CN103239488A - Application of crude extract product of Ardisia mamillata Hance - Google Patents
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Abstract
The invention relates to an application of a crude extract product of Ardisia mamillata Hance in the technical field of medicines. According to the invention, Ardisia mamillata Hance is extracted through using a 0-95% ethanol solution to obtain the crude extract product, and the crude extract product can be directly used for preparing antitumor medicines without refining. A problem that effective antitumor components can be obtained through refining the crude extract product of Ardisia mamillata Hance in the prior art is overcome, a new use of the crude extract product of Ardisia mamillata Hance in the direct preparation of the antitumor medicines is provided, the resource is saved, and the cost is saved.
Description
Affiliated technical field
The invention belongs to medical technical field, be specifically related to the application of Ardisia mamillata crude extract.
Background technology
Ardisia mamillata be Myrsinacea Ardisa plant Ardisia mamillata (
Ardisia mamillataHance), its branch and leaf and rhizome all are used as medicine.Be grown in the in the shade woods of 200 ~ 400 meters of height above sea level, happiness is cloudy, all living creatures.There is kind more than 300 in this platymiscium whole world, and China has 68 kinds, 12 mutation.Be distributed widely in wide geographic area on the south the Changjiang river, reserves are abundant.Especially distribute comparatively concentrated with Yunnan and area, Hainan.Ardisia mamillata is considered to have clearing away heat-damp and promoting diuresis in Chinese medicine, the effect of blood circulation promoting and blood stasis dispelling.According to record in " Chinese medicine voluminous dictionary ", this plant can be used for treating diseases such as dysentery, hepatitis, cholecystitis, rheumatism, traumatic injury, hemoptysis, furuncle swell and ache.From Ardisia mamillata, separated first in 1984 obtain being the saponin of parent nucleus with Cyclamiritin A since, reported from this plant to separate to obtain about ten kinds of saponin compounds that wherein the overwhelming majority is to be parent nucleus with pentacyclic triterpene Cyclamiritin A.Ardisia mamillata B (Ardisiacrispin B) is the highest a kind of saponin of content wherein.The preparation process of existing Ardisia mamillata saponin adopts water or ethanol extraction to obtain crude extract, and crude extract obtains refining thing through resin or the absorption of other parting materials, eluting again.Refining thing has anti-tumor activity.
Summary of the invention
The objective of the invention is to overcome Ardisia mamillata crude extract in the prior art and must be directly used in the application of antitumor medicine for people provide a kind of Ardisia mamillata crude extract through the refining deficiency that just obtains the antitumor effective ingredient.
The objective of the invention is to realize by following technical proposals.
The application of Ardisia mamillata crude extract of the present invention is characterized in that the crude extract that Ardisia mamillata extracts with 0～95% alcoholic solution, and without refining, crude extract directly prepares the application of antitumor drug.
In the such scheme, it is that the conventional pharmaceutic adjuvant drug preparation technique routinely of crude extract interpolation is made compositions that described crude extract directly prepares antitumor drug.
In the such scheme, described pharmaceutic adjuvant is filler, adhesive, disintegrating agent, lubricant or/and antiseptic, and described compositions is tablet, capsule, granule, effervescent tablet or the oral liquid of crude extract dry product content 10～1000mg unit dose.
In the such scheme, described antitumor drug is anti-hepatocarcinoma, anti-renal carcinoma, anti-pulmonary carcinoma, anti-people's mammary gland duct carcinoma or antileukemie medicine.
Usually, crude extract is because crude extract is removed a large amount of impurity through refining purification, thereby makes refining thing have the effect more much higher than crude extract only for the preparation of refining thing.But, the inventor finds in comparative study, and the Ardisia mamillata crude extract has and makes with extra care the effect that thing is more or less the same at anti-tumor aspect, and this shows that the Ardisia mamillata crude extract is in subtractive process, what remove is not only impurity, but has comprised the effective ingredient of suitable component.Further analyze, this result of study prompting not necessarily only limits to the pentacyclic triterpene saponin compound about the composition that has anti-tumor activity in the Ardisa plant.Therefore, the present invention has broken through people about having the mistaken ideas of anti-tumor active ingredient in the Ardisa plant, provides the Ardisia mamillata crude extract to be directly used in the new purposes of antitumor medicine, economizes on resources, and has reduced cost.
Further specify the present invention below by embodiment, the present invention is not limited only to described embodiment.
The specific embodiment:
Embodiment one
Get Ardisia mamillata medical material 10 kg, add water 100 kg, extracted 2 hours, extract altogether 3 times, merge extractive liquid, filters, and filtrate decompression concentrates, and drying obtains the Ardisia mamillata water extract dry extract of about 1.3 kg.Dry extract contains Ardisia mamillata B 21.5 %.
With 0.25% trypsinization monolayer HepG2 cultured cell, be made into the individual cells suspension with containing 10% hyclone RPMI1640 culture fluid, with every hole 10
3-10
4Individual cell inoculation in 96 well culture plates, every pore volume 200 μ l.Culture plate is put into CO
2Incubator, at 37 ℃, 5%CO
2And under the saturated humidity condition, behind the cultivation 24h, add the culture fluid that contains the above-mentioned dry extract medicine of variable concentrations and 5-fluorouracil respectively, wherein, the concentration of high dose, middle dosage, low dosage and positive group is respectively 70,45,30,20 mg/kg.Every hole 180 μ l, blank group adds isopyknic culture fluid.37 ℃, 5%CO
2And under the saturated humidity condition, cultivate 48h after, every hole adds MTT solution (5mg/ml) 20 μ l, 37 ℃ are continued to hatch 4h, stop cultivating, and carefully discard in the hole culture supernatant night.Every hole adds 150 μ l DMSO, vibration 10min.Select the 490nm wavelength, measure each hole absorbance value at enzyme-linked immunosorbent assay instrument, calculate suppression ratio and the results are shown in Table one.
Table one
| ? | Positive group | High dose | Middle dosage | Low dosage |
| Suppression ratio (%) | 69.1 | 66.6 | 70.5 | 64.8 |
5-fluorouracil positive controls and Ardisia mamillata water extract group are compared with matched group after handling the HepG2 cell, and the proliferation activity of cell obviously is suppressed.
The dry extract of getting above-mentioned Ardisia mamillata water extract is raw material,
Prescription dry extract 150g
Carboxymethyl starch sodium 20g
Microcrystalline cellulose 30g
Make 1000
Preparation method: take by weighing carboxymethyl starch sodium and the microcrystalline cellulose of recipe quantity, and the Ardisia mamillata dry extract mixing of recipe quantity, add an amount of 75% ethanol, stir the granulation of sieving, drying, granulate.Get the granule for preparing, tablet forming heavily is the tablet of 0.2g.Every contains Ardisia mamillata dry extract 0.15g.
The dry extract of getting above-mentioned Ardisia mamillata water extract is raw material;
Prescription dry extract 15g
Ethylparaben 0.5g
Citric acid 1g
Stevioside 1g
Distilled water is an amount of
Make 1000ml
Preparation method: dry extract, citric acid, stevioside is soluble in water, filter, get ethylparaben and be dissolved in the small amount of ethanol, add filtrate, add the water standardize solution, be packed as 10ml/ bottle specification behind the mix homogeneously, every bottle of oral liquid contains Ardisia mamillata dry extract 0.15g.
Embodiment two
Ardisia mamillata medical material 10 kg, alcoholic solution 100 kg of adding 50%, reflux, extract, 2 hours is extracted 3 times altogether, and merge extractive liquid, filters, and filtrate decompression concentrates, and drying obtains the Ardisia mamillata ethanol crude extract dry extract of about 1.5 kg.Dry extract contains Ardisia mamillata B 23.7 %.
With 0.25% trypsinization monolayer people hepatocarcinoma SMMC7721 cultured cell, be made into the individual cells suspension with containing 10% hyclone RPMI1640 culture fluid, with every hole 10
3-10
4Individual cell inoculation in 96 well culture plates, every pore volume 200 μ l.Culture plate is put into CO
2Incubator, at 37 ℃, 5%CO
2And under the saturated humidity condition, behind the cultivation 24h, add the culture fluid that contains the above-mentioned dry extract medicine of variable concentrations and 5-fluorouracil respectively, wherein, the concentration of high dose, middle dosage, low dosage and positive group is respectively 70,45,30,20 mg/kg.Every hole 180 μ l, blank group adds isopyknic culture fluid.37 ℃, 5%CO
2And under the saturated humidity condition, cultivate 48h after, every hole adds MTT solution (5mg/ml) 20 μ l, 37 ℃ are continued to hatch 4h, stop cultivating, and carefully discard in the hole culture supernatant night.Every hole adds 150 μ l DMSO, vibration 10min.Select the 490nm wavelength, measure each hole absorbance value at enzyme-linked immunosorbent assay instrument, calculate suppression ratio and the results are shown in Table two.
Table two
| ? | Positive group | High dose | Middle dosage | Low dosage |
| Suppression ratio (%) | 43.0 | 39.8 | 39.2 | 32.1 |
Behind 5-fluorouracil positive controls and the Ardisia mamillata crude extract group handler hepatocarcinoma SMMC7721 cell, compare with matched group, the proliferation activity of cell obviously is suppressed.
Get people's hepatocarcinoma SMMC7721 cell suspension 0.2ml/ and only be inoculated in the nude mice oxter, totally 10.Treat to get when tumor growth is good tumor tissues and be used for the foundation of transplanted tumor in nude mice model.Take out tumor tissues in sterile working's mode, with an amount of DMEM complete culture solution suspension cell again, get this cell suspension 0.2ml/ and only be inoculated in the nude mice oxter, totally 90.Behind the inoculated tumour cell suspension, pressed normalized form and calculate gross tumor volume with vernier caliper measurement transplanted tumor major diameter and minor axis 1 time, and treated that most of animal tumor volume reached 100mm in per 3 days
3During the left and right sides, press the gross tumor volume random packet, 10 ~ 15 every group is respectively model control group, positive controls, refining thing group and crude extract group.Calculate tumour inhibiting rate and the results are shown in Table three.Wherein, the administering mode of positive controls, refining thing group and crude extract group is gastric infusion; Refining thing contains Ardisia mamillata B 92.7 %, and crude extract contains Ardisia mamillata B 23.7 %.
Table three
| Group | Number of animals | Tumour inhibiting rate (%) |
| Model group | 11 | － |
| Positive controls 0.2mg/kg | 10 | 39.4 |
| Refining thing 25mg/kg | 11 | 53.5 |
| Crude extract 30mg/kg | 11 | 36.7 |
Illustrate: nude mice death under this dosage occurs in the refining thing group administration process, point out its certain toxicity.Both tumour inhibiting rate differences are as described in the table three, and the dosage of its Ardisia mamillata B content crude extract group only is 30.7% of refining thing group relatively, and tumour inhibiting rate then reaches 68.6% of refining thing group, and does not have nude mice death.The anti-SMMC7721 performance of proof crude extract is better than refining thing on the contrary.
The dry extract of getting above-mentioned Ardisia mamillata ethanol crude extract is raw material,
Prescription dry extract 150g
Carboxymethyl starch sodium 20g
Microcrystalline cellulose 30g
Pregelatinized Starch 100g
Magnesium stearate 1.5g
Make 1000
Preparation method: take by weighing carboxymethyl starch sodium, microcrystalline cellulose and the pregelatinized Starch of recipe quantity, and the Ardisia mamillata dry extract mixing of recipe quantity, add an amount of 75% ethanol, stir the granulation of sieving, drying, granulate, the magnesium stearate mixing of adding recipe quantity.Adorn capsule No. 1.Every capsules contains Ardisia mamillata dry extract 0.15g.
Embodiment three
Get Ardisia mamillata medical material 10 kg, add 70% alcoholic solution 100 kg, reflux, extract, 2 hours is extracted 3 times altogether, and merge extractive liquid, filters, and filtrate decompression concentrates, and drying obtains the Ardisia mamillata ethanol crude extract dry extract of about 1.6 kg.Dry extract contains Ardisia mamillata B 25.6 %.
Get frozen murine sarcoma H22 cell, cell suspension is made with normal saline in the recovery back, only is inoculated in mouse peritoneal with 0.5ml/, treat to carry out again going down to posterity between Mus after ascites tumor forms after 3 generations, when treating that obvious ascites appears in mice, aseptic extraction peritoneal fluid, platform are expected blue repelling attack living cell counting number.Be made into 1.0 * 10 with normal saline
7Cell/ml suspension, subcutaneous vaccination 0.2ml/ in right side of mice oxter only inoculates second day, be divided into 5 groups at random by body weight, 12 every group, above-mentioned Ardisia mamillata ethanol crude extract high and low dose group is irritated stomach by 100mg/kg, 50mg/kg dosage respectively and is given the respective concentration medicine, every day 1 time, continuous 10 days; Fluorouracil Injection the test the 1st, 2,4,5,7,8 day, with 20mg/kg dosage lumbar injection, every day 1 time, totally 6 times.After the last administration 24 hours, whole mices are put to death in the cervical vertebra dislocation, and the separation tumor mass is also weighed.Calculate tumour inhibiting rate and see Table four.
Table four
| Group | Dosage (mg/kg) | Number of animals | Tumor is heavy | Tumour inhibiting rate (%) |
| Model group | 0 | 12 | 1.671±0.397 | - |
| The Fluorouracil Injection group | 20 | 12 | 0.837±0.197 | 50 |
| The extract high dose group | 100 | 11 | 1.092±0.331 | 36.5 |
| The extract low dose group | 50 | 10 | 1.357±0.266 | 19.3 |
As shown in Table 4, Ardisia mamillata ethanol crude extract has inhibitory action to rat liver cancer H22 cell.
Get frozen murine sarcoma H22 cell, cell suspension is made with normal saline in the recovery back, only is inoculated in mouse peritoneal with 0.5ml/, treat to carry out again going down to posterity between Mus after ascites tumor forms after 3 generations, when treating that obvious ascites appears in mice, aseptic extraction peritoneal fluid, platform are expected blue repelling attack living cell counting number.Be made into 1.0 * 107 cells/ml suspension with normal saline, right side of mice oxter subcutaneous vaccination 0.2ml/ only, inoculate second day, be divided into 7 groups at random by body weight, every group 10, wherein, refining thing group (refining thing contains Ardisia mamillata B 92.7 %) and two groups of medicine groups of crude extract group (crude extract contains Ardisia mamillata B 25.6 %) are irritated stomach and are given the variable concentrations medicine, every day 1 time, continuous 10 days; The cisplatin group is with 4mg/kg dosage lumbar injection, and 1 time every other day, continuous 5 times.After the last administration 24 hours, mice is put to death in the cervical vertebra dislocation, and the separation tumor mass is also weighed.Calculate tumour inhibiting rate.The results are shown in Table five.
Table five
| Group | Number of animals | Tumour inhibiting rate (%) |
| Model group | 10 | － |
| Cisplatin group 4mg/kg | 8 | 77.0 |
| Refining thing group 40mg/kg | 10 | 37.5 |
| Crude extract group 100mg/kg | 11 | 34.6 |
Table five shows, the dosage of its Ardisia mamillata B content crude extract group is 69% of refining thing group relatively, tumour inhibiting rate then reaches 92.3% of refining thing group, proves refining afterproduct and crude extract to the tumour inhibiting rate of H22 cell, and its Ardisia mamillata B content there is no great gap relatively.
The Ardisia mamillata ethanol crude extract of this example preparation is in anticancer experiment in vitro, acute T chronic myeloid leukemia cell Jurkat, human renal carcinoma cell OS-RG-2, human lung cancer cell A549, people's mammary gland duct carcinoma cell MDA-MB-435 all there are stronger resistance, show that the Ardisia mamillata crude extract has the wide characteristics of anticancer spectrum.
The dry extract of getting above-mentioned Ardisia mamillata ethanol crude extract is raw material;
Prescription dry extract 150g
Dextrin 400g
Microcrystalline cellulose 50g
Soluble starch 300g
Mannitol 100g
Make 1000g
Preparation method: take by weighing dextrin, soluble starch, the mannitol of recipe quantity, and the Ardisia mamillata dry extract mixing of recipe quantity, add an amount of 85% ethanol, stir, the granulation of sieving, drying, granulate is made granule.Pack in the aluminium foil bag with granule racking machine branch, every packed 1.0g contains Ardisia mamillata dry extract 0.15g.
Embodiment four
Get Ardisia mamillata medical material 10 kg, add 95% alcoholic solution 100 kg, reflux, extract, 2 hours is extracted 3 times altogether, and merge extractive liquid, filters, and filtrate decompression concentrates, and drying obtains the Ardisia mamillata ethanol crude extract dry extract of about 1.5 kg.Dry extract contains Ardisia mamillata B 28.1 %.
Get 2 of the nude mices of 18～22g in healthy 6 ages in week, go down to posterity 4 times HepG2 cell with 0.2ml(about 4 * 10 with cultivation
6) to be inoculated in the right side of mice oxter subcutaneous, the aseptic tumor body that takes off when treating the tumor bulk-growth to the 2cm left and right sides shreds and homogenate, makes suspension with normal saline, with 0.2ml(about 10
7) to be inoculated in 30 nude mice right side of mice oxters subcutaneous, inoculated tumour reaches 30mm
3About after, and 30 mices are divided into the high, medium and low dosage group of dry extract of model group, positive controls, above-mentioned Ardisia mamillata ethanol crude extract at random by gross tumor volume, 6 every group, begin administration simultaneously.The 5-fluorouracil of positive group lumbar injection 20mg/kg body weight, 0.5ml/, per 3 days 1 time, continuous 20 days; Ardisia mamillata ethanol crude extract drug test group is irritated stomach Ardisia mamillata ethanol crude extract (70mg/kg, 45mg/kg, 30mg/kg) respectively, and 0.5ml/, every day 1 time, continuous 20 days; Model group is irritated the aquesterilisa of the same volume of stomach.Surveyed the line of apsides of 1 tumor in per 3 days.In the end 24h puts to death animal after 1 administration, gets tumor, measures size and weight.Calculate tumor tissues volume size with formula.The processing that takes statistics, with positive control relatively.The results are shown in Table six.
Table six
5-fluorouracil positive controls and Ardisia mamillata ethanol crude extract medicine group gross tumor volume after administration obviously dwindle, and the tumor tissues volume and weight is starkly lower than model group.Show that Ardisia mamillata ethanol crude extract medicine can effectively suppress tumor growth.
The dry extract of getting above-mentioned Ardisia mamillata ethanol crude extract is raw material;
Prescription dry extract 300g
Dextrin 120g
Microcrystalline cellulose 150g
Magnesium stearate 50g
Tartaric acid 140g
Sodium bicarbonate 130g
Mannitol 50g
Make 1000g
Preparation method: the tartaric acid, sodium bicarbonate, Ardisia mamillata dry extract, dextrin, microcrystalline cellulose, the mannitol that take by weighing recipe quantity respectively.Its mesotartaric acid, dextrin, microcrystalline cellulose, mannitol mixing are ground into fine powder, use 95% alcohol granulation, drying, and granulate, standby.Sodium bicarbonate mixes with the Ardisia mamillata dry extract, pulverizes 80 mesh sieves, with the granulates mixing, and adds the magnesium stearate of recipe quantity before tabletting, and tablet forming heavily is the effervescent tablet of 0.5g, and every contains Ardisia mamillata dry extract 0.15g.
Claims (4)
1. the application of an Ardisia mamillata crude extract is characterized in that the crude extract that Ardisia mamillata extracts with 0～95% alcoholic solution, and without refining, crude extract directly prepares the application of antitumor drug.
2. the application of Ardisia mamillata crude extract according to claim 1 is characterized in that it is that the conventional pharmaceutic adjuvant drug preparation technique routinely of crude extract interpolation is made compositions that described crude extract directly prepares antitumor drug.
3. the application of Ardisia mamillata crude extract according to claim 2, it is characterized in that described pharmaceutic adjuvant is filler, adhesive, disintegrating agent, lubricant or/and antiseptic, described compositions is tablet, capsule, granule, effervescent tablet or the oral liquid of crude extract dry product content 10～1000mg unit dose.
4. the application of Ardisia mamillata crude extract according to claim 1 is characterized in that described antitumor drug is anti-hepatocarcinoma, anti-renal carcinoma, anti-pulmonary carcinoma, anti-people's mammary gland duct carcinoma or antileukemie medicine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104914203A (en) * | 2015-05-27 | 2015-09-16 | 西南民族大学 | Detection method for Ardisia crispa herb or extract thereof |
| CN105116059A (en) * | 2015-05-27 | 2015-12-02 | 西南民族大学 | Detection method for medicinal material Ardisia mamillata Hance or extract thereof |
| CN106110015A (en) * | 2016-06-29 | 2016-11-16 | 张月媚 | A kind of traditional herbal medicine treating cervical lymphadenitis and using method thereof |
| CN113730426A (en) * | 2021-11-01 | 2021-12-03 | 中山市中医院 | Application of Ardisiacripin B in preparation of medicine for preventing or treating inflammatory bowel disease |
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| CN101157717A (en) * | 2006-10-04 | 2008-04-09 | 薛永新 | Preparation method of Ardisia mamillata B and uses thereof |
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| CN1634180A (en) * | 2003-12-29 | 2005-07-06 | 陈宝川 | Medicine for treating malignant tumor and its processing method |
| CN101157717A (en) * | 2006-10-04 | 2008-04-09 | 薛永新 | Preparation method of Ardisia mamillata B and uses thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104914203A (en) * | 2015-05-27 | 2015-09-16 | 西南民族大学 | Detection method for Ardisia crispa herb or extract thereof |
| CN105116059A (en) * | 2015-05-27 | 2015-12-02 | 西南民族大学 | Detection method for medicinal material Ardisia mamillata Hance or extract thereof |
| CN106110015A (en) * | 2016-06-29 | 2016-11-16 | 张月媚 | A kind of traditional herbal medicine treating cervical lymphadenitis and using method thereof |
| CN113730426A (en) * | 2021-11-01 | 2021-12-03 | 中山市中医院 | Application of Ardisiacripin B in preparation of medicine for preventing or treating inflammatory bowel disease |
| CN113730426B (en) * | 2021-11-01 | 2023-02-21 | 中山市中医院 | Application of Ardisiacripin B in preparation of medicine for preventing or treating inflammatory bowel disease |
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Application publication date: 20130814 |