CN101507741A - Red beet extract and composite and preparation method and use thereof in preparing medicine for restraining tumor growth - Google Patents
Red beet extract and composite and preparation method and use thereof in preparing medicine for restraining tumor growth Download PDFInfo
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Abstract
The invention relates to the technical field of extracts and compositions of plants and application of the same in preparing medicines for inhibiting the growth of tumor, and discloses extracts and a composition of red beet, a preparation method of the same, and application of the same in preparing the medicines for inhibiting the growth of the tumor. The extracts of the red beet or/and the composition of the extracts of the red beet have the characteristics of not containing saccharose and improving the concentration of effective substances; and the nano-filtration is adopted for preparation at normal temperature, thereby avoiding the degradation and degeneration of the extracts, being favorable for the requirement of environmental protection, and being suitable for the need of mass production. The extracts of the red beet have inhibition effect on the growth of Hela cells of epithelial cancer cell lines of cervices, the composition of the extracts of the red beet can cause the necrosis of cancer cells of tumor bodies, and the extracts of the red beet or/and the composition of the extracts of the red beet can be applied to the preparation of the medicines for inhibiting the growth of the tumor. And the composition of the extracts of the red beet is safe and nontoxic.
Description
Technical field
The present invention relates to extract and the compositions of plant and be applied in the technical field that preparation suppresses the medicine of tumor growth, is a kind of red beet extract and composition and method of making the same and the medicine that is applied in preparation inhibition tumor growth.
Background technology
The Herba Gynurae bicoloris of Chenopodiaceae (BetaVulgarisL.Var.rubra) is a kind of mutation of Radix Betae, claims filiale again, is commonly called as leaf beet, and tool leads to the intestinal therapeutic method to keep the adverse QI flowing downwards, opens the effect of chest and diaphragm, and can control vomiting and diarrhoea and drive parasite; Be edible vegetable, but can eat raw, also prepared food, output is higher, low price, contain the amount of pigment height, wherein contained red pigment is a natural edible red pigment, and mainly Radix Betae cyanine and the yellow institute of xanchromatic Radix Betae by redness forms, and main betanin in the Radix Betae cyanine, account for the 75%-95% of red pigment, its main component is the beet red glycosides, belongs to carbohydrate derivative, and molecular formula is C
24H
26N
2O
13, molecular weight 550.48.The U.S. allowed to use Herba Gynurae bicoloris concentrated solution and dehydration sugar beet powder as food coloring since nineteen sixty, and the FAO/WHO of the United Nations food additive joint specialist committee has drawn up the standard of Herba Gynurae bicoloris pigment in 1976, and corresponding regulation has also been done by Japan.At present, America and Europe and Japan are all in further investigation Herba Gynurae bicoloris pigment and application thereof.Strong, nontoxic, the safety of Herba Gynurae bicoloris pigment colored ability, be widely used in the processing that beverage, ice cream, wine, confection, cake are mounted products such as flower, canned food, and the liver function of improvement is arranged, effect (the Cheng Dayou that facilitating digestion absorbs, guest's power, Liu Qiaohong etc. the nutrition of Herba Gynurae bicoloris and function. Chinese beet sugar industry .2006 (1): 50-52; VonElbe, Food Sciences such as J.H., 1974,39:334).
Contained red pigment adopts membrance separation, microbe fermentation method and resin method usually in the preparation Herba Gynurae bicoloris.Because contained red pigment is the easy oxidation pigment of thermal sensitivity in the Herba Gynurae bicoloris, heat treated is disadvantageous for pigment itself, and membrance separation is a kind of new separation technology that carries out at normal temperatures, by different membrane aperture and membrance separation parameter are set, major impurity can be removed effectively; He Chongyan, Wang Hui, people such as Feng Chunsheng adopt twice partition method of ultrafiltration nanofiltration (beet red pigment is refining, stabilization technology research. Food Additives Used in China .1998 (2): 10-13), effectively to red pigment component separating contained in the Herba Gynurae bicoloris, refining and concentrated, and be applied to produce product quality (Powdered) (percentage absorptivity E
1% 1cmBe 8.8) GB8271-87 that is up to state standards (percentage absorptivity E
1% 1cmBe not less than 3) requirement, developed complex natural antioxidant simultaneously, obviously improved the stability of red pigment to light, heat.Sugar, nitrate and the nitrite that remove effectively under the less situation in the crude extract can influenced with microbe fermentation method to red pigment; Drdak ferments with the beet red extracting solution inoculation of 7 kinds of beer yeast after to pasteurize, monitor the average content of betanin, class betanin, betaindin, class betaindin and saccharide with HPLC, behind the fermentation 40h, red pigment keeps ideal level, the content of sugar then has bigger reduction (Drdak.M, Altamirano.R.C, Rajniakova.A, Simko.P, Karovicova.J, Benkovska.D. Herba Gynurae bicoloris pigment composition: the Fermentation of different cereuisiae fermentum. Food Science magazine 1992,57 (4): 935-936.); Grajek studies show that Paracoccus denitrificans ATCC 19367 and Ochrobactrum anthrop i ATCC 21909 with what denitrifying bacteria was removed nitrate and nitrous acid;
These two kinds of bacterial strains remove the efficient of nitrate, nitrite higher (Grajek.W, Walkowiak-Tomczak.D, Lewandowska.M, Mueller.A, Stefanski.R. utilize denitrifying bacteria to reduce nitrate in the red beet juice.
Przemysl Spozywczy.1997,51 (7): 40-43; Grajek.W, Walkowiak-Tomczak.D. influence the factor of denitrifying bacteria red beet juice denitration speed. agricultural and Food Chemistry magazine .1997,45 (5): 1963-1966); But, Czap ski research thinks that be disadvantageous with the denitrifying bacteria fermentation to red pigment, and flavochrome is unaffected substantially, abnormal smells from the patient to pigment also has certain influence (Czapski.J simultaneously, Maksymiuk.M, Grajek.W. analyze the condition of the biological denitrification hydrochlorate of red beet juice with replying surperficial method. agricultural and Food Chemistry magazine .1998,46 (11) 4702-4705.).With the pigment crude extract through the macroporous resin treatment roguing, through salt acidify ethanol exchange and concentrate the pure product of pigment that can make (Gong Min, Chen Qingxi. macroporous adsorbent resin concentrates the research of Hylocereus undatus pigment. Xiamen University's journal (natural science edition) .2004 (43): 44-46.).
Still not having at present the cytology of relevant Herba Gynurae bicoloris and extract thereof inhibition tumor growth effect or the research data or the data of laboratory animal openly reported.
Summary of the invention
The invention provides a kind of red beet extract and composition and method of making the same and be applied in the medicine that preparation suppresses tumor growth, above-mentioned the deficiencies in the prior art have been overcome, improved concentration of active substance, its red beet extract and compositions have the effect that suppresses tumor growth.
One of technical scheme of the present invention realizes by following measure: a kind of red beet extract, adopt UV-VIS spectrophotometry that this red beet extract liquid is measured, its absworption peak is 535nm, in solid content (solid content is the dry thing of 105 ℃ of oven dry of red beet extract solution gained), its percentage absorptivity E
1% 1cmBeing 45～55, is the acetic acid sodium acetate buffer of pH5.4 and measure used diluent; Wherein, this red beet extract liquid obtains by the following method: through Herba Gynurae bicoloris clean, broken juice, filtration, adding stabilizing agent, resin chromatography purification, washing remove impurity (wash the resin chromatographic column with water, dispel impurity), eluting, eluent nanofiltration obtain red beet extract liquid after concentrating.
Two of technical scheme of the present invention realizes by following measure: a kind of preparation method of red beet extract, it is characterized in that carrying out as follows: through Herba Gynurae bicoloris clean, broken juice, filtration, adding stabilizing agent, resin chromatography purification, washing remove impurity, eluting, eluent nanofiltration obtain red beet extract liquid after concentrating, adopt UV-VIS spectrophotometry to measure to this red beet extract liquid, its absworption peak is 535nm, in solid content, its percentage absorptivity E
1% 1cmBe 45～55, wherein measure acetic acid-sodium acetate buffer that used diluent is pH5.4.
Also can further do following optimization or selection at two of one of technical scheme of the present invention or technical scheme:
The aforementioned stable agent can be one or more in vitamin C, sodium benzoate, sodium thiosulfate, sodium sulfite, the different vitamin sodium of D-, and consumption is to add stabilizing agent 5mg to 50mg in every 100ml red beet juice filtrate.
Above-mentioned resin is selected anion exchange resin or macroporous resin for use, and eluant is the hydrochloric acid of 0.1-1% (W/V), the acetic acid of 1-10% (W/V), the Fructus Citri Limoniae aqueous acid of 10-50% (W/V), or the alcoholic solution of the 10-50% of above acid (V/V);
Above-mentioned eluent adopts the film of 100 to 500 molecular weight, carries out nanofiltration at ambient temperature and concentrates.
Three of technical scheme of the present invention realizes by following measure: the compositions of above-mentioned red beet extract, it adds lactose in above-mentioned red beet extract liquid, starch, dextrin, maltodextrin, cyclodextrin, mannitol, xylitol, protein sugar, acesulfame potassium, sucralose, methylcellulose, in the arabic gum one or more, total consumption is to add 2g to 10g in every 100ml red beet extract liquid, carry out the reduced vacuum drying, spray drying or lyophilization, get the red beet extract compositions, adopt ultraviolet visible spectrophotometry that the red beet extract compositions is measured, its absworption peak is 535nm, percentage absorptivity E
1% 1cmBeing 15～35, is acetic acid-sodium acetate buffer of pH5.4 and measure used diluent.
Four of technical scheme of the present invention realizes by following measure: the preparation of compositions method of above-mentioned red beet extract, it is undertaken by following step: add lactose in red beet extract liquid, starch, dextrin, maltodextrin, cyclodextrin, mannitol, xylitol, protein sugar, acesulfame potassium, sucralose, methylcellulose, in the arabic gum one or more, total consumption is to add 2g to 10g in every 100ml red beet extract liquid, carry out the reduced vacuum drying, spray drying or lyophilization, get the red beet extract compositions, adopt ultraviolet visible spectrophotometry that the red beet extract compositions is measured, its absworption peak is 535nm, percentage absorptivity E
1% 1cmBeing 15～35, is the acetic acid sodium acetate buffer of pH5.4 and measure used diluent.
Five of technical scheme of the present invention realizes by following measure: above-mentioned red beet extract is applied in the medicine that preparation suppresses tumor growth.
Six of technical scheme of the present invention realizes by following measure: the compositions of above-mentioned red beet extract is applied in the medicine that preparation suppresses tumor growth.
Beet extract of the present invention is or/and the red beet extract compositions has the advantages that not contain sucrose, improved concentration of active substance, the preparation at normal temperatures owing to adopt nanofiltration, thereby avoid extract degraded and degeneration, help environmental requirement, be suitable for the needs of large-scale production.Red beet extract antithetical phrase epithelium of cervix uteri cancerous cell line Hela cell growth of the present invention is inhibited, red beet extract compositions of the present invention can cause the necrosis of tumor body cancerous cell, and red beet extract of the present invention is or/and the red beet extract compositions can be applicable to the medicine that preparation suppresses tumor growth.And this red beet extract compositions safety non-toxic.
Description of drawings
Accompanying drawing 1 is the photo 1 of lotus Hela glucagonoma nude mice oncocytology test of the present invention (matched group).
Accompanying drawing 2 is the photo 2 of lotus Hela glucagonoma nude mice oncocytology test of the present invention (13mg/ daily dose group).
Accompanying drawing 3 is the photo 3 of lotus Hela glucagonoma nude mice oncocytology test of the present invention (26mg/ daily dose group).
The specific embodiment
The present invention is not subjected to the restriction of following embodiment, can determine concrete embodiment according to technical scheme of the present invention and practical situation.
One, red beet extract (liquid) and preparation method thereof
Embodiment 1: Herba Gynurae bicoloris, clean, broken juice, filter paper coarse filtration, the sodium sulfite of adding 5mg in every 100ml red beet juice filtrate, by AB-8 type low pole macroporous resin chromatography purification, with the purified water of 8-15 times of column volume, washing remove impurity, the alcoholic solution eluting of 0.5% cobefrin hydrochloride 0% of 8-12 times of column volume, eluent adopts and can concentrate by the film nanofiltration of 100 molecular weight, gets red beet extract (liquid).
Embodiment 2: Herba Gynurae bicoloris, clean, broken juice, filter paper coarse filtration, the sodium thiosulfate of adding 6mg in every 100ml red beet juice filtrate, by AB-8 type low pole macroporous resin chromatography purification, with the purified water of 8-15 times of column volume, washing remove impurity, the 5% acetic acid-20% alcoholic solution eluting of 8-12 times of column volume, eluent adopts and can concentrate by the film nanofiltration of 200 molecular weight, gets red beet extract (liquid).
Embodiment 3: Herba Gynurae bicoloris, clean, broken juice, plate-and-frame filtration adds the 5mg vitamin C in every 100ml red beet juice filtrate, by DEAE-cellulose (DEAE-25) resin chromatography purification, with the purified water of 10-20 times of column volume, washing remove impurity, 20% citric acid eluting of 8-12 times of column volume, eluent adopts and can concentrate by the film nanofiltration of 500 molecular weight, gets red beet extract (liquid).
Embodiment 4: Herba Gynurae bicoloris, clean, broken juice, filter paper coarse filtration, the sodium sulfite of adding 10mg in every 100ml red beet juice filtrate, by the polar macroporous resin chromatography purification of S-8 type, with the purified water of 8-10 times of column volume, washing remove impurity, the alcoholic solution eluting of 0.1% hydrochloric acid-40% of 8-12 times of column volume, eluent adopts and can concentrate by the film nanofiltration of 200 molecular weight, gets red beet extract (liquid).
Embodiment 5: Herba Gynurae bicoloris, clean, broken juice, gauze coarse filtration, the sodium thiosulfate of adding 10mg in every 100ml red beet juice filtrate, by DEAE-cellulose (DEAE-52) resin chromatography purification, with the purified water of 10-20 times of column volume, washing remove impurity, 1% hydrochloric acid eluting of 8-12 times of column volume, eluent adopts and can concentrate by the film nanofiltration of 250 molecular weight, gets red beet extract (liquid).
Embodiment 6: Herba Gynurae bicoloris, clean, broken juice, filter paper coarse filtration, the vitamin C of adding 40mg in every 100ml red beet juice filtrate, by S-8 type macroporous resin chromatography purification, with the purified water of 8-10 times of column volume, washing remove impurity, the alcoholic solution eluting of 20% citric acid-20% of 8-12 times of column volume, eluent adopts and can concentrate by the film nanofiltration of 300 molecular weight, gets red beet extract (liquid).
Embodiment 7: Herba Gynurae bicoloris, clean, broken juice, filter paper coarse filtration, the sodium benzoate of adding 10mg in every 100ml red beet juice filtrate, by the polar macroporous resin chromatography purification of X-5 type, with the purified water of 8-10 times of column volume, washing remove impurity, the alcoholic solution eluting of 0.2% cobefrin hydrochloride 0% of 8-12 times of column volume, eluent adopts and can concentrate by the film nanofiltration of 350 molecular weight, gets red beet extract (liquid).
Embodiment 8: Herba Gynurae bicoloris, clean, broken juice, filter paper coarse filtration, the vitamin C of adding 20mg in every 100ml red beet juice filtrate, by the polar macroporous resin chromatography purification of S-8 type, with the purified water of 8-10 times of column volume, washing remove impurity, the alcoholic solution eluting of 40% citric acid-20% of 8-12 times of column volume, eluent adopts and can concentrate by the film nanofiltration of 400 molecular weight, gets red beet extract (liquid).
The embodiment gained red beet extract (liquid) of above red beet extract (liquid) and preparation method thereof adopts ultraviolet visible spectrophotometry to measure, and its absworption peak is 535nm, in solid content, and its percentage absorptivity E
1% 1cmBe 45-55 (measuring used diluent is acetic acid-sodium acetate buffer of pH5.4) that the concentration of active substance of this red beet extract is apparently higher than standard GB 8271-87 (percentage absorptivity E
1% 1cmBe not less than 3) and bibliographical information (percentage absorptivity E
1% 1cmBe 8.8) (He Chongyan, Wang Hui, Feng Chunsheng etc. beet red pigment is refining, stabilization technology research. Food Additives Used in China .1998 (2): 10-13).
Two, red beet extract compositions
Embodiment 1: above embodiment gained red beet extract (liquid), and maltodextrin and the 0.05-0.5g protein sugar of adding 2-8g among every 100ml, spray drying gets the red beet extract dried powder.
Embodiment 2: above embodiment gained red beet extract (liquid), add the starch of 0.2-2g and lactose and the 0.05-0.5g acesulfame potassium of 3-7g among every 100ml, and lyophilization gets the red beet extract compositions.
Embodiment 3: above embodiment gained red beet extract (liquid), add maltodextrin, the beta-schardinger dextrin-of 0.5-3g, the methylcellulose of 0.05-0.5g, the arabic gum of 0.05-0.5g and the acesulfame potassium of 0.05-0.5g of 2-5g among every 100ml, spray drying gets the red beet extract dried powder.
Embodiment 3: above embodiment gained red beet extract (liquid), add the starch of 0.2-2g, the lactose of 3-6g, the acesulfame potassium of 0.05-0.5g, the sucralose of 1-10mg among every 100ml, and spray drying gets the red beet extract compositions.
Embodiment 4: above embodiment gained red beet extract (liquid), add the xylitol of 2-6g, the starch of 0.2-2g, the protein sugar of 0.05-0.5g among every 100ml, and lyophilization gets the red beet extract compositions.
Embodiment 5: above embodiment gained red beet extract (liquid), add mannitol, the starch of 0.2-2g, the maltodextrin of 3-5g, the arabic gum of 0.05-0.5g and the acesulfame potassium of 0.05-0.5g of 0.3-2g among every 100ml, lyophilization gets the red beet extract compositions.
Embodiment 6: above embodiment gained red beet extract (liquid), and dextrin, the xylitol of 2-6g and the methylcellulose of 0.05-0.5g of adding 1-2g among every 100ml, lyophilization gets the red beet extract compositions.
Embodiment 7: above embodiment gained red beet extract (liquid), add the lactose of 3-7g, the acesulfame potassium of 0.05-0.5g among every 100ml, and spray drying gets the red beet extract compositions.
Embodiment 8: above embodiment gained red beet extract (liquid), and the xylitol of adding 2-10g among every 100ml, lyophilization gets the red beet extract compositions.
Above embodiment gained red beet extract compositions adopts UV-VIS spectrophotometry to measure, and its absworption peak is 535nm, percentage absorptivity E
1% 1cmBe 15-35 (measuring used diluent is acetic acid-sodium acetate buffer of pH5.4) that the concentration of active substance of this red beet extract compositions is apparently higher than standard GB 8271-87 (percentage absorptivity E
1% 1cmBe not less than 3) and bibliographical information (percentage absorptivity E
1% 1cmBe 8.8) (He Chongyan, Wang Hui, Feng Chunsheng etc. beet red pigment is refining, stabilization technology research. Food Additives Used in China .1998 (2): 10-13).
Three, the detection method of red beet extract (liquid) and compositions
1, the detection method of red beet extract (liquid)
Red beet extract (liquid) is an aubergine liquid, and available ultraviolet-visible spectrophotometer detects, and its absworption peak is 535nm.Concrete detection method is: the accurate 1ml red beet extract (liquid) of drawing, acetic acid-sodium acetate buffer with pH5.4 is diluted to 200ml, with the absorbance (A) of ultraviolet-visible spectrophotometric determination at the 535nm place, calculate red beet extract (liquid) at the percentage absorptivity at 535nm place (when referring at wavelength necessarily, solution quality concentration is 1% (W/V), thickness is the absorbance of 1cm, uses E
1% 1cmExpression).
Be folded to the solid content in the red beet extract (liquid), promptly in solid content, red beet extract (liquid) is at the percentage absorptivity E at 535nm place
1% 1cmComputing formula be:
E
1％ 1cm＝A/(C×L)
The absorbance at A------535nm place
C------measures the percentage concentration (g/100ml) of liquid
L------is the cuvette sample path length
The assay method of the solid content in the red beet extract (liquid): get red beet extract (liquid) 10ml, 105 ℃ of dryings, constant weight claims dry thing weight, calculates the solid content (W/V) of red beet extract (liquid).
2, the detection method of red beet extract compositions
The red beet extract compositions is aubergine pressed powder or granule, and available ultraviolet-visible spectrophotometer detects, and its absworption peak is 535nm.Concrete detection method is: precision takes by weighing 0.2000g red beet extract compositions, with the acetic acid of pH5.4-sodium acetate buffer dissolving and be diluted to 100ml, redilution is 10 times behind the mixing, with the absorbance (A) of ultraviolet-visible spectrophotometric determination, calculate the percentage absorptivity (E of red beet extract compositions then at the 535nm place at the 535nm place
1% 1cm).Computing formula is:
E
1％ 1cm＝A/(C×L)
The absorbance at A------535nm place
C------measures the percentage concentration (g/100ml) of liquid
L------is the cuvette sample path length
Four, the identification experiment of sucrose in the red beet extract
Get red beet extract (liquid), add the 0.05mol/L sulfuric acid solution, after boiling,, add alkaline cupric tartrate test solution again and (get cupric sulphate crystal 6.93g, be dissolved in water into 100ml with the neutralization of 0.1mol/L sodium hydroxide solution; Get sodium potassium tartrate tetrahydrate crystallization 34.6g and sodium hydroxide 10g, add water and make and be dissolved into 100ml; Time spent, promptly), heating did not see that the red precipitate of Red copper oxide generates, and illustrates in the red beet extract (liquid) not contain sucrose with two liquid mixed in equal amounts.
Five, red beet extract and compositions thereof suppress the cytology and the laboratory animal test of tumor growth effect
About Herba Gynurae bicoloris and extract thereof, still there are not the relevant cytology of tumor growth effect or the research data or the data of laboratory animal of suppressing at present.Below the foregoing description gained red beet extract and compositions thereof are carried out pharmacodynamics test, as follows:
1, red beet extract (liquid) is to external Hela cyto-inhibition result of the test
With human cervical cancer 1 epithelial cancer cell line Hela cell with the RPMI-1640 that contains 10% new-born calve serum in 96 orifice plates at 37 ℃, 5%CO
2Cultivate under the condition, the red beet extract that adds after adherent 2 hours through the filtering variable concentrations of 0.2 μ m continues to cultivate 24 and 48 hours, and red beet extract sees Table 1 to the influence of Hela cell growth counting.Red beet extract increases and strengthens gradually with concentration the inhibitory action of Hela cell growth, in 500ppm concentration, red beet extract average suppression ratio to Hela cell growth during 24 hours is 87%, be 70% during 48 hours, be the computing formula of Hela inhibitory rate of cell growth:
Suppression ratio=((matched group porocyte number-test group porocyte number)/(matched group porocyte number-0 o'clock matched group porocyte number)) * 100%
2, the red beet extract compositions is to inoculation Hela cell nude mice tumor body inhibitory action result of the test
1. to inoculating the influence of Hela cell nude mice tumor growth
With the Hela cell cultivated with trypsinization after, with 1640 culture medium (containing new calves 10% serum) with cell dilution to 1 * 10
6/ ml is injected in subcutaneous 0.2ml/ day of nude mice right side omoplate and cervical region then.Be divided into three groups of A, B and C at random, feed to red beet extract compositions 0mg, 13mg and 26mg every day respectively.Measure oral red beet extract compositions and inoculate Hela cell nude mice tumor size during 8 weeks, and weigh getting the tumor body behind the sacrifice of animal, the result shows, the size of tumor body is relevant with the dosage of weight and the oral red beet extract of nude mice, at 26mg/ during day, its tumor body volume ratio matched group little by 40% (P＜0.05), weight is hanged down 29% (P＜0.05), and in 13mg/ difference not significantly (table 2) during day.
2. lotus Hela glucagonoma nude mice oncocytology is observed
Feed and cytology's image of tumor body is seen photo 1,2 and 3 to the red beet extract compositions.Photo 1 is cytology's section of A group (matched group) lotus Hela glucagonoma nude mice tumor body, has more typical tumor cell feature, be that cell is an oval, endochylema is few, nucleus mostly is oval, visible little kernel in the part nuclear, part nuclear hyperchromatism, rounded, irregular shape illustrates that growth of tumour cell is not suppressed; Photo 2 is the 13mg/ day red beet extract compositions of feeding to B group nude mice, cytology's section of lotus Hela glucagonoma nude mice tumor body, and cell section visible cell obscurity boundary, the part cellularity is unclear, and prompting has tumor cell to necrose; Photo 3 is the 26mg/ day red beet extract compositions of feeding to C group nude mice, cytology's section of lotus Hela glucagonoma nude mice tumor body, and cell section visible cell nuclear disappears, and cellularity is unclear, and prompting has large stretch of tumor cell necrosis.Above result shows that the red beet extract compositions can cause the necrosis of tumor body cancerous cell, and the degree of its effect is relevant with dosage.
Photo 1: the cell section of matched group inoculation Hela cell tumor bearing nude mice tumor body, HE dyeing, 400 times.Visible cell is an oval, and endochylema is few, and nucleus mostly is oval, visible little kernel in the part nuclear, part nuclear hyperchromatism, rounded, irregular shape.Caryoplasm is out of proportion.And visible karyokinesis picture.Illustrate that growth of tumour cell is good.
Photo 2: the cell section of tumor bearing nude mice tumor body during the red beet extract compositions of feeding (13mg/ day/Mus), HE dyeing, 400 times.The visible cell obscurity boundary, the part cellularity is unclear.Results suggest has tumor cell to necrose.
Photo 3: the cell section of tumor bearing nude mice tumor body during the red beet extract compositions of feeding (26mg/ day/Mus), HE dyeing, 400 times.Visible cell nuclear disappears, and cellularity is unclear.Results suggest has large stretch of tumor cell necrosis.
Can think that by above result of the test the red beet extract compositions can cause the necrosis of tumor body cancerous cell, and the degree of its effect is relevant with dosage.
The red beet extract compositions shows inoculation Hela cell nude mice tumor body inhibitory action result of the test: inoculation Hela cell nude mice during day, has the effect (P＜0.05) of obvious suppression tumor growth to 26mg/ feeding.According to the effective dose of above nude mice, press literature method (chief editor such as Xu Shuyun. the pharmacological experiment methodology. People's Health Publisher, 2005) reckoning, the consumption that is equivalent to be grown up is 10g/ day (26 * 387.9/1000=10g/ day).
Six, red beet extract compositions acute toxicity testing
1, the acute toxic reaction after the oral red beet extract compositions of experiment purpose observation mice
2, experiment material Kunming mouse, male and female half and half, body weight 20 ± 2g, medical experiment animal supply center provides by Xinjiang, the red beet extract compositions, red powder to inoculation Hela cell nude mice tumor body inhibitory action result of the test gained nude mice effective dose, calculates that adult's consumption is equivalent to 10g/ day according to the red beet extract compositions.Preservation condition: room temperature.Face with preceding and be mixed with desired concn with dissolved in purified water.
3, experimental technique and result:
1. preliminary experiment: choose 10 of mices, body weight 20.32 ± 0.71g, fasting be can't help water 12 hours, the red beet extract compositions is mixed with Cmax (50%), irritates stomach with the 0.4ml/10g capacity, observes 24 hours, there is no death, and spirit and active state also no abnormal.Because of 0.4ml/10g is the maximum gastric capacity of irritating of mice, maximum tolerated dose (MTD) experiment is further carried out in the no abnormality seen reaction.
2. the mensuration of maximum tolerated dose (MTD): choose 60 of healthy mices, male and female half and half, body weight 20.58 ± 0.76g, be divided into three groups of the low dose of administration group of matched group, red beet extract compositions, the heavy dose of administration groups of red beet extract compositions at random according to the body weight equilibrium, every group of 20 animals, male and female half and half, mice fasting be can't help water 12 hours.Matched group (pure water) is pressed the 0.4ml/10g capacity and is irritated stomach twice (6 hours at interval); The low dose of administration group of red beet extract compositions 20g/kg (be equivalent to be grown up per kilogram consumption every day 120 times) with Cmax (50%), press 0.4ml/10g capacity filling stomach; The heavy dose of administration group of red beet extract compositions 40g/kg (be equivalent to be grown up per kilogram consumption every day 240 times) with Cmax (50%), press the 0.4ml/10g capacity and irritates stomach twice at (interval 6 hours).Give a certain amount of water of mice and food every day after the administration, and observe 7 days (being d) continuously, the response situation of record animal, diet, body weight change and death condition, the timely postmortem of dead animal, 7 days (being d) back surviving animals sacrificed by decapitation, core, liver,spleen,kidney, brain, testis, ovary weigh, and calculates organ coefficient.
The result does not see dead mouse, and spirit and active state also no abnormal.The mice average weight was 27.88 ± 2.30g when experiment finished.So the maximum tolerated dose of the oral red beet extract compositions of mice is 40.0g/kg, is equivalent to 240 times of per day for adults per kilogram consumption.Internal organs postmortem and histological examination no abnormality seen are not seen difference between the organ coefficient group.See table 3,4 for details.This proves this red beet extract compositions safety non-toxic.
Table 1 red beet extract (liquid) is to the influence (ten thousand cells/ml, 6 test meansigma methodss) of Hela cell growth counting
| Incubation time (hour) | 0 | 24 | 48 |
| Pigment concentration ppm | |||
| 0 (matched group) | 7.0±1.5 | 15.9±1.7 | 24.9±1.5 |
| 15.6 | 13.9±1.5 | 21.9±2.4 | |
| 31.2 | 12.0±1.0 | 20.0±1.9 | |
| 62.5 | 10.7±1.2 | 17.0±2.0 | |
| 125.0 | 10.0±1.1 | 14.5±2.1 | |
| 250.0 | 9.3±1.2 | 13.7±2.7 | |
| 500.0 | 8.2±1.0 | 12.4±1.6 |
Table 2 red beet extract compositions is to the influence (cm of inoculation Hela cell nude mice gross tumor volume and weight
3, g)
Annotate: it is not remarkable not put on target colleague data representation difference, has the different target colleague data representation significant differences (P＜0.05) of going up
The mtd test the weight of animals of table 3 red beet extract compositions filling stomach mice (x ± s)
Annotate: when 0d is administration, more equal P between group〉0.05.
The mtd test that table 4 red beet extract compositions is irritated stomach mice organ coefficient after 7 days (x ± s)
Annotate: more equal P between group 0.05
Claims (10)
- [claim 1] 1, a kind of red beet extract, it is characterized in that adopting ultraviolet visible spectrophotometry that this red beet extract liquid is measured, its absworption peak is 535nm, in solid content, its percentage absorptivity is 45 to 55, is acetic acid-sodium acetate buffer of pH5.4 and measure used diluent; Wherein, this red beet extract liquid obtains by the following method: through Herba Gynurae bicoloris clean, broken juice, filtration, adding stabilizing agent, resin chromatography purification, washing remove impurity, eluting, eluent nanofiltration obtain red beet extract liquid after concentrating.
- [claim 2] 2, red beet extract according to claim 1, it is characterized in that stabilizing agent is one or more in vitamin C, sodium benzoate, sodium thiosulfate, sodium sulfite, the different vitamin sodium of D-, consumption is to add stabilizing agent 5mg to 50mg in every 100ml red beet juice filtrate.
- [claim 3] 3, red beet extract according to claim 1 and 2, it is characterized in that resin selects anion exchange resin or macroporous resin for use, eluant is 0.1% to 1% hydrochloric acid, 1% to 10% acetic acid, 10% to 50% Fructus Citri Limoniae aqueous acid, or 10% to 50% alcoholic solution of above acid; Eluent adopts the film of 100 to 500 molecular weight, carries out nanofiltration at ambient temperature and concentrates.
- The preparation method of [claim 4] 4, a kind of red beet extract, it is characterized in that carrying out as follows: through Herba Gynurae bicoloris clean, broken juice, filtration, adding stabilizing agent, resin chromatography purification, washing remove impurity, eluting, eluent nanofiltration obtain red beet extract liquid after concentrating, adopt ultraviolet visible spectrophotometry to measure to this red beet extract liquid, its absworption peak is 535nm, in solid content, its percentage absorptivity is 45 to 55, wherein measures acetic acid-sodium acetate buffer that used diluent is pH5.4.
- The preparation method of [claim 5] 5, red beet extract according to claim 4, it is characterized in that stabilizing agent is one or more in vitamin C, sodium benzoate, sodium thiosulfate, sodium sulfite, the different vitamin sodium of D-, consumption is to add stabilizing agent 5mg to 50mg in every 100ml red beet juice filtrate.
- [claim 6] 6, according to the preparation method of claim 4 or 5 described red beet extracts, it is characterized in that resin selects anion exchange resin or macroporous resin for use, eluant is 0.1% to 1% hydrochloric acid, 1% to 10% acetic acid, 10% to 50% Fructus Citri Limoniae aqueous acid, or 10% to 50% alcoholic solution of above acid; Eluent adopts the film of 100 to 500 molecular weight, carries out nanofiltration at ambient temperature and concentrates.
- [claim 7] 7, a kind of compositions according to claim 3 red beet extract, it is characterized in that adding lactose in the red beet extract liquid, starch, dextrin, maltodextrin, cyclodextrin, mannitol, xylitol, protein sugar, acesulfame potassium, sucralose, methylcellulose, in the arabic gum one or more, total consumption is to add 2g to 10g in every 100ml red beet extract liquid, carry out the reduced vacuum drying, spray drying or lyophilization, get the red beet extract compositions, adopt ultraviolet visible spectrophotometry that the red beet extract compositions is measured, its absworption peak is 535nm, percentage absorptivity is 15 to 35, is acetic acid-sodium acetate buffer of pH5.4 and measure used diluent.
- [claim 8] 8, a kind of preparation of compositions method of red beet extract according to claim 7, it is characterized in that being undertaken: in red beet extract liquid, add lactose by following step, starch, dextrin, maltodextrin, cyclodextrin, mannitol, xylitol, protein sugar, acesulfame potassium, sucralose, methylcellulose, in the arabic gum one or more, total consumption is to add 2g to 10g in every 100ml red beet extract liquid, carry out the reduced vacuum drying, spray drying or lyophilization, get the red beet extract compositions, adopt ultraviolet visible spectrophotometry that the red beet extract compositions is measured, its absworption peak is 535nm, percentage absorptivity is 15 to 35, is acetic acid-sodium acetate buffer of pH5.4 and measure used diluent.
- [claim 9] 9, a kind of red beet extract according to claim 3 are applied in the medicine that preparation suppresses tumor growth.
- The compositions of [claim 10] 10, a kind of red beet extract according to claim 7 is applied in the medicine that preparation suppresses tumor growth.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110041727A (en) * | 2019-05-17 | 2019-07-23 | 石河子大学 | A kind of method of ultrasound assisted extraction beet red pigment |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110041727A (en) * | 2019-05-17 | 2019-07-23 | 石河子大学 | A kind of method of ultrasound assisted extraction beet red pigment |
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