of 400 times Changes in form.

result:

Figure 26 shows the morphological changes of three kinds of human cancer cells before and after 1, 3, 5, and 8 hours of action with the water-soluble extract of yellow eggplant (30g/ml) of the present invention, wherein row A is Liver cancer cells (Hep3B), row B are lung cancer cells (H441) and row C are breast cancer cells (MCF-7). The control group is the cells not treated with the extract, and the arrows indicate typical cell morphology changes.

It can be seen from Fig. 26 that for the three cancer cells treated with the water-soluble extract of yellow eggplant of the present invention, the reduction of the nucleus, the condensation of chromatin and the generation of apoptotic bodies can be observed within 1 hour. The growth of these cancer cells becomes more obvious, and finally the cell membrane ruptures.

Since Sub-G1 waves, chromatin condensation and the generation of apoptotic bodies are typical characteristics of apoptosis, the results of this pharmacological test and the above pharmacological test 3 clearly show that the water-soluble extract according to the present invention can activate cancer cells Apoptotic mechanism, resulting in the death of cancer cells.

Pharmacological test 5. In vivo animal model test of the yellow water eggplant water-soluble extract of the present invention

In order to confirm whether the water-soluble extract according to the present invention can produce anti-cancer effect in vivo, this embodiment further conducts an in vivo animal model test with nude mice.

experimental method:

First, human lung cancer cells H441 (2×10 ⁷ cells) were implanted into the back of nude mice (BABL/c-nu-nu, 8 weeks old, weighing about 20-25 g) by subcutaneous injection, and the tumor was treated. After forming and starting to grow (about 10 days), the nude mice with tumors were randomly divided into groups (about 6-7 nude mice in each group).

For the nude mice in the injection group, use a 0.8mm injection needle to intraperitoneally inject 220 μg of water-soluble extract dissolved in water once a day, inject continuously for 3 days and then stop the drug for 4 days, record the body weight of nude mice every two days, and The size change of the tumor on the back of the nude mice was measured with a micrometer, and then the next course of treatment was continued, and the observation was continued for more than two months. The calculation method of tumor size: tumor size=length×width×(height/2).

For the oral group, the water-soluble extract (600 μg) dissolved in water was orally administered to each nude mouse using a feeder, fed once a day, fed continuously for 5 days, and then stopped for 2 days. The body weight of the nude mice and the tumor size were measured in the same manner as the injection group. Continue to feed and observe for more than two months. Tumor size was calculated in the same way as in the injected group.

result:

Figure 27, Figure 28 and Figure 29 respectively show nude mice transplanted with human lung cancer cells on the back, without administering the water-soluble extract of eggplant according to the present invention, and administering the water-soluble extract of eggplant according to the present invention with feeding And with intraperitoneal injection administration according to the observed results of the eggplant water-soluble extract of the present invention, wherein Fig. 27 represents the control group (6 nude mice), Fig. 28 represents the oral experimental group (7 nude mice) and Fig. 29 represents the tumor changes in the injection experimental group (7 nude mice).

As can be seen from the results shown in Fig. 27, Fig. 28 and Fig. 29, the tumor growth rate in the nude mice in the nude mice in the control group without administration of the water-soluble extract of yellow water eggplant according to the present invention is fast, and can grow to about 100% in 1 month. 6 times the original size; most of the tumors in the nude mice that were orally administered with the water-soluble extract of Eggplant japonica of the present invention shrunk or disappeared, and a few grew slightly, but the growth rate was much lower than that of the control group; The tumors in the nude mice that were intraperitoneally injected with the water-soluble extract of Solanum solani according to the present invention completely shrunk or disappeared. These results show that administration of the water-soluble extract according to the present invention by oral or intraperitoneal injection can effectively slow down the growth, reduce or even eliminate the tumors transplanted in nude mice, and the effect of intraperitoneal injection Better than oral effect.

Pharmacological test 6. Anticancer test in vitro of solanine and solanine obtained from the further purification of the yellow water eggplant water-soluble extract of the present invention

In this experiment, the biological activity of purified solanine and solanine obtained from the water-soluble extract of eggplant yellow water of the present invention was further studied by HPLC.

experimental method:

With reference to the HPLC method described in the above-mentioned Example 2, the two compounds of solanine and solanine can be further separated and purified from the water-soluble extract of eggplant in yellow water according to the present invention, and they can also be directly dissolved in the water.

Referring to Pharmacological Test 1, the cytotoxicity test was performed according to the tetrazolium salt analysis method. Human lung cancer cells (H441) were treated with different doses of purified solanine and solanine obtained from the water-soluble extract of Solanum solani of the present invention for 12 hours, and then according to the manufacturer's operating procedures [CellTiter 96 TM AQ (Promega, Madison, USA)] for colorimetric assay of cell viability. The experimental data are expressed as the mean ± standard deviation of three experiments.

result:

Figure 30 shows the growth inhibitory effects of solanine and solanine purified from the water-soluble extract of Solanum solanum of the present invention on human lung cancer cells. From the results shown in FIG. 30, it can be seen that solanine and solanine purified from the water-soluble extract of Eggplant japonica of the present invention have significant anticancer activity.

Based on the above experimental results, the applicant has successfully obtained the water-soluble extract of Solanum plants, and at the same time established the scientific analysis conditions of the water-soluble extract in the extraction process and found out the main components, so that the quality can be effectively achieved. Control. In addition, the water-soluble extract according to the present invention has been proved to have the activity of inhibiting the growth of cancer cells and initiating the natural apoptosis mechanism of cancer cells, which fully shows that the water-soluble extract according to the present invention can inhibit the growth of cancer cells and cancer cells. Development of cell assays.

All patents and literature cited in this specification are hereby incorporated by reference in their entirety. In case of conflict, the detailed description of the case (including definitions) will prevail.

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.

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