CN109651233A - A kind of alkaloid compound and its method and application that separation is extracted from clavus dendrobium nobile - Google Patents
A kind of alkaloid compound and its method and application that separation is extracted from clavus dendrobium nobile Download PDFInfo
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- CN109651233A CN109651233A CN201910111694.9A CN201910111694A CN109651233A CN 109651233 A CN109651233 A CN 109651233A CN 201910111694 A CN201910111694 A CN 201910111694A CN 109651233 A CN109651233 A CN 109651233A
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- C07—ORGANIC CHEMISTRY
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- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/56—Ring systems containing three or more rings
- C07D209/80—[b, c]- or [b, d]-condensed
- C07D209/94—[b, c]- or [b, d]-condensed containing carbocyclic rings other than six-membered
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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Abstract
This programme discloses a kind of alkaloid compound in effective ingredients in plant extractive technique field and its extracts the method and application of separation from clavus dendrobium nobile.It is characteristic of the invention that the method for modern chromatographic partition method purification isolates and purifies such more complicated ingredient from clavus dendrobium nobile using extraction and column chromatography enriched composition.A kind of isolated alkaloid component is extracted in preparation method success provided by the invention from clavus dendrobium nobile, names are as follows: clavus dendrobine A, this ingredient have preferable extracorporeal anti-tumor cytotoxic activity, have exploitation at the prospect of anti-tumor drug.
Description
Technical field
The present invention relates to effective ingredients in plant extractive technique fields, and in particular to a kind of alkaloid compound and its from stick
The method and application of separation are extracted in arthrolith dry measure used in former times.
Background technique
Modern pharmacological studies have shown that dendrobium nobile have adjust immunity, antitumor, hypoglycemic, antibacterial, anti-platelet aggregation,
The effects of anti-oxidant, blood pressure lowering.Clavus dendrobium nobile (Dendrobium findlayanum Par.et Rchb.f.) is a kind of happiness yin
Cool herbaceos perennial has higher medical value, but less for the research of its effective component.Alkaloid compound
It is a kind of nitrogenous alkaline organic compound, most of to have complicated cyclic structure, nitrogen is included in ring more, there is significant life
Object activity, is one of effective component important in Chinese herbal medicine.The present invention is isolated a kind of with certain anti-from clavus dendrobium nobile
The alkaloid compound of tumor promotion, the compound structure is so far there are no relevant report.However in Chinese herbal medicine clavus dendrobium nobile
The general content of active constituent is low, is difficult to obtain.
Summary of the invention
The invention is intended to provide a kind of alkaloid compound and its extract from clavus dendrobium nobile the method and application of separation,
To develop one of active constituent in clavus dendrobium nobile.The separation method has only used a silica gel column chromatography, reduces sample
Product loss carries out essence point using half preparation HPLC, and controllability and favorable reproducibility, trace active ingredient not easily run off, and compound is pure
Degree is high, easy to operate.
One of this programme alkaloid compound, the chemical structural formula of the alkaloid compound are as follows:
The present invention also provides the method for separating the alkaloid compound, including following step are extracted from clavus dendrobium nobile
It is rapid:
1) after taking the dry stem of clavus dendrobium nobile to crush, with methanol eddy extraction 1~4 time of volumetric concentration 80%~95%, often
Secondary to extract 1~3 hour, extracting solution is concentrated under reduced pressure to give methanol extract by combined extract；
2) it disperses gained methanol extract in step 1) in after aqueous solution is sufficiently stirred, successively uses petroleum ether, ethyl acetate
With each extraction of n-butanol difference 1~4 time, it is concentrated under reduced pressure to give each position medicinal extract；
3) ethyl acetate extract medicinal extract in step 2) and n-butanol portion medicinal extract are separated by silica gel column chromatography, uses petroleum
Ether-ethyl acetate carries out gradient elution with volume ratio 100:0~0:100, then carries out TLC detection and analysis, is divided into 12~50
Component；
4) it is separated after merging component 10~12 in step 3) through HPLC, the first methanol ladder for being 70%~100% with concentration
Degree elution, elutes 12 subfractions；
5) the 7th component of component in step 4) is carried out to methanol isocratic elution that is secondarily purified, being 66% with concentration with HPLC,
Obtain target compound.
Further, the step 3) is with petroleum ether-ethyl acetate with volume ratio 100:0；5:1；2:1；1:1；0:100 is carried out
Gradient elution.
Further, HPLC column chromatography used is 10 μm in the step 5), 10mm × 250mm, C18Column chromatography, using concentration
For 66% methanol isocratic elution.
Further, the eluant, eluent methanol in the step 5) can be used concentration and be replaced by 55% acetonitrile.
The advantages of present invention extraction alkaloid compound, is, first by silica gel column chromatography, is combined with TLC method purple
Then outer carry out rough segmentation recycles HPLC chromatogram to carry out secondary separation and purifies to obtain monomer, whole process has only used a silicon
Rubber column gel column chromatography reduces sample loss, and separation method uses HPLC controllability and favorable reproducibility, and micro constitutent not easily runs off, chemical combination
Object purity is high, operation are also relatively simple.
Further, the alkaloid compound is preparing the application in anticancer drug and anti-leukemia medicine.
Detailed description of the invention
Fig. 1 is the structure chart of clavus dendrobine A of the present invention；
The HR-ESI-MS that Fig. 2 is clavus dendrobine A of the present invention schemes；
Fig. 3 is clavus dendrobine A's of the present invention1H-NMR figure；
Fig. 4 is clavus dendrobine A's of the present invention13C-NMR figure；
Fig. 5 is clavus dendrobine A's of the present invention1H-1H COSY figure；
The HSQC that Fig. 6 is clavus dendrobine A of the present invention schemes；
The HMBC that Fig. 7 is clavus dendrobine A of the present invention schemes.
Specific embodiment
Below by the further details of explanation of specific embodiment:
Alkaloid compound of the present invention, the chemical structural formula of the alkaloid compound are as follows:
Hereinafter referred to as the alkaloid compound is clavus dendrobine A.
Embodiment 1:
The method for separating above-mentioned alkaloid compound is extracted from clavus dendrobium nobile, comprising the following steps:
1) the dry stem 3.0kg of clavus dendrobium nobile is taken, after crushed, with 95% methanol heating and refluxing extraction of volumetric concentration 4 times, often
Secondary 3h merges 4 extracting solutions, extracting solution is carried out to be concentrated under reduced pressure to give methanol extract 0.5kg；
2) it disperses methanol extract in step 1) in after being sufficiently stirred in aqueous solution, successively with petroleum ether, ethyl acetate, just
Butanol respectively extracts 4 times, is concentrated under reduced pressure to give each position medicinal extract；
3) ethyl acetate extract medicinal extract in step 2) and n-butanol portion medicinal extract are separated by silica gel column chromatography, uses petroleum
Ether-ethyl acetate is with volume ratio 100:0；5:1；2:1；1:1；0:100 gradient elution is divided into 50 groups in conjunction with TLC detection method
Point: Fr.1~Fr.50；
4) the 10th~12 component in step 3) is merged and is separated through HPLC, the methanol-water for being 70%~100% with concentration
Solution gradient elution, chromatographic column C18, 20mm × 250mm, flow velocity 20.0mL/min gradient elution, the secondary color of 3~30min of collection
Spectral peak, Detection wavelength 230nm are divided into 12 subfractions: Fr.1~Fr.12；
5) the 7th component Fr.7 is isolated and purified by HPLC in step 4), the methanol isocratic elution for being 66% with concentration, color
Compose column C18, 10mm × 250mm, flow velocity 6.0mL/min isocratic elution, collect 16~18min between chromatographic peak, Detection wavelength
230nm obtains clavus dendrobine A.
Embodiment 2: the difference from embodiment 1 is that
Refluxing extraction concentration is methanol solution refluxing extraction 3 times of 85% in step 1)；Extraction 2 times in step 2)；Step
It is rapid 4) in replaced with concentration by 70%~100% acetonitrile-aqueous solution, Detection wavelength 240nm, 260nm；With dense in step 5)
Degree is replaced by 66% acetonitrile-aqueous solution, Detection wavelength 240nm, 260nm.Remaining step can also be obtained with embodiment 1
Beneficial effect of the present invention.
Embodiment 3: the difference from embodiment 1 is that
The methanol eddy that refluxing extraction concentration is 90% in step 1) extracts 4 times；Extraction 4 times in step 2)；Step 4)
It is middle to be replaced with concentration by 70%~100% acetonitrile-aqueous solution, Detection wavelength 240nm, 260nm；It is with concentration in step 5)
66% acetonitrile-aqueous solution replaces, Detection wavelength 240nm, 260nm.Remaining step can also obtain this hair with embodiment 1
The bright beneficial effect.
The present invention carries out the condition that TLC inspection is known: solvent is petroleum ether-ethyl acetate system and methylene chloride-methanol system
System, color developing agent a: observes fluorescence under ultraviolet lamp (254nm)；Color developing agent b: iodine cylinder colour developing.
Structural Identification: as shown in Figure 2 to 7, the spectral technique utilized, clavus dendrobine A mainly includes nuclear magnetic resoance spectrum1H-NMR、13The structure of C-NMR, HSQC, HMBC and mass spectral analysis HR-ESI-MS authenticating compound.
The compound is red oil.HR-ESI-MS[M+H]+M/z 276.1764 (calculated value 276.1600), in conjunction with
H NMR spectroscopy determines that the molecular formula of compound is C16H21NO3, by spectral technique, determine that the compound is clavus dendrobine A.Its core
Magnetic data is shown in Table 1.
Nuclear magnetic data (400/100MHz, the CDCl of 1 clavus dendrobine A of table3):
Embodiment 4
1. measuring clavus dendrobium nobile A to people's glioblastoma cells (A172), human neuroblastoma cells (SHSY5Y)
With the inhibiting effect of human cervical carcinoma cell (Hela)
Using conventional mtt assay, the above-mentioned cell of logarithmic growth phase is chosen, adjusts cell with containing 10% hyclone nutrient solution
Concentration is 10 × 105A/mL is inoculated into the flat tissue culture plate in 96 holes, is placed in 37 DEG C, 5%CO with every 100 μ L of hole2Incubator
It is respectively 0.9375,1.875,3.75,7.5,15,30 μm of ol/L that the concentration of sample, which is added, in middle culture afterwards for 24 hours, if 3 multiple holes.?
37 DEG C, 5%CO2Continue to discard the culture solution of carry sample after cultivating 48h in incubator, is supplemented 100 μ L culture solutions.Then every hole
20 μ L MTT solution (5mg/mL) are added to continue to cultivate 4h, discards supernatant liquid after centrifugation, after being rinsed 2~3 times with PBS, adds
Culture solution containing MTT.Culture is terminated, the culture solution in hole is sucked.Finally, 100 μ L DMSO, low-speed oscillation 15min are added in every hole
It dissolves crystal sufficiently, tissue culture plate is placed in microplate reader, measure the light absorption value (A) at 490nm wavelength, i.e. OD value,
Calculate growth inhibition ratio.Experimental result is recorded, data are handled.Cis-platinum is positive control.Growth inhibition ratio=(1- experimental group is average
A/ control group average A-value) × 100% using sample concentration as abscissa, inhibiting rate is that ordinate draws inhibiting rate curve graph, is found out
The concentration of sample when inhibiting rate is 50%.Clavus dendrobine A is to people's glioblastoma cells (A172), human neuroblastoma
The IC of cell (SHSY5Y) and human cervical carcinoma cell (Hela)50Respectively 10.42 μM, 8.90 μM, 10.63 μM.
2. measuring clavus dendrobium nobile A to the inhibiting effect of people's Acute Monocytic Leukemia Cell Line mv4-11.
Experiment stepWith above-mentioned stepThe difference is that: the concentration of drug is respectively as follows: 3.33,10,30 μm of ol/L, often
Kind drug is measured in parallel 3 times.IC of the clavus dendrobine A to people's Acute Monocytic Leukemia Cell Line mv4-1150It is 12.3 μM.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (6)
1. a kind of alkaloid compound, it is characterised in that: the chemical structural formula of the alkaloid compound are as follows:
2. extracting the method for separating alkaloid compound as described in claim 1 from clavus dendrobium nobile, it is characterised in that: packet
Include following steps:
1) after taking the dry stem of clavus dendrobium nobile to crush, with methanol heating and refluxing extraction 1~4 time of volumetric concentration 80%~95%, often
Secondary to extract 1~3 hour, extracting solution is concentrated under reduced pressure to give methanol extract by combined extract；
2) it disperses gained methanol extract in step 1) in after aqueous solution is sufficiently stirred, successively with petroleum ether, ethyl acetate and just
Butanol respectively extracts 1~4 time, is concentrated under reduced pressure to give each position medicinal extract；
3) ethyl acetate extract medicinal extract in step 2) and n-butanol portion medicinal extract are separated by silica gel column chromatography, with petroleum ether-
Ethyl acetate carries out gradient elution with volume ratio 100:0~0:100, then carries out TLC detection and analysis, is divided into 12~50 groups
Point；
4) it is separated after merging the 10th~12 component in step 3) through HPLC, carries out ladder with the methanol that concentration is 70%~100%
Degree elution, elutes 12 subfractions；
5) the 7th component in step 4) is carried out to methanol isocratic elution that is secondarily purified, being 66% with concentration with HPLC, obtains target
Compound.
3. extraction separation method according to claim 2, it is characterised in that: the step 3) petroleum ether-ethyl acetate
With volume ratio 100:0；5:1；2:1；1:1；0:100 carries out gradient elution.
4. extraction separation method according to claim 3, it is characterised in that: HPLC column chromatography used is in the step 5)
10 μm, 10mm × 250mm, C18Column chromatography, use concentration for 66% methanol isocratic elution.
5. the extraction separation method according to any one of claim 2~4, it is characterised in that: washing in the step 5)
De- agent methanol can be used concentration and be replaced by 55% acetonitrile.
6. alkaloid compound according to claim 1 is preparing the application in anticancer drug and anti-leukemia medicine.
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| CN201910111694.9A CN109651233B (en) | 2019-02-12 | 2019-02-12 | Alkaloid compound, and method for extracting and separating alkaloid compound from dendrobium clavatum and application of alkaloid compound |
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| CN201910111694.9A CN109651233B (en) | 2019-02-12 | 2019-02-12 | Alkaloid compound, and method for extracting and separating alkaloid compound from dendrobium clavatum and application of alkaloid compound |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112457261A (en) * | 2020-12-12 | 2021-03-09 | 中国科学院昆明植物研究所 | Preparation method and application of dendrobium officinale leaf alkaloid |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05331141A (en) * | 1992-05-25 | 1993-12-14 | Taisho Pharmaceut Co Ltd | Production of dendrobine derivative |
| CN107021947A (en) * | 2017-05-31 | 2017-08-08 | 遵义医学院 | A noval chemical compound and its extraction separation method in HERBA DENDROBII |
| CN108912091A (en) * | 2018-08-22 | 2018-11-30 | 遵义医学院 | A kind of HERBA DENDROBII bibenzyl based component and extract separation and chiral separation method |
| CN109096232A (en) * | 2018-08-08 | 2018-12-28 | 遵义医学院 | A kind of dibenzyl dimer class compound and its method and application that separation is extracted from HERBA DENDROBII |
-
2019
- 2019-02-12 CN CN201910111694.9A patent/CN109651233B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05331141A (en) * | 1992-05-25 | 1993-12-14 | Taisho Pharmaceut Co Ltd | Production of dendrobine derivative |
| CN107021947A (en) * | 2017-05-31 | 2017-08-08 | 遵义医学院 | A noval chemical compound and its extraction separation method in HERBA DENDROBII |
| CN109096232A (en) * | 2018-08-08 | 2018-12-28 | 遵义医学院 | A kind of dibenzyl dimer class compound and its method and application that separation is extracted from HERBA DENDROBII |
| CN108912091A (en) * | 2018-08-22 | 2018-11-30 | 遵义医学院 | A kind of HERBA DENDROBII bibenzyl based component and extract separation and chiral separation method |
Non-Patent Citations (2)
| Title |
|---|
| GUI-YUAN LIU ET AL.: "Dendrobine-type alkaloids and bibenzyl derivatives from Dendrobium findlayanum", 《FITOTERAPIA》 * |
| HIROSHI MORITA ET AL.: "New Picrotoxinin-type and Dendrobine-type Sesquiterpenoidsfrom Dendrobium Snowflake "Red Star"", 《TETRAHEDRON》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112457261A (en) * | 2020-12-12 | 2021-03-09 | 中国科学院昆明植物研究所 | Preparation method and application of dendrobium officinale leaf alkaloid |
| CN112457261B (en) * | 2020-12-12 | 2023-07-14 | 中国科学院昆明植物研究所 | Preparation method and application of dendrobium candidum leaf alkaloid |
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