CN108853274A - A kind of extraction of the total phenols of radix tetrastigme and purification process - Google Patents
A kind of extraction of the total phenols of radix tetrastigme and purification process Download PDFInfo
- Publication number
- CN108853274A CN108853274A CN201810871115.6A CN201810871115A CN108853274A CN 108853274 A CN108853274 A CN 108853274A CN 201810871115 A CN201810871115 A CN 201810871115A CN 108853274 A CN108853274 A CN 108853274A
- Authority
- CN
- China
- Prior art keywords
- radix tetrastigme
- extraction
- total phenols
- ethyl alcohol
- purification process
- 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.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Engineering & Computer Science (AREA)
- Pain & Pain Management (AREA)
- Biotechnology (AREA)
- Cardiology (AREA)
- Rheumatology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Alternative & Traditional Medicine (AREA)
- Heart & Thoracic Surgery (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Medicines Containing Plant Substances (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention belongs to phenols extraction and purification process technical field, the extraction of specially a kind of total phenols of radix tetrastigme and purification process, include the following steps：Step 1 crushes the radix tetrastigme leaf after drying, obtain radix tetrastigme leaf medicinal powder, ethyl alcohol is added, the additional amount of the ethyl alcohol is 10-35 times of radix tetrastigme leaf medicinal powder weight, selection refluxing extraction, temperature extraction takes or any one extracting method of ultrasonic extraction, extracting solution filter under conditions of 50-80 DEG C；Step 2 obtains the extracting solution removing ethyl alcohol of step 1 to extract concentrate, concentrate will be extracted to be splined on large pore resin absorption column, after loading, first eluted with removal of impurities solvent, the sour water that the removal of impurities solvent is pH=3, it is eluted again with ethanol solution, collects the eluent containing total phenols, the total phenols of radix tetrastigme that eluent is concentrated to dryness to purify.Preparation method of the present invention is simple, easy to operate, has obtained the total phenols extraction of optimal radix tetrastigme and purification process.
Description
Technical field
It is extracted the invention belongs to phenols and purification process technical field, the extraction of specially a kind of total phenols of radix tetrastigme and pure
Change method.
Background technique
Radix tetrastigme category Vitaceae Tetrastigma plant tetratigma hemsleyanum (Tetrastigma hemsleyanum Diels et
Gilg) section plant, be China use a kind of more long medicinal plant, gold thread hoist, silk thread hang Jin Zhong, the flat rattan of three leaves,
Stone mouse etc. is the existing alias of radix tetrastigme；Its is cool in nature, bitter, pungent, nontoxic.It is recorded according to ancient Chinese medical book, radix tetrastigme warp
It is commonly used to the diseases such as treatment hyperpyretic convulsion, pneumonia, hepatitis, irregular menstruation.
Modern research shows that radix tetrastigme have antitumor, anti-inflammatory, antipyretic, analgesia, liver protection, it is antiviral the effects of, be China spy
The Chinese herbal medicine for having and being commonly used.In civil, radix tetrastigme all herbal medicine, since the resource of leaf is sustainable, regenerated resources, experiment
The study found that radix tetrastigme leaf phenols component content is also higher, it include phenols class and phenolic acid class chemical combination through the qualitative phenols component of liquid matter
Object is main active in radix tetrastigme leaf.And through research confirm radix tetrastigme leaf pharmacological activity include it is antitumor, anti-oxidant,
Treat cardiovascular disease, anti-inflammatory, antiviral, anti-infective etc..
Related radix tetrastigme, which was studied, in recent years has been related to many aspects, but the extraction for phenols total in radix tetrastigme leaf
And purification process research is accomplished best there is no deeply investigating.And majority research object is all radix tetrastigme root tuber at present, for
The research of radix tetrastigme leaf is less, and extraction, the purification process research of phenols total for radix tetrastigme Ye are rarely reported, and are usually to utilize
Ultraviolet spectrophotometry is measured total phenols content to optimize technique, cannot targetedly guarantee the extraction of effective component
Rate.
Summary of the invention
The technical problem to be solved by the present invention is to：Extraction and the purification process of a kind of total phenols of radix tetrastigme are provided.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is：A kind of extraction of the total phenols of radix tetrastigme and
Purification process includes the following steps：
Step 1 crushes the radix tetrastigme leaf after drying, obtains radix tetrastigme leaf medicinal powder, and ethyl alcohol is added, and the ethyl alcohol adds
Entering amount is 10-35 times of radix tetrastigme leaf medicinal powder weight, select refluxing extraction, temperature extract take or ultrasonic extraction any one
Extracting method, extracting solution filter under conditions of 50-80 DEG C；
Step 2 obtains the extracting solution removing ethyl alcohol of step 1 to extract concentrate, carries out wet process dress with macroporous absorbent resin
Column will extract concentrate and be splined on large pore resin absorption column, after loading, first be carried out with removal of impurities solvent with the flow velocity of 2-6BV/h
Elution, the sour water that the removal of impurities solvent is pH=2-6, dosage 1-5BV, then with the ethanol solution of 50%-90% with 2-6BV/h
Flow velocity eluted, collect the eluent containing total phenols, eluent be concentrated to dryness purifying the total phenols of radix tetrastigme.
The beneficial effects of the present invention are：The extraction of the total phenols of radix tetrastigme provided by the invention and purification process, using height
Effect liquid phase chromatogram method and ultraviolet spectrophotometry combine, and are measured to the phenols component content in radix tetrastigme leaf extract,
High performance liquid chromatography has high reproducibility and high sensitivity, so that experimental result is more accurate, and comprehensive analysis its rate of extract,
Based on single factor exploration, the total phenols extraction of optimal radix tetrastigme and purification process are obtained in conjunction with orthogonal experiment method；When all suction filtrations
It filters under conditions of being maintained at 50-80 DEG C, is found during Study on Extraction Method, by the different volumes score of certain volume
When ethyl alcohol is added in radix tetrastigme leaf powder, when concentration of alcohol is lower than 50%, radix tetrastigme powder is flown on ethyl alcohol liquid level, cannot
It infiltrates immediately.Bottom is sunken to after being kept for slightly boiled extraction a period of time.When extracting completion suction filtration acquisition supernatant, concentration is lower than
50% ethyl alcohol is difficult to filter, and extraction efficiency is bad.Concentration of alcohol is inadequate at this time, the radix tetrastigme polysaccharide that is extracted and viscous
The ingredients such as liquid matter are more so that extracting solution is more sticky, whens all suction filtrations be maintained at 50-80 DEG C as far as possible under conditions of filter, subtract
The loss of few filtrate volume.Removal of impurities solvent is used as using sour water, total phenols in three leafiness will not follow sour water elution to be eluted,
Almost without leakage, impurity is eluted, and improves the purification effect of the total phenols of radix tetrastigme.
Detailed description of the invention
Fig. 1 is the HPLC map of reference substance solution in the specific embodiment of the invention；
Fig. 2 is radix tetrastigme extracting solution sample HPLC map in the specific embodiment of the invention；
Fig. 3 is the total phenols assay result broken line of radix tetrastigme purification process elution curve in the specific embodiment of the invention
Figure；
Fig. 4 is the HPLC map of sour water elution in the specific embodiment of the invention；
Fig. 5 is the HPLC map of 10% ethanol solution elution in the specific embodiment of the invention；
Fig. 6 is the HPLC map of 40% ethanol solution elution in the specific embodiment of the invention；
The HPLC map for the ethanol solution elution that Fig. 7 is in the specific embodiment of the invention 50%；
The HPLC map for the ethanol solution elution that Fig. 8 is in the specific embodiment of the invention 60%；
The HPLC map for the ethanol solution elution that Fig. 9 is in the specific embodiment of the invention 70%；
The HPLC map for the ethanol solution elution that Figure 10 is in the specific embodiment of the invention 80%；
The HPLC map for the ethanol solution elution that Figure 11 is in the specific embodiment of the invention 90%；
Figure 12 is the radix tetrastigme HPLC map after purification of embodiment 1 in the specific embodiment of the invention；
Figure 13 is the radix tetrastigme HPLC map after purification of embodiment 2 in the specific embodiment of the invention；
Figure 14 is the radix tetrastigme HPLC map after purification of embodiment 3 in the specific embodiment of the invention；
Figure 15 is the radix tetrastigme HPLC map after purification of embodiment 4 in the specific embodiment of the invention；
Figure 16 is the radix tetrastigme HPLC map after purification of embodiment 5 in the specific embodiment of the invention；
Figure 17 is the radix tetrastigme HPLC map after purification of embodiment 6 in the specific embodiment of the invention；
Figure 18 is the radix tetrastigme HPLC map after purification of embodiment 7 in the specific embodiment of the invention.
Label declaration：
1, neochlorogenic acid；2, chlorogenic acid；3, Cryptochlorogenic acid；4, Lutonaretin；5, orientoside；
6, vitexin rhamnoside；7, Vitexin；8, isovitexin.
Specific embodiment
To explain the technical content, the achieved purpose and the effect of the present invention in detail, below in conjunction with embodiment and cooperate attached
Figure is explained.
The design of most critical of the present invention is:Radix tetrastigme leaf is mentioned using high performance liquid chromatography and ultraviolet spectrophotometry
Total phenols content in object is taken to be measured, its rate of extract of comprehensive analysis, different methods of extraction, extracting parameter and purifying are big
Hole resin carrier and purifying parameter, obtain the total phenols method for extraction and purification of optimal radix tetrastigme.
Please refer to Fig. 1~Figure 18, a kind of extraction of the total phenols of radix tetrastigme and purification process include the following steps：
Step 1 crushes the radix tetrastigme leaf after drying, obtains radix tetrastigme leaf medicinal powder, and ethyl alcohol is added, and the ethyl alcohol adds
Entering amount is 10-35 times of radix tetrastigme leaf medicinal powder weight, select refluxing extraction, temperature extract take or ultrasonic extraction any one
Extracting method, extracting solution filter under conditions of 50-80 DEG C；
Step 2 obtains the extracting solution removing ethyl alcohol of step 1 to extract concentrate, carries out wet process dress with macroporous absorbent resin
Column will extract concentrate and be splined on large pore resin absorption column, after loading, first be carried out with removal of impurities solvent with the flow velocity of 2-6BV/h
Elution, the sour water that the removal of impurities solvent is pH=2-6, dosage 1-5BV, then with the ethanol solution of 50%-90% with 2-6BV/h
Flow velocity eluted, collect the eluent containing total phenols, eluent be concentrated to dryness purifying the total phenols of radix tetrastigme.
As can be seen from the above description, the beneficial effects of the present invention are：The extraction of the total phenols of radix tetrastigme provided by the invention and
Purification process carries out total phenols content in radix tetrastigme leaf extract using high performance liquid chromatography and ultraviolet spectrophotometry
Measurement, high performance liquid chromatography has high reproducibility and high sensitivity so that experimental result is more accurate, and comprehensive analysis its obtain
Cream rate is based on single factor exploration, obtains the total phenols extraction of optimal radix tetrastigme and purification process in conjunction with orthogonal experiment method；All pumpings
It filters under conditions of being maintained at 50-80 DEG C when filter, is found during Study on Extraction Method, by the different volumes of certain volume point
When several ethyl alcohol is added in radix tetrastigme leaf powder, when concentration of alcohol is lower than 50%, radix tetrastigme powder is flown on ethyl alcohol liquid level,
It cannot infiltrate immediately.Bottom is sunken to after being kept for slightly boiled extraction a period of time.When extracting completion suction filtration acquisition supernatant, concentration
It is difficult to filter lower than 50% ethyl alcohol, and extraction efficiency is bad.Concentration of alcohol is inadequate at this time, the radix tetrastigme polysaccharide extracted
And the ingredients such as mucilaginous substance are more so that extracting solution is more sticky, whens all suction filtrations be maintained at 50-80 DEG C as far as possible under conditions of take out
Filter, reduces the loss of filtrate volume.Using sour water as removal of impurities solvent, sour water can inhibit the ionization of phenols component, reinforce phenol
Retention rate of the constituents on macroreticular resin, total phenols in three leafiness will not follow sour water elution to be eluted, almost without letting out
Dew, impurity can be eluted, and improve the purification effect of the total phenols of radix tetrastigme.
Further, the extraction of the total phenols of above-mentioned radix tetrastigme and purification process, the step 1 are specially：After drying
Radix tetrastigme leaf crushes, and obtains radix tetrastigme leaf medicinal powder, and the additional amount of the ethyl alcohol is 25 times of radix tetrastigme leaf medicinal powder weight,
The volume fraction of Extraction solvent ethyl alcohol is 50%-90%, selects refluxing extraction, reflux extracting time 30-150min, and reflux mentions
Taking number is 1-3 times, and extracting solution filters under conditions of 50-80 DEG C.
Further, the extraction of the total phenols of above-mentioned radix tetrastigme and purification process, the step 1 are specially：After drying
Radix tetrastigme leaf crushes, and obtains radix tetrastigme leaf medicinal powder, and ethyl alcohol is added, and the additional amount of the ethyl alcohol is radix tetrastigme leaf medicinal powder weight
25 times of amount, the volume fraction of Extraction solvent ethyl alcohol are 70%, select refluxing extraction, reflux extracting time 120min, reflux
Extraction time is 3 times, and extracting solution filters under conditions of 50-80 DEG C.
Further, the extraction of the total phenols of above-mentioned radix tetrastigme and purification process, the step 2 are specially：By mentioning for step 1
It takes liquid to remove ethyl alcohol to obtain extracting concentrate, carries out wet method dress post with macroporous absorbent resin, concentrate will be extracted and be splined on macropore
It on adsorption resin column, is first eluted with removal of impurities solvent with the flow velocity of 6BV/h, the removal of impurities solvent is the sour water of pH=3, then uses
The ethanol solution that volume parts are 50% is eluted with the flow velocity of 2BV/h, and the pH of the ethanol solution is 2-6, is collected containing total
The eluent of phenols, the total phenols position of radix tetrastigme that eluent is concentrated to dryness to purify.
Seen from the above description, with volume fraction be after 50% ethanol elution in macroporous absorbent resin almost without total phenols
Residual, is eluted with the ethanol solution of pH=2-6, and total phenols is easier to be eluted by concentration, and effect is better than with being not added
Sour ethyl alcohol.
Further, the extraction of the total phenols of above-mentioned radix tetrastigme and purification process, the large pore resin absorption column of the step 2 are
HP20, HPD300 or D101, it is 1 that the diameter height of the large pore resin absorption column, which compares,:15-1:5.
Seen from the above description, three kinds of best macroreticular resins are：HPD300, D101, HP20, wherein HP20 is pharmaceutical grade
Macroporous absorbent resin, with before needing not move through pre-treatment, and every organic residue all meets《Chinese Pharmacopoeia》2015 editions requirements,
Experiment shows that HP20 macroporous absorbent resin is still higher than 90%, HP20 resin to the rate of transform of total phenols after using 7 times and at least may be used
Using 8 times, therefore select HP20 macroporous absorbent resin as the Purification Resin of radix tetrastigme extracting solution.
Further, the extraction of the total phenols of above-mentioned radix tetrastigme and purification process, the high ratio of the diameter of the large pore resin absorption column
It is 1:5.
Embodiment 1
A kind of extraction of the total phenols of radix tetrastigme and purification process, include the following steps：
Radix tetrastigme leaf after drying is crossed 40 meshes by step 1 after pulverizer beats powder, weighs radix tetrastigme leaf medicinal powder 5g,
The additional amount of ethyl alcohol is 175ml, and the volume fraction of Extraction solvent ethyl alcohol is 100%, select refluxing extraction, temperature extraction to take or surpass
Any one extracting method that sound extracts, extraction time are 1 time, and extracting solution filters under conditions of 50-80 DEG C；
Step 2 obtains the extracting solution removing ethyl alcohol of step 1 to extract concentrate, carries out wet process dress with macroporous absorbent resin
Column will extract concentrate and be splined on large pore resin absorption column, after loading, first be carried out with removal of impurities solvent with the flow velocity of 2-6BV/h
Elution, the removal of impurities solvent is the sour water of pH=3, then is eluted with 50% ethanol solution with the flow velocity of 2-6BV/h, and collection contains
The eluent of total phenols, the total phenols of radix tetrastigme that eluent is concentrated to dryness to purify, radix tetrastigme HPLC map after purification are figure
12。
Embodiment 2
A kind of extraction of the total phenols of radix tetrastigme and purification process, include the following steps：
Step 1, will it is dry after radix tetrastigme leaf cross 40 meshes after pulverizer beats powder, selection refluxing extraction, when refluxing extraction
Between be 120min, refluxing extraction number is 1 time, and extracting solution filters under conditions of 50-80 DEG C, recycles ethyl alcohol, it is dry after
Cream 1.2135g, the rate of extract 24.26%, it is that 1.028%, the UV survey total phenols of extracting solution contains that HPLC, which surveys the total phenols content of extracting solution,
Amount is 1.043%；
Step 2 obtains the extracting solution removing ethyl alcohol of step 1 to extract concentrate, carries out wet process dress with macroporous absorbent resin
Column will extract concentrate and be splined on large pore resin absorption column, first be eluted with removal of impurities solvent with the flow velocity of 2-6BV/h, institute
The sour water that removal of impurities solvent is pH=2 is stated, then is eluted with the ethanol solution that volume parts are 50% with the flow velocity of 2-6BV/h,
Collect the eluent containing total phenols, the total phenols of radix tetrastigme that eluent is concentrated to dryness to purify, radix tetrastigme HPLC figure after purification
Spectrum is Figure 13.
Embodiment 3
Radix tetrastigme leaf after drying is crossed 40 meshes by step 1 after pulverizer beats powder, obtains radix tetrastigme leaf medicinal powder 5g, second
The additional amount of alcohol is 100ml, and the volume fraction of Extraction solvent ethyl alcohol is 70%, selects refluxing extraction, reflux extracting time is
90min, refluxing extraction number are 1 time, and extracting solution filters under conditions of 50-80 DEG C；Ethyl alcohol is recycled, obtains to obtain cream after dry
1.2196g, the rate of extract 24.38%, it is that 1.245%, UV surveys the total phenols content of extracting solution that HPLC, which surveys the total phenols content of extracting solution,
It is 1.646%；
Step 2 obtains the extracting solution removing ethyl alcohol of step 1 to extract concentrate, carries out wet process dress with macroporous absorbent resin
Column will extract concentrate and be splined on large pore resin absorption column, first be eluted with removal of impurities solvent with the flow velocity of 2BV/h, described
Clean the sour water that solvent is pH=6, then is eluted with the ethanol solution that volume fraction is 70% with the flow velocity of 4BV/h, collects
Eluent containing total phenols, the total phenols of radix tetrastigme that eluent is concentrated to dryness to purify, radix tetrastigme HPLC map after purification are
Figure 14.
Embodiment 4
Radix tetrastigme leaf after drying is crossed 40 meshes by step 1 after pulverizer beats powder, weighs radix tetrastigme leaf medicinal powder 5g,
The additional amount of the ethyl alcohol is 100ml, and the volume fraction of Extraction solvent ethyl alcohol is 60%, selects refluxing extraction, when refluxing extraction
Between be 60min, refluxing extraction number be 2 times, extracting solution filters under conditions of 50-80 DEG C；Ethyl alcohol is recycled, obtains to obtain cream after dry
1.3462g, the rate of extract 26.91%, it is that 1.303%, UV surveys the total phenols content of extracting solution that HPLC, which surveys the total phenols content of extracting solution,
It is 1.600%；
Step 2 obtains the extracting solution removing ethyl alcohol of step 1 to extract concentrate, carries out wet process dress with macroporous absorbent resin
Column, large pore resin absorption column are HP20 large pore resin absorption column, and it is 1 that the diameter height of the HP20 large pore resin absorption column, which compares,:15-1:
5.Concentrate will be extracted to be splined on large pore resin absorption column, first eluted with removal of impurities solvent with the flow velocity of 4BV/h, it is described to remove
Miscellaneous solvent is the sour water of pH=3, then is eluted with volume parts for 90% ethanol solution with the flow velocity of 6BV/h, the second
Alcoholic solution is added after formic acid is adjusted to pH=2 and is eluted, and collects the eluent containing total phenols, and eluent is concentrated to dryness to obtain purifying
The total phenols of radix tetrastigme, radix tetrastigme HPLC map after purification is Figure 15.
Embodiment 5
Radix tetrastigme leaf after drying is crossed 40 meshes by step 1 after pulverizer beats powder, weighs radix tetrastigme leaf medicinal powder 5g,
The additional amount of the ethyl alcohol is 125ml, and the volume fraction of Extraction solvent ethyl alcohol is 70%, selects refluxing extraction, when refluxing extraction
Between be 120min, refluxing extraction number be 3 times, extracting solution filters under conditions of 50-80 DEG C；Using high performance liquid chromatography and
Ultraviolet spectrophotometry is measured total phenols content in radix tetrastigme leaf extract, and HPLC surveys the content of the total phenols of extracting solution
It is 1.5757% for 1.3462%, the UV content for measuring total phenols, the rate of extract of extracting solution is 30.63%；
Step 2 obtains the extracting solution removing ethyl alcohol of step 1 to extract concentrate, carries out wet process dress with macroporous absorbent resin
Column, large pore resin absorption column are HP20 large pore resin absorption column, and it is 1 that the diameter height of the HP20 large pore resin absorption column, which compares,:15.It will
It extracts concentrate to be splined on large pore resin absorption column, first be eluted with removal of impurities solvent with the flow velocity of 6BV/h, the removal of impurities is molten
Agent is the sour water of pH=3, the removal of impurities solvent first acid for adjusting pH, then the ethanol solution for being 50% with volume parts with 2BV/h
Flow velocity eluted, the ethanol solution is added after formic acid is adjusted to pH=4 and is eluted, and collects the elution containing total phenols
Liquid, the total phenols of radix tetrastigme that eluent is concentrated to dryness to purify.Using high performance liquid chromatography and ultraviolet spectrophotometry to three
The green total phenols content of leaf is measured, and it is that 98.75%, UV measures total phenols rate of transform and is that HPLC, which measures total phenols rate of transform,
97.58%, radix tetrastigme HPLC map after purification is Figure 16.
Embodiment 6
Radix tetrastigme leaf after drying is crossed 40 meshes by step 1 after pulverizer beats powder, weighs radix tetrastigme leaf medicinal powder 5g,
The additional amount of the ethyl alcohol is 125ml, and the volume fraction of Extraction solvent ethyl alcohol is 70%, selects refluxing extraction, when refluxing extraction
Between be 120min, refluxing extraction number be 3 times, extracting solution filters under conditions of 50-80 DEG C；Using high performance liquid chromatography and
Ultraviolet spectrophotometry is measured total phenols content in radix tetrastigme leaf extract, and HPLC surveys the content of the total phenols of extracting solution
It is 1.5757% for 1.3462%, the UV content for measuring total phenols, the rate of extract of extracting solution is 30.63%；
Step 2 obtains the extracting solution removing ethyl alcohol of step 1 to extract concentrate, carries out wet process dress with macroporous absorbent resin
Column, large pore resin absorption column are HP20 large pore resin absorption column, and it is 1 that the diameter height of the HP20 large pore resin absorption column, which compares,:10.It will
It extracts concentrate to be splined on large pore resin absorption column, first be eluted with removal of impurities solvent with the flow velocity of 2BV/h, the removal of impurities is molten
Agent is the sour water of pH=3, the removal of impurities solvent first acid for adjusting pH, then the ethanol solution for being 50% with volume parts with 4BV/h
Flow velocity eluted, the ethanol solution is added after formic acid is adjusted to pH=6 and is eluted, and collects the elution containing total phenols
Liquid, the total phenols of radix tetrastigme that eluent is concentrated to dryness to purify.Using high performance liquid chromatography and ultraviolet spectrophotometry to three
The green total phenols content of leaf is measured, and it is that 96.97%, UV measures total phenols rate of transform and is that HPLC, which measures total phenols rate of transform,
97.12%, radix tetrastigme HPLC map after purification is Figure 17.
Embodiment 7
Radix tetrastigme leaf after drying is crossed 40 meshes by step 1 after pulverizer beats powder, weighs radix tetrastigme leaf medicinal powder 5g,
The additional amount of the ethyl alcohol is 125ml, and the volume fraction of Extraction solvent ethyl alcohol is 70%, selects refluxing extraction, when refluxing extraction
Between be 120min, refluxing extraction number is 3 times, and extracting solution filters under conditions of 50-80 DEG C, using high performance liquid chromatography and
Ultraviolet spectrophotometry is measured total phenols content in radix tetrastigme leaf extract, and HPLC surveys the content of the total phenols of extracting solution
It is 1.5757% for 1.3462%, the UV content for measuring total phenols, the rate of extract of extracting solution is 30.63%；
Step 2 obtains the extracting solution removing ethyl alcohol of step 1 to extract concentrate, carries out wet process dress with macroporous absorbent resin
Column, large pore resin absorption column are HP20 large pore resin absorption column, and it is 1 that the diameter height of the HP20 large pore resin absorption column, which compares,:5.It will
It extracts concentrate to be splined on large pore resin absorption column, first be eluted with removal of impurities solvent with the flow velocity of 6BV/h, the removal of impurities is molten
Agent is the sour water of pH=3, the removal of impurities solvent first acid for adjusting pH, the ethanol solution addition formic acid adjusting that volume parts are 50%
It is eluted after to pH=3 with the flow velocity of 2BV/h, collects the eluent containing total phenols, what eluent was concentrated to dryness to purify
The total phenols of radix tetrastigme is measured the total phenols content of radix tetrastigme using high performance liquid chromatography and ultraviolet spectrophotometry,
It is that measure total phenols rate of transform be 100% to 94.02%, UV that HPLC, which measures total phenols rate of transform, radix tetrastigme HPLC figure after purification
Spectrum is Figure 18.
The present invention uses orthogonal experiment method combination single factor exploration, passes through ultraviolet spectrophotometry and high performance liquid chromatography
Assay is carried out to the total phenols of radix tetrastigme, has obtained the total phenols method for extraction and purification of optimal radix tetrastigme.
1, reagent
Reference substance：Radix tetrastigme reference substance information is shown in Table 1, and following reference substance is measured through area normalization method, and purity is all larger than
98%.
1 radix tetrastigme reference substance information of table
Sample：Radix tetrastigme leaf is prosperous Fujian No.1, is the dried leaf of radix tetrastigme (Tetrastigmahemsleyanum).
2, the total phenols the rate of extract measurement of radix tetrastigme and content assaying method
(1) the rate of extract measures
The filtered radix tetrastigme extracting solution of 25ml is drawn with pipette precision, is placed in dry to constant weight by standards of pharmacopoeia
In evaporating dish, 105 DEG C of continuous drying 3h of baking oven are placed in, taking-up, which is put in drier, after slightly cooling down lets cool to room temperature, precise weighing.
The rate of extract is calculated by formula 1-1.
The rate of extract=(weight after the empty evaporating dish constant weight of weight-after (sample+evaporating dish) constant weight)/sample weight × V × 100% (1-
1) wherein V is extracting liquid volume.
(2) extracting method investigates the assay of the total phenols of radix tetrastigme
A, chromatographic condition
Chromatographic column Welch Ultimate XB-C18 (2.7 μm of 4.6 × 150mm)；0.1% aqueous formic acid of mobile phase
(A)-acetonitrile (B), gradient elution：98%A (0~4min), 98%A → 90%A (4~15min), 90%A → 88%A (15~
30min), 82%A → 80%A (30~51min), 80%A → 98%A (51~65min)；Flow velocity 0.4mL/min, column temperature 30
DEG C, 5.0 μ L of sample volume.
B, the preparation of reference substance：Take reference substance under table 1 appropriate respectively, accurately weighed, addition methanol is made into certain dense respectively
The stock solution of degree.Respectively with 50% methanol dilution to concentration (μ gmL-1) be 8.0,6.21,6.16,9.8,5.876,4.987,
4.4,22.76 single reference substance solution, by above-mentioned chromatographic condition sample introduction, HPLC chromatogram such as Fig. 1, three leafiness extracting solution samples
HPLC chromatogram such as Fig. 2.
(3) ultraviolet content assaying method：The total phenols of radix tetrastigme uses ultraviolet specrophotometer after triethylamine set process develops the color 30min
Its absorbance A value is measured at λ=400nm.And standard curve is established by standard items of Vitexin, obtain Vitexin concentration C (ug/
Ml) with absorbance A regression equation：A=0.06601C+0.00177, correlation coefficient r2=0.99998.
3, extracting method is investigated
Take 6 250ml round-bottomed flasks, number ultrasound -1, ultrasound -2, reflux -1, reflux -2, temperature leaching -1, temperature leaching -2.It weighs
Radix tetrastigme leaf medicinal powder 5g, totally three groups, every group parallel two parts.By solid-liquid ratio (radix tetrastigme leaf medicinal powder weight：Ethyl alcohol body
Product) 1:25 are added corresponding times of 95 ethyl alcohol of amount, and refluxing extraction 1h, 60 DEG C of temperature extractions take 3h, ultrasonic extraction 30min, extracting solution respectively
It filters while hot, measures filtrate volume, as a result such as table 2 and table 3.
2 Different Extraction Method the rate of extract result of table
| Serial number | Sample name | It must cream weight/g | The rate of extract % |
| 1 | Ultrasound _ 1 | 0.636882 | 12.74% |
| 2 | Ultrasound _ 2 | 0.481184 | 9.90% |
| 3 | Reflux _ 1 | 1.237296 | 24.72% |
| 4 | Reflux _ 2 | 1.22255 | 24.43% |
| 5 | Temperature leaching _ 1 | 1.122212 | 22.44% |
| 6 | Temperature leaching _ 2 | 1.121044 | 22.42% |
The total phenols assay result of 3 Different Extraction Method of table
| Serial number | Sample name | HPLC surveys total phenols content % | UV surveys total phenols content % |
| 1 | Ultrasound _ 1 | 0.75% | 0.78% |
| 2 | Ultrasound _ 2 | 0.56% | 0.59% |
| 3 | Reflux _ 1 | 1.17% | 1.53% |
| 4 | Reflux _ 2 | 1.21% | 1.49% |
| 5 | Temperature leaching _ 1 | 1.20% | 1.38% |
| 6 | Temperature leaching _ 2 | 1.22% | 1.33% |
According to result it is found that total phenols content is highest in the rate of extract and extracting solution of refluxing extraction, so final choosing
Refluxing extraction, which is determined, as the total phenols extraction process of radix tetrastigme investigates method.
4, single factor exploration radix tetrastigme extraction process
4.1 Extraction solvents measure investigation again
4.1.1 experimental method
Weigh radix tetrastigme leaf powder 5g respectively, six groups, every group two parts to 500ml round-bottomed flask.It is separately added into corresponding material
Liquor ratio (radix tetrastigme leaf medicinal powder weight：Ethyl alcohol volume) 95 alcohol refluxs extract 1h, filtrate volume is measured after suction filtration and is remembered
Record, as a result such as table 4 and table 5
4 different solvents of table measure radix tetrastigme the rate of extract result again
| Serial number | Sample name | It must cream weight/g | The rate of extract % |
| 1 | Solid-liquid ratio 1：10 | 1.033884 | 23.10% |
| 2 | Solid-liquid ratio 1：15 | 1.145556 | 22.90% |
| 3 | Solid-liquid ratio 1：20 | 1.15752 | 23.14% |
| 4 | Solid-liquid ratio 1：25 | 1.263082 | 25.25% |
| 5 | Solid-liquid ratio 1：30 | 1.28352 | 25.67% |
| 6 | Solid-liquid ratio 1：35 | 1.229937 | 24.59% |
5 different solvents of table measure the total phenols assay result of radix tetrastigme again
| Serial number | Sample name | HPLC surveys total phenols content % | UV surveys total phenols content % |
| 1 | Solid-liquid ratio 1：10 | 1.1218% | 1.3954% |
| 2 | Solid-liquid ratio 1：15 | 1.2143% | 1.5252% |
| 3 | Solid-liquid ratio 1：20 | 1.2210% | 1.5297% |
| 4 | Solid-liquid ratio 1：25 | 1.2967% | 1.6897% |
| 5 | Solid-liquid ratio 1：30 | 1.2882% | 1.6596% |
| 6 | Solid-liquid ratio 1：35 | 1.2857% | 1.6247% |
According to result it is found that when solvent measures less or more again, the total phenols content of the radix tetrastigme of extraction is respectively less than extracted molten
Agent be 25 times amount when as a result, therefore selection solid-liquid ratio (radix tetrastigme leaf medicinal powder weight：Ethyl alcohol volume) 1：25 carry out radix tetrastigme
The investigation of extraction time.
4.2 extraction times were investigated
4.2.1 experimental method
Weigh radix tetrastigme leaf powder 5g respectively, five groups, every group two parts, until in 250ml volumetric flask.25 times of 95% second of amount are added
Alcohol distinguishes refluxing extraction 30min, 60min, 90min, 120min, 150min, filtrate volume is measured after suction filtration and is recorded.As a result
Such as table 6 and table 7.
The different extraction time radix tetrastigme the rate of extract results of table 6
The different extraction time total phenols assay results of radix tetrastigme of table 7
According to result it is found that extraction efficiency is best when extraction time is 120min, total phenols content highest, so to extract
120min is as the optimum extraction time.
4.3 Extraction solvent volume fraction of ethanol are investigated
4.3.1 experimental method
Weigh radix tetrastigme leaf powder 5g respectively, six groups, every group two parts, until 25 times of amount water are added in 250ml round-bottomed flask,
10%, 30%, 50%, 70%, 90% ethyl alcohol extracts 120 minutes.Filtrate volume is measured after filtering while hot and is recorded.As a result such as table
8 and table 9.
The different volume fraction of ethanol radix tetrastigme the rate of extract measurement results of table 8
The total phenols assay result of the different volume fraction of ethanol radix tetrastigmes of table 9
According to result it is found that extraction efficiency is best when volume fraction of ethanol is 70%, total phenols contains in radix tetrastigme extracting solution
Highest is measured, so determining best volume fraction of ethanol 70%.
5, orthogonal experiment method investigates radix tetrastigme extraction process
Factor level is screened by experiment of single factor, passes through L9 (34) orthogonal trial experimental program, preferably optimum extraction work
Skill.
5.1 experimental method
Weigh radix tetrastigme leaf powder 5g respectively, nine groups, every group parallel two parts, until in 250ml round-bottomed flask, by L9 (34) just
It hands over experiment table to carry out refluxing extraction, filtrate volume is measured after filtering while hot and records.As a result such as table 10, table 11 and table 12.
The total phenols of 10 radix tetrastigme of table extracts orthogonal experiment the rate of extract measurement result
Total phenols assay (HPLC) result of 11 orthogonal experiment of table
Total phenols assay (UV) statistical result of 12 radix tetrastigme extraction process orthogonal experiment of table
Orthogonal experiment obtains cream yield and total phenols assay result is imported in orthogonal experiment statistical software and calculated, and obtains pole
Poor R and K value, very poor R analysis is the result shows that maximum to the total phenols extraction rate impact of radix tetrastigme in four influence factors is ethyl alcohol
Percentage by volume is followed successively by from small to large：A extraction time < B extraction time < C solid-liquid ratio < D volume fraction of ethanol, while by
It is known that the difference of extraction time is mainly manifested among once and twice in K value, extract 2 times it is almost the same with 3 results,
So selective extraction 2 times and 3 times carry out confirmatory experiment simultaneously.The rate of extract result is determined by experiment it is recognised that 70% ethyl alcohol
The three groups of total phenols the rate of extract extracted are higher, and total main body of phenols content difference of extraction is between 50% and 70%, and 60%
Gained is extracted without very big difference with 70% ethyl alcohol, selection selection concentration of alcohol 60% and 70% carries out confirmatory experiment simultaneously,
It determines best.Compare extraction time result it is recognised that in radix tetrastigme extracting solution total phenols content with the increase of extraction time and
Increase, but 90min and 120min difference is smaller, so final choice extraction time 120min and 90min carry out verifying in fact simultaneously
It tests, determines best.Finally, solvent measures the influence maximum extracted to radix tetrastigme again, but main difference is embodied in 15 times of amounts and 20 times
It is 20 times smaller with 25 times of amount differences between amount, therefore the final verifying technique preferably gone out is table 13, orthogonal verification result is 14 He of table
Table 15.
The orthogonal verification test table of 13 radix tetrastigme extracting method of table
| Test serial number | Extraction time | Extraction time | Solid-liquid ratio | Volume fraction of ethanol |
| 1 | 120(min) | 3 | 1：25 | 70 |
| 2 | 90(min) | 2 | 1：20 | 60 |
| 3 | 90(min) | 2 | 1：20 | 70 |
The orthogonal verification test the rate of extract measurement result of 14 radix tetrastigme extraction process of table
| Serial number | Sample name | Obtain cream weight/g | The rate of extract |
| 1 | Verify 1-1 | 1.4918 | 29.83% |
| 2 | Verify 1-2 | 1.5178 | 30.34% |
| 3 | Verify 1-3 | 1.5324 | 30.63% |
| 4 | Verify 2-1 | 1.3593 | 27.17% |
| 5 | Verify 2-2 | 1.3671 | 27.34% |
| 6 | Verify 2-3 | 1.3578 | 27.15% |
| 7 | Verify 3-1 | 1.3583 | 27.16% |
| 8 | Verify 3-2 | 1.3261 | 26.51% |
| 9 | Verify 3-3 | 1.3346 | 26.68% |
The total phenols assay result of the orthogonal verification test of 15 radix tetrastigme extraction process of table
| Serial number | Sample name | HPLC surveys total phenols content | UV surveys total phenols content |
| 1 | Verify 1-1 | 1.3079% | 1.5756% |
| 2 | Verify 1-2 | 1.3189% | 1.5757% |
| 3 | Verify 1-3 | 1.3462% | 1.5214% |
| 4 | Verify 2-1 | 1.2934% | 1.5084% |
| 5 | Verify 2-2 | 1.3146% | 1.5218% |
| 6 | Verify 2-3 | 1.3543% | 1.4484% |
| 7 | Verify 3-1 | 1.3772% | 1.5097% |
| 8 | Verify 3-2 | 1.3186% | 1.5376% |
| 9 | Verify 3-3 | 1.3487% | 1.5760% |
By confirmatory experiment result it is found that its rate of extract of verification test 1 and the extraction gained total phenols content of percentage are highest,
So determining that the total phenols of radix tetrastigme extracts optimum process and is：Extraction time 120min is extracted 3 times, solid-liquid ratio 1：25, ethyl alcohol volume
Score 70%.
6, the total phenols purifying process research of radix tetrastigme
The screening of 6.1 macroreticular resins
By investigate identical Static Adsorption time D101, AB-8, HP20, HPD100, HPD300, HPD450, HPD600,
The macroreticular resin of eight kinds of different models of HPD700 extracts the adsorbance and parsing amount of total phenols in concentrate to radix tetrastigme, preferably
Optimal macroreticular resin model out.
8 25ml cleaning EP pipes are taken, are numbered with resin model.Weigh pharmaceutical grade macroreticular resin D101, AB-8,
Each 1g of HP20, HPD100, HPD300, HPD450, HPD600, HPD700 is loaded in corresponding EP pipe, accurate respectively that radix tetrastigme is added
Concentrate 20ml is extracted, in 37 DEG C of shaking tables for 24 hours with 120 turns/min of revolving speed oscillation, takes out raffinate ultrapure water after absorption respectively
It is settled to as in 50ml volumetric flask, shakes up.95 ethyl alcohol of 20ml is added with pipette precision in resin to be resolved, is placed on 37 DEG C of perseverances
It is attached for 24 hours with 120 turns/min of revolving speed parsing in warm shaking table, 95 ethyl alcohol constant volume of desorbed solution is taken out in 50ml capacity after being cooled to room temperature
In bottle, shake up.Total phenols content in measurement absorption extraction raffinate and desorbed solution respectively, it is attached by comparing raffinate and parsing after its absorption
The height of total phenols content preferably goes out best macroreticular resin model in liquid, as a result such as table 16.
16 macroreticular resin the selection result of table
As can be seen from Table 16, preferable three kinds of macroreticular resins are：HPD300, D101, HP20；HPD300 is best, but
HPD300 is non-medicinal rank resin, HP20 adsorbance and parsing amount and HPD300 without larger difference, and HP20 is that pharmaceutical grade is big
Hole resin, with before needing not move through pre-treatment, and every organic residue all meets 2015 editions pharmacopoeial requirements, therefore final choice HP20
Purification Resin of the macroreticular resin as radix tetrastigme extracting solution.
6.2 total phenols elution curves are investigated
Take HP20 resin 40g (1BV=60ml), loading 60ml medical fluid, after Dynamic Adsorption, respectively with water, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90% ethyl alcohol (with formic acid tune pH=2~6) elutes resin with 2BV/h flow velocity
Column is first eluted with sour water, each gradient elution 4BV, and every 1BV collects 1 flow point, measures the rate of extract, total phenols content respectively
And elution curve is drawn, obtain the total phenols assay result figure 3 of radix tetrastigme purification process elution curve.
According to Fig. 4~Figure 11's, the result shows that, total phenols will not follow sour water elution to be eluted, almost without leakage.With
When 10% ethanol elution, total phenols starts to get off with 10% ethanol elution, but speed is slow and measures very few.Start to use
After 40% ethanol elution, it can be seen that total phenols colour band apparent motion, most total phenols is with 40% ethyl alcohol quilt in extracting solution
Elution；Almost without total phenols residual in resin after 50% ethanol elution.Therefore sour water is selected to purify as radix tetrastigme extracting solution
Clean solvent, and 50%~90% ethyl alcohol is solvent-rich.
6.3 orthogonal experiment methods optimize the total phenols purifying process of radix tetrastigme
By orthogonal arrage L9 (33) contrived experiment scheme, removal of impurities solvent flow rate, solvent-rich stream are investigated using orthogonal experiment method
Speed, diameter height ratio, finally determine optimal purification process condition.Orthogonal design table is shown in Table 17.
17 L9 (3 of table3) orthogonal design factor level table
18 Orthogonal Experiment and Design of table and result
Table 19
According to experimental result it is found that from 1~No. 9 orthogonal experiments in table 18 it is found that purifying active component every time
Total phenols rate of transform all 94% or more, very poor R the result shows that, in the horizontal extent of setting, total phenols rate of transform is regarded
Evaluation index, the smallest influence of three factors to purifying process is the high ratio of diameter, influences maximum to be removal of impurities solvent flow rate, removal of impurities
Flow velocity too slow (2BV/h) will lead to total phenols rate of transform decline；The rate of transform of solvent-rich flow velocity (2BV/h) total phenols when slower
It is higher.It is analyzed by orthogonal software it is found that optimum combination is：Clean solvent flow rate 6BV/h, solvent-rich flow velocity 2BV/h, the high ratio of diameter
It is 1：5(HPLC)；Clean solvent flow rate 6BV/h, and solvent-rich flow velocity 2BV/h, diameter height is than 1：10(UV)；As shown in Table 19, most
Excellent purifying process is：Clean solvent flow rate 6BV/h, and solvent-rich flow velocity 2BV/h, diameter height is than 1：5.
In conclusion extraction and the purification process of the total phenols of radix tetrastigme provided by the invention, using high performance liquid chromatography
Total phenols content in radix tetrastigme leaf extract is measured with ultraviolet spectrophotometry, high performance liquid chromatography has Gao Chong
Existing property and high sensitivity, so that experimental result is more accurate, and comprehensive analysis its rate of extract, it is based on single factor exploration, in conjunction with just
Experimental method is handed over to obtain the total phenols extraction of optimal radix tetrastigme and purification process；It is maintained under conditions of 50-80 DEG C when all suction filtrations
It filters, is found during Study on Extraction Method, radix tetrastigme leaf powder is added in the ethyl alcohol of the different volumes score of certain volume
When middle, when concentration of alcohol is lower than 50%, radix tetrastigme powder is flown on ethyl alcohol liquid level, cannot be infiltrated immediately.It keeps slightly boiled to mention
Bottom is sunken to after taking a period of time.When extracting completion suction filtration acquisition supernatant, concentration is difficult to filter lower than 50% ethyl alcohol, and
And extraction efficiency is bad.Concentration of alcohol is inadequate at this time, and the ingredients such as the radix tetrastigme polysaccharide extracted and mucilaginous substance are more, so that
Extracting solution is more sticky, filters under conditions of being maintained at 50-80 DEG C as far as possible when so filtering, reduces the loss of filtrate volume.It adopts
Use sour water as removal of impurities solvent, total phenols in three leafiness will not follow sour water elution to be eluted, and almost without leakage, mention
The high purification effect of the total phenols of radix tetrastigme.
With volume fraction to be remained in macroporous absorbent resin after 50% ethanol elution almost without total phenols, with pH=3's
Ethanol solution is eluted, and total phenols is easier to be eluted.It tests and compares 8 kinds of macroreticular resins altogether, respectively HPD100,
HPD300, HPD450, HPD600, HPD700, D101, AB-8, HP20.Comparison discovery HPD300, D101 and HP20 absorption parsing
Attached effect is suitable.But HP20 is pharmaceutical grade macroporous absorbent resin, and with before needing not move through pre-treatment, and every organic residue all accords with
It closes《Chinese Pharmacopoeia》2015 editions requirements, experiment show HP20 macroporous absorbent resin after using 7 times to the rate of transform of total phenols
Being still higher than 90%, HP20 resin at least can be used 8 times, therefore select HP20 macroporous absorbent resin as the purifying of radix tetrastigme extracting solution
Resin.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalents made by bright specification and accompanying drawing content are applied directly or indirectly in relevant technical field, similarly include
In scope of patent protection of the invention.
Claims (6)
1. extraction and the purification process of a kind of total phenols of radix tetrastigme, which is characterized in that include the following steps：
Step 1 crushes the radix tetrastigme leaf after drying, obtains radix tetrastigme leaf medicinal powder, ethyl alcohol, the additional amount of the ethyl alcohol is added
It is 10-35 times of radix tetrastigme leaf medicinal powder weight, selection refluxing extraction, temperature extraction take or any one extraction of ultrasonic extraction
Method, extracting solution filter under conditions of 50-80 DEG C；
Step 2 obtains the extracting solution removing ethyl alcohol of step 1 to extract concentrate, carries out wet method dress post with macroporous absorbent resin, will
It extracts concentrate to be splined on large pore resin absorption column, after loading, first be eluted with removal of impurities solvent with the flow velocity of 2-6BV/h,
The sour water that the removal of impurities solvent is pH=2-6, dosage 1-5BV, then with the ethanol solution of 50%-90% with the stream of 2-6BV/h
Speed is eluted, and the eluent containing total phenols, the total phenols of radix tetrastigme that eluent is concentrated to dryness to purify are collected.
2. extraction and the purification process of the total phenols of radix tetrastigme according to claim 1, which is characterized in that step 1 tool
Body is：Radix tetrastigme leaf after drying is crushed, radix tetrastigme leaf medicinal powder is obtained, the additional amount of the ethyl alcohol is radix tetrastigme leaf medicinal material
25 times of powder weight, the volume fraction of Extraction solvent ethyl alcohol are 10%-90%, select refluxing extraction, reflux extracting time is
30-150min, refluxing extraction number are 1-3 times, and extracting solution filters under conditions of 50-80 DEG C.
3. according to claim 1 or the extraction of 2 described in any item total phenols of radix tetrastigme and purification process, which is characterized in that institute
Stating step 1 is specially：Radix tetrastigme leaf after drying is crushed, radix tetrastigme leaf medicinal powder is obtained, ethyl alcohol is added, the ethyl alcohol adds
Enter 25 times that amount is radix tetrastigme leaf medicinal powder weight, the volume fraction of Extraction solvent ethyl alcohol is 70%, selects refluxing extraction, returns
Stream extraction time is 120min, and refluxing extraction number is 3 times, and extracting solution filters under conditions of 50-80 DEG C.
4. extraction and the purification process of the total phenols of radix tetrastigme according to claim 1, which is characterized in that step 2 tool
Body is：The extracting solution of step 1 is removed ethyl alcohol to obtain extracting concentrate, wet method dress post is carried out with macroporous absorbent resin, will extract
Concentrate is splined on large pore resin absorption column, is first eluted with removal of impurities solvent with the flow velocity of 6BV/h, the removal of impurities solvent is
The sour water of pH=3, then eluted with the ethanol solution that volume parts are 50% with the flow velocity of 2BV/h, the ethanol solution
PH is 2-6, collects the eluent containing total phenols, the total phenols of radix tetrastigme that eluent is concentrated to dryness to purify.
5. according to claim 1 or the extraction of 4 described in any item total phenols of radix tetrastigme and purification process, which is characterized in that institute
The large pore resin absorption column for stating step 2 is HP20, HPD300 or D101, and it is 1 that the diameter height of the large pore resin absorption column, which compares,:15-
1:5。
6. extraction and the purification process of the total phenols of radix tetrastigme according to claim 5, which is characterized in that the macroporous absorption
It is 1 that the diameter height of resin column, which compares,:5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810871115.6A CN108853274A (en) | 2018-08-02 | 2018-08-02 | A kind of extraction of the total phenols of radix tetrastigme and purification process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810871115.6A CN108853274A (en) | 2018-08-02 | 2018-08-02 | A kind of extraction of the total phenols of radix tetrastigme and purification process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108853274A true CN108853274A (en) | 2018-11-23 |
Family
ID=64307506
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810871115.6A Pending CN108853274A (en) | 2018-08-02 | 2018-08-02 | A kind of extraction of the total phenols of radix tetrastigme and purification process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108853274A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109303819A (en) * | 2018-11-24 | 2019-02-05 | 郎国忠 | A kind of processing technology of radix tetrastigme |
| CN115837052A (en) * | 2022-12-11 | 2023-03-24 | 浙江武义汇美中药材有限公司 | Efficient preparation method of radix tetrastigme stem and leaf blood fat reducing polyphenol, product and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104435401A (en) * | 2014-11-13 | 2015-03-25 | 福建中医药大学 | Method for purifying general flavone from aboveground part of radix tetrastigme |
| CN106526043A (en) * | 2016-12-12 | 2017-03-22 | 福建中医药大学 | RRLC-Q-TOF-MS method for detecting diversified chemical components in leaves of tetrastigma hemsleyanum diels et gilg |
-
2018
- 2018-08-02 CN CN201810871115.6A patent/CN108853274A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104435401A (en) * | 2014-11-13 | 2015-03-25 | 福建中医药大学 | Method for purifying general flavone from aboveground part of radix tetrastigme |
| CN106526043A (en) * | 2016-12-12 | 2017-03-22 | 福建中医药大学 | RRLC-Q-TOF-MS method for detecting diversified chemical components in leaves of tetrastigma hemsleyanum diels et gilg |
Non-Patent Citations (5)
| Title |
|---|
| 窦德强: "《中药化学》", 30 April 2012, 湖南科学技术出版社 * |
| 范世明等: "三叶青叶化学成分鉴定及其总黄酮含量测定研究", 《药物分析杂志》 * |
| 范世明等: "三叶青叶指纹图谱研究及8种酚类成分含量测定", 《中国中药杂志》 * |
| 辽宁省卫生局: "《辽宁省医院制剂规范 1982年版》", 31 December 1982, 辽宁省卫生局 * |
| 郑建仙: "《功能性食品学》", 30 June 2009, 中国轻工业出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109303819A (en) * | 2018-11-24 | 2019-02-05 | 郎国忠 | A kind of processing technology of radix tetrastigme |
| CN115837052A (en) * | 2022-12-11 | 2023-03-24 | 浙江武义汇美中药材有限公司 | Efficient preparation method of radix tetrastigme stem and leaf blood fat reducing polyphenol, product and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102641326B (en) | Astragalus extract, as well as preparation and application methods thereof | |
| CN103776926B (en) | The foundation of rabdosia lophanthide HPLC-FPS | |
| CN102631414B (en) | SepHaniadelavayi Diels total alkaloid extraction and purification technology | |
| CN106727806B (en) | A kind of preparation method of sanchi leaf total saposins | |
| CN102133363A (en) | Quality detection method for chinarue cough particle | |
| CN101966223A (en) | Fingerprint detection method for compound wintercreeper preparation | |
| CN108853274A (en) | A kind of extraction of the total phenols of radix tetrastigme and purification process | |
| CN104725450A (en) | Method for extracting high-purity oleuropein from jasminum grandiflorum | |
| CN104391072A (en) | Quality control method of traditional Chinese medicine compound preparation for treating osteoporosis | |
| CN103830306B (en) | A kind of preparation method of folium lonicerae effective extract | |
| CN111948305B (en) | Quality control method and preparation method for producing Qingda granules and compound traditional Chinese medicine based on Qingda granules | |
| CN101926889B (en) | Method for detecting white paeony root-medlar particles | |
| CN102068649B (en) | Quality control method for gastric condition-regulating pill as traditional Chinese preparation | |
| CN101632722B (en) | Wild buckwheat rhizome polyphenol extract and preparation method thereof | |
| CN103006721A (en) | Method for extracting phenylethanoid glycoside and flavonoid C-glycoside from corallodiscus flabellata | |
| CN108042590A (en) | A kind of residual extract of Radix Ginseng stem and leaf of low agriculture and preparation method thereof | |
| CN101703669B (en) | Smilax china effective fractions and extraction as well as purification process thereof | |
| CN104274727B (en) | The quality determining method of clear battalion's oral liquid | |
| CN102924545B (en) | A kind of enrichment of akebiasaponin D and purification process | |
| CN105616946A (en) | Preparation for treating cough, preparation method and quality control method thereof | |
| CN109912582A (en) | The method of mangiferin is extracted from mango leaf | |
| CN104111295A (en) | Method for controlling quality of Chinese herbal preparation | |
| CN109045087A (en) | A kind of enriching and purifying technique of Chinese sumac active component | |
| CN104614480B (en) | A kind of Herba Rabdosiae Lophanthoidis water solublity total flavones and fingerprint atlas detection method thereof | |
| CN104697950A (en) | Method for detecting pseudo-ginseng content and optimally extracting and purifying |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |