CN102000158A - Extraction method of Dicranostigma leptodum (Maxim.) Fedde alkaloid and extract obtained by same - Google Patents
Extraction method of Dicranostigma leptodum (Maxim.) Fedde alkaloid and extract obtained by same Download PDFInfo
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Abstract
The invention discloses an extraction method of Dicranostigma leptodum (Maxim.) Fedde alkaloid and an extract prepared by the method. The method comprises the following steps: extracting the whole Dicranostigma leptodum (Maxim.) Fedde with water, precipitating with ethanol, concentrating, and performing freeze-drying treatment to obtain Dicranostigma leptodum (Maxim.) Fedde alkaloid. The extract obtained by the method has high alkaloid content; and the method has the advantages of high extraction rate and good reproducibility and is suitable for large-scale industrialization.
Description
Technical field
The present invention relates to Chinese medicine extract, relate in particular to the extracting method of Herba dicranostigmae and the extract that obtains by this method.
Background technology
Herba dicranostigmae [Dicranostigma leptopodum (Maxim.) Fedde], have another name called Herba Dicranostigmatis Leptopodi, the baldhead spends, reins in the horse back, belong to the biennial or herbaceos perennial of Papaveraceae tinea alba Pittosporum, be recited as Adiantum monochlamys in the Compendium of Material Medica, grow in the hills of height above sea level 600-1300m, on grass slope, roadside, the ridge, farmland or the wall, roof etc. locates.Herba dicranostigmae is at all herbal medicine among the people, and energy heat-clearing and toxic substances removing, reducing swelling and alleviating pain, parasite killing are treated tonsillitis, throat pain, lymphoid tuberculosis (flat pestilence, lymphoid tuberculosis), bald scar, diseases such as scabies.
The bibliographical information Herba dicranostigmae contains multiple isoquinoline alkaloid, herb mainly contain isocorydine (having another name called the dextrorotation isocorydine) (d-isocorydine), corydaline (Corydine), magnoline (Magnflorine), different corydaline (lsocorydine), protopine (Protopine).Smooth if separation such as row, purify and identified different corydaline, corydaline, the protopine in the Herba dicranostigmae, see by " pharmacy circular " 1981 (2): 52 and " pharmaceutical analysis magazine ", 1982,2 (5): 273-277.
The total alkaloids effective ingredient of extraction separation has multiple pharmacological effect from Herba dicranostigmae.Chen Zhengshan etc. have reported the influence of Herba dicranostigmae extract to functions of murine peritoneal macrophages, " Shanghai IMMUNOLOGY KEY WORDS INDEX; 2001,21 (4): 216-218; Mao Aihong etc. have reported the influence of Herba dicranostigmae extract to the mouse immune liver damage, " Chinese Pharmacological Bulletin; 2004,20 (8): 940-943; Gong Yanni etc. have reported the Herba dicranostigmae extract to lotus Mus Immune Effects, " Lanzhou University's journal (medicine) ", 2008,34 (3): 48-52; Wang Tingpu etc. have reported that the Herba dicranostigmae extract is inhibited to the pathogen Orf virus of sheep infective pustule, " Chinese veterinarian's medical magazine ", 1998,1 (1): 9-11; Xue slaps woods and has reported that the Herba dicranostigmae extract is inhibited to avian infectious bronchitis virus, " Gansu animal and veterinary ", 2004,174 (1): 16-17; Zhao Qiang etc. have reported that the Herba dicranostigmae extract is inhibited to escherichia coli, staphylococcus aureus, bacillus subtilis, excrement enterobacteria, " Chinese veterinary science ", 2008,38 (12): 1098-1101.In addition, Chen Quan etc. summarize the pharmacologically active of Herba dicranostigmae extract, disclose the Herba dicranostigmae extract to therapeutical effect lungy, " sky and water normal college's journal ", 2008,28 (2): 49-52.
The extracting method of the Herba dicranostigmae total alkaloids of bibliographical information has water extraction and alcohol extracting method.The processing step of water extraction mainly comprises: Herba dicranostigmae powder water extraction, and with 75% ethanol precipitation impurity, remove ethanol and obtain Herba dicranostigmae alkaloid concentrated solution; See " Chinese veterinarian's medical magazines " such as Wang Ting uncut jades, 2008, the 6 phases, " Anhui agricultural sciences " 2008,36 (17): 7294-96 such as 41-44 page or leaf and Wang Tingpu.The processing step of alcohol extracting method comprises: 75% ethanol extraction Herba dicranostigmae powder, and 85% ethanol precipitation impurity is removed ethanol and is obtained the Herba dicranostigmae concentrated solution; See " Chinese veterinarian's medical magazines " such as Wang Ting uncut jades, 2008, the 6 phases, 41-44 page or leaf.
In the disclosed extracting method of prior art, alcohol extracting method cost height, water extraction then yield is lower, and the Herba dicranostigmae concentrated solution volume that two kinds of methods obtain is bigger, be not suitable for large-scale production, so this area presses for, and cost is low, yield is high, the Herba dicranostigmae alkaloid extracting method of suitable large-scale production.
Summary of the invention
It is lower that technical problem to be solved by this invention provides a kind of cost, is fit to the Herba dicranostigmae alkaloid extracting method of large-scale production and the extract that is obtained by this method.
Technical problem to be solved by this invention realizes by following method.
A kind of Herba dicranostigmae alkaloid extraction method is characterized in that: with Herba dicranostigmae herb water extraction, and filtration or centrifugal; Filtrate adds alcoholic solvent precipitation, filtration or centrifugal; Get filtrate and be concentrated into 1/10 of former liquid measure, become the extractum state, carry out lyophilizing and handle.
Described Herba dicranostigmae herb can be in advance through processing such as section or pulverizing, to increase contacting of medical material and water.
Described water extraction mode includes but not limited to merceration extraction method, ultrasonic extraction, soxhlet extraction etc., preferred ultrasonic extraction.Wherein supersound extraction is 0.5～3 hour, preferred 1 hour.
Described water extraction temperature is 30-80 ℃, preferred 60-80 ℃, and more preferably 80 ℃.
Described extraction water with the liquid material of Herba dicranostigmae herb than (volume/weight) is: 10: 1-50: 1, preferred 30: 1-45: 1, and more preferably 30: 1.
Described alcoholic solvent includes but not limited to methanol, ethanol, isopropyl alcohol or butanols etc., preferred alcohol.The volume fraction of alcoholic solvent is 35-95%, preferred 75%.
Described filtering technique includes but not limited to: normal pressure or filtration under diminished pressure, microfiltration, ultrafiltration.
Described filtrate concentrates under the condition of normal pressure or decompression, and decompression state wherein is 0.09-0.1MPa.Further, described filtrate can concentrate under the condition of room temperature or heating.For example, adopt vacuum rotary evaporator to realize concentrating.
Described freeze-drying process, pre-freeze temperature are-20 to-70 ℃, and be preferred :-40 ℃ to-70 ℃, more preferably-70 ℃.Described vacuum lyophilization process can be procedural cooling, also can be the freezing of fixed temperature, and temperature range is-61 ℃.The vacuum of described lyophilisation is the lyophilizing vacuum condition of those skilled in the art's routine, preferred 0.011MPa.
The Herba dicranostigmae alkaloid of the method for the invention preparation can add biological acceptable auxiliary and further make dosage forms such as tablet, capsule.
The present invention also comprises the Herba dicranostigmae alkaloid that is obtained by said method.
The present invention prepares method extraction ratio height, the favorable reproducibility of Herba dicranostigmae alkaloid with respect to traditional water extraction process, and does not need to use in a large number organic solvents such as ethanol, has greatly reduced cost, is suitable for suitability for industrialized production Herba dicranostigmae alkaloid.
Description of drawings
The UV spectrum of Fig. 1, sample and reference substance
Fig. 2, Herba dicranostigmae total alkaloids standard curve
Fig. 3, different ultrasonic time are to the influence of biology total alkali extraction ratio in the Herba dicranostigmae
The influence of biology total alkali extraction ratio in Fig. 4, the different liquid material comparison Herba dicranostigmae
Fig. 5, different pH value are to the influence of biology total alkali extraction ratio in the Herba dicranostigmae
Fig. 6, temperature are to the influence of biology total alkali extraction ratio in the Herba dicranostigmae
Fig. 7, different ethanol volume fraction are to the influence of biology total alkali extraction ratio in the Herba dicranostigmae
The specific embodiment
Crude drug: Herba dicranostigmae herb wild or plantation is gathered in the crops afterwash, is dried, chopping, and it is standby to make powder.
The alkaloidal discriminating of Herba dicranostigmae: the Herba dicranostigmae extract is mixed with aqueous solution, adds 3 of bismuth potassium iodide solution, generates light-yellow precipitate.
Herba dicranostigmae is alkaloidal quantitatively
Isocorydine (isocorydine) has another name called (+)-isocorydine, isocorydine, is one of main active of Herba dicranostigmae.The present invention's Isocorydine hydrochloride (ISOCORYDINEHYDROCHLORIDE), CAS number: 13552-72-2 is product (purchasing in the special biochemical technology company limited of Changsha Foucault) in contrast, are used for the quantitative of Herba dicranostigmae total alkaloids.
Reference substance (isocorydine) solution and sample solution are scanned in the 190-500nm wave-length coverage, to determine the maximum absorption wavelength of Herba dicranostigmae total alkaloids.Definite through testing, Herba dicranostigmae total alkaloids extracting solution has absorption maximum at the 210nm place, sees Fig. 1 (curve 1 is reference substance solution, and curve 2 is a sample solution).
Accurately take by weighing alkaloid reference substance 4mg, adding deionized water 4ml formulation concentration is the contrast liquid mother solution of 1mg/ml.Add deionized water during mensuration and be diluted to that concentration is respectively 0.01,0.02,0.03,0.04,0.05,0.06mg/ml.With the deionized water is blank, surveys absorbance O.D value at 210nm wavelength place, and measurement result sees Table 1.With concentration (mg/ml) is abscissa (X), and the absorbance A value is vertical coordinate (Y), obtains standard curve, sees Fig. 2.Regression equation by the standard curve match is: Y=0.43956X-0.00546.
Table 1 production standard curve data
Accurately take by weighing each extract sample 4mg and be dissolved in the 4ml deionized water, 6 times of dilutions, every group each parallel three parts, leave standstill, cross microporous filter membrane (0.22nm), survey absorbance O.D value and be averaged, with measured O.D value substitution standard curve regression equation, calculate biology total alkali content, get the biology total alkali extraction ratio.
Alkaloid percentage ratio is in the extract:
Alkaloid (%)=test fluid alkaloid concentration (mg/ml) * 6 times * 4ml ÷ 4mg * 100%
The alkaloid extraction ratio is:
The total extraction ratio of alkaloid (%)=extract gross mass (g) * alkaloid (%) ÷ crude drug quality (g) * 100%.
Herba dicranostigmae herb powder 10g, adding the deionization water logging by 1: 15 (weight/volume) puts and spends the night, high speed centrifugation (3500r/min) 10min gets supernatant, getting supernatant, to add volume fraction be that 95% ethanol to original solution alcoholic strength reaches 80%, standing over night, room temperature high speed centrifugation (3500r/min) 10min gets supernatant, and 50 ℃ of rotary evaporations reclaim ethanol and are concentrated into 20ml.Concentrated solution-70 ℃ freeze overnight, vacuum lyophilization (61 ℃ 0.011Mpa) got Herba dicranostigmae alkaloid goods in 24 hours.
Detect: precision is weighed, get each goods respectively 4mg be dissolved in the 4ml deionized water, microporous filter membrane (0.22nm) is crossed in 6 times of dilutions, surveys absorbance O.D value.
Herba dicranostigmae herb powder 10g presses and aids in the ultrasonic treatment instrument at 60 ℃ of processing 1h after (weight/volume) adds the deionized water immersion at 1: 15, and high speed centrifugation (3500r/min) 10min gets supernatant.Getting supernatant, to add volume fraction be that 95% ethanol reaches 80% to the original solution alcoholic strength, standing over night, and room temperature high speed centrifugation (3500r/min) 10min gets supernatant, and 50 ℃ of rotary evaporations reclaim ethanol and also are concentrated into 20ml.Concentrated solution-70 ℃ freeze overnight, vacuum lyophilization (61 ℃ 0.011Mpa) got Herba dicranostigmae alkaloid goods in 24 hours.
Detect: precision is weighed, and each goods 4mg respectively are dissolved in the 4ml deionized water, and microporous filter membrane (0.22nm) is crossed in 6 times of dilutions, surveys absorbance O.D value.
Herba dicranostigmae herb powder 10g, add deionized water at 1: 15 by (weight/volume) in the flask, electric jacket adds thermal siphon eight times, stop heating and get flask filtrate, adding volume fraction is that 95% ethanol to original solution alcoholic strength reaches 80%, standing over night, room temperature high speed centrifugation (3500r/min) 10min gets supernatant, and 50 ℃ of rotary evaporations reclaim ethanol and are concentrated into 20ml.Concentrated solution-70 ℃ freeze overnight, vacuum lyophilization (61 ℃ 0.011Mpa) got Herba dicranostigmae alkaloid goods in 24 hours.
Detect: precision is weighed, get each goods respectively 4mg be dissolved in the 4ml deionized water, microporous filter membrane (0.22nm) is crossed in 6 times of dilutions, surveys absorbance O.D value.
The Herba dicranostigmae total alkaloids extract measurement result that three kinds of extracting modes obtain sees Table 2, and as can be seen from the results, the ultrasonic extraction effect is better.
The different extracting modes of table 2 to Herba dicranostigmae biology total alkali extraction effect relatively
Based on the ultrasonic extraction of embodiment 1, factors such as extraction time, liquid material ratio, pH value, temperature, ethanol volume fraction are optimized.
Tested for 1 extraction time to extracting the influence of Herba dicranostigmae biology total alkali
Accurately take by weighing Herba dicranostigmae herb powder 10g respectively, add at 15: 1 than (weight/volume) by the liquid material and to aid in the ultrasonic treatment instrument respectively after deionized water soaks and handle 0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h successively for 60 ℃, other steps are with embodiment 1 ultrasonic extraction.The results are shown in Table 3, Fig. 3, effect was best when extraction time was 0.5h.
Table 3 extraction time influences result and analysis to Herba dicranostigmae biology total alkali extraction ratio
| Ultrasonic time | 0h | 0.5h | 1h | 1.5h | 2h | 2.5h | 3h |
| The O.D value | 1.755 | 1.774 | 1.765 | 1.761 | 1.759 | 1.754 | 1.755 |
| Extraction ratio/% | 4.094 | 4.14 | 4.118 | 4.11 | 4.104 | 4.092 | 4.094 |
Accurately take by weighing each 10g of Herba dicranostigmae herb powder sample respectively, added in 10: 1,15: 1,20: 1,25: 1,30: 1,35: 1,40: 1,45: 1,50: 1 than (weight/volume) by the liquid material respectively and aid in the ultrasonic treatment instrument respectively after deionized water soaks handle 1h under 60 ℃ of conditions, other steps are with embodiment 1 ultrasonic extraction.The results are shown in Table 4, Fig. 4, solid-liquid ratio (weight/volume) is that 30: 1 o'clock effects are best.
The influence of table 4 liquid material comparison Herba dicranostigmae total alkaloids extraction ratio
Test 3pH value is to extracting the influence of Herba dicranostigmae biology total alkali
Accurately take by weighing each 10g of Herba dicranostigmae herb powder sample respectively, respectively add at 15: 1 than (weight/volume) by the liquid material and to aid in the ultrasonic treatment instrument respectively after pH2,3,4,5,6,7,8,9 deionized water soak handle 1h under 60 ℃ of conditions, other steps are with embodiment 1 ultrasonic extraction.The results are shown in Table 5, Fig. 5, pH value is 7 to be that effect is best.
Table 5, pH value are to the influence of Herba dicranostigmae biology total alkali extraction ratio
Accurately take by weighing each 10g of Herba dicranostigmae herb powder sample respectively, respectively add at 15: 1 than (weight/volume) by the liquid material and to aid in the ultrasonic treatment instrument after deionized water soaks and handle 1h respectively under 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ conditions, other steps are with embodiment 1 ultrasonic extraction.The results are shown in Table 6, Fig. 6, visible biology total alkali extraction ratio raises with temperature.
Table 6, temperature are to the influence of biology total alkali extraction ratio in the Herba dicranostigmae
| Temperature/ |
30 | 40 | 50 | 60 | 70 | 80 |
| The O.D value | 1.726 | 1.728 | 1.734 | 1.745 | 1.751 | 1.762 |
| Extraction ratio | 4.026 | 4.03 | 4.044 | 4.07 | 4.086 | 4.112 |
| /％ |
Accurately take by weighing each 10g of Herba dicranostigmae herb powder sample respectively, respectively add at 15: 1 than (weight/volume) by the liquid material and aid in the ultrasonic treatment instrument after deionized water soaks handle 1h under 60 ℃ of conditions, high speed centrifugation (3500r/min) 10min gets supernatant.Get supernatant and add ethanol respectively, make the ethanol volume fraction reach 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, other steps are with embodiment 1 ultrasonic extraction.The results are shown in Table 7, Fig. 7, the ethanol volume fraction is that 75% o'clock effect is best.
Table 7, ethanol volume fraction are to the influence of Herba dicranostigmae biology total alkali extraction ratio
Test of the influence of 1 different precooling temperatures to biology total alkali extraction ratio in the Herba dicranostigmae
Accurately take by weighing Herba dicranostigmae herb powder 10g respectively, add at 30: 1 than (weight/volume) by the liquid material and to aid in the ultrasonic treatment instrument respectively after deionized water soaks and handle each 1h successively for 80 ℃, getting, treatment fluid high speed centrifugation (3500r/min) 5min gets supernatant, getting supernatant respectively adds dehydrated alcohol and reaches 80% to the original solution alcoholic strength, standing over night.Room temperature high speed centrifugation (3500r/min) 5min gets supernatant, and 50 ℃ of water-bath rotary evaporations reclaim ethanol and concentrated supernatant to 20ml.
Three groups of experiments of parallel as stated above design, the concentrated solution of gained are respectively-20 ℃ ,-40 ℃ ,-70 ℃ freeze overnight, vacuum lyophilization (61 ℃, 0.011Mpa) 48 hours dry Herba dicranostigmae biology total alkali goods, precision is weighed.Get each goods 4mg respectively and be dissolved in the 4ml deionized water, 6 times of dilutions, every group each parallel three parts, leave standstill, cross microporous filter membrane (0.22nm), survey absorbance O.D value and be averaged.It is the highest to the results are shown in Table 8 ,-70 ℃ of precooling temperature total alkali extraction ratios.
Table 8, different precooling temperature are to Herba dicranostigmae biology total alkali extraction effect relatively
Table 9, different pre-cool time are to Herba dicranostigmae biology total alkali extraction effect relatively
Test of the influence of 3 different freeze-drying times to biology total alkali extraction ratio in the Herba dicranostigmae
Table 10, different freeze-drying time are to Herba dicranostigmae biology total alkali extraction effect relatively
The extract obtained alkaloid content determination of embodiment 4 small lot extraction processes
Take by weighing Herba dicranostigmae herb powder 10g, add at 30: 1 than (weight/volume) by the liquid material and to aid in the ultrasonic treatment instrument respectively after deionized water soaks and handle each 1h successively for 80 ℃, getting, treatment fluid high speed centrifugation (3500r/min) 5min gets supernatant, get respectively and reset and add dehydrated alcohol and reach 75% to the original solution alcoholic strength, standing over night.Room temperature high speed centrifugation (3500r/min) 5min gets supernatant, and 50 ℃ of water-bath rotary evaporations reclaim ethanol and concentrated supernatant to 20ml.The concentrated solution of gained is-70 ℃ of freeze overnight, vacuum lyophilization (61 ℃, 0.011Mpa) 48 hours dry Herba dicranostigmae biology total alkali extract.
85.83%, 79.80%, 89.98%, 78.60%, 90.11% carry out repeated trials altogether 8 times, the alkaloid of extract is measured and calculated to the extract of gained by the method for specific embodiment part, and the result is respectively:,
88.13%, 81.99%, 86.79%, meansigma methods is 85.15%.
Take by weighing Herba dicranostigmae herb powder 5000g, parallel three parts is that 7 deionized water soaks to put in 80 ℃ and spends the night by adding pH at 1: 25.With 6 layers of gauze coarse filtration, be evaporated to 2.5L, adding volume fraction is that 95% ethanol reaches 75% to the original solution alcoholic strength, standing over night, room temperature high speed centrifugation (6500r/min) 5min gets supernatant, and 55 ℃ of rotary evaporation in vacuo reclaim ethanol and are concentrated into 1000ml.Concentrated solution-75 ℃ freeze overnight, vacuum lyophilization (61 ℃ 0.011Mpa) got Herba dicranostigmae alkaloid goods in 24 hours.The result extracts from the 5000g raw material and obtains the 318.1805g extract.
By the method mensuration of specific embodiment part and alkaloid and total extraction ratio of calculating extract, the alkaloid of extract is 75.0082% as a result, and total extraction ratio is 4.77%.
Alkaloid changes before and after testing 2 lyophilizing
Table 11: heavy dose of test lyophilizing front and back lyophilizing rate measurement result table that extracts
Extract the result as can be seen from heavy dose, extraction ratio is about 4.77%, is higher than the extraction ratio of each monomial factor and low dose of test; Alkaloid is about 75%, is lower than the alkaloid of low dose of test slightly; Vacuum lyophilization lyophilizing rate average out to 97.74%, loss was less when vacuum lyophilization was described.All in all, heavy dose of extraction process of the present invention is suitable for the suitability for industrialized production that the Herba dicranostigmae biology total alkali extracts.
Claims (10)
1. Herba dicranostigmae alkaloid extraction method is characterized in that: with Herba dicranostigmae herb water extraction, filter or centrifugal; Filtrate adds alcoholic solvent precipitation, filtration or centrifugal; Get filtrate and be concentrated into 1/10 of former liquid measure, become the extractum state, carry out lyophilizing and handle.
2. the method for claim 1, it is characterized in that: described extraction water is 10: 1～50: 1 with the liquid material of Herba dicranostigmae herb than (volume/weight).
3. method as claimed in claim 2 is characterized in that, described extraction water is 30: 1 with the liquid material of Herba dicranostigmae herb than (volume/weight).
4. the method for claim 1, it is characterized in that: described water extraction process is carried out under the condition of 30～80 ℃ of temperature.
5. the method for claim 1 is characterized in that: described water extraction process usefulness supersound process 0.5～3 hour.
6. the method for claim 1 is characterized in that, described alcoholic solvent precipitation is meant that adding the final volume mark is that 35～95% ethanol precipitates.
7. method as claimed in claim 6 is characterized in that, described alcoholic solvent precipitation is meant that adding the final volume mark is that 75% ethanol precipitates.
8. the method for claim 1 is characterized in that: described lyophilizing is handled and is comprised precooling and vacuum lyophilization.
9. method as claimed in claim 9 is characterized in that: described lyophilizing is handled and is meant-70～-20 ℃ of freeze overnight, lyophilization 24～48 hours under-61 ℃, 0.011Mpa condition then.
10. a Herba dicranostigmae alkaloid is characterized in that described Herba dicranostigmae alkaloid obtains by extracting as any described method of claim 1-9.
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| CN107029158B (en) * | 2017-05-01 | 2020-02-28 | 天水师范学院 | Compound Chinese herbal medicine external preparation for treating rabbit mange ringworm |
| CN107715810A (en) * | 2017-09-05 | 2018-02-23 | 中国农业科学院兰州畜牧与兽药研究所 | A kind of preparation method of the red careless essential oil microcapsules of cyclopentadienyl |
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