CN1120708C - Medicine prepared from bolostemmatoside B for preventing and curing tumor - Google Patents
Medicine prepared from bolostemmatoside B for preventing and curing tumor Download PDFInfo
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- CN1120708C CN1120708C CN 99106201 CN99106201A CN1120708C CN 1120708 C CN1120708 C CN 1120708C CN 99106201 CN99106201 CN 99106201 CN 99106201 A CN99106201 A CN 99106201A CN 1120708 C CN1120708 C CN 1120708C
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Abstract
The present invention relates to rhizoma bolbostemmae tubeimoside II and rhizoma bolbostemmae saponin containing the tubeimosides II, which is used for preparing, preventing and treating tumors. The tubeimoside II is a chemical component extracted from the rhizoma bolbostemmae and is a natural ramification of tubeimoside I; the rhizoma bolbostemmae saponin is saponin component in the rhizoma bolbostemmae, wherein the saponin component does not contain rhizoma bolbostemmae tubeimoside III. The tubeimoside II and the saponin have a special antiinflammation function, can obviously inhibit the growth of human malignant cells and mouse transplant tumors so as to prolong the life time of tumor-bearing mice; the present invention has inducing differentiation effect on HL-60 cells and can resist the promoter action of a tumor promoter for mouse cutaneous tumors. The tubeimoside II and the saponin can be used for preventing and treating mammals, especially people's various tumors.
Description
The present invention relates to bolbstemmatoside B and contain the Rhizoma Bolbostematis saponin preparation prevention of bolbstemmatoside B and the medicine of treatment tumor.
As everyone knows, malignant tumor is a kind of commonly encountered diseases, frequently-occurring disease of serious harm health.Statistical result shows that malignant tumor is the human second largest cause of the death that is only second to cardio-cerebrovascular disorder.For malignant tumor, conventional method has surgical operation therapy, radiotherapy, chemotherapy and biological regulator treatment etc.Chemotherapeutics at present commonly used has stronger side effect more, and natural effective antitumor medicine high price not only, and patient is difficult to bear in the clinical use, and some raw material belongs to rare protection species.
The natural derivative Lobatoside H of having described bolbstemmatoside B in Chinese patent description 94104124.7,95113751.4 cures mainly vaginitis, cervicitis, cervical erosion, cervical polyp, proctitis, rectal cancer etc.Lobatoside H can also kill and wound the leukaemia and the leukaemia is had induction of differentiation.Above-mentioned patent application at this as a reference.
Bolbstemmatoside B is a kind of monomer effective ingredient of separating from the Chinese medicine Rhizoma Bolbostematis, and molecular formula is C63H98O30, and molecular weight is 1334, and chemical constitution is seen Fig. 1.The difference of it and Lobatoside H is: on 16 carbon of Lobatoside H aglycon is hydrogen, and is hydroxyl on 16 carbon of bolbstemmatoside B aglycon.Bolbstemmatoside B is the same with Lobatoside H, and good water solubility, stability are strong, and only yield is than glycosides first low (glycosides second about 0.5%, glycosides first about 1.8%).The Rhizoma Bolbostematis saponin means the saponin component in the Rhizoma Bolbostematis that does not contain Rhizoma Bolbostematis glycosides third, and preparation is simple for it, and yield is also higher, about 2.2%.
The extracting method of bolbstemmatoside B or Rhizoma Bolbostematis saponin is referring to document (Wang Yongqing, Yu Lijian etc.: Shaanxi new medicine, 10:55,1981; Kasai, Retal, Phytochemistry, 27:1439-1446,1988)
The present invention relates to bolbstemmatoside B or Rhizoma Bolbostematis saponin as the pharmaceutical composition that prevents and treat various tumors, this pharmaceutical composition comprises bolbstemmatoside B or Rhizoma Bolbostematis saponin and pharmaceutically useful carrier.
The present invention relates to as the defined preparation of drug combination method of claim 1, this method comprises that bolbstemmatoside B or Rhizoma Bolbostematis saponin mix with pharmaceutically useful carrier separately or dissolve.
The present invention relates to bolbstemmatoside B or Rhizoma Bolbostematis saponin as prevention with treat various malignant tumor medicines and carry out application in the mammal of this prevention or treatment at needs.
Bolbstemmatoside B or Rhizoma Bolbostematis saponin can mix with any pharmaceutically useful carrier or dissolve, as in skin, mucosa, gastrointestinal and pharmaceutically suitable carrier of parenteral.This pharmaceutical composition uses with the form of conventional pharmaceutical formulation, as tablet, granule, powder, capsule, the cachet of solid form, fasten agent etc., or the ointment of semi-solid form, ointment etc., or injection of liquid form, suspending agent, syrup, Emulsion, liniment etc.Use pharmaceutically useful excipient and additive in this pharmaceutical composition, these pharmaceutically useful excipient and additive comprise nontoxic compatible filler, binding agent, disintegrating agent, buffer agent, antiseptic, antioxidant, lubricant, correctives, thickening agent, coloring agent, emulsifying agent, stabilizing agent etc.As lactose, citric acid, stearic acid, magnesium stearate Gypsum Fibrosum powder, sucrose, corn starch, Pulvis Talci, gelatin, agar, pectin, Oleum Arachidis hypogaeae semen, cocoa butter, ethylene glycol, glucose, procaine hydrochloride, lidocaine hydrochloride, ascorbic acid etc.This pharmaceutical composition can be by the common process preparation of various preparations.
The present invention relates to bolbstemmatoside B or Rhizoma Bolbostematis saponin medicine as the prevention malignant tumor, can be used as food additive, cosmetics additive joins in food or the cosmetics, also can be used as the various precancerous lesions of medicine composite for curing, as esophagus hypertrophy, leukoplakia vulvae, atrophic gastritis, chronic cervicitis, cervical polyp, rectal polyp etc.
The present invention relates to bolbstemmatoside B or Rhizoma Bolbostematis saponin pharmaceutical composition as the treatment malignant tumor, can be used for leukemia, also can be used for various entity swollen body tumors, as esophageal carcinoma, hepatocarcinoma, gastric cancer, cancer of pancreas, colon cancer, rectal cancer, cervical cancer, ovarian cancer, breast carcinoma, Folium Nicotianae preparatum cancer, oral cancer, lip cancer, skin carcinoma, bladder cancer, chorionic epithelioma, carcinoma of parotid gland, lymphoma, osteoma, melanoma and various intracranial tumors etc.The content of bolbstemmatoside B or Rhizoma Bolbostematis saponin is the about 0.001-100mg of every kg body weight every day in the made various combination of oral medication, preferably about 0.01-50mg, more excellent about 0.05-30mg, best about 0.1-10mg.Divide every day and take for 2-4 time, 10-30 days is a course of treatment, and general medication 3-6 course of treatment, each, the interval was 10-30 days course of treatment.Made medicinal composition for injections can supply intramuscular injection or intravenous drip, and wherein the content of bolbstemmatoside B or Rhizoma Bolbostematis saponin is every kg body weight 0.001-50mg every day, preferably about 0.01-25mg/kg, more excellent about 0.05-15mg, best about 0.1-3mg.Divide 1-3 use every day, 10-30 days is a course of treatment, and general medication 3-6 course of treatment, each, the interval was 10-30 days course of treatment.Yet accurate dose should be determined by the doctor, depends primarily on patient's age, body weight, the state of an illness, reaction etc.
We find in experiment, bolbstemmatoside B or Rhizoma Bolbostematis saponin show special anti-inflammatory effect, can significantly suppress the growth of human malignant lesion's cell and mice transplanted tumor, prolong tumor-bearing mice life cycle, human promyelocytic leukemia cell (HL-60 cell) is induced differentiation effect, can resist carcinogenic promoting agent the tumorigenic facilitation of mouse skin.
Enumerate experiment content and result thereof below, can be used for preparing the evidence of various preventions and treatment malignant tumor medicine as bolbstemmatoside B or Rhizoma Bolbostematis saponin.One. the test of pesticide effectiveness
1. anti-inflammatory edema test
7 age in week female ICR Mice Auricle two sided coatings 12-0-myristoyl Fo Bo-13-acetas (hereinafter to be referred as TPA, 2 μ g are dissolved in 20 μ l acetone) bring out edema.The bolbstemmatoside B solution of 30 minutes topical application variable concentrations before the coating TPA.The reagent matched group only is coated with the solvent (acetone) with volume.With vernier caliper measurement Mus ear thickness, be accurate to 0.01 after TPA handles 5 hours, dropsy of ear is represented with the thickness increase of ear.TPA handles that the increase of Mus ear thickness reaches maximum after 5 hours under the above-mentioned experiment condition.The consumption of bolbstemmatoside B is divided into 4 dosage groups: 7.5 * 10
-3μ mol/ ear, 3.7 * 10
-2μ mol/ ear, 7.5 * 10
-2μ mol/ ear, 1.1 * 10
-1μ mol/ ear.The result shows that the Mus dropsy of ear that bolbstemmatoside B brings out TPA has obvious suppression effect (seeing Table 1), and the dose-effect dependence is remarkable.The anti-inflammatory edema effect of Rhizoma Bolbostematis saponin slightly is inferior to bolbstemmatoside B, but is better than Lobatoside H (data does not show).Bolbstemmatoside B is calculated as follows the suppression ratio of Mus otitis edema:
The increase suppression ratio P value of the inhibitory action experiment condition Mus ear thickness of the Mus otitis edema that table 1 bolbstemmatoside B topical application is brought out TPA
bThe P value
c
(* 10
-2Mm) a (%) contrast (TPA 2 μ g/ ears) 10.6 ± 0.76
Lobatoside H 7.5 * 10-3 μ mol/ ear 7.8 ± 0.51 24.8＜0.053.7 * 10-2 μ mol/ ear 6.8 ± 0.48 35.2＜0.017.5 * 10-2 μ mol/ ear 5.6 ± 0.55 46.7＜0.0011.1 * 10-1 μ mol/ ear 1.8 ± 0.22 83.8＜0.001
Bolbstemmatoside B 7.5 * 10-3 μ mol/ ear 6.8 ± 0.48 38.1＜0.01＞0.053.7 * 10-2 μ mol/ ear 4.6 ± 0.43 59.0＜0.001＜0.017.5 * 10-2 μ mol/ ear 1.6 ± 0.34 86.3＜0.001＜0.0011.1 * 10-1 μ mol/ ear 0.0 ± 0.23 100.0＜0.001＜0.05a, meansigma methods ± standard error.B, P value (t check) compare with matched group.C, P value (t check) compare with same dose Lobatoside H group.
2. to the inhibitory action of human malignant lesion's cell growth
Trial drug: bolbstemmatoside B
Test tumor strain: human promyelocytic leukemia cell (HL-60).
Test method: in diameter was the plastics saucer of 35mm, culture medium was to contain 1640 of 10% hyclone with the HL-60 cell inoculation, placed 37 ℃, the incubator of moisture-saturated, 95% air, 5% carbon dioxide to cultivate.The bolbstemmatoside B solution of variable concentrations is added in the culture medium, observe inhibition effect respectively tumor cell growth.Meanwhile, the physiological saline solution of adding equal volume compares experiment in same culture.In 1-3 days, use trypan blue method living cell counting number every day behind the adding bolbstemmatoside B.
Result of the test: Fig. 2 represents the inhibition effect of bolbstemmatoside B to the HL-60 cell strain growth.Bolbstemmatoside B is better than Lobatoside H (referring to patent CN 1146895A) to the inhibitory action of H-60 cell growth.The Rhizoma Bolbostematis saponin slightly is inferior to bolbstemmatoside B to the inhibitory action of HL-60 cell strain growth, but is better than Lobatoside H (data does not show).
3. to growth of mice transplantable tumor and the influence of animal survival phase
Trial drug: bolbstemmatoside B
Experimental animal: BALB/c mouse
Test tumor: the portable sarcoma 180 (S of mice
180), ehrlich carcinoma (Ehrlich ascitescarcinoma).
Test method: random packet.Each is organized mice and weighs and the subcutaneous or intraperitoneal injection 10 at left groin in experiment beginning day
7Individual tumor cell.Inoculate the bolbstemmatoside B solution that began muscle or lumbar injection variable concentrations in back second day.Negative control group and positive controls inoculation similar number oncocyte, physiological saline solution or the 5-fluorouracil solution or the Lobatoside H solution of muscle or lumbar injection equal volume simultaneously.Can lay one's hand on 5-6 day and the tumor piece behind the subcutaneous vaccination tumor cell.Behind the subcutaneous vaccination tumor cell 13 days, crane one after weighing and put to death mice, the complete tumor of peeling off is weighed.Calculate and respectively organize the heavy and standard error of average tumor.Tumour inhibiting rate is calculated as follows: tumour inhibiting rate (%)=(1-A/B) * 100
It is heavy that wherein A represents the average tumor of test group, and B represents the average tumor of matched group heavy.
Abdominal cavity inoculation oncocyte person, the death condition of record tumor animal, behind tumor inoculation 35 days.
Result of the test: table 2 shows the influence of bolbstemmatoside B to the animal tumor growth.Table 3 shows that bolbstemmatoside B is to the tumor animal prolongation of life cycle.The Rhizoma Bolbostematis saponin slightly is inferior to bolbstemmatoside B to the inhibitory action of animal tumor growth with to the prolongation effect of tumor animal life cycle, but is better than Lobatoside H (data does not show).Table 2: go up Bulbus Fritillariae Uninbracteatae glycosides second to mice transplanted tumor sarcoma 180 (S
180) growth inhibitory action
Moving dosage (mg/ group thing Kg/ daily weight
b(g) tumor is heavy
b(g) tumour inhibiting rate
cP
d
The a few days number
) a(%) contrasts 10 18.5 ± 0.31 19.6 ± 0.54 2.68 ± 0.285 fluorouracil 10 3 * 12 18.6 ± 0.28 18.6 ± 0.95 1.95 ± 0.24 27.2＜0.05 Bolbostemma paniculatum glucoside A 10 1 * 12 18.5 ± 0.24 20.0 ± 0.61 2.06 ± 0.25 23.3＜0.05 Bolbostemma paniculatum glucoside A 10 2 * 12 18.5 ± 0.24 19.3 ± 0.57 1.85 ± 0.13 31.3＜0.05 Bolbostemma paniculatum glucoside A 10 3 * 12 18.7 ± 0.36 18.1 ± 0.55 1.26 ± 0.31 52.9＜0.01 bolbstemmatoside B 10 1 * 12 18.4 ± 0.19 20.2 ± 0.43 1.52 ± 0.21 43.3＜0.01 bolbstemmatoside B 10 2 * 12 18.5 ± 0.24 19.2 ± 0.52 1.35 ± 0.17 49.8＜0.01 bolbstemmatoside B 10 3 * 12 18.5 ± 0.27 18.0 ± 0.47 1.07 ± 0.13 60.1＜0.001a, abdominal cavity medication after processing pre-treatment. B, meansigma methods ± standard error.C, tumour inhibiting rate (%)=(1-A/B) * 100, A represent the average tumor of experimental group heavy, and B represents the average tumor of matched group heavy.D, P value (t check) compare with matched group.Table 3 bolbstemmatoside B influences tumor kind group dosage to lotus tumor BALB/c mouse life cyclea(mg/Kg/ number of mice The average survival time number of days T/C
b
Day * number of days (only) (mean value SD) ehrlich carcinoma contrasts 0 20 18.13 ± 3.24
Bolbstemmatoside B 2.5 * 10 20 32.52 ± 4.16 1.79
Lobatoside H 2.5 * 10 20 27.20 ± 3.11 1.50S180 contrast 0 20 14.40 ± 1.88
Bolbstemmatoside B 2.5 * 10 20 27.60 ± 3.64 1.92
Intramuscular injection medicinal liquid next day of behind Lobatoside H 2.5 * 10 20 20.30 ± 1.95 1.41a. abdominal cavities inoculation oncocyte.B.T/C: wherein T represents the The average survival time number of days of experimental group, and C represents the The average survival time number of days of matched group.
4. to the induction of differentiation of HL-60 cell
Trial drug: bolbstemmatoside B
Test method: the bolbstemmatoside B aqueous solution that in the HL-60 cell culture fluid, adds 2.4 μ mol/L, cultivated 5 days by 2 described conditions, observe metamorphosis, and the HL-60 cell is carried out nitroblue tetrazolium (NBT) reduction test, zymosan phagocytosis test, and nonspecific esterase stain test.Matched group is divided into positive control and negative control group.The Lobatoside H aqueous solution that in the HL-60 cell culture fluid, adds 1mmol/L retinoic acid or 2.4 μ mol/L, and experimentize by the method for experimental group, be positive control.
Result of the test: the similar normal metamyelocyte of cellular morphology behind the HL-60 cell differentiation of hatching jointly with bolbstemmatoside B, the nonspecific esterase stain feminine gender, and negative control group is induced the cell of differentiation to be no more than 6%.Result of the test (seeing Table 4) shows that bolbstemmatoside B is induced differentiation effect to the HL-60 cell.The Rhizoma Bolbostematis saponin slightly is inferior to bolbstemmatoside B to the differentiation effect of inducing of HL-60 cell, but is better than Lobatoside H (data does not show).Table 4 bolbstemmatoside B is to the induction of differentiation of HL-60 cell
a
Group is cultivated metamyelocyte or neutral nitrogen blue tetrazolium reduction zymosan phagocytic rate
Natural law granulocyte (%)
b(%)
b(%)
b
Contrast 3 4.44 ± 0.41 5.77 ± 0.50 2.97 ± 0.21
The average result of 5 4.74 ± 0.35 6.21 ± 0.42 3.75 ± 0.34 vitamin A acids 3 68.27 ± 8.11 65.57 ± 6.60 63.56 ± 6.14 (1mmol/L) 5 65.26 ± 5.08 52.06 ± 4.55 61.54 ± 4.68 bolbstemmatoside Bs 3 33.40 ± 2.98 27.35 ± 2.56 32.46 ± 3.86 (2.4 μ mol/L), 5 65.56 ± 5.21 57.89 ± 6.86 44.86 ± 3.76 Bolbostemma paniculatum glucoside As 3 28.60 ± 3.35 22.84 ± 2.64 28.96 ± 4.49 (2.4 μ mol/L) three parts of parallel tests of 5 60.84 ± 3.64 53.16 ± 6.33 40.58 ± 3.76a.. B. meansigma methods ± standard error.Two. toxicological test
1. acute toxicity test
Select for use the ICR mice to test with muscle and intraperitoneal injection method.
Result of the test: the LD of intramuscular injection bolbstemmatoside B
50Be 45.66mg/Kg, the LD of Lobatoside H
50Be 40.28mg/Kg, the LD of Rhizoma Bolbostematis saponin
50Be 42.36mg/Kg.The LD of the bolbstemmatoside B of intraperitoneal administration
50Be 20.9mg/Kg, the LD of Lobatoside H
50Be 18.7mg/Kg, the LD of Rhizoma Bolbostematis saponin
50Be 19.6mg/kg.
2. to the toxic action of normal cultured cell
Adopt colorimetry to test the toxic action of bolbstemmatoside B to the T-Lymphoblastoid strain (MT-2 cell) of In vitro culture, result of the test (seeing Table 5) shows, when the concentration of bolbstemmatoside B is reduced to 40 μ g/ml by 80 μ g/ml, cytotoxicity significantly descends, when concentration was lower than 40 μ g/ml, toxicity was very low.The CD of Rhizoma Bolbostematis Yi Xibao poison
50Be 68 μ g/ml, the cytotoxic CD of Lobatoside H
50Be 59 μ g/ml.The cytotoxic CD of Rhizoma Bolbostematis saponin
50Be 64 μ g/ml.Table 5 bolbstemmatoside B is to the toxic action of MT-2 cell
Concentration (μ g/ml) cytotoxic percentage rate (%) bolbstemmatoside B 10 1.5
20 2.0
40 10.0
80 84.6 Lobatoside H 10 2.2
20 6.5
40 13.0
80 96.4
3. anti-cancer test
Adopt the classical experimental model that brings out mice two-stage cutaneous papilloma, swash cancer with dimethylbenzanthracene, TPA urgees cancer.Experimental group every Mus in short cancer is smeared skin of back with 0.5mg bolbstemmatoside B solution, 18 weeks of Therapy lasted at every turn.
Result of the test: skin of back is smeared bolbstemmatoside B and is suppressed to occur on the mouse skin tumor fully and (use the effect of 1mg Lobatoside H identical with every Mus at every turn, referring to patent CN 1146895A), and the band ratio of outflow of matched group is 80%, and every Mus average tumor number is 11.36 (Fig. 3).The application of bolbstemmatoside B does not hinder the increase of mice body weight.The anti-cancer effect of Rhizoma Bolbostematis saponin slightly is inferior to bolbstemmatoside B, but is better than Lobatoside H (data does not show).Three, prevent and treat the test of the malignant tumor mechanism of action
1, to the synthetic influence of tumor cell DNA (deoxyribonucleic acid) (DNA)
With
3The H-thymidine method of mixing is measured bolbstemmatoside B and Lobatoside H to people's pancreas cancer and the synthetic influence of stomach cancer cell DNA.Pancreas cancer or stomach cancer cell were cultivated 24 hours, added bolbstemmatoside B as experimental group or add its solvent and organize in contrast.Pancreas cancer or stomach cancer cell were cultivated after 24,48,72 hours, then with
3H-thymidine labelling 1 hour, and measure the radioactivity that it mixes acid-insoluble substances.Result of the test shows that bolbstemmatoside B suppresses
3The H-thymidine mixes DNA component (seeing Table 6), points out the normal synthetic and ripe inhibition that has been subjected to bolbstemmatoside B of these tumor cells DNA.Result of the test shows that also the synthetic inhibition of different carcinoma cell DNA occurs in different times.The Rhizoma Bolbostematis saponin slightly is inferior to bolbstemmatoside B to the synthetic inhibitory action of tumor cell DNA, but is better than Lobatoside H (data does not show).Table 6 bolbstemmatoside B is to people's pancreas cancer and the synthetic influence of stomach cancer cell DNA
*
It is synthetic that the 3H-thymidine mixes DNA
Cultivate date (day) cell condition 123 pancreas cancers contrast 3.96 ± 0.17 4.03 ± 0.13 6.54 ± 0.71
(100) (100) (100)
Bolbstemmatoside B 3.32 ± 0.46 3.16 ± 0.38 2.48 ± 0.42
(3μg/ml) (93.3) (87.1) (50.7，P＜0.001)
Lobatoside H 3.85 ± 0.41 3.77 ± 0.31 3.92 ± 0.42
(3 μ g/ml) (97.1) (93.3) (59.9) gastric cancer contrast 9.18 ± 0.51 11.35 ± 0.35 8.33 ± 0.21
(100) (100) (100)
Bolbstemmatoside B 5.96 ± 0.68 5.88 ± 0.76 4.98 ± 0.65
(10μg/ml) (69.7) (58.5) (66.4)
Lobatoside H 6.51 ± 0.38 7.57 ± 0.66 6.26 ± 0.45
The value that provides in (10 μ g/ml) (70.9) (66.7) (75.2) * table is represented the meansigma methods of three parts of parallel tests, and the value in the bracket is to contrast the percent as 100%
2,
3H mixes cultured cell C3H10T1/2 and clones the test of 8 phospholipid
C3H10T1/2 is cloned 8 cell inoculations in culture bottle,, place 37 ℃, the incubator of moisture-saturated, 95% air, 5% carbon dioxide to cultivate with containing the DMEM culture medium of 10% hyclone.Cell is transferred in the plastics saucer that diameter is 35mm after pancreatin is handled, and making its density is 2 * 10
5Cell/ml.Cultivate after 48 hours, bolbstemmatoside B adds with the form of aqueous solution, makes its final concentration in culture medium be respectively 5 μ g/ml, 10 μ g/ml, 15 μ g/ml.Meanwhile, matched group adds the aquae destilIata sterilis of equal volume, cultivates under the same conditions.Cell after cultivating in 1 hour, and test group and matched group add TPA (5 * 10 simultaneously
-8Mol/L) and
3H choline (4 μ ci/ dish) continues to cultivate 4 hours.
With
3The cell that the H choline was hatched is together handled the back collection through pancreatin, and rinses with phosphate buffer and to be suspended in the phosphate buffer of 150 μ l after washing.Lipid in the cell extracts with improved Bligh and Dyer method.After being about to cell freeze thawing once, (1/2, v/v), wherein methanol contains 2% acetic acid to add the chloroform/methanol of 300 μ l.Cell suspension is handled with ice bath type supersonic generator, adds 100 μ l chloroforms again, and vibration mixing, mixed liquor are divided into chloroform phase and liquid phase after centrifuge is centrifugal.Radioactivity with liquid flashing counter measuring chloroform phase.Protein content is measured with the LowryShi method of improvement.
Result of the test: as shown in Figure 4, bolbstemmatoside B has the TPA of inhibition to be strengthened
3H mixes the effect of cell phospholipid, still can measure this effect when the concentration of bolbstemmatoside B is lower than 5 μ g/ml, and shows the dose-effect dependence.Bolbstemmatoside B suppresses TPA to be strengthened
3The effect that H mixes cell phospholipid is than Lobatoside H strong (referring to patent CN1146895A).The Rhizoma Bolbostematis saponin suppresses TPA to be strengthened
3The effect that H mixes cell phospholipid slightly is inferior to bolbstemmatoside B, but is better than Lobatoside H (data does not show).
3. the intracellular receptor experiment of Lobatoside H and TPA competition HL-60
HL-60 cell 1640 culture medium culturings that contain 10% hyclone.
Test method: the HL-60 cell that centrifugal collection is cultivated, after phosphate buffer (PBS) washing, be suspended in the buffer that contains 50mM Tris-HCl, handle with the ultrasonic cell disintegration instrument, collect supernatant, get cell extract, add the Tris-HCl buffer and adjust protein concentration to 200 μ/ml in cell extraction, the bolbstemmatoside B that adds variable concentrations again reaches
3The TPA of H labelling puts into incubator, hatches 20 minutes for 39 ℃, adds the filter membrane sucking filtration immediately, behind the acetone rinsing filter membrane, filter membrane is put into scintillation vial, with its radioactivity of liquid flashing counting determining.
Result of the test: bolbstemmatoside B competitive inhibition TPA is at the intracellular receptor of HL-60.Four, the pharmacokinetics of bolbstemmatoside B test
1. the absorption test of bolbstemmatoside B
Get 14 of NIH mices, irritate stomach (100mg/kg) administration with bolbstemmatoside B solution, different time is respectively put to death 2 mices after the administration, take out whole gastrointestinal tract and content, make homogenate, with making blank with method, promptly put to death mice after the matched group administration, measure the medicament contg that remains in digestive tract and the content.
Result of the test: the response rate of locating bolbstemmatoside B in the dead's gastrointestinal after the administration at once is 99.0%, and 0.5,1,2,4,8,12 hour the response rate is respectively 88.8%, 79.3%, 56.7%, 38.0%, 16.6% and 0 after the administration.
2. tissue distribution assays
Get 20 of NIH mices, 4 of rats, every Mus intramuscular injection bolbstemmatoside B 60mg/kg after 1 hour, cores, liver, spleen, lung, kidney and cerebral tissue 1 gram, adds normal saline 3ml homogenate, measures the content of bolbstemmatoside B in each tissue.
Result of the test: bolbstemmatoside B is the highest liver and spleen intensive amount, takes second place at blood, lung and heart intensive amount, and is minimum at kidney and brain intensive amount.
3. the test that combines of bolbstemmatoside B and plasma protein, histone
Adopt equilibrium dialysis mensuration bolbstemmatoside B and blood plasma, liver, the proteic combination rate of nephridial tissue.
Result of the test: bolbstemmatoside B and plasma protein and liver, the proteic combination rate of nephridial tissue are respectively
16.3％、26.0％、18.1％。
4. excretion test
Get 10 of NIH mices, intramuscular injection 60mg/kg bolbstemmatoside B is collected twenty-four-hour urine liquid and is got bile, measures wherein bolbstemmatoside B content.
Result of the test: do not have bolbstemmatoside B in bile and urine, the supposition bolbstemmatoside B may be discharged from urine or bile with the form of metabolite.
Five, clinical test result just
1. the inventor adopts the Rhizoma Bolbostematis saponin suppository that contains bolbstemmatoside B of the present invention embodiment 4 preparations to carry out the clinical trial observation.
Case is selected
Cervicitis patient's 36 examples, cervical polyp patient 10 examples become patient's 3 examples, cervical cancer patient's 8 examples between cervix uteri.
Using method
Change, cervical cancer patient between chronic cervicitis, cervical polyp, cervix uteri: clean secretions with aseptic cotton rod earlier, then suppository is inserted the cervix uteri place.The next day once, ten times is a course of treatment.Treat 1-5 the course of treatment, decide on the state of an illness.
Result of the test
The present invention is very remarkable to the curative effect of chronic cervicitis, and effective percentage reaches 99%.To the complication of chronic cervicitis, significant curative effect is arranged as cervical polyp, cervical erosion, cervix uteri plumpness etc.The present invention can make to become between cervix uteri and take a turn for the worse, and early cervical carcinoma is had tangible curative effect.No local irritation does not cause allergic reaction.
2. the inventor adopts the Rhizoma Bolbostematis saponin tablet that contains bolbstemmatoside B of the present invention embodiment 1 preparation to carry out the clinical trial observation.
Case is selected
Advanced esophageal cancer patient 8 examples, esophageal carcinoma patients after surgery 12 examples, late gastric cancer patient's 7 examples, gastric cancer patients after surgery 9 examples.
Using method
Take every day 3 times, each 1 (containing Rhizoma Bolbostematis saponin 50mg), 20 days is a course of treatment, 4 courses of treatment of medication, 10 days at interval each course of treatment.
Result of the test
The advanced esophageal cancer patient on average prolongs 126 days life cycle, and the esophageal carcinoma patients after surgery on average prolongs 626 days life cycle.Late gastric cancer patient on average prolongs 183 days life cycle, and the gastric cancer patients after surgery on average prolongs 742 days life cycle.
Fig. 1: the chemical structural formula of Lobatoside H, glycosides second and glycosides third
Fig. 2: the topical application bolbstemmatoside B is to the inhibitory action of the short cancer process of mice.Abscissa is all numbers of short cancer among the figure, and vertical coordinate represents to be with tumor Mus number (%) among the A figure, and vertical coordinate is represented tumor number/Mus among the B figure.Zero--zero, represent matched group, ●--●, represent experimental group.
Fig. 3: bolbstemmatoside B strengthens the inhibition effect that 3H mixes cultured cell phospholipid to TPA.Abscissa is represented the concentration (μ g/ml) of bolbstemmatoside B among the figure, and vertical coordinate is suppression ratio (%).
Fig. 4: bolbstemmatoside B is to the inhibitory action of HL-60 cell growth.Abscissa is for cultivating natural law among the figure, and vertical coordinate is a viable count (viable count/ml).Zero--zero representative contrast (adding simultaneously with the volume solvent in the culture fluid), ●--● represent bolbstemmatoside B consumption 1.9 μ mol/L, *-* represents bolbstemmatoside B consumption 3.8 μ mol/L, △--△ represents bolbstemmatoside B consumption 7.6 μ mol/L, and ■--■ represents bolbstemmatoside B consumption 15.2 μ mol/L.
Six, embodiment
Following formula examples has illustrated dosage form of the present invention.
Embodiment 1: tablet,The NO amounts of components
Mg/ sheet mg/ sheet 1 bolbstemmatoside B (or Rhizoma Bolbostematis saponin) 50 100 2 lactose USP 122 123 3 rice starches, food stage is 30 40
10% pure water slurry 4 corn starchs, food stage 95 130 5 magnesium stearate 37
Add up to 300 400
Manufacture method
The 1st and the 2nd component of mixing is 15 minutes in suitable mixer, the 3rd component and mixture granulation.If desired, grind moist granule, and make it dry by a primary dcreening operation.If desired, dried granules is sieved, and mixed 10-15 minute with the 4th.Adding the 5th mixed 1-3 minute.In suitable tablet machine, mixture is pressed into suitable size and weight.
Embodiment 2: capsuleThe NO amounts of components
Mg/ grain mg/ grain 1 bolbstemmatoside B (or Rhizoma Bolbostematis saponin) 50 100 2 lactose USP 106 120 3 corn starchs, food stage 40 73 4 magnesium stearate NF 47
Add up to 200 300
Manufacture method
The 1st, 2 and the 3rd were mixed 10-15 minute in suitable mixer, add the 4th and mixed 1-3 minute.On suitable encapsulation machine with in the capsule that it is suitable that this mixture is packed into.
Embodiment 3: injectionBolbstemmatoside B (or Rhizoma Bolbostematis saponin) 10g or 50g glucose 490g or 450g water for injection add to 10000ml
Embodiment 4: suppositoryWith 10000 of production invention products is that used raw material of example and proportioning thereof are: bolbstemmatoside B (or Rhizoma Bolbostematis saponin) 300g or 600g Tween 80 800g or 800g cacao bean ester (or semi-synthetic fatty acid glycerine) add to the 10000g manufacture method to be mixed: the proportioning by present embodiment takes by weighing bolbstemmatoside B (or Rhizoma Bolbostematis saponin), Tween 80 and cacao bean ester (or semi-synthetic fatty acid glycerine), cacao bean ester (or semi-synthetic fatty acid glycerine) is put into stainless-steel pan heating in water bath to major part to be dissolved, stop heating, add Tween 80 and bolbstemmatoside B (or Rhizoma Bolbostematis saponin) then respectively, be stirred to mix homogeneously with the rustless steel blender, just obtain hybrid medicine of the present invention.The preparation of lubricant:, make lubricant with green soap and each abundant mixing and stirring of 80% medical alcohol a and 5 times of glycerol.Molding: on the bolt mould, coat the lubricant of having prepared.Hybrid medicine of the present invention is poured in the bolt mould to overflowing die orifice, allowed its natural cooling.After treating to solidify fully, cut with cutter and to overflow part.Opening mold takes out suppository from mould then.Aseptic packaging: quality examination is carried out in the prepared agent of fastening, and every weight should be 1g, contains bolbstemmatoside B (or Rhizoma Bolbostematis saponin) 30mg or 60mg.Pack cobalt 60 sterilizations after the passed examination.
Claims (1)
1. bolbstemmatoside B or the Rhizoma Bolbostematis saponin that contains bolbstemmatoside B are in the purposes of preparation prevention and treatment people and the various tumour medicines of mammal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99106201 CN1120708C (en) | 1999-05-05 | 1999-05-05 | Medicine prepared from bolostemmatoside B for preventing and curing tumor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99106201 CN1120708C (en) | 1999-05-05 | 1999-05-05 | Medicine prepared from bolostemmatoside B for preventing and curing tumor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1273113A CN1273113A (en) | 2000-11-15 |
| CN1120708C true CN1120708C (en) | 2003-09-10 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99106201 Expired - Fee Related CN1120708C (en) | 1999-05-05 | 1999-05-05 | Medicine prepared from bolostemmatoside B for preventing and curing tumor |
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| Country | Link |
|---|---|
| CN (1) | CN1120708C (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106236616A (en) * | 2015-06-05 | 2016-12-21 | 于立坚 | Cosmetics new raw material all tobeimosides |
| CN108003214B (en) * | 2017-12-22 | 2020-05-19 | 成都普思生物科技股份有限公司 | Saponin compound extracted from rhizoma bolbostemmae, and its preparation method and application |
| CN108175769A (en) * | 2017-12-22 | 2018-06-19 | 南京师范大学 | Bolbostemma paniculatum glucoside A is preparing the application in treating medicine for treating rheumatoid arthritis |
| CN108553474A (en) * | 2018-05-02 | 2018-09-21 | 陕西科技大学 | Glucosides tobeimosides third are preparing the application in treating special hypotype leukemia medicament |
| CN111675747B (en) * | 2020-07-30 | 2021-04-27 | 中国医学科学院医药生物技术研究所 | Antitumor drug and application |
-
1999
- 1999-05-05 CN CN 99106201 patent/CN1120708C/en not_active Expired - Fee Related
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| CN1273113A (en) | 2000-11-15 |
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