CN1276751C - Application of scutellaria root extract in preparation of anti-stomach cancer medicine - Google Patents
Application of scutellaria root extract in preparation of anti-stomach cancer medicine Download PDFInfo
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- CN1276751C CN1276751C CN 03109942 CN03109942A CN1276751C CN 1276751 C CN1276751 C CN 1276751C CN 03109942 CN03109942 CN 03109942 CN 03109942 A CN03109942 A CN 03109942A CN 1276751 C CN1276751 C CN 1276751C
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Abstract
The present invention discloses the application of scutellaria extracts to the preparation of medicine for resisting stomach cancer, wherein scutellaria extracts particularly refer to extracts with a flavone mother body structure, can be scutellarin or scutellaroside, or can be a mixture of scutellarin and scutellaroside and salts prepared from scutellaria extracts acceptable to pharmacy. The medicine prepared from scutellaria extracts for resisting stomach cancer can directly inhibit the division of cancer cells, and performs the function of strongly inducing the apoptosis of cancer cells. The medicine has no obvious effect on normal cell growth period and apoptosis. Moreover, the medicine also performs the function of inhibiting vascularization inside stomach cancer, particularly revascularization in oxygen deficiency environment; consequently, the blood supply of stomach cancer is effectively cut off so that stomach cancer is denatured and necrosed. The medicine obviously inhibits and kills the growth of transplanted stomach cancer, and has no toxic or side effect. The scutellaria extracts have very positive practical significance for preparing medicine for treating or preventing stomach cancer.
Description
Technical field
The present invention relates to the application in the anti-gastric cancer medicine of preparation of the purposes of Radix Scutellariae extract, particularly Radix Scutellariae extract.
Technical background
The sickness rate of gastric cancer has downward trend year by year in worldwide, but at the mortality rate of China's gastric cancer still in the first place, according to incompletely statistics, mortality of gastric carcinoma is occupied the 2%-2.6% in people's death, in the malignant tumor cause of the death, account for 23%, because chemotherapy is with great toxicity, and gastric cancer is relatively poor to the sensitivity of ray, so the application of radiotherapy is very restricted in curing gastric cancer, the treatment of gastric cancer is at present still based on surgery, chemotherapy and radiation only as a kind of means of auxiliary or palliative treatment, though Therapeutic Method is updated in recent years, still pace up and down at 20-30% by 5 years total survival rates.Even by the radiotherapy combined chemotherapy, its 5 years survival rates are also only below 30%.Existence person more than 5 years also was greatly affected for quality of life in later stage, and recurrence especially easily.So can not treat gastric cancer effectively at present.
Because the histopathology of gastric cancer is very complicated, as include: papillary adenocarcinoma, tubular adenocarcinoma, poorly differentiated adenocarcinoma, mucinous adenocarcinoma, draw and guard against cell carcinoma, undifferentiated carcinoma and special type cancer etc., different parts and dissimilar also brings very big difficulty to its treatment and medication.
China applies for a patent CN-1352965A and discloses a kind of medicine for the treatment of esophagus, stomach, pulmonary carcinosis, it adopts a large amount of Chinese crude drugs is that raw material is prepared, though the inventor introduces multiple cancer total effective rate is reached 98.3%, but so many Chinese crude drug is formed, component and content is the branch of weight not all, be difficult in the actual therapeutic thus and apply in a flexible way, suit the remedy to the case at different condition of illness.
Baikal skullcap root (Scutellaria baicalensis Georgi) head is stated from Shennong's Herbal, has another name called Huang Wen, no a kind of reed mentioned in ancient books etc.According to the report of existing document, the pharmacological effect bitter in the mouth of Radix Scutellariae, cold in nature, function is let out excess-fire, except that damp and hot, detoxifcation, hemostasis, antiabortive, various bacteria all there is inhibitory action, to influenza virus, dermatophytes also has inhibitory action, and heat extraction is arranged, detoxifcation, calmness, blood pressure lowering, function of gallbladder promoting, smooth muscle spasm is removed in diuresis, suppresses effects such as intestinal tube wriggling.The acetone extract of Radix Scutellariae also is expected to become the Natural antioxidant of preserving processed food.Root, the leaf extract of Radix Scutellariae have antiulcer activity.
Baicalin in the Radix Scutellariae extract and baicalin, existing its pharmacological action of bibliographical information is antiinflammatory, anti-allergy action; The hepatic cholagogic effect; Blood pressure lowering, diuresis, calmness, refrigeration function; The aldose reductase inhibitory action.Clinical practice is mainly used in infectious hepatitis, acute biliary infection, lead poisoning, heat-clearing and toxic substances removing, vomiting and nausea uncomfortable in chest, cough due to lung-heat, frequent fetal movement and sore etc.With regard to baicalin, the in vitro study report that is confined at breast cancer cell is arranged also.But these researchs all also are in very early stage, and are also fuzzy for the antitumaous effect mechanism of Radix Scutellariae extract.
Summary of the invention
The objective of the invention is provides a kind of preparation to have efficiently for the deficiency that solves above-mentioned technology, uses Radix Scutellariae extract in the anti-gastric cancer medicine that has no side effect, i.e. the application of Radix Scutellariae extract in the anti-gastric cancer medicine of preparation.
In order to achieve the above object, the invention provides a kind of Radix Scutellariae extract, use the Radix Scutellariae extract drug prepared and be used for the treatment of or prevent gastric cancer in the application of preparation in the anti-gastric cancer medicine.Radix Scutellariae extract refers in particular to the extract with flavone precursor structure, can be baicalin, also can be baicalin, can also be both mixture, and both proportionings can be that any proportioning is mixed in the mixture.Described Radix Scutellariae extract can be the pharmaceutically acceptable salt that Radix Scutellariae extract is made, as with following acid: similar acid-addition salts such as maleic acid, fumaric acid, succinic acid, tartaric acid, acetic acid, hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid; Can add suitable solvent when needing, as analog such as water, methanol, ethanol, ether, oxolanes.Radix Scutellariae extract and pharmaceutically acceptable salt can oral, drug administration by injection, as powder, granule, tablet, piller, capsule, Emulsion, syrup, injection etc.Described preparation can prepare with conventional method.
Described Radix Scutellariae extract with flavone precursor structure, its flavone precursor structure is as shown below:
Described baicalin (baicalein) is baicaligenin again, and flavone compound is one of main active of Radix Scutellariae.Molecular weight 270.25 can be obtained by the baicalin hydrolysis.
The structure of baicalin: on the flavone precursor structure 4 ', 5,6,7 four hydrogen base-H are replaced by hydroxyl-OH.
The structure of baicalin (baicalin) is: on the flavone precursor structure 4 ', 5,6 three hydrogen base-H are replaced 7 hydroxyl-OH and glucuronic acid condensation, i.e. baicalin-7-O-glucuronic acid by hydroxyl-OH.
The application of Radix Scutellariae extract of the present invention in preparation gastric cancer medicine discloses the new purposes of Radix Scutellariae extract in pharmacy.Radix Scutellariae extract, particularly baicalin have tangible inhibitory or killing effect to stomach cancer cell.
Apoptosis is to be prevalent in intravital a kind of spontaneous, the process of cell death initiatively of cell biological, be that organism is kept the metastable intrinsic mechanism of cell quantity, this mechanism is if having obstacle or take place unusual, just might cause tumor or other pathological changes, tumor is that a kind of apoptosis is crossed slow and bred too much disease, if can suppress the propagation of tumor cell and induce its apoptosis, just can control the generation and the development of tumor effectively.In addition, when diameter of tumor during in the 1-2 mm square, because tumor body central part anoxia, tumor cell can produce angiogenesis factor, like this, tumor can obtain required nutrition of its spontaneous growth and oxygen, by to this neovascularity inhibition of proliferation, help to suppress the continued growth of tumor cell, therefore, suppress the growth of tumor cell, need to seek and to suppress the propagation of tumor cell and induce its apoptosis and have the material of angiogenesis inhibiting activity.
And of the present invention discovering, baicalin in the Radix Scutellariae extract, its special mechanism of action are directly to suppress the division and the hypertrophy of stomach cancer cell, and have the effect of induced strong cancer cell-apoptosis, and normal cell growth cycle and apoptosis are not had obvious effect; Another mainly acts on is to suppress the gastric cancer internal blood vessel to generate, particularly under anaerobic environment revascularization, thereby the blood supply of cutting off tumor effectively makes its degeneration necrosis.Particularly in the isolated cells culture experiment, show as the inhibition of stomach cancer cell release angiogenesis factor and the apoptosis of induction of vascular endothelial cell.In addition, in living animal (nude mice) experiment, discovery can press down the growth of killing transplantation tumor significantly, the highlyest effectively reaches 95%, and without any side effects.
Radix Scutellariae extract has been excavated in the application of Radix Scutellariae extract of the present invention in pharmacy, particularly baicalin and the baicalin new purposes in the production field of curing gastric cancer and prophylactic agent.The drug toxicity of Radix Scutellariae extract of the present invention, particularly baicalin, baicalin preparation is low, and human body is had no side effect.Radix Scutellariae extract of the present invention, particularly baicalin, baicalin raw material sources are extensive, and cost is lower.The present invention has great realistic significance for curing gastric cancer and capturing early of this difficult problem of prevention.
In order to understand essence of the present invention better, the present invention is further described below in conjunction with contrast pharmacological evaluation and result.
As shown in Figure 1, be that the baicalin (baicalein) in the Radix Scutellariae extract is induced the stomach cancer cell karyorhexis, apoptosis starts from nuclear cracking.Show among the figure that tangible nucleus cracking (arrow indication among the right figure) appears in stomach cancer cell after baicalin is handled 2 days.Baicalin does not have this effect (left figure) to normal cell.DAPI immunostaining (special cells nuclear staining method) is adopted in this experiment.Control is undressed cellular control unit.
Experimental result 2:
As shown in Figure 2, be the apoptosis situation that baicalin is induced stomach cancer cell.Special immunohistochemical staining method (tunel Stain) is adopted in this experiment.The method is the optimum cell apoptosis identification of means of generally acknowledging in the present world wide, apoptosis or just after apoptotic cells is dyed, be displaing yellow or crocus, and normal cell then manifests redness.Photo demonstration stomach cancer cell has been induced the apoptosis of these cells after baicalin (baicalein) is handled 3 days, then unaffected through the same normal cell of handling.
As shown in Figure 3, this photo is the apoptosis situation of baicalin induction of vascular endothelial cell.Special immunohistochemical staining method (tunel stain) is adopted in this experiment. and apoptosis or just presenting yellow after apoptotic cells is dyed, normal cell then presents redness.Shown two kinds of blood vessel endotheliums in the picture, promptly criticized chrotoplast (HAEC) in Human umbilical vein endothelial cells (HUVEC) and the human artery, induced the apoptosis of these cells after baicalin (baicalein) is handled 3 days, control is undressed cellular control unit.
As shown in Figure 4, this figure is that baicalin suppresses the experimental status that stomach cancer cell discharges angiogenesis factor.
Experimental technique: 1 * 10
5Stomach cancer cell is planted on the culture plate that diameter is 15mm, and culture fluid contains 10% calf serum.After treating cell attachment in 24 hours, change culture fluid into serum-free medium.Change beginning dosing in 24 hours behind the liquid.After different time is handled, culture fluid is collected, with the content of ELISA mensuration culture fluid medium vessels somatomedin, measured content needs to proofread and correct through actual cell number.Stomach cancer cell is after baicalin is handled 2 days, and angiogenesis factor discharges obviously and reduces.(1%O in anoxic environment
2), the baicalin inhibitory action is especially remarkable.
As shown in Figure 5, this photo shows that baicalin suppresses nude mice and goes up the angiogenesis situation of transplanting in the gastric cancer.
Experimental technique: it is subcutaneous earlier stomach cancer cell line to be seeded in nude mice.From the 2nd week beginning, baicalin is through subcutaneous administration, 3 times weekly.In 12 weeks of Therapy lasted to the, measure the surface area of a tumor weekly.Experiment separates tumor after finishing on nude mice, after chemical fixation and frozen section, carry out immunohistochemical staining with eight factors as antibody.Brown fragment is the tumor vessel section in the picture.Left figure sample is taken from not medication group, and right figure sample is taken from the medication group, dosage 1mg/kg body weight, and visible vessels quantity obviously reduces.
Experimental result 6:
Fig. 6 is the DNA division situation that baicalin suppresses stomach cancer cell.Visible stomach cancer cell is after baicalin is handled among the figure, and the cell number that is in DNA synthesis stage (S phase) obviously reduces, the presynthetic phase (G0/G1) cell number then obviously increase.Since these cells the presynthetic phase G0/G1 accumulation, thereby suppressed this proliferation of cells effectively.Experimental technique: stomach cancer cell is after baicalin is handled, again through fluorescent labeling, with the cell cycle analysis method cell number that is in each different division stages is carried out quantitative assay at last, the ratio that measured numerical value is accounted for total cell number compares and obtains chart.
As shown in Figure 7, be that baicalin (baicalein), baicalin (baicalin) and matched group usefulness Semen Coicis extract (wogonin) press down the curative effect comparison of killing stomach cancer cell.In stripped sex culture experiment, but baicalin, baicalin and Semen Coicis extract all have the effect of stomach cancer cell extremely, but action intensity is slightly variant.Relative intensity: baicalin＞baicalin＞Semen Coicis extract.
The result tests 8
As Fig. 8, shown in Figure 9, be that baicalin (baicalein) has suppressed the expression (Fig. 8) of Cyclooxygenase (COX-2) and synthetic (Fig. 9) of prostaglandin.COX-2 is the enzyme that a kind of catalysis prostaglandin generates, and this enzyme and its product prostaglandin have played important effect in the generation of tumor and development.One of baicalin main mechanism is to play the effect of cancer inhibitting and killing cell by its these inhibitory action.Experimental technique: Table A adopts West Blot Analysia method to measure COX-2 protein expression level, and the right numeral is pointed out the proteinic molecular weight of COX-2.Table B adopts the ELISA method to measure by stomach cancer cell and is released into content of prostaglandin in the culture fluid, and does to take statistics after the alignment with actual cell number and learn processing.
Experimental result 9:
As shown in figure 10, this chart is that baicalin presses down the dose curve that kills stomach cancer cell growth in the cell culture experiments in vitro.Curve 1 expression normal cell, curve 2 expression breast cancer cells, curve 3 expression stomach cancer cells.
Experimental technique: identical condition of culture is all adopted in three kinds of experimental cell strains.1 * 10
5Cell is planted on the culture plate that diameter is 15mm, and culture fluid contains 10% calf serum.After treating cell attachment in 24 hours, change culture fluid the influence of serum-free medium into the somatomedin cell growth avoiding existing in the serum.Change beginning dosing in 24 hours behind the liquid.Dosing was made cell counting after 5 days.Before the counting earlier with Tai Panlan with cell dyeing to get rid of the influence of cell of dying in heaven to experimental result.Every bit is the meansigma methods of 3 experiments in the curve.
As seen, be 0.1 to have begun effect (20% suppress) with concentration from curve, when concentration was 0.5 μ g/ml, the inhibition of stomach cancer cell growth was 50%.And when concentration reached 10 μ g/ml, inhibition can reach 95%.Baicalin does not have obvious effect to normal cell.As seen baicalin can press down the vitro growth of gastric cancer cell of killing In vitro culture effectively.
Last table is that baicalin presses down the growth of killing transplanting gastric cancer on the living animal.Experimental technique: it is subcutaneous earlier stomach cancer cell line to be seeded in nude mice, through 1-2 after week tumor begin growth.The baicalin subcutaneous injection is from the beginning of second week, 3 times weekly.Laboratory animal is divided into matched group of 3 test dose combinations, every group of 10 nude mices at random.In 12 weeks of Therapy lasted to the, measure the surface area of a gastric cancer tumor weekly.Experiment separates the gastric cancer tumor after finishing on nude mice, and does to preserve to handle and prepare against as pharmacology, SABC and biochemical analysis.Show as Figure 11: baicalin presses down the metering curve that kills transplantation tumor.Show as Figure 12: but baicalin has obviously transplantation tumor kills effect.
As shown in figure 13, experiment shows in the photo be tumor substantially specimen separate from the nude mice of 3 various dose families and matched group.Stomach cancer cell was transplanted to nude mice after last 12 weeks, and the gastric cancer tumor on the not medication group nude mice has been grown to and has been exceeded 250mm
2(surface area), the tumor of medication group nude mice then present significantly with the increase of dosage and dwindle.Every group of 4 tumors cardinal principle specimen is to obtain at random from 10 specimen among the figure.
Experimental result 12:
When following experiment is independent medication of baicalin (baicalein) and baicalin (baicalin) or mixed reagent the stomach cancer cell of isolated culture and the effect of mastocarcinoma cell are compared.
Experimental technique: 1 * 10
5Stomach cancer cell and mastocarcinoma cell are planted on the culture plate that diameter is 15mm, and culture fluid contains 10% calf serum.Treat (about 24 hours) behind the cell attachment, change culture fluid the influence of serum-free medium into the somatomedin cell growth avoiding existing in the serum.Change beginning dosing in 24 hours behind the liquid.Dosing was made cell counting after 5 days.Before the counting earlier with Tai Panlan with cell dyeing to get rid of the influence of cell of dying in heaven to experimental result.As every bit in Figure 14 table is the meansigma methods of three experiments.Figure 15 is the experiment statistics table.
Experimental result:
Separately during medication, the action intensity of baicalin be baicalin 2-3 doubly.The IC of baicalin
50Be about 0.15 μ g/ml, and the IC of baicalin
50Be about 0.45 μ g/ml.When baicalin and baicalin mixed reagent (1: 1), it has slight increase to the inhibitory action of stomach cancer cell growth when the low dose, but after dosage increases, and its effect is greater than baicalin (medication separately), but less than baicalin (medication separately).Matched group has increased the vitro growth of gastric cancer cell of isolated culture, does not have any inhibitory action.
Above-mentioned experimental result Radix Scutellariae extract as can be seen is used to prepare anti-gastric cancer medicine, can obviously suppress the division of stomach cancer cell, and has the effect of induced strong cancer cell-apoptosis, and normal cell growth cycle and apoptosis are not had obvious effect; And tool inhibition gastric cancer internal blood vessel generation, the particularly revascularization under anaerobic environment, thereby the blood supply of cutting off tumor effectively makes its degeneration necrosis.Particularly the baicalin medicine that is used to prepare treatment or prevention gastric cancer has very active operation significance.
Description of drawings
Fig. 1 is that baicalin (baicalein) is induced stomach cancer cell karyorhexis experiment photo;
Fig. 2 is the apoptosis situation experiment photo that baicalin is induced stomach cancer cell;
Fig. 3 is the apoptosis situation experiment photo of baicalin induction of vascular endothelial cell;
Fig. 4 is that baicalin suppresses the experimental status photo that stomach cancer cell discharges angiogenesis factor;
Fig. 5 is that baicalin suppresses the upward interior angiogenesis situation of transplanting gastric cancer of nude mice;
Fig. 6 is the DNA division situation that baicalin (baicalein) suppresses stomach cancer cell;
Fig. 7 presses down the curative effect of killing stomach cancer cell to compare;
Fig. 8 is that baicalin (baicalein) has suppressed the expression of Cyclooxygenase (COX-2);
Fig. 9 is the synthetic situation that baicalin (baicalein) has suppressed prostaglandin;
Figure 10 is that baicalin presses down the growth of killing transplanting gastric cancer on the living animal;
Figure 11 is that baicalin presses down the metering curve that kills transplantation tumor;
Figure 12 is that baicalin kills effect to pressing down of transplantation tumor;
Figure 13 is that stomach cancer cell is transplanted to nude mice last 12 all backs tumor growths relatively;
Figure 14 be baicalin (baicalein) and baicalin (baicalin) separately when medication or mixed reagent to the effect of the stomach cancer cell of isolated culture relatively;
Figure 15 is that Radix Scutellariae extract compares stomach cancer cell and mastocarcinoma cytosis.
The specific embodiment
Present embodiment provides the extracting method of Radix Scutellariae extract, and at first decocting boils Radix Scutellariae coarse powder twice, adds acid and transfers to PH1-2, and the 70-80 insulation was left standstill 12-24 hour, filtered.Precipitate adds the water stirring makes into suspension, transfers PH7-7.5 to dissolving with alkali, adds ethanol and stirs, filtration.Filtrate is the Radix Scutellariae extracting solution with effective Radix Scutellariae extract composition.
Embodiment 2:
Present embodiment provides the medicine that utilizes the existing goods baicalin to be used to prepare anti-gastric cancer, takes by weighing baicalin (commercially available) 300 grams, mannitol 75 grams, sodium citrate 250 grams, powder pin preparation technology with routine makes 1000 powder pins, and every contains baicalin 300mg, as anti-gastric cancer medicine.
Embodiment 3:
Present embodiment provides and will obtain baicalin after the baicalin hydrolysis, take by weighing 100 gram baicalin raw materials, add lactose 66g, pregelatinized Starch 64g, microcrystalline Cellulose 66g, magnesium stearate 3g, film-making technology routinely makes 1000 in tablet, be equivalent to every and contain 100 milligrams of baicalins, as anti-gastric cancer medicine.
Embodiment 4:
Present embodiment provides baicalin and baicalin 1: 1 mixing by weight.Take by weighing 100 gram mixed materials, add lactose 66g, pregelatinized Starch 64g, microcrystalline Cellulose 66g, magnesium stearate 3g makes 1000 capsules with the capsules preparation technique of routine, and every capsules contains baicalin and baicalin mixture 100mg, as anti-gastric cancer medicine.
Claims (1)
1. the application of Radix Scutellariae extract in the anti-gastric cancer medicine of preparation, described Radix Scutellariae extract is the mixture of baicalin or baicalin or baicalin and baicalin.
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