CN108276400A - A kind of method for high yield extraction of rotundine - Google Patents
A kind of method for high yield extraction of rotundine Download PDFInfo
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- C07D455/00—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
- C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
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Abstract
The present invention relates to a kind of method for high yield extraction of rotundine, are related to bulk pharmaceutical chemicals field.Extracting method includes：At least once by hydrochloric acid cold soaking extraction of the mountain tortoise medicine materical crude slice through 1.5 2.5wt%, first time filtrate after each hydrochloric acid cold soaking extraction merges the first filtrate through being obtained by filtration for the first time every time, slowly reinforces alkali to be adjusted to pH value being 9 10, standing separation must precipitate.At least once by alcohol reflux extraction of the precipitation through 80 95vt%, it is filtered for second after each alcohol reflux extraction, concentrates, crystallize after merging the second filtrate through being obtained by filtration for the second time every time, filter.Its is easy to operate, effectively improves the recovery rate of rotundine, improves the utilization rate of raw material.
Description
Technical field
The present invention relates to bulk pharmaceutical chemicals fields, and more particularly to a kind of method for high yield extraction of rotundine.
Background technology
Mountain tortoise, alias Stephania epigaea, Menispermaceae, golden Calamus.It is frequently grown in Stone Mountain, is the famous Chinese traditional herbs in Yunnan.Its
Root tuber is flourishing, usually floating open country face, while root tuber is rich in alkaloid, and the alkaloid in the tortoise of mountain is up to 3-4%, wherein main
To be isoquinoline alkaloid, wherein content highest and analgesic effect optimal components is 2,3,9,10- tetramethoxies -5,8,
13,13a- tetrahydrochysene -6H- dibenzo [a, g] quinolizines,Also known as rotundine.
Studies have shown that rotundine has analgesia, calm, hypnosis and stable effect.Its analgesic activity is weaker than piperazine
For pyridine, it is better than general analgesic-antipyretic.The apnea inhibiting effect under therapeutic dose, does not also cause gastrointestinal smooth muscle spasmus.
It is preferable to Chronic persistent pain and internal organ dull pain effect, to acute sharp pain (such as postoperative pain, treatment of traumatic pain etc.), late period
Cancer pain effect is poor.While generating analgesic activity, calm and hypnosis can be caused.Not with existing mountain tortoise resource
Foot, how to improve the recovery rate of rotundine becomes a problem, while Stephania delavayi extract contains palmatine and the right side
Revolve tetrahydropalmatine, prior art purifying process extraction product purity not enough, cannot effectively reduce objectionable impurities palmatine and
Dextrorotation tetrahydropalmatine is removed, making preparation using the raw material, there are clinical safety risks.
Invention content
It is easy to operate the purpose of the present invention is to provide a kind of method for high yield extraction of rotundine, have
Effect improves the recovery rate of rotundine, improves the utilization rate of raw material.
The present invention solves its technical problem using following technical scheme to realize.
The present invention proposes a kind of method for high yield extraction of rotundine comprising：
At least once by hydrochloric acid cold soaking extraction of the mountain tortoise medicine materical crude slice through 1.5-2.5wt%, the after the extraction of each hydrochloric acid cold soaking
Primary filtering, merges the first filtrate through being obtained by filtration for the first time every time, slowly reinforces aqueous slkali to be adjusted to pH value being 9-10, quiet
Separation is set, must be precipitated.
At least once by alcohol reflux extraction of the precipitation through 80-95vt%, it is filtered for second after each alcohol reflux extraction,
It concentrates, crystallizes after merging the second filtrate through being obtained by filtration for the second time every time, filter.
The advantageous effect of the extracting method of rotundine is in the mountain tortoise of the embodiment of the present invention：
The hydrochloric acid of 1.5-2.5wt% effectively extracts the rotundine in the tortoise medicine materical crude slice of mountain, at the same cold soaking extract to
It is few primary, fully rotundine is made to be leached from medicinal material, meanwhile, it is filtered after extraction, the leachate prevented contains
Impurity interferes the efficiency of preparation.Strong base solution is it is possible to prevente effectively from security risk caused by the short time is highly exothermic, neutralizes simultaneously
Acidic materials, meanwhile, strong base solution displaces rotundine from leachate, while pH value is 9-10, improves left
The stability of tetrahydropalmatine in the solution is revolved, prevents it from being combined with acidic materials.The alcohol reflux extraction operation of 80-95vt%
Simply, while reflowing result is good, while ethyl alcohol is convenient for removing from follow-up crude product.To sum up, rotundine is effectively improved
Recovery rate.
Description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the hydrogen nuclear magnetic resonance spectrogram for the rotundine that test example provides；
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram for the rotundine that test example provides；
Fig. 3 be the rotundine that provides of test example carbon-13 nmr spectra (13C-NMR) figure；
Fig. 4 is the carbon-13 nmr spectra figure for the rotundine that test example provides.
Specific implementation mode
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, builds according to normal condition or manufacturer
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
The extracting method of rotundine in the mountain tortoise of the embodiment of the present invention is specifically described below.
The present invention provides a kind of method for high yield extraction of rotundine, including：
S1. by mountain tortoise medicine materical crude slice, the hydrochloric acid cold soaking through 1.5-2.5wt% extracts at least once, after each hydrochloric acid cold soaking extraction
It filters for the first time, merges the first filtrate through being obtained by filtration for the first time every time, obtain leaching liquor.
Optionally, mountain tortoise medicine materical crude slice is crushed before hydrochloric acid cold soaking, crosses 2-5 mesh sieve, obtains the coarse granule of mountain tortoise, effectively
The specific surface area for increasing mountain tortoise, makes it fully be infiltrated on hydrochloric acid, extraction effect is good.
The hydrochloric acid of a concentration of 1.5-2.5wt% effectively makes rotundine effectively dissolve, and leaches in solution, improves
Leaching effect.Preferably, in preferred embodiments of the present invention, in order to make 1.5-2.5wt% hydrochloric acid leaching extraction effect more
It is good, it is preferable that the volume ratio of the additive amount of mountain tortoise medicine materical crude slice and the additive amount of hydrochloric acid is 1：5.5-7.
In order to make under above-mentioned condition, leaching effect is further increased, it is preferable that hydrochloric acid cold soaking is extracted from 0.02MPa-
It is carried out under conditions of 0.05MPa, 30-35 DEG C, the immersion mountain tortoise for effectively going deep into hydrochloric acid, improves leaching content, simultaneously
0.02MPa-0.05MPa, 30-35 DEG C are worked in coordination, and the efficiency of leaching is effectively facilitated, further with a concentration of 1.5-2.5wt%
Hydrochloric acid coordinated, improve leaching effect.
Optionally, in preferred embodiments of the present invention.Further include surpassing between being filtered to first time after the extraction of hydrochloric acid cold soaking
Sound wave extracts 5-10min, wherein it is preferred that the power of ultrasonic wave is 1600-1700w, frequency 38-40KHz.Pass through ultrasonic wave extraction
It works in coordination with hydrochloric acid cold soaking, effectively improves leaching effect.Supersound process keeps its institutional framework more fluffy, is carried out convenient for follow-up
Secondary cold soaking and filtering, ultrasound makes leaching effect more preferably after salt Ore Leaching.
At least once by hydrochloric acid cold soaking extraction of the mountain tortoise medicine materical crude slice through 1.5-2.5wt%, wherein preferably drink mountain tortoise
Hydrochloric acid cold soaking extraction of the piece through 1.5-2.5wt% is three times, wherein between being filtered to first time after each hydrochloric acid cold soaking extraction, all
Ultrasonic wave extraction can be carried out, can also be after the extraction of first time hydrochloric acid cold soaking between the first filtrate optionally, into
Row ultrasonic wave extraction, and the remaining auxiliary that can be configured or be not provided with twice ultrasonic wave extraction according to actual conditions.Wherein,
The judder of ultrasonic wave and cavitation effect can be such that solvent penetrates into crude drug cell, to accelerate the dissolution of active ingredient, carry
High extracting efficiency.
It is filtered for the first time after each hydrochloric acid cold soaking extraction, it is preferable that filtered 90-100 mesh sieve for the first time effectively removes
To the first filtered fluid in impurity, prevent the interference of impurity, while improving subsequent extraction efficiency.
Merge the first filtrate through being obtained by filtration for the first time every time, obtains leaching liquor.
S2. slowly reinforce alkali in leaching liquor to be adjusted to pH value being 9-10, standing separation must precipitate.
Preferably, highly basic is NaOH solution, effectively adjusts pH value, neutralizes hydrochloric acid and some organic acidic materials, while strong
Alkali replaces alkaloid.
Since the process heat release of highly basic is added, it is preferred that slowly reinforcing alkali process with stirring, with 35-45r/min
Speed stir at least 25 minutes.It is effectively prevent the security risk caused by very exothermic, while heat release promotes the generation of precipitation.
Optionally, 12 hours are stood to after precipitating completely, extraction supernatant liquid to channel separator, isolated first
Partly precipitated；Lower layer's suspension is put into link-suspended basket centrifuge, isolated second part precipitation, merge first part's precipitation with
Second part precipitates, and must precipitate.
S3. by precipitation, at least once, second of filtering will every time pass through second the alcohol reflux extraction through 80-95vt%
The second filtrate being obtained by filtration concentrates after merging, and crystallizes, and filters.
Refluxing extraction includes the following steps：First precipitation is heated to reflux after the ethyl alcohol of 85-95vt% impregnates 20-40min
1-2.5h。
Due to refluxing extraction be at least twice after, the content of active principle precipitated when extracting for the first time is higher, meanwhile, it is more
After secondary refluxing extraction, the content of the active ingredient of precipitation is seldom, carries out refluxing extraction again at this time, and cost is higher, therefore preferably
Ground, refluxing extraction are at least twice, 1.8-2.5h to be heated to reflux in refluxing extraction for the first time, remaining after for the first time is heated to reflux
In be heated to reflux 1-1.5h.
It is further preferred that the volume ratio of the additive amount of the additive amount and ethyl alcohol of precipitation is 1：4-5 makes precipitation be filled with ethyl alcohol
Sub-dip is steeped, and the efficiency of subsequent ethanol refluxing extraction is improved.
Preferably, it was filtered into 110-125 mesh sieve for the second time, effectively removes impurity.
Preferably, will after the second filtrate after ethyl alcohol heating and refluxing extraction merges, do not need it is cold set, directly after merging, into
Row concentration optionally, is closed wherein the second filtrate after merging is concentrated into 0.48-0.52 times of the second filtrate volume after merging
The second filtrate after and is concentrated under reduced pressure in vacuum suck outer circulation single-effect concentrator, and concentrated effect is good, and efficiency is good, and concentration is completed
Concentrate afterwards has certain temperature.
Optionally, concentrate is put into crystallizing tank while hot, is placed such as 24 hours, crystallization is precipitated, filtered, obtained thick
Product.While hot the reason of, is, with the reduction of temperature, saturation degree reduces, is more easy to precipitate crystal.Obtained crystal is yellowish to yellow
The crystallization of color；It is odorless, it is tasteless.
After suction filtration, obtained crystal is dried, material label is sticked, indicates the name of an article, lot number, quantity, production time
Deng.According to《Sampling standard rule of management/SMP-QC008》It is sampled, inspection, crude product shines《Rotundin quality standard/QS-
CP220》Crude product quality standard test examines qualification that crude product is transferred to subsequent processing.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
A kind of method for high yield extraction of rotundine comprising：
S1. mountain tortoise medicine materical crude slice is packed into extraction filling, the hydrochloric acid cold soaking extraction through 2wt% is primary, after the extraction of hydrochloric acid cold soaking
It sieves with 100 mesh sieve and carries out first time filtering, obtain leaching liquor.
Wherein, the volume ratio of the additive amount of the additive amount and hydrochloric acid of mountain tortoise medicine materical crude slice is 1：6.
S2. leaching liquor is put into settling tank, slowly adds NaOH solution wherein in leaching liquor, slowly reinforce alkali process companion
It stirs 30 minutes, is thoroughly mixed uniformly with the speed of 40r/min, when to adjust pH value be 9, stand and place 12 hours to heavy
It forms sediment complete, extracts supernatant liquid to channel separator, isolated first part's precipitation；Lower layer's suspension be put into 3-foot from
In scheming, isolated second part precipitation merges first part's precipitation and second part precipitation, must precipitate.
S3. alcohol reflux of the precipitation through 95vt% is extracted once, crosses 120 mesh sieve and carry out second of filtering, by what is obtains
Second filtrate is concentrated into 0.48 times of the second filtrate original volume, is put into crystallizing tank while hot, places 24 hours, and crystallization is precipitated, and takes out
Filter gained.
The volume ratio of the additive amount of precipitation and the additive amount of ethyl alcohol is 1：4.
Embodiment 2
A kind of method for high yield extraction of rotundine comprising：
S1. mountain tortoise medicine materical crude slice is packed into extraction filling, three times, each hydrochloric acid cold soaking carries the hydrochloric acid cold soaking extraction through 2wt%
It is sieved with 100 mesh sieve after taking and carries out first time filtering, merged the first filtrate through being obtained by filtration for the first time every time, obtain leaching liquor.
Wherein, the volume ratio of the additive amount of the additive amount and hydrochloric acid of mountain tortoise medicine materical crude slice is 1：6.
S2. leaching liquor is put into settling tank, slowly adds NaOH solution wherein in leaching liquor, slowly reinforce alkali process companion
It stirs 30 minutes, is thoroughly mixed uniformly with the speed of 40r/min, when to adjust pH value be 9, stand and place 12 hours to heavy
It forms sediment complete, extracts supernatant liquid to channel separator, isolated first part's precipitation；Lower layer's suspension be put into 3-foot from
In scheming, isolated second part precipitation merges first part's precipitation and second part precipitation, must precipitate.
S3. will precipitation through 95vt% ethyl alcohol impregnate 30min after, heating and refluxing extraction three times, in refluxing extraction for the first time
It is heated to reflux 2.5h, remaining after for the first time is heated to reflux 1h in being heated to reflux.Meanwhile crossing 120 after each alcohol reflux extraction
Mesh sieve carries out second and filters, after merging the second filtrate through being obtained by filtration for the second time every time, the second filtrate after merging is dense
It is reduced to 0.48 times of the second filtrate volume after merging, is put into crystallizing tank while hot, is placed 24 hours, crystallization is precipitated, filters institute
.
The volume ratio of the additive amount of precipitation and the additive amount of ethyl alcohol is 1：4.
Embodiment 3
A kind of method for high yield extraction of rotundine comprising：
S1. mountain tortoise medicine materical crude slice is packed into extraction filling, the hydrochloric acid cold soaking through 2wt% carries under conditions of 0.03MPa, 35 DEG C
It takes three times, through power is 1650w after the extraction of each hydrochloric acid cold soaking, is sieved with 100 mesh sieve after the ultrasonic wave extraction 25min of frequency 39KHz
First time filtering is carried out, merges the first filtrate through being obtained by filtration for the first time every time, obtains leaching liquor.
Wherein, the volume ratio of the additive amount of the additive amount and hydrochloric acid of mountain tortoise medicine materical crude slice is 1：6.
S2. leaching liquor is put into settling tank, slowly adds NaOH solution wherein in leaching liquor, slowly reinforce alkali process companion
It stirs 30 minutes, is thoroughly mixed uniformly with the speed of 40r/min, when to adjust pH value be 9, stand and place 12 hours to heavy
It forms sediment complete, extracts supernatant liquid to channel separator, isolated first part's precipitation；Lower layer's suspension be put into 3-foot from
In scheming, isolated second part precipitation merges first part's precipitation and second part precipitation, must precipitate.
S3. it by precipitation after the ethyl alcohol of 95vt% impregnates 30min, then carries out heating and refluxing extraction three times, flows back in for the first time
2.2h is heated to reflux in extraction, remaining after for the first time is heated to reflux 1h in being heated to reflux.Meanwhile after each alcohol reflux extraction
It crosses 120 mesh sieve and carries out second of filtering, after merging the second filtrate through being obtained by filtration for the second time every time, by the second filter after merging
Liquid is concentrated into 0.48 times of the second filtrate volume after merging, is put into crystallizing tank while hot, places 24 hours, and crystallization is precipitated, and takes out
Filter gained.
The volume ratio of the additive amount of precipitation and the additive amount of ethyl alcohol is 1：4.
Embodiment 4
A kind of method for high yield extraction of rotundine comprising：
S1. mountain tortoise medicine materical crude slice is packed into extraction filling, the hydrochloric acid cold soaking through 2wt% carries under conditions of 0.03MPa, 35 DEG C
It takes three times, ultrasonic wave extraction 10min is carried out after each hydrochloric acid cold soaking extraction, while being sieved with 100 mesh sieve after the extraction of hydrochloric acid cold soaking every time
First time filtering is carried out, merges the first filtrate through being obtained by filtration for the first time every time, obtains leaching liquor.
Wherein, the volume ratio of the additive amount of the additive amount and hydrochloric acid of mountain tortoise medicine materical crude slice is 1：6.
S2. leaching liquor is put into settling tank, slowly adds NaOH solution wherein in leaching liquor, slowly reinforce alkali process companion
It stirs 30 minutes, is thoroughly mixed uniformly with the speed of 40r/min, when to adjust pH value be 9, stand and place 12 hours to heavy
It forms sediment complete, extracts supernatant liquid to channel separator, isolated first part's precipitation；Lower layer's suspension be put into 3-foot from
In scheming, isolated second part precipitation merges first part's precipitation and second part precipitation, must precipitate.
S3. it by precipitation after the ethyl alcohol of 95vt% impregnates 40min, then carries out heating and refluxing extraction three times, flows back in for the first time
2.2h is heated to reflux in extraction, remaining after for the first time is heated to reflux 1h in being heated to reflux.Meanwhile after each alcohol reflux extraction
It crosses 120 mesh sieve and carries out second of filtering, after merging the second filtrate through being obtained by filtration for the second time every time, by the second filter after merging
Liquid is concentrated into 0.48 times of the second filtrate volume after merging, is put into crystallizing tank while hot, places 24 hours, and crystallization is precipitated, and takes out
Filter gained.
The volume ratio of the additive amount of precipitation and the additive amount of ethyl alcohol is 1：4.
Embodiment 5
A kind of method for high yield extraction of rotundine comprising：
S1. mountain tortoise medicine materical crude slice is packed into extraction filling, the hydrochloric acid through 2.1wt% is cold under conditions of 0.035MPa, 33 DEG C
Extraction takes three times, ultrasonic wave extraction 7min is carried out after each hydrochloric acid cold soaking extraction, while crossing 95 mesh after the extraction of hydrochloric acid cold soaking every time
Sieve carries out first time filtering, merges the first filtrate through being obtained by filtration for the first time every time, obtains leaching liquor.
Wherein, the volume ratio of the additive amount of the additive amount and hydrochloric acid of mountain tortoise medicine materical crude slice is 1：6.
S2. leaching liquor is put into settling tank, slowly adds NaOH solution wherein in leaching liquor, slowly reinforce alkali process companion
It stirs 40 minutes, is thoroughly mixed uniformly with the speed of 43r/min, when to adjust pH value be 9, stand and place 12 hours to heavy
It forms sediment complete, extracts supernatant liquid to channel separator, isolated first part's precipitation；Lower layer's suspension be put into 3-foot from
In scheming, isolated second part precipitation merges first part's precipitation and second part precipitation, must precipitate.
S3. it by precipitation after the ethyl alcohol of 90vt% impregnates 35min, then carries out heating and refluxing extraction three times, flows back in for the first time
2.3h is heated to reflux in extraction, remaining after for the first time is heated to reflux 1.3h in being heated to reflux.Meanwhile each alcohol reflux extraction
123 mesh sieve is crossed afterwards and carries out second of filtering, after merging the second filtrate through being obtained by filtration for the second time every time, by second after merging
Filtrate is concentrated into 0.52 times of the second filtrate volume after merging, is put into crystallizing tank while hot, places 24 hours, and crystallization is precipitated,
Filter gained.
Wherein, the volume ratio of the additive amount of the additive amount and ethyl alcohol of precipitation is 1：4.5.
Comparative example 1
After mountain tortoise medicine materical crude slice is infiltrated with 10% ammonium hydroxide, after 1 week is impregnated in benzene, with the salt acid extraction of 2wt%, extraction
Liquid is placed, and precipitation, filtering is precipitated, and precipitation is filtered with after hot water dissolving, after filtrate is alkalized with ammonium hydroxide, extracted using ether again,
The ether extraction liquid being evaporated, is added the mixed liquor crystallization of chloroform-ethyl alcohol in residue, and filtering obtains a left side for sheet
Revolve tetrahydropalmatine.
Test example
1) detection of rotundine
Using rotundine provided by the invention as sample, production method is：By mountain tortoise medicine materical crude slice through 2wt%'s
Hydrochloric acid cold soaking extracts three times, is filtered for the first time after each hydrochloric acid cold soaking extraction, merges first every time through being obtained by filtration for the first time
Filtrate slowly reinforces aqueous slkali to be adjusted to pH value being 9, and standing separation must precipitate；The first alcohol reflux through 95vt% will be precipitated
Extraction three times, is filtered for second after the extraction of each alcohol reflux, the after merging the second filtrate through being obtained by filtration for the second time every time
Primary concentration, crystallizes, and filters, obtains the crude product of rotundine.
Three times by second alcohol reflux extraction of the crude product through 95vt%, it filters, closes for the third time after each alcohol reflux extraction
And the third filtrate through being obtained by filtration for the third time, second of concentration are stood still for crystals every time, will be used obtained crystal after suction filtration
After the third ethyl alcohol washing at least twice of 95vt%, laying depth is the crystal layer of 3cm, dry under the conditions of 55 DEG C of hot wind
4h, it is cooling.
With Nat'l Pharmaceutical ＆ Biological Products Control Institute；The rotundine that lot number is 100452-200301 is control group.
Using rotundine provided by the invention as sample, elemental analysis, infrared absorption spectrum, ultraviolet is carried out
Absorption spectrum, high performance liquid chromatography, nuclear magnetic resonance spectroscopy, carbon-13 nmr spectra, mass spectrum (MS) and heat analysis.With Chinese drug
Biological products assay institute；The rotundin that lot number is 100452-200301 is control group.
1. elemental analysis：Using instrument model：U.S.'s thermoelectricity IRIS1000ICP-AES type elemental analysers, test method：
Combustion method carries out elemental analysis, and the results are shown in Table 1.
1 elemental analysis test data of table
| Element | C | H | N | O |
| First time measured value (%) | 70.94 | 7.04 | 3.91 | 18.01 |
| Second of measured value (%) | 70.89 | 7.03 | 3.95 | 18.03 |
| Third time measured value (%) | 70.91 | 7.05 | 3.96 | 18.08 |
| Average value (%) | 70.91 | 7.04 | 3.94 | 18.04 |
| Theoretical value (%) | 70.90 | 7.03 | 3.94 | 18.01 |
Conclusion：The measured value of elemental analysis three times of sample is almost the same, and is consistent substantially with theoretical value.Elemental analysis result
Show that the content of each element and the theoretical value of the rotundine calculated by molecular formula are almost the same in sample.
2. examination of infrared spectrum：Using instrument model：Shimadzu FTIR-7600/BV0190 infrared spectrometers, test condition：
KCl pressed disc methods carry out rotundin examination of infrared spectrum provided by the invention, and the results are shown in Table 2.
2 sample examination of infrared spectrum data of table
It can be obtained by table 2, the feature structures such as methylene, phenyl ring, methoxyl group for being connected containing tertiary amine, with tertiary amine in the sample,
It is consistent with rotundin molecular structure information.Then, the verification of infrared absorption spectrum data is carried out to above-mentioned sample, as a result such as table 3
It is shown：
3 verification result of table
Verification result further demonstrates that the infrared signature bands of a spectrum of sample are consistent with rotundin.
3. ultraviolet spectral analysis：Test sample is prepared, composing analysis TU1810 ultraviolet-uisible spectrophotometers using Beijing carries out
Ultraviolet spectral analysis.Specific test sample includes neutral solution：Weigh appropriate amount of sample be configured to 95% ethanol solution it is appropriate dense
The solution of degree.Containing the mobile phase solution under quantifier：Appropriate amount of sample is weighed to be configured to appropriate concentration containing the mobile phase under quantifier
Solution.Acid solution：Weigh the solution that appropriate amount of sample is configured to appropriate concentration with 0.1mol/L hydrochloric acid.Alkaline solution：It weighs suitable
Amount sample is configured to the solution of appropriate concentration with 0.1mol/L sodium hydroxides, methanol solution.The results are shown in Table 4.
4 ultraviolet spectrum data of table
In four kinds of solvents, sample provided by the invention is consistent with rotundin reference substance, near 210nm and 281nm
There is absorption peak.
4. high performance liquid chromatography：Using instrument model：1100 series of high efficiency liquid chromatograph of Agilent, using test side
Method：(method is numbered：XEA-QC-006) in the chromatogram recorded under assay item, compare the reservation of test solution main peak
Whether the time should consistent with the retention time of rotundin reference substance solution main peak.
Conclusion：It is tested using rotundin reference substance, in the chromatogram recorded under assay item, rotundin control
The retention time of product solution is 12.999 minutes, and the retention time of test solution main peak is 12.927 minutes, retention time one
It causes.
5. nuclear magnetic resonance spectroscopy is tested：With test equipment：BRUKER-400M, test condition：Solvent：CDCl3；Internal standard：
TMS carries out nuclear magnetic resonance spectroscopy test, as a result as shown in table 5 and Fig. 1-2.
It is parsed for the ease of the information to nuclear magnetic resonance spectroscopy and carbon-13 nmr spectra, to the hydrogen in rotundin molecule
Atom and carbon atom position and ownership illustrate, and spy is numbered each skeletal atom in rotundin structural formula, structural formula：
(nonsystematic is named, only for structural identification)
Table 51H- modal datas and analysis result
It can be obtained by table 5 and Fig. 2, molecular formula：C21H25NO4, 18-, 19-, 20- and 21- methoxyl group Hydrogen Protons
With similar chemical environment, close proximity is answered in chemical shift；7-, 10- fragrant Hydrogen Protons are without adjacent hydrogen atom, Ying Cheng
Reveal unimodal；1-, 2- Hydrogen Protons it is adjacent, 2 heavy peaks should all be showed, all Hydrogen Protons should all be in molecular structure1H-
Appearance in nuclear magnetic resoance spectrum.
By pair1The parsing of H- nuclear magnetic resoance spectrums, we have confirmed that sample molecule contains 25 Hydrogen Protons, with rotundin molecule
Formula is consistent；6.621~6.894ppm be phenyl ring virtue Hydrogen Proton, totally 4；4.229~4.268ppm is 1 methine Hydrogen Proton；
3.848~3.890ppm is methoxyl group Hydrogen Proton, totally 4 methoxyl groups, 12 Hydrogen Protons；Other peaks are methylene Hydrogen Proton, I
Each proton peak is belonged to, result meets the molecular structure information of rotundin sodium.
6. carbon-13 nmr spectra (13C-NMR)
Test equipment：BRUKER-400M
Test condition：Solvent：CDCl3；Internal standard：TMS
It is parsed for the ease of the information to nuclear magnetic resonance spectroscopy and carbon-13 nmr spectra, as a result such as table 6 and Fig. 3-4
It is shown.To in rotundin molecule hydrogen atom and carbon atom position and ownership illustrate, it is special by each bone in rotundin structural formula
Frame atom is numbered, structural formula：(nonsystematic is named, only for structural identification)
Table 613C- modal datas and analysis result
| Carbon sequence number | Chemical shift (ppm) | Carbon sequence number | Chemical shift (ppm) |
| 12 | 28.977 | 1 | 111.238 |
| 4 | 36.192 | 2 | 123.794 |
| 13 | 51.419 | 15 | 126.655 |
| 14 | 53.894 | 11 | 127.584 |
| 21 | 55.749 | 3 | 128.495 |
| 18 | 55.774 | 6 | 129.523 |
| 19 | 55.987 | 17 | 144.950 |
| 20 | 59.220 | 8 | 147.332 |
| 5 | 60.098 | 9 | 147.387 |
| 10 | 108.477 | 16 | 150.181 |
| 7 | 110.853 | - | - |
By the parsing to 13C- nuclear magnetic resoance spectrums, we have confirmed that sample molecule contains 21 carbon atoms, residing chemistry
Environment is consistent with rotundin structure.6.621~6.894ppm be phenyl ring virtue Hydrogen Proton, totally 4；4.229~4.268ppm is 1
Methine Hydrogen Proton；3.848~3.890ppm is methoxyl group Hydrogen Proton, totally 4 methoxyl groups, 12 Hydrogen Protons；Other peaks are Asia
Methyl Hydrogen Proton, we belong to each proton peak, and result meets the molecular structure information of rotundin sodium.
7. mass spectrum (MS)
Preparation method：In the round-bottomed flask that rotundin finished product is put into right amount, 4 times of 95% ethyl alcohol of amount, refluxing extraction 2 is added
Hour, filtering, merging filtrate.Filtrate is sucked in rotary evaporator after being concentrated under reduced pressure into 0.48~0.52 times of liquid volume,
It is placed at room temperature for 24 hours, crystallization is precipitated, filter, 0.2~0.3 times of 95% ethyl alcohol wash crystallization is used in combination, obtained highly finished product are put
In stainless steel disc, under the conditions of 50~55 DEG C, hot air circulation is dried 4 hours, is let cool to room temperature, loaded in clean polybag
After the assay was approved up to structural confirmation sample.Test equipment：BRUKER-400M.Test method：The sources ESI, positive ion mode exist
In ESI-MS positive ion mass spectrum figures, the quasi-molecular ions at mass-to-charge ratio (m/z) 356 is [M+H]+peak of sample, derives the molecule of sample
Amount is 355, consistent with rotundin molecular weight.
Heat analysis 8. (TG, DCS) measuring unit：Weapon industry Natural Environmental Test center, test equipment：Q600SDT is same
Walk thermal analyzer, test condition：Under High Purity Nitrogen atmosphere protection, 10 DEG C/min of heating rate, temperature range is room temperature to 400
℃.Parsing：
Thermogravimetric testing result：Room temperature is to 120 DEG C of weightlessness 3.9%, room temperature to 140 DEG C of weightlessness 4.8%, and room temperature is lost to 200 DEG C
4.9% is weighed, thermogravimetric testing result and moisture, the fusing point of rotundin mutually echo.
Differential scanning calorimetry testing result：In temperature-rise period, occur 3 endothermic peaks successively, summit temperature is respectively
109.7℃、126.0℃、145.3℃。
To sum up, molecular formula infers that sample provided by the invention is：C21H25NO4, molecular weight be 355.43 left-handed corydalis tuber
B prime.By1H-NMR、1H-1Hcosy、13C-NMR, HMQC, HMBC provide specific ownership to the peak C, H all in spectrogram, right
C-H incidence relations, which give, adequately and reasonably to be parsed, in conjunction with the functional group's characteristic absorption peak provided in infrared spectrum ultraviolet spectra,
The molecular formula released by mass spectrum, elemental analysis, the structure that the character description provided by TG, DSC can release institute's sample are：
2) embodiment 1-5 is repeated, the extraction of rotundine in the tortoise of mountain is carried out, as test example 1-5, pilot scale
It tests a 1-5 and is all made of the mountain tortoise of 250kg and tested.Using the mountain tortoise of 250kg using in pharmacopeia (2010) corydalis tuber item
The method of record as a control group, i.e., using 95% ethyl alcohol as solvent, carries out the extraction of rotundine.
By in embodiment 1-5 and control group 1, the crude product of every group of obtained rotundine according to《Rotundin matter
Amount standard/QS-CP220》Crude product quality standard test, passer record, and failure records.
It should be noted that the above test group and control group, every group is repeated 5 times, wherein obtained crude product be it is odorless,
The tasteless yellowish crystallization to yellow.The yield of every group of obtained rotundine crude product is as shown in table 7.
7 yield results of table
It can be obtained by table 7, rotundine meets made from method provided by the invention《Rotundin quality standard/
QS-CP220》Crude product quality standard test, meanwhile, the ethyl alcohol in pharmacopeia (2010) corydalis tuber item 95% is solvent, is carried out left
The extraction comparison of tetrahydropalmatine is revolved, average yield provided by the invention is better than control group.Contrast test group 1-5 simultaneously, can be with
Find out, number, the condition of minute-pressure extraction and the technology of ultrasonic wave assisted extraction of extraction can be effectively increased yield.
4) palmatine detection experiment
Chromatographic condition and assay method are as follows：
Chromatographic condition:Chromatographic column：Kromasil100-5C18250x4.6mm5μm；Mobile phase：(0.05mol/L di(2-ethylhexyl)phosphates
Hydrogen potassium solution and 0.05mol/L heptane sulfonic acid sodium salts (1:1), contain 0.2% triethylamine, with phosphoric acid tune PH to 6.5 ± 0.05)-
Methanol (35:65)；Detection wavelength：280nm；Flow velocity：1ml/min；Sample size：20μl；Column temperature：35℃；Run time：30 points
Clock.
Solution is prepared:Blank solution：Methanol：Mobile phase (1:9, v:v).
Test solution：Example 1-5 and comparative example 1 each about 20mg, it is accurately weighed, it sets in 100ml measuring bottles, is added
10ml methanol, which is ultrasonically treated 5 minutes, makes dissolving, is diluted to scale with mobile phase, mixing filters to obtain the final product.(test sample concentration：
0.2mg/ml).Contrast solution：Precision measures the above-mentioned test solutions of 1ml and is placed in 200ml volumetric flasks, is diluted to mobile phase
Scale, mixing filter to obtain the final product.(rotundin concentration：0.001mg/ml), palmatine inspection is carried out to embodiment 1-5 and comparative example 1
It surveys, the results are shown in Table 8.
8 palmatine of table detects test result
| Embodiment | Palmatine (%) |
| Embodiment 1 | <0.1 |
| Embodiment 2 | <0.1 |
| Embodiment 3 | <0.1 |
| Embodiment 4 | <0.1 |
| Embodiment 5 | <0.1 |
| Comparative example 1 | 0.6 |
As shown in Table 8, rotundine made from the method that the present invention uses effectively reduces palmatine content.
5) dextrorotation tetrahydropalmatine detection experiment
Chromatographic condition and assay method are as follows：
Chromatographic condition：Chromatographic column：KcromasilCellucoat (4.6mm × 250mm, 5 μm)；Mobile phase：Absolute ethyl alcohol；
Survey wavelength：230nm；Flow velocity：0.5ml/min；Sample size：20μl；Column temperature：30℃
Solution is prepared：System suitability solution：Precision weighs rotundine reference substance and dextrorotation tetrahydropalmatine
Appropriate reference substance is added mobile phase dissolved dilution and a concentration of 0.3mg/ml of rotundine, dextrorotation tetrahydropalmatine is made
The mixed solution of a concentration of 0.45 μ g/ml.
Test solution：The each about 15mg of the product of Example 1-5 and comparative example 1 respectively, it is accurately weighed, set 50ml amounts
In bottle, flowing phased soln is added and is diluted to scale, mixing filters to obtain the final product.(test sample concentration：0.3mg/ml)
Own control solution：Precision measures the above-mentioned test solutions of 3ml and is placed in 100ml volumetric flasks, with flowing phase dilution
To scale, mixing；It takes 1ml to be placed in 20ml volumetric flasks again, is diluted to scale with mobile phase, mixing filters to obtain the final product, and (rotundin is dense
Degree：0.45 μ g/ml), test result is referring to table 9：
9 dextrorotation tetrahydropalmatine test result of table
It can be obtained by table 9, method provided by the invention effectively removes dextrorotation tetrahydropalmatine.
In conclusion in the mountain tortoise of the embodiment of the present invention rotundine extracting method, it is easy to operate, have
Effect improves the recovery rate of rotundine, improves the utilization rate of raw material.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (10)
1. a kind of method for high yield extraction of rotundine, which is characterized in that including：
At least once by hydrochloric acid cold soaking extraction of the mountain tortoise medicine materical crude slice through 1.5-2.5wt%, after each hydrochloric acid cold soaking extraction for the first time
Filtering, the first filtrate through being obtained by filtration for the first time, slow aqueous slkali of reinforcing are adjusted to pH value as 9-10, stand and divide every time for merging
From must precipitate；
At least once by alcohol reflux extraction of the precipitation through 80-95vt%, it is filtered for second after each alcohol reflux extraction,
It concentrates, crystallizes after merging the second filtrate through being obtained by filtration for the second time every time, filter.
2. extracting method according to claim 1, which is characterized in that refluxing extraction includes the following steps：It first will be described heavy
It forms sediment after the ethyl alcohol impregnates 20-40min, is heated to reflux 1-2.5h.
3. extracting method according to claim 2, which is characterized in that refluxing extraction is at least twice, to be carried in reflux for the first time
1.8-2.5h is heated to reflux in taking, remaining after for the first time is heated to reflux 1-1.5h in being heated to reflux.
4. extracting method according to claim 2, which is characterized in that the addition of the additive amount of the precipitation and the ethyl alcohol
The volume ratio of amount is 1：4-5.
5. extracting method according to claim 1, which is characterized in that the first time mistake after the hydrochloric acid cold soaking extraction
Between filter, further include through power be 1600-1700w, the ultrasonic wave extraction 20-30min of frequency 38-40KHz.
6. extracting method according to claim 1, which is characterized in that hydrochloric acid cold soaking is extracted from 0.02MPa-0.05MPa,
It is carried out under conditions of 30-35 DEG C.
7. extracting method according to claim 1, which is characterized in that the additive amount of the mountain tortoise medicine materical crude slice and the hydrochloric acid
Additive amount volume ratio be 1：5.5-7.
8. extracting method according to claim 1, which is characterized in that the highly basic is NaOH solution；
Preferably, slowly reinforce alkali process to stir at least 25 minutes with the speed of 35-45r/min.
9. extracting method according to claim 1, which is characterized in that filtered 90-100 mesh sieve for the first time；
Preferably, it was filtered into 115-125 mesh sieve for the second time.
10. extracting method according to claim 1, which is characterized in that second filtrate after merging is concentrated into conjunction
0.48-0.52 times of second filtrate volume after and.
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| CN109400599A (en) * | 2018-11-29 | 2019-03-01 | 四川旭阳药业有限责任公司 | A kind of extracting method of tetrahydropalmatine |
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| CN101227917A (en) * | 2005-07-26 | 2008-07-23 | 山西亚宝药业集团股份有限公司 | Medicament with drug rehabilitation function and preparing method thereof |
| CN105949185A (en) * | 2016-05-24 | 2016-09-21 | 南宁圣特生物科技有限公司 | Method for preparing L-tetrahydropalmatine hydrochloride from Stephania kwangsiensis |
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| CN101227917A (en) * | 2005-07-26 | 2008-07-23 | 山西亚宝药业集团股份有限公司 | Medicament with drug rehabilitation function and preparing method thereof |
| CN105949185A (en) * | 2016-05-24 | 2016-09-21 | 南宁圣特生物科技有限公司 | Method for preparing L-tetrahydropalmatine hydrochloride from Stephania kwangsiensis |
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| CN109400599A (en) * | 2018-11-29 | 2019-03-01 | 四川旭阳药业有限责任公司 | A kind of extracting method of tetrahydropalmatine |
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