CN102247393B - Preparation method of oleanolic acid saponin component and application thereof - Google Patents
Preparation method of oleanolic acid saponin component and application thereof Download PDFInfo
- Publication number
- CN102247393B CN102247393B CN 201110142210 CN201110142210A CN102247393B CN 102247393 B CN102247393 B CN 102247393B CN 201110142210 CN201110142210 CN 201110142210 CN 201110142210 A CN201110142210 A CN 201110142210A CN 102247393 B CN102247393 B CN 102247393B
- Authority
- CN
- China
- Prior art keywords
- parts
- group
- water
- tumor
- component
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a preparation method of an oleanolic acid saponin component, namely 3-O-alpha-L-rhamnopyranonose-(1->2)[beta-D-glucopyranose-(1->4)]-alpha-L-arabopyranose oleanolic acid saponin, and application of the component in tumor resistance. The invention also relates to application of an extract which is prepared from the root of pulsatillachinensis (bunge) regel and contains the compound for resisting tumor, including lung cancer, liver cancer, stomach cancer, colon cancer, glioma, breast cancer, cervical cancer, leukaemia, lymph cancer and the like.
Description
Technical field
The invention belongs to medicine, veterinary drug technical field, the preparation method and this composition anti-tumor application thereof that relate to a kind of oleanolic acid saponin constituents 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin are as being used for pulmonary carcinoma, hepatocarcinoma, gastric cancer, colon cancer, glioma, breast carcinoma, cervical cancer, leukemia, lymphatic cancer etc.The invention still further relates to the purposes from the anti-above-mentioned tumor of extract conduct that contains this chemical compound of the Radix Pulsatillae (Pulsatilla chinensis (Bunge) Regel) rhizome.
Background technology
Radix Pulsatillae medical material is taken from the dry root of the Ranunculaceae Pulsatilla plant Radix Pulsatillae, and bitter in the mouth is cold in nature.Return stomach, large intestine channel, tool heat-clearing and toxic substances removing, the merit of eliminating pathogenic heat from blood to cure dysentery.Be used for toxic-heat and blood stasis, pudendal pruritus leukorrhagia, amebic dysentery.According to the literature, contain protoanemonin (Protoanemonin), Anemonin (Anemonin), Radix Pulsatillae spirit (Okinalin in the Radix Pulsatillae, C32H46O2), Radix Pulsatillae English (Okinalein, C4H602) etc., recent domestic scholar isolation identification from the Pulsatilla plant goes out tens kinds of pentacyclic triterpene saponin compositions, and these saponin are mainly two kinds on oleanane type and lupinane type.The Pulsatilla plant has widely pharmacology and biological activity, is mainly manifested in the effects such as antitumor, anti-inflammatory, antibacterial, antiviral, apoptosis, antibiont enzyme, parasite killing, spermicidal.
Through repeatedly research discovery, the water extraction liquid of the Radix Pulsatillae, alcohol extract and basic hydrolysis product thereof all have anti-tumor activity, basic hydrolysis product especially, and activity is stronger, through further component analysis research, find wherein all to contain a series of oleanolic acid saponin constituents.
Summary of the invention
The present invention utilizes modern separation technology and identification of means that the chemical constituent that the Radix Pulsatillae carries out system is separated, therefrom separate and obtain a kind of oleanolic acid saponin constituents: 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin, by the anti tumor activity in vitro screening study, find that this chemical compound has significant antitumor action, yet the content of this composition in Radix Pulsatillae medical material is not high; Simultaneously research is found, the water extract of the Radix Pulsatillae and ethanol extract all have certain antitumor action, and before the activity of its basic hydrolysis product obviously is better than hydrolysis, component analysis is found, greatly improves before the content of 3-O-α in the basic hydrolysis afterproduct-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin is hydrolyzed.Radix Pulsatillae herb resource is abundant, and hydrolyzate is active clear and definite, and preparation is simple, is fit to industrial mass production, can be used as the preparation antitumor drug, has wide market prospect and is worth with exploitation.
Technical scheme of the present invention is as follows:
1, the application of 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin in the preparation antitumor drug.
2, contain the application of Radix Pulsatillae extract in the preparation antitumor drug of 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
3, the application of 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin in the anti-pulmonary carcinoma of preparation, hepatocarcinoma, gastric cancer, colon cancer, glioma, breast carcinoma, cervical cancer, leukemia, lymphatic cancer medicine.
4, contain the application of Radix Pulsatillae extract in the anti-pulmonary carcinoma of preparation, hepatocarcinoma, gastric cancer, colon cancer, glioma, breast carcinoma, cervical cancer, leukemia, lymphatic cancer medicine of 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
5, the preparation method of 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin may further comprise the steps:
The first step becomes coarse powder with Radix Pulsatillae pulverizing medicinal materials, water or 0.1-95% soak with ethanol 0.5-2h, and 6-12 doubly measures heating and refluxing extraction 2-3 time, and each 1-3h filters merging filtrate.Above-mentioned filtrate is evaporated to 0.5-1.0g raw medicinal herbs/mL in 50-70 ℃, and without the alcohol flavor, 0-4 ℃ of cold preservation is spent the night, and analyses glue, filters, and namely gets Radix Pulsatillae extract.
Second step adds sodium hydroxide solution and is transferred to PH10-13 in the Radix Pulsatillae extract aqueous solution, and ebuillition of heated 4-10 hour, every 30-60 minute survey PH, remain on PH10-13, let cool, add hydrochloric acid solution, transfer PH 3-7, centrifugal, go precipitation, namely get the Radix Pulsatillae extract hydrolyzed solution
The 3rd step, the Radix Pulsatillae extract hydrolyzed solution, through D101 or AB-8 or ADS-17 macroporous resin adsorption, water, 30-40% ethanol, 50-80% ethanol, 95% ethanol elution are collected 50-80% ethanol elution part successively, namely get the BTW58 component.Through the experiment of antitumor pharmacology, prove that the BTW58 component is Radix Pulsatillae anti-tumor effective component.
The 4th step, Radix Pulsatillae anti-tumor effective component is through ODS reverse phase silica gel post, carries out gradient elution with the mixed liquor of first alcohol and water (6:4-9:1), and gradient eluent has five groups successively, and its volume proportion is respectively
6 parts of first group of methanol: 4 parts in water;
7 parts of second group of methanol: 3 parts in water;
8 parts of the 3rd group of methanol: 2 parts in water;
85 parts of the 4th group of methanol: 15 parts in water;
9 parts of the 5th group of methanol: 1 part in water;
85 parts of the 5th step, collection methanol: the eluting part that water is 15 parts, through Sephadex LH-20 post methanol-water 7:3 eluting, obtain Compound I, relatively reach the LC/MS/MS test through reference substance, this chemical compound is 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
Oleanolic acid saponin constituents of the present invention is the chemical compound of single component, and its chemical name is 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
Description of drawings:
Fig. 1 is the molecular structural formula of 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin;
Fig. 2 is the HPLC analysis chart of BTW58 component;
Fig. 3 is the hydrogen nuclear magnetic resonance spectrogram of 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin;
Fig. 4 is the carbon-13 nmr spectra figure of 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin;
Specific embodiment:
The preparation method of 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin is:
Case study on implementation 1
The first step becomes coarse powder with Radix Pulsatillae pulverizing medicinal materials, the 0.5-2h that is soaked in water, and 6-12 doubly measures heating and refluxing extraction 2-3 time, and each 1-3h filters merging filtrate.Above-mentioned filtrate is evaporated to 0.5-1.0g raw medicinal herbs/mL in 50-70 ℃, and 0-4 ℃ of cold preservation is spent the night, and analyses glue, filters, and namely gets Radix Pulsatillae extract.
[0014] second step adds sodium hydroxide solution and is transferred to PH10-13 in the Radix Pulsatillae extract aqueous solution, and ebuillition of heated 4-10 hour, every 30-60 minute survey PH, remain on PH10-13, let cool, add hydrochloric acid solution, transfer PH 3-7, centrifugal, go precipitation, namely get the Radix Pulsatillae extract hydrolyzed solution
The 3rd step, the Radix Pulsatillae extract hydrolyzed solution, through D101 or AB-8 or ADS-17 macroporous resin adsorption, water, 30-40% ethanol, 50-80% ethanol, 95% ethanol elution are collected 50-80% ethanol elution part successively, namely get the BTW58 component.Through the experiment of antitumor pharmacology, prove that the BTW58 component is Radix Pulsatillae anti-tumor effective component.
[0015] the 4th step, Radix Pulsatillae anti-tumor effective component is through ODS reverse phase silica gel post, carries out gradient elution with the mixed liquor of first alcohol and water (6:4-9:1), and gradient eluent has five groups successively, and its volume proportion is respectively
6 parts of first group of methanol: 4 parts in water;
7 parts of second group of methanol: 3 parts in water;
8 parts of the 3rd group of methanol: 2 parts in water;
85 parts of the 4th group of methanol: 15 parts in water;
9 parts of the 5th group of methanol: 1 part in water;
85 parts of the 5th step, collection methanol: the eluting part that water is 15 parts, through Sephadex LH-20 post methanol-water 7:3 eluting, obtain Compound I, relatively reach the LC/MS/MS test through reference substance, this chemical compound is 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
[0016] case study on implementation 2
The first step becomes coarse powder with Radix Pulsatillae pulverizing medicinal materials, and with 10% soak with ethanol 0.5-2h, 6-12 doubly measures heating and refluxing extraction 2-3 time, and each 1-3h filters merging filtrate.Above-mentioned filtrate is evaporated to 0.5-1.0g raw medicinal herbs/mL in 50-70 ℃, and without the alcohol flavor, 0-4 ℃ of cold preservation is spent the night, and analyses glue, filters, and namely gets Radix Pulsatillae extract.
[0017] second step adds sodium hydroxide solution and is transferred to PH10-13 in the Radix Pulsatillae extract aqueous solution, and ebuillition of heated 4-10 hour, every 30-60 minute survey PH, remain on PH10-13, let cool, add hydrochloric acid solution, transfer PH 3-7, centrifugal, go precipitation, namely get the Radix Pulsatillae extract hydrolyzed solution
The 3rd step, the Radix Pulsatillae extract hydrolyzed solution, through D101 or AB-8 or ADS-17 macroporous resin adsorption, water, 30-40% ethanol, 50-80% ethanol, 95% ethanol elution are collected 50-80% ethanol elution part successively, namely get the BTW58 component.Through the experiment of antitumor pharmacology, prove that the BTW58 component is Radix Pulsatillae anti-tumor effective component.
[0018] the 4th step, Radix Pulsatillae anti-tumor effective component is through ODS reverse phase silica gel post, carries out gradient elution with the mixed liquor of first alcohol and water (6:4-9:1), and gradient eluent has five groups successively, and its volume proportion is respectively
6 parts of first group of methanol: 4 parts in water;
7 parts of second group of methanol: 3 parts in water;
8 parts of the 3rd group of methanol: 2 parts in water;
85 parts of the 4th group of methanol: 15 parts in water;
9 parts of the 5th group of methanol: 1 part in water;
85 parts of the 5th step, collection methanol: the eluting part that water is 15 parts, through Sephadex LH-20 post methanol-water 7:3 eluting, obtain Compound I, relatively reach the LC/MS/MS test through reference substance, this chemical compound is 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
[0019] case study on implementation 3
The first step becomes coarse powder with Radix Pulsatillae pulverizing medicinal materials, and with 20% soak with ethanol 0.5-2h, 6-12 doubly measures heating and refluxing extraction 2-3 time, and each 1-3h filters merging filtrate.Above-mentioned filtrate is evaporated to 0.5-1.0g raw medicinal herbs/mL in 50-70 ℃, and without the alcohol flavor, 0-4 ℃ of cold preservation is spent the night, and analyses glue, filters, and namely gets Radix Pulsatillae extract.
[0020] second step adds sodium hydroxide solution and is transferred to PH10-13 in the Radix Pulsatillae extract aqueous solution, and ebuillition of heated 4-10 hour, every 30-60 minute survey PH, remain on PH10-13, let cool, add hydrochloric acid solution, transfer PH 3-7, centrifugal, go precipitation, namely get the Radix Pulsatillae extract hydrolyzed solution
The 3rd step, the Radix Pulsatillae extract hydrolyzed solution, through D101 or AB-8 or ADS-17 macroporous resin adsorption, water, 30-40% ethanol, 50-80% ethanol, 95% ethanol elution are collected 50-80% ethanol elution part successively, namely get the BTW58 component.Through the experiment of antitumor pharmacology, prove that the BTW58 component is Radix Pulsatillae anti-tumor effective component.
[0021] the 4th step, Radix Pulsatillae anti-tumor effective component is through ODS reverse phase silica gel post, carries out gradient elution with the mixed liquor of first alcohol and water (6:4-9:1), and gradient eluent has five groups successively, and its volume proportion is respectively
6 parts of first group of methanol: 4 parts in water;
7 parts of second group of methanol: 3 parts in water;
8 parts of the 3rd group of methanol: 2 parts in water;
85 parts of the 4th group of methanol: 15 parts in water;
9 parts of the 5th group of methanol: 1 part in water;
85 parts of the 5th step, collection methanol: the eluting part that water is 15 parts, through Sephadex LH-20 post methanol-water 7:3 eluting, obtain Compound I, relatively reach the LC/MS/MS test through reference substance, this chemical compound is 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
[0022] embodiment 4,
The first step becomes coarse powder with Radix Pulsatillae pulverizing medicinal materials, and with 40% soak with ethanol 0.5-2h, 6-12 doubly measures heating and refluxing extraction 2-3 time, and each 1-3h filters merging filtrate.Above-mentioned filtrate is evaporated to 0.5-1.0g raw medicinal herbs/mL in 50-70 ℃, and without the alcohol flavor, 0-4 ℃ of cold preservation is spent the night, and analyses glue, filters, and namely gets Radix Pulsatillae extract.
[0023] second step adds sodium hydroxide solution and is transferred to PH10-13 in the Radix Pulsatillae extract aqueous solution, and ebuillition of heated 4-10 hour, every 30-60 minute survey PH, remain on PH10-13, let cool, add hydrochloric acid solution, transfer PH 3-7, centrifugal, go precipitation, namely get the Radix Pulsatillae extract hydrolyzed solution
The 3rd step, the Radix Pulsatillae extract hydrolyzed solution, through D101 or AB-8 or ADS-17 macroporous resin adsorption, water, 30-40% ethanol, 50-80% ethanol, 95% ethanol elution are collected 50-80% ethanol elution part successively, namely get the BTW58 component.Through the experiment of antitumor pharmacology, prove that the BTW58 component is Radix Pulsatillae anti-tumor effective component.
[0024] the 4th step, Radix Pulsatillae anti-tumor effective component is through ODS reverse phase silica gel post, carries out gradient elution with the mixed liquor of first alcohol and water (6:4-9:1), and gradient eluent has five groups successively, and its volume proportion is respectively
6 parts of first group of methanol: 4 parts in water;
7 parts of second group of methanol: 3 parts in water;
8 parts of the 3rd group of methanol: 2 parts in water;
85 parts of the 4th group of methanol: 15 parts in water;
9 parts of the 5th group of methanol: 1 part in water;
85 parts of the 5th step, collection methanol: the eluting part that water is 15 parts, through Sephadex LH-20 post methanol-water 7:3 eluting, obtain Compound I, relatively reach the LC/MS/MS test through reference substance, this chemical compound is 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
[0025] embodiment 5,
The first step becomes coarse powder with Radix Pulsatillae pulverizing medicinal materials, and with 60% soak with ethanol 0.5-2h, 6-12 doubly measures heating and refluxing extraction 2-3 time, and each 1-3h filters merging filtrate.Above-mentioned filtrate is evaporated to 0.5-1.0g raw medicinal herbs/mL in 50-70 ℃, and without the alcohol flavor, 0-4 ℃ of cold preservation is spent the night, and analyses glue, filters, and namely gets Radix Pulsatillae extract.
[0026] second step adds sodium hydroxide solution and is transferred to PH10-13 in the Radix Pulsatillae extract aqueous solution, and ebuillition of heated 4-10 hour, every 30-60 minute survey PH, remain on PH10-13, let cool, add hydrochloric acid solution, transfer PH 3-7, centrifugal, go precipitation, namely get the Radix Pulsatillae extract hydrolyzed solution
The 3rd step, the Radix Pulsatillae extract hydrolyzed solution, through D101 or AB-8 or ADS-17 macroporous resin adsorption, water, 30-40% ethanol, 50-80% ethanol, 95% ethanol elution are collected 50-80% ethanol elution part successively, namely get the BTW58 component.Through the experiment of antitumor pharmacology, prove that the BTW58 component is Radix Pulsatillae anti-tumor effective component.
[0027] the 4th step, Radix Pulsatillae anti-tumor effective component is through ODS reverse phase silica gel post, carries out gradient elution with the mixed liquor of first alcohol and water (6:4-9:1), and gradient eluent has five groups successively, and its volume proportion is respectively
6 parts of first group of methanol: 4 parts in water;
7 parts of second group of methanol: 3 parts in water;
8 parts of the 3rd group of methanol: 2 parts in water;
85 parts of the 4th group of methanol: 15 parts in water;
9 parts of the 5th group of methanol: 1 part in water;
85 parts of the 5th step, collection methanol: the eluting part that water is 15 parts, through Sephadex LH-20 post methanol-water 7:3 eluting, obtain Compound I, relatively reach the LC/MS/MS test through reference substance, this chemical compound is 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
[0028] embodiment 6,
The first step becomes coarse powder with Radix Pulsatillae pulverizing medicinal materials, and with 70% soak with ethanol 0.5-2h, 6-12 doubly measures heating and refluxing extraction 2-3 time, and each 1-3h filters merging filtrate.Above-mentioned filtrate is evaporated to 0.5-1.0g raw medicinal herbs/mL in 50-70 ℃, and without the alcohol flavor, 0-4 ℃ of cold preservation is spent the night, and analyses glue, filters, and namely gets Radix Pulsatillae extract.
[0029] second step adds sodium hydroxide solution and is transferred to PH10-13 in the Radix Pulsatillae extract aqueous solution, and ebuillition of heated 4-10 hour, every 30-60 minute survey PH, remain on PH10-13, let cool, add hydrochloric acid solution, transfer PH 3-7, centrifugal, go precipitation, namely get the Radix Pulsatillae extract hydrolyzed solution
The 3rd step, the Radix Pulsatillae extract hydrolyzed solution, through D101 or AB-8 or ADS-17 macroporous resin adsorption, water, 30-40% ethanol, 50-80% ethanol, 95% ethanol elution are collected 50-80% ethanol elution part successively, namely get the BTW58 component.Through the experiment of antitumor pharmacology, prove that the BTW58 component is Radix Pulsatillae anti-tumor effective component.
[0030] the 4th step, Radix Pulsatillae anti-tumor effective component is through ODS reverse phase silica gel post, carries out gradient elution with the mixed liquor of first alcohol and water (6:4-9:1), and gradient eluent has five groups successively, and its volume proportion is respectively
6 parts of first group of methanol: 4 parts in water;
7 parts of second group of methanol: 3 parts in water;
8 parts of the 3rd group of methanol: 2 parts in water;
85 parts of the 4th group of methanol: 15 parts in water;
9 parts of the 5th group of methanol: 1 part in water;
85 parts of the 5th step, collection methanol: the eluting part that water is 15 parts, through Sephadex LH-20 post methanol-water 7:3 eluting, obtain Compound I, relatively reach the LC/MS/MS test through reference substance, this chemical compound is 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
[0031] embodiment 7,
The first step becomes coarse powder with Radix Pulsatillae pulverizing medicinal materials, and with 95% soak with ethanol 0.5-2h, 6-12 doubly measures heating and refluxing extraction 2-3 time, and each 1-3h filters merging filtrate.Above-mentioned filtrate is evaporated to 0.5-1.0g raw medicinal herbs/mL in 50-70 ℃, and without the alcohol flavor, 0-4 ℃ of cold preservation is spent the night, and analyses glue, filters, and namely gets Radix Pulsatillae extract.
[0032] second step adds sodium hydroxide solution and is transferred to PH10-13 in the Radix Pulsatillae extract aqueous solution, and ebuillition of heated 4-10 hour, every 30-60 minute survey PH, remain on PH10-13, let cool, add hydrochloric acid solution, transfer PH 3-7, centrifugal, go precipitation, namely get the Radix Pulsatillae extract hydrolyzed solution
The 3rd step, the Radix Pulsatillae extract hydrolyzed solution, through D101 or AB-8 or ADS-17 macroporous resin adsorption, water, 30-40% ethanol, 50-80% ethanol, 95% ethanol elution are collected 50-80% ethanol elution part successively, namely get the BTW58 component.Through the experiment of antitumor pharmacology, prove that the BTW58 component is Radix Pulsatillae anti-tumor effective component.
[0033] the 4th step, Radix Pulsatillae anti-tumor effective component is through ODS reverse phase silica gel post, carries out gradient elution with the mixed liquor of first alcohol and water (6:4-9:1), and gradient eluent has five groups successively, and its volume proportion is respectively
6 parts of first group of methanol: 4 parts in water;
7 parts of second group of methanol: 3 parts in water;
8 parts of the 3rd group of methanol: 2 parts in water;
85 parts of the 4th group of methanol: 15 parts in water;
9 parts of the 5th group of methanol: 1 part in water;
85 parts of the 5th step, collection methanol: the eluting part that water is 15 parts, through Sephadex LH-20 post methanol-water 7:3 eluting, obtain Compound I, relatively reach the LC/MS/MS test through reference substance, this chemical compound is 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
[0034]
Research is found, in the ethanol extraction of the Radix Pulsatillae, contain 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin, but content is starkly lower than the BTW58 component after basic hydrolysis.
[0035] BTW58 component HPLC analyzes:
Chromatographic column does not add pre-column, and pressure is bigger than normal: Kromasil(C18,250*4.6mm, 5um); Flow velocity: 1ml/min; Column temperature: 35 ℃; Sample size: 20ul; Elution system: methanol-water system
ELSD:SHIMADZU ELSD-LT II; Column temperature: 40 ℃; Press:350Kpa
Gradient condition: time (min) methanol (%)
0.01 65
10 70
30 70
50 100
55 100
55.01 STOP
The Compound I structure elucidation:
The white amorphous powder, sulphuric acid ethanol displaing amaranth speckle, acetic anhydride-strong sulfuric acid response is positive, and the Molish reacting positive points out this chemical compound may be saponins compound.Compound I obtains oleanolic acid saponin unit and monosaccharide with the complete acid hydrolysis of 2N TFA, and sugar carries out GC behind derivatization analyzes, and detects the existence of L-arabinose, L-rhamnose and D-Glucose.
[0036]
13C NMR spectrum shows 47 carbon signals altogether, comprises 30 aglycon carbon signals.Wherein aglycon carbon signal and oleanolic acid aglycon are basically identical.88.9,28 carbonyl carbon signals are at δ 180.4 to low field displacement to δ for 3 carbon, and the sugar that this chemical compound is described is to be connected on 3 of aglycon.
133 sugared signals occurred in the C NMR spectrum, the GC analysis result in conjunction with behind NMR data and the hydrolysis derivatization proves that it is respectively L-arabinose, L-rhamnose and D-Glucose.In addition,
13Show in the C NMR spectrum, 3 end group carbon signals that sugar is corresponding occur at δ 106.5,105.1 and 101.9 positions.Through above-mentioned analysis, and and disclosed document [Opinya A.Ekabo, Norman R. Farnsworth, Henderson T., et al. J Nat Prod, 1996,59:431-435] in data compare, the structure that proves Compound I is 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
13C NMR spectrum attribution data sees Table 1.
[0037] carbon of table 1 Compound I spectrum data (Pyridine-d
6)
| No | δ (ppm) | No | δ (ppm) | No | δ (ppm) |
| 1 | 39.0 | 17 | 46.8 | 3′ | 74.2 |
| 2 | 26.8 | 18 | 42.1 | 4′ | 79.7 |
| 3 | 88.9 | 19 | 46.6 | 5′ | 64.6 |
| 4 | 39.9 | 20 | 31.1 | Rha-1′′ | 101.9 |
| 5 | 56.1 | 21 | 34.4 | 2′′ | 72.6 |
| 6 | 18.7 | 22 | 33.4 | 3′′ | 72.4 |
| 7 | 33.4 | 23 | 28.2 | 4′′ | 74.2 |
| 8 | 39.6 | 24 | 17.2 | 5′′ | 69.9 |
| 9 | 48.2 | 25 | 15.9 | 6′′ | 18.8 |
| 10 | 37.2 | 26 | 17.5 | Glc-1′′′ | 106.5 |
| 11 | 23.9 | 27 | 26.3 | 2′′′ | 75.6 |
| 12 | 122.7 | 28 | 180.4 | 3′′′ | 78.7 |
| 13 | 145.0 | 29 | 33.4 | 4′′′ | 71.4 |
| 14 | 42.3 | 30 | 23.9 | 5′′′ | 78.9 |
| 15 | 28.5 | Ara-1′ | 105.1 | 6′′′ | 62.7 |
| 16 | 23.9 | 2′ | 76.5 |
The research of antitumor pharmacology:
From whole, cellular level, the vivo and vitro experiment combines, complete observation the antitumor action of BTW58 component and Compound I, experiment in vitro is the result show, BTW58 component, Compound I all have preferably inhibitory action to 17 kinds of tumor cells such as vitro human pulmonary carcinoma, hepatocarcinoma, gastric cancer, colon cancer, glioma, breast carcinoma, leukemia, it is to inhibition concentration (IC50) scope of partly imitating of various subject cells: 1.48-18.1 μ g/ml, basically all less than 10ug/ml; In vivo test shows, BTW58 component, Compound I present stronger active anticancer to nude mice heteroplastic transplantation tumor in the bodies such as people's pulmonary carcinoma, hepatocarcinoma, gastric cancer, colon cancer, glioma, breast carcinoma, leukemia, cervical cancer, and to nude mice heteroplastic transplantation tumor in the bodies such as people's renal carcinoma, human nasopharyngeal carcinoma without obvious active anticancer.
[0038] 1. Compound I and BTW58 component are partly imitated inhibition concentration to vitro human 17 tumor cells
Adopt mtt assay, select 15 kinds of human tumor cell lines, investigate the Compound I of variable concentrations and the impact that the BTW58 component is bred human tumor cells.Collect each tumor cell of logarithmic (log) phase, adjust concentration of cell suspension to 1 * 10
6Individual/mL, add 100 μ L in the every hole of 96 orifice plates, place 5% CO
2, hatch 24 h in 37 ℃ of incubators after, be paved with the hole to cell monolayer at the bottom of (96 hole flat underside), add the medicine saponin (1.5625,3.125,6.25,12.5,25 μ g/mL) of Concentraton gradient, each concentration is established 5 multiple holes.Behind medicine and cytosis 48 h, calculate the suppression ratio (%) of medicine cell growth.
[0039] experimental result prompting, Compound I and BTW58 component all present good concentration dependence to the growth inhibitory effect of various subject cells, inhibition concentration (IC50) scope of partly imitating to various subject cells: 1.48-18.1 μ g/ml see Table 2, can find out that from experimental result BTW58 component and Compound I partly imitate inhibition concentration basically all less than 10ug/ml to these Growth of Cells, suitable with the cisplatin effect, some cell strain such as glioma U251, SHG-44, the inhibitions such as mouse melanoma B16 all are better than cisplatin, the result of partly imitating inhibition concentration of comprehensive each subject cell can point out external pulmonary carcinoma, hepatocarcinoma, gastric cancer, colon cancer, breast carcinoma, glioma, the growth inhibited effect of the cell strains such as leukemia is more remarkable.
[0040] table 2 Compound I and BTW58 component are partly imitated inhibition concentration (IC50, μ g/mL) to the growth of various subject cells
| Cell strain | The BTW58 component | Compound I |
| Pulmonary carcinoma A549 | 5.43 | 3.01 |
| Pulmonary carcinoma NCI-H460 | 5.24 | 3.15 |
| Human glioma cells SHG-44 | 1.48 | 3.11 |
| Human hepatocellular carcinoma BEL-7402 | 9.1 | 5.46 |
| Human hepatocellular carcinoma SMMC-7721 | 4.03 | 3.25 |
| Human glioma cells U251 | 3.04 | 3.18 |
| Human erythroleukemia cell K-562 | 2.69 | 2.45 |
| Differentiation adenocarcinoma of stomach SGC-7901 among the people | 6.84 | 3.12 |
| Human breast carcinoma SK-BR-3 | 8.42 | 6.24 |
| Human cervical carcinoma Hela | 5.52 | 5.28 |
| The low differentiation of people gastric gland BGC-823 | 3.87 | 3.47 |
| People's promyelocytic leukemia HL-60 | 8.23 | 6.35 |
| Nasopharyngeal carcinoma | 15.32 | 7.62 |
| People 786-O kidney clear cell adenocarcinoma cell | 18.1 | 7.44 |
| Human colon adenocarcinoma HCT-116 | 5.03 | 1.32 |
| Human colon adenocarcinoma HT-29 | 8.21 | 1.69 |
| Human breast carcinoma MCF-7 | 7.52 | 5.35 |
| Murine melanoma B-16 | 2.22 | 2.04 |
| Carcinoma of prostate | 12.21 | 5.21 |
2. utilized mice transplanted tumor scale-model investigation Compound I and BTW58 component is on the impact of hepatocarcinoma H22.
[0041] get the mice that inoculates H22 sarcoma cell 7d, take off cervical vertebra and put to death, extract ascites under the aseptic condition, adjusting oncocyte concentration with physiological saline solution is 2 * 10
7/ ml, subcutaneous vaccination 0.2 ml tumor cell suspension after every right side of mice axillary fossa sterilization.Begin administration behind mouse inoculation oncocyte 24 h, normal group and lotus tumor model group only gavage PBS 0.5ml/ every day; Positive controls intraperitoneal injection of cyclophosphamide 40mg/kg, the next day 1 time; Compound I 50mg/kg, BTW58 component 100mg/kg, successive administration 14 days.24 h after the last administration, body weight, thymic weight, the thymic weight of KM Mus respectively organized in weighing, peel off tumor tissues and weigh, and the aseptic spleen of getting weighed.
[0042] experimental result prompting, it is heavy that the BTW58 component can obviously reduce the H22 mouse tumor, and suppression ratio is up to 75.6%; It is heavy that Compound I also can obviously reduce the H22 mouse tumor, up to 81%, suitable with the cyclophosphamide effect to the suppression ratio of rat liver cancer growth.See Table 3.
[0043] table 3 Compound I and BTW58 component on H22 mice with tumor tumor heavily reach tumour inhibiting rate impact (n=10,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Matched group | / | 1.89±0.76 | / |
| | 100 | 0.46±0.38** | 75.7% |
| The Compound I group | 50 | 0.36±0.26** | 81% |
| The | 40 | 0.18±0.15** | 90.5% |
Annotate: compare * P＜0.05, * * P＜0.01 with matched group
3. utilized mice transplanted tumor scale-model investigation Compound I and BTW58 component is on the impact of S180
Get the mice of inoculation S180 sarcoma cell 7d, take off cervical vertebra and put to death, extract ascites under the aseptic condition, adjusting oncocyte concentration with physiological saline solution is 2 * 10
6/ ml, subcutaneous vaccination 0.2 ml tumor cell suspension after every right side of mice axillary fossa sterilization.Begin administration behind mouse inoculation oncocyte 24 h, normal group and lotus tumor model group only gavage PBS 0.5ml/ every day; Positive controls intraperitoneal injection of cyclophosphamide 40mg/kg, the next day 1 time; Compound I 50mg/kg, BTW58 component 100mg/kg, successive administration 14 days.24 h after the last administration, body weight, thymic weight, the thymic weight of KM Mus respectively organized in weighing, peel off tumor tissues and weigh, and the aseptic spleen of getting weighed.
[0044] experimental result prompting, it is heavy that the BTW58 component can obviously reduce the S180 mouse tumor, and suppression ratio is up to 64.1%; It is heavy that Compound I also can obviously reduce the H22 mouse tumor, up to 67.1%, suitable with the cyclophosphamide effect to the suppression ratio of rat liver cancer growth.See Table 2.
[0045] table 3 Compound I and BTW58 component on S180 mice with tumor tumor heavily reach tumour inhibiting rate impact (n=10,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Model group | / | 2.37±0.68 | / |
| | 100 | 0.85±0.74* | 64.1% |
| The Compound I group | 50 | 0.78±0.31** | 67.1% |
| The | 40 | 0.25±0.42** | 89.5% |
Annotate: compare * P＜0.05, * * P＜0.01 with matched group
4. utilize the interior nude mice heteroplastic transplantation tumor model observation Compound I of body and BTW58 component on the impact of people's hepatocarcinoma SMMC-7721
Aseptic taking-up tumor mass under people's cancer kind Corium Mus, with the clean blood of normal saline flushing, select well-grown tumor tissues to be cut into small pieces, about grain of rice size, it is subcutaneous that small tissue blocks is inoculated in nude mice right side axillary fossa, after the inoculation until Growth of Tumors Transplanted (100mm after a certain size
3More than) begin the administration of dividing into groups, by the grouping of tumor volume, sub-model group, positive drug cyclophosphamide group (40mg/kg, ip), Compound I 50mg/kg, BTW58 component 100mg/kg, every group each 7.When experiment finishes, strip the tumor piece, claim tumor heavy, calculate the heavy suppression ratio of tumor.
[0046] result shows, Compound I and BTW58 component inhibitory rate 75% and 77.7%, effect are better than cyclophosphamide (61.1%) effect, and relatively there were significant differences with model group, and visible Compound I and BTW58 component all have preferably inhibitory action to hepatocarcinoma.
[0047] table 4 Compound I and BTW58 component on SMMC-7721 animal tumor heavily reach tumour inhibiting rate impact (n=7,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Model group | / | 1.57±0.31 | / |
| | 100 | 0.39±0.24** | 75% |
| The Compound I group | 50 | 0.35±0.21 | 77.7% |
| The | 40 | 0.61±0.24** | 61.1% |
Annotate: * and model group compare P＜0.05 * * and model group compares P＜0.01
5. utilize the interior nude mice heteroplastic transplantation tumor model observation Compound I of body and BTW58 component on the impact of people's glioma SHG-44
Aseptic taking-up tumor mass under people's cancer kind Corium Mus, with the clean blood of normal saline flushing, select well-grown tumor tissues to be cut into small pieces, about grain of rice size, it is subcutaneous that small tissue blocks is inoculated in nude mice right side axillary fossa, after the inoculation until Growth of Tumors Transplanted (100mm after a certain size
3More than) begin the administration of dividing into groups, by the grouping of tumor volume, sub-model group, positive drug cyclophosphamide group (40mg/kg, ip), Compound I 50mg/kg, BTW58 component 100mg/kg, every group each 7.When experiment finishes, strip the tumor piece, claim tumor heavy, calculate the heavy suppression ratio of tumor.
[0048] see Table, Compound I and BTW58 component inhibitory rate 79.3%, 81.1%, effect is better than cyclophosphamide (66.5%), and relatively there were significant differences with model group, and visible Compound I and BTW58 component have preferably inhibitory action to glioma.
[0049] table 5 Compound I and BTW58 component on SHG-44 animal tumor heavily reach tumour inhibiting rate impact (n=7,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Model group | / | 1.27±0.15 | / |
| | 100 | 0.26±0.13** | 79.3% |
| The Compound I group | 50 | 0.24±0.19 | 81.1% |
| The | 40 | 0.42±0.28** | 66.5% |
Annotate: * and model group compare P＜0.05 * * and model group compares P＜0.01
6. utilize the interior nude mice heteroplastic transplantation tumor model observation Compound I of body and BTW58 component on the impact of people's gastric cancer SGC-7901
Aseptic taking-up tumor mass under people's cancer kind Corium Mus, with the clean blood of normal saline flushing, select well-grown tumor tissues to be cut into small pieces, about grain of rice size, it is subcutaneous that small tissue blocks is inoculated in nude mice right side axillary fossa, after the inoculation until Growth of Tumors Transplanted (100mm after a certain size
3More than) begin the administration of dividing into groups, by the grouping of tumor volume, sub-model group, positive drug cyclophosphamide group (40mg/kg, ip), Compound I 50mg/kg, BTW58 component 100mg/kg, every group each 7.When experiment finishes, strip the tumor piece, claim tumor heavy, calculate the heavy suppression ratio of tumor.
[0050] see Table, Compound I and BTW58 component inhibitory rate 60.1%, 61.6%, relatively there were significant differences with model group, and visible Compound I and BTW58 component have preferably inhibitory action to gastric cancer.
[0051] table 6 Compound I and BTW58 component on SGC-7901 animal tumor heavily reach tumour inhibiting rate impact (n=7,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Model group | / | 1.38±0.45 | / |
| | 100 | 0.55±0.28* | 60.1% |
| The Compound I group | 50 | 0.53±0.36* | 61.6% |
| The | 40 | 0.41±0.36* | 70.5% |
Annotate: * and model group compare P＜0.05 * * and model group compares P＜0.01
7. utilize the interior nude mice heteroplastic transplantation tumor model observation Compound I of body and BTW58 component on the impact of human breast carcinoma MCF-7
Aseptic taking-up tumor mass under people's cancer kind Corium Mus, with the clean blood of normal saline flushing, select well-grown tumor tissues to be cut into small pieces, about grain of rice size, it is subcutaneous that small tissue blocks is inoculated in nude mice right side axillary fossa, after the inoculation until Growth of Tumors Transplanted (100mm after a certain size
3More than) begin the administration of dividing into groups, by the grouping of tumor volume, sub-model group, positive drug cyclophosphamide group (40mg/kg, ip), Compound I 50mg/kg, BTW58 component 100mg/kg, every group each 7.When experiment finishes, strip the tumor piece, claim tumor heavy, calculate the heavy suppression ratio of tumor.
[0052] see Table, Compound I and BTW58 component inhibitory rate 62.5%, 68.3%, relatively there were significant differences with model group, and visible Compound I and BTW58 component have preferably inhibitory action to breast carcinoma.
[0053] table 7 Compound I and BTW58 component on MCF-7 animal tumor heavily reach tumour inhibiting rate impact (n=7,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Model group | / | 1.83±1.19 | / |
| | 100 | 0.69±0.54* | 62.5% |
| The Compound I group | 50 | 0.58±0.43* | 68.3% |
| The | 40 | 0.39±0.25** | 78.6% |
Annotate: * and model group compare P＜0.05 * * and model group compares P＜0.01
8. utilize the interior nude mice heteroplastic transplantation tumor model observation Compound I of body and BTW58 component on the impact of human leukaemia K562
Aseptic taking-up tumor mass under people's cancer kind Corium Mus, with the clean blood of normal saline flushing, select well-grown tumor tissues to be cut into small pieces, about grain of rice size, it is subcutaneous that small tissue blocks is inoculated in nude mice right side axillary fossa, after the inoculation until Growth of Tumors Transplanted (100mm after a certain size
3More than) begin the administration of dividing into groups, by the grouping of tumor volume, sub-model group, positive drug cyclophosphamide group (40mg/kg, ip), Compound I 50mg/kg, BTW58 component 100mg/kg, every group each 7.When experiment finishes, strip the tumor piece, claim tumor heavy, calculate the heavy suppression ratio of tumor.
[0054] see Table, Compound I and BTW58 component inhibitory rate 85.2%, 77.2%, relatively there were significant differences with model group, and visible Compound I and BTW58 component have preferably inhibitory action to leukemia.
[0055] table 8 Compound I and BTW58 component on K562 animal tumor heavily reach tumour inhibiting rate impact (n=7,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Model group | / | 1.45±1.19 | / |
| | 100 | 0.21±0.35** | 85.2% |
| The Compound I group | 50 | 0.33±0.46** | 77.2% |
| The | 40 | 0.46±0.27** | 68.2% |
Annotate: * and model group compare P＜0.05 * * and model group compares P＜0.01
9. utilize the interior nude mice heteroplastic transplantation tumor model observation Compound I of body and BTW58 component on the impact of human cervical carcinoma Hela
Aseptic taking-up tumor mass under people's cancer kind Corium Mus, with the clean blood of normal saline flushing, select well-grown tumor tissues to be cut into small pieces, about grain of rice size, it is subcutaneous that small tissue blocks is inoculated in nude mice right side axillary fossa, after the inoculation until Growth of Tumors Transplanted (100mm after a certain size
3More than) begin the administration of dividing into groups, by the grouping of tumor volume, sub-model group, positive drug cyclophosphamide group (40mg/kg, ip), Compound I 50mg/kg, BTW58 component 100mg/kg, every group each 7.When experiment finishes, strip the tumor piece, claim tumor heavy, calculate the heavy suppression ratio of tumor.
[0056] see Table, Compound I and BTW58 component inhibitory rate 75.9%, 73.8%, relatively there were significant differences with model group, and visible Compound I and BTW58 component have preferably inhibitory action to cervical cancer.
[0057] table 9 Compound I and BTW58 component on human cervical carcinoma Hela animal tumor heavily reach tumour inhibiting rate impact (n=7,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Model group | / | 1.87±1.24 | / |
| | 100 | 0.45±0.38* | 75.9% |
| The Compound I group | 50 | 0.49±0.34* | 73.8% |
| The | 40 | 0.41±0.46* | 78.1% |
Annotate: * and model group compare P＜0.05 * * and model group compares P＜0.01
10. utilize the interior nude mice heteroplastic transplantation tumor model observation Compound I of body and BTW58 component on the impact of human colon carcinoma HCT-116
Aseptic taking-up tumor mass under people's cancer kind Corium Mus, with the clean blood of normal saline flushing, select well-grown tumor tissues to be cut into small pieces, about grain of rice size, it is subcutaneous that small tissue blocks is inoculated in nude mice right side axillary fossa, after the inoculation until Growth of Tumors Transplanted (100mm after a certain size
3More than) begin the administration of dividing into groups, by the grouping of tumor volume, sub-model group, positive drug cyclophosphamide group (40mg/kg, ip), Compound I 50mg/kg, BTW58 component 100mg/kg, every group each 7.When experiment finishes, strip the tumor piece, claim tumor heavy, calculate the heavy suppression ratio of tumor.
[0058] see Table, Compound I and BTW58 component inhibitory rate 74.5%, 80.9%, relatively there were significant differences with model group, and visible Compound I and BTW58 component have preferably inhibitory action to colon cancer.
[0059] table 10 Compound I and BTW58 component on human colon carcinoma HCT-116 animal tumor heavily reach tumour inhibiting rate impact (n=7,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Model group | / | 2.04±1.75 | / |
| | 100 | 0.52±0.36** | 74.5% |
| The Compound I group | 50 | 0.39±0.56** | 80.9% |
| The | 40 | 0.44±0.43** | 78.4% |
Annotate: * and model group compare P＜0.05 * * and model group compares P＜0.01
11. utilize the interior nude mice heteroplastic transplantation tumor model observation Compound I of body and BTW58 component on the impact of people's pulmonary carcinoma A549
Aseptic taking-up tumor mass under people's cancer kind Corium Mus, with the clean blood of normal saline flushing, select well-grown tumor tissues to be cut into small pieces, about grain of rice size, it is subcutaneous that small tissue blocks is inoculated in nude mice right side axillary fossa, after the inoculation until Growth of Tumors Transplanted (100mm after a certain size
3More than) begin the administration of dividing into groups, by the grouping of tumor volume, sub-model group, positive drug cyclophosphamide group (40mg/kg, ip), Compound I 50mg/kg, BTW58 component 100mg/kg, every group each 7.When experiment finishes, strip the tumor piece, claim tumor heavy, calculate the heavy suppression ratio of tumor.
[0060] see Table, Compound I and BTW58 component inhibitory rate 74.5%, 80.9%, relatively there were significant differences with model group, and visible Compound I and BTW58 component have preferably inhibitory action to pulmonary carcinoma.
[0061] table 11 Compound I and BTW58 component on people's pulmonary carcinoma A549 animal tumor heavily reach tumour inhibiting rate impact (n=7,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Model group | / | 1.97±1.25 | / |
| | 100 | 0.55±0.29** | 72.1% |
| The Compound I group | 50 | 0.50±0.31** | 74.6% |
| The | 40 | 0.56±0.28** | 71.6% |
Annotate: * and model group compare P＜0.05 * * and model group compares P＜0.01
12. utilize the interior nude mice heteroplastic transplantation tumor model observation Compound I of body and BTW58 component on the impact of people's kidney clear cell adenocarcinoma 786-O
Aseptic taking-up tumor mass under people's cancer kind Corium Mus, with the clean blood of normal saline flushing, select well-grown tumor tissues to be cut into small pieces, about grain of rice size, it is subcutaneous that small tissue blocks is inoculated in nude mice right side axillary fossa, after the inoculation until Growth of Tumors Transplanted (100mm after a certain size
3More than) begin the administration of dividing into groups, by the grouping of tumor volume, sub-model group, positive drug cyclophosphamide group (40mg/kg, ip), Compound I 50mg/kg, BTW58 component 100mg/kg, every group each 7.When experiment finishes, strip the tumor piece, claim tumor heavy, calculate the heavy suppression ratio of tumor.
[0062] the results are shown in following table, Compound I and BTW58 component obvious tumor-inhibiting action do not occur to renal carcinoma.
[0063] table 12 Compound I and BTW58 component on 786-O animal tumor heavily reach tumour inhibiting rate impact (n=7,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Model group | / | 1.45±1.19 | / |
| | 100 | 1.7±1.35 | -17.4% |
| The Compound I group | 50 | 1.5±1.23 | -0.03% |
| The | 40 | 0.58±0.25* | 60.2% |
Annotate: * and model group compare P＜0.05 * * and model group compares P＜0.01
13. utilize the interior nude mice heteroplastic transplantation tumor model observation Compound I of body and BTW58 component on the impact of human nasopharyngeal carcinoma CNE
Aseptic taking-up tumor mass under people's cancer kind Corium Mus, with the clean blood of normal saline flushing, select well-grown tumor tissues to be cut into small pieces, about grain of rice size, it is subcutaneous that small tissue blocks is inoculated in nude mice right side axillary fossa, after the inoculation until Growth of Tumors Transplanted (100mm after a certain size
3More than) begin the administration of dividing into groups, by the grouping of tumor volume, sub-model group, positive drug cyclophosphamide group (40mg/kg, ip), Compound I 50mg/kg, BTW58 component 100mg/kg, every group each 7.When experiment finishes, strip the tumor piece, claim tumor heavy, calculate the heavy suppression ratio of tumor.
[0064] the results are shown in following table, Compound I and BTW58 component obvious tumor-inhibiting action do not occur to nasopharyngeal carcinoma.
[0065] table 13 Compound I and BTW58 component on CNE animal tumor heavily reach tumour inhibiting rate impact (n=7,
± s)
| Group | Dosage (mg/kg) | Tumor heavy (g) | Tumour inhibiting rate |
| Model group | / | 1.45±1.36 | / |
| | 100 | 1.53±1.02 | -5.4% |
| The Compound I group | 50 | 1.38±1.11 | 4.8% |
| The | 40 | 0.54±0.64* | 62.8% |
Annotate: * and model group compare P＜0.05 * * and model group compares P＜0.01.
Claims (1)
1.3-O-the preparation method of α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin is characterized in that may further comprise the steps:
The first step becomes coarse powder with Radix Pulsatillae pulverizing medicinal materials, water or 0.1-95% soak with ethanol 0.5-2h, and 6-12 doubly measures heating and refluxing extraction 2-3 time, and each 1-3h filters merging filtrate;
Above-mentioned filtrate is evaporated to 0.5-1.0g raw medicinal herbs/mL in 50-70 ℃, and without the alcohol flavor, 0-4 ℃ of cold preservation is spent the night, and analyses glue, filters, and namely gets Radix Pulsatillae extract;
Second step adds sodium hydroxide solution and is transferred to pH10-13 in the Radix Pulsatillae extract aqueous solution, and ebuillition of heated 4-10 hour, every 30-60 minute survey pH, remain on pH10-13, let cool, add hydrochloric acid solution, transfer pH 3-7, centrifugal, go precipitation, namely get the Radix Pulsatillae extract hydrolyzed solution;
The 3rd step, the Radix Pulsatillae extract hydrolyzed solution, through D101 or AB-8 or ADS-17 macroporous resin adsorption, water, 30-40% ethanol, 50-80% ethanol, 95% ethanol elution are collected 50-80% ethanol elution part, and be get final product successively
The BTW58 component
Through the experiment of antitumor pharmacology, prove that the BTW58 component is Radix Pulsatillae anti-tumor effective component;
The 4th step, Radix Pulsatillae anti-tumor effective component is through ODS reverse phase silica gel post, carries out gradient elution with the mixed liquor of first alcohol and water, and gradient eluent has five groups successively, and its volume proportion is respectively
5 parts of first group of methanol: 5 parts in water;
6 parts of second group of methanol: 4 parts in water;
7 parts of the 3rd group of methanol: 3 parts in water;
8 parts of the 4th group of methanol: 2 parts in water;
9 parts of the 5th group of methanol: 1 part in water;
8 parts of the 5th step, collection methanol: the eluting part that water is 2 parts through Sephadex LH-20 post methanol-water 7:3 eluting, obtains
Compound I, relatively reaching the LC/MS/MS test through reference substance, this chemical compound is 3-O-α-L-pyrans rhamnose-(1 → 2) [β-D-Glucopyranose .-(1 → 4)]-α-L-arabopyranose oleanolic acid saponin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110142210 CN102247393B (en) | 2011-05-30 | 2011-05-30 | Preparation method of oleanolic acid saponin component and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110142210 CN102247393B (en) | 2011-05-30 | 2011-05-30 | Preparation method of oleanolic acid saponin component and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102247393A CN102247393A (en) | 2011-11-23 |
| CN102247393B true CN102247393B (en) | 2013-03-27 |
Family
ID=44975200
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110142210 Active CN102247393B (en) | 2011-05-30 | 2011-05-30 | Preparation method of oleanolic acid saponin component and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102247393B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102504004A (en) * | 2011-09-29 | 2012-06-20 | 江西本草天工科技有限责任公司 | Preparation process of oleanolic acid tridesmosidic saponin ingredients |
| CN103169723A (en) * | 2011-12-22 | 2013-06-26 | 江西本草天工科技有限责任公司 | Preparation method and application of oleanolic acid disaccharide saponin component |
| CN103169724A (en) * | 2011-12-23 | 2013-06-26 | 江西本草天工科技有限责任公司 | Preparation method and application of oleanolic acid trisaccharide saponin component |
| CN102603856B (en) * | 2011-12-31 | 2015-03-25 | 沈阳药科大学 | Anti-tumor saponin in anemone plants and preparation method thereof as well as application |
| CN102793761B (en) * | 2012-08-27 | 2014-03-05 | 江西本草天工科技有限责任公司 | Windflower extract, preparation method and application thereof |
| CN105534995B (en) * | 2016-02-24 | 2018-05-18 | 上海交通大学医学院附属仁济医院 | Tyrosine kinase inhibitor is preparing the application in treating chronic myelocytic leukemia drug to compound Hu-17 alone or in combination |
| CN111875662B (en) * | 2020-08-19 | 2022-08-16 | 中国人民解放军空军军医大学 | Extraction method and application of clematis midriflora stem chemical extract |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100337635C (en) * | 2004-09-28 | 2007-09-19 | 徐东铭 | Medicine containing general pasqueflower saponin composition and its preparation method and uses |
| CN102068509B (en) * | 2010-04-30 | 2013-05-01 | 江西中医学院 | Method for preparing pulsatilla chinensis (Bunge) Regel active ingredient, method for preparing preparation of pulsatilla chinensis (Bunge) Regel active ingredient and application of pulsatilla chinensis (Bunge) Regel active ingredient in preparation of antineoplastic medicament |
-
2011
- 2011-05-30 CN CN 201110142210 patent/CN102247393B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| Debiton E. et al.In addition to membrane injury |
| Debiton, E. et al.In addition to membrane injury, an affinity for melanin might be involved in the high sensitivity of human melanoma cells to hederacolchiside A1.《Melanoma Research》.2004,第14卷(第2期),97-105. * |
| Gerkens, Pascal C. et al.Apoptosis and cytolysis induced by giganteosides and hederacolchisides in HL-60 cells.《Anticancer Research》.2007,第27卷(第4B期),2529-2534. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102247393A (en) | 2011-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102247393B (en) | Preparation method of oleanolic acid saponin component and application thereof | |
| CN101242850A (en) | Composition, function and use of xanthoceras sorbifolia extract and compound isolated from same, method for preparing same | |
| CN106674311B (en) | A kind of benzofuran glycosides compound and its preparation method and application | |
| CN101824067A (en) | Barrigenol-type triterpenoid saponins compound, preparation method and application thereof | |
| CN102178688B (en) | Preparation method of ivy saponins ingredient and application of the ingredient to resisting tumors | |
| Lee et al. | Novel dammarane-type triterpene saponins from Panax ginseng root | |
| Huang et al. | Genus Periploca (Apocynaceae): a review of its classification, phytochemistry, biological activities and toxicology | |
| CN103169723A (en) | Preparation method and application of oleanolic acid disaccharide saponin component | |
| CN101880306A (en) | Stauntonia brachyanthera Hand-Mazz saponins components as well as preparation method and application thereof | |
| CN105061545B (en) | Triterpene saponin componds and its preparation method and application in shiny-leaved yellowhorn | |
| CN103610682B (en) | The preparation method of 3 Alpha-hydroxy-30-olive-12,20 (29)-diene-28-acid and preparing the application in antitumor drug | |
| CN103169724A (en) | Preparation method and application of oleanolic acid trisaccharide saponin component | |
| CN102295677B (en) | In the salicornia europaeal of North America, triterpenoid saponin and its production and use falls in one | |
| CN113214214B (en) | Preparation method and application of terpenoid in Atractylodes lancea | |
| CN102772501A (en) | Rheum emodi Wall. extract and its preparing method | |
| CN110204589B (en) | Effective component of feather cockscomb seed, extraction method and application thereof in preparing neuroprotective medicament | |
| CN103739660B (en) | A kind of compound, its extracting method, its application preparing antitumor drug and antitumor drug of preparation thereof | |
| CN108676054B (en) | A kind of triterpene compound and its preparation method and application | |
| CN101468950B (en) | Novel compound separated from immature exocarp of Juglans mandshurica Maxim, and preparation and use thereof | |
| CN105801634A (en) | Preparation method and application of new straight chain alcohol and glucoside compound in walnut green husks | |
| CN105418725B (en) | Pentacyclic triterpenoid and its application | |
| Lee et al. | Anti-inflammatory and cytotoxic compounds from solanum macaonense | |
| CN103739657B (en) | A kind of Sasanguasaponin compound, its preparation method, the antitumor drug applying and prepare | |
| CN114014899B (en) | Preparation method of anticancer compound | |
| CN103665090B (en) | Ilex cornuta saponin compound, its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: HARBIN SANCTITY BIOPHARMACEUTICAL CO., LTD. Effective date: 20140814 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20140814 Address after: 330006 No. 56 Yangming East Road, Jiangxi, Nanchang Patentee after: Bencao Tian'gong Science & Tech. Co., Ltd., Jiangxi Patentee after: HARBIN SANCTITY BIOLOGICAL PHARMACEUTICAL CO., LTD. Address before: 330006 No. 56 Yangming East Road, Jiangxi, Nanchang Patentee before: Bencao Tian'gong Science & Tech. Co., Ltd., Jiangxi |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20211227 Address after: 330000 788 Torch Road, high tech Development Zone, Nanchang, Jiangxi Patentee after: JIANGXI HERBFINE HI-TECH Co.,Ltd. Address before: 330006 No. 56, Yangming East Road, Nanchang City, Jiangxi Province Patentee before: JIANGXI HERBFINE HI-TECH Co.,Ltd. Patentee before: Harbin Shengtai biopharmaceutical Co., Ltd |
|
| TR01 | Transfer of patent right |