CN1962665B - Extraction method of antineoplastic composition bhang flavone A from bhang - Google Patents
Extraction method of antineoplastic composition bhang flavone A from bhang Download PDFInfo
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Abstract
The invention discloses an extracting method of dolichnin A with excellent antineoplastic activity from hemp, which comprises the following steps: (1) extracting for 2-3 times through alcohol; combining extract; condensing into concrete; (2) adsorbing through large-hole resin; eluting through alcohol; combining approximate component; evaporating alcohol; (3) separating to purify rough extract through high-speed reflux chromatogram; forming solvent system of solid phase and fluid phase through skellysolve B, acetic ester, carbinol and water; filling solid phase in the chromatographic column; rotating column to pump fluid phase in the column; sampling; eluting to obtain high-purity product.
Description
Technical field
The invention belongs to the separation technology field of material, relate to the separation method that comprises with sorbent treatment liquid, to be specially with the resin be sorbent material, be that sorbent material replaces by the method for metathetical liquid and comes effective constituent in the separating solid substances with the mobile solvent.Simultaneously, also relate to anti-tumor activity medicine.
Background technology
Cancer is the major disease of serious harm people's life health, has every year more than 600 ten thousand people to die from malignant tumour in the world.The medicine that uses clinically mainly is traditional cell toxicant class chemosynthesis antitumor drug at present, though part medicine antitumor action is obvious, toxic side effect is big, and selectivity is relatively poor, is easy to generate resistance.Traditional Chinese medicine and pharmacy is the practical experience crystallization that an accumulation Chinese nation prevents and cures diseases for thousands of years, has distinct theoretical system, can embody the life science of determination of treatment based on pathogenesis obtained through differentiation of symptoms and signs and individualized treatment characteristics of the times again.The traditional Chinese medical science can be distinguished the disease that doctor trained in Western medicine is not clear, and Chinese medicine can be controlled Western medicine refractory, incurable disease.Therefore the attention and the favor of domestic and international the world of medicine more and more received in the research of antitumor Chinese medicine.
Root or leaf of Falcate dolichos is the root of leguminous plants dolichos falcatus Klein [Dolichos tenuicaulis (Baker) Craibs], is distributed in ground such as Guizhou and Yunnan, has swelling and pain relieving, detoxify and drain pus and effect such as antitumor.Less [the Tian Chengguo of research report about Root or leaf of Falcate dolichos chemical ingredients and pharmacologically active, Deng. the herbal medicine communication, 1977, (2): 8～12. yellow thick engaging, etc. Acta Pharmacologica Sinica, 1983, (4): 286～288.], therefore the world of medicine wishes to obtain this effective ingredients in plant in a hurry, and has gained some understanding to its chemical structure, pharmacology, the property of medicine with at antineoplastic action, yet does not see the report that extracts effective constituent wherein and put down in writing its chemical structure so far.
Summary of the invention
Defective at the prior art existence, it has the method for the component of obvious anti-tumor activity to the purpose of this invention is to provide a kind of extraction from national medicinal plant Root or leaf of Falcate dolichos, separation, purifying, analyze, test and definite its chemical structure, and measure its antineoplastic activity.
Embodiment of the present invention comprise: medicinal material extract, the separation and purification three process of crude extract preparation and crude extract: operation (1) adopts certain density extraction using alcohol medicinal material to obtain extract, operation (2) adopts macroporous adsorption resin chromatography to obtain crude extract, operation (3) adopts high-speed countercurrent chromatography that crude extract is carried out separation and purification, it comprises that preparation constitutes stationary phase, the solvent system of moving phase, make in the counter current chromatograph pillar and be full of stationary phase, pillar is rotated, again moving phase is pumped in the post, enter the crude extract sample for preparing by sampling valve, according to detecting collection of illustrative plates receiving target component.Concrete grammar is:
1. medicinal material extract
After getting the suitable pulverizing of Root or leaf of Falcate dolichos, extract 2～3 times with 75%～95% ethanol heat of 6～8 times of amounts, each 1.0～2.0h, united extraction liquid is evaporated to medicinal extract.The medicinal material of pulverizing is that 0.5～20.0mm is advisable with particle diameter.When heat is extracted with extraction effect under the reflux state for well.
2. crude extract preparation,
Good low-pole macroporous adsorbent resin installs in the chromatography column to get a certain amount of pre-treatment, (resin and medicinal material amount ratio were 0.5～1: 1) with full formula extraction medicinal extract after water washed repeatedly, after the absorption fully, with impurity such as abundant sweetening off of big water gaging and inorganic salt, use 30%～50% ethanol elution of 3～5 times of medicinal material amounts more successively, 75%～95% ethanol elution of 4～6 times of medicinal material amounts receives corresponding ethanol elution stream part, supplies raw materials as further separation and purification sample.Wherein preferably use 3～4 times of medicinal material amount 40～50% ethanol and 4～5 times of medicinal material amount 85～95% ethanolic soln wash-outs, the elutriant break into portions, with the wherein close several parts of merging of component, underpressure distillation is also reclaimed the dry target component that gets of ethanol final vacuum.Macroporous resin with comprise 1300, D101 or AB-8 exist
In kind for well.Be that eluting water is colourless to adsorbing complete back sign with impurity such as abundant sweetening off of big water gaging and inorganic salt in operation, water consumption is approximately 6～7 times of medicinal material amount.Be divided into 35～45 equal portions when ethanol eluate accesses in the practice, the 3rd～10 part of merging removes the ethanol effect under reduced pressure for well in the middle of getting.
3. separation and purification
Use the high speed adverse current chromatogram separation and purification, it comprises step: preparation constitutes the solvent system of stationary phase, moving phase, make in the counter current chromatograph pillar and be full of stationary phase, pillar is rotated, again moving phase is pumped in the post, by the sampling valve sample introduction, according to detecting spectrogram receiving target composition, the solvent for use system is made up of normal hexane, ethyl acetate, methyl alcohol, water, and its amount ratio is 1: 1.8～2.2: 1.8～2.2: 1.
4. the purity detecting of isolate and structure are identified
Adopt high performance liquid chromatography that separating obtained component is carried out purity detecting (peak area normalization method) purity greater than 98.0%, volatilize carry out behind the solvent MS,
1H NMR and
13C NMR analyzes, and carries out structural confirmation according to the gained data.Its chemical structure is:
Its chemical name is: (2S)-5,2 ', 6 '-trihydroxy--3
, 8-diisoamyl thiazolinyl-2
, 2
-dimethyl pyrans-[5
, 6
: 6,7]-2 " " ', 4 " " '-cyclohexadiene-1 ' " "-ketone-[2 ' " ", 3 ' " ": 3 ', 4 ']-flavanone.Belong to flavonoid compound (hereinafter to be referred as Root or leaf of Falcate dolichos flavone A dolichnin A).
5. the anti-tumor activity test of gained compound
Adopt mtt assay to investigate the external growth-inhibiting effect of gained compound to 5 kinds of human tumor cell lines (BEL-7402, SGC-7901, MCF-7, HT-29 and A498).As a result this compound to the half-inhibition concentration of selected 5 kinds of human tumor cell lines all less than 4.5 μ g/ml.
Also carried out simultaneously of the restraining effect test of this compound to S180 transplanted sarcoma growth in the mouse body.
Set up S180 transplanted sarcoma model in the mouse body, this compound of abdominal injection, and with the positive control drug cyclophosphamide-a control, calculate the tumour inhibiting rate of this compound, observe of immunity system and the leukocytic influence of this compound simultaneously to mouse.The result shows that when intraperitoneal injection 10mg/kg, the inhibitory rate of this compound is to more than 61%.
The present invention is rationally effective to the technology of extraction from medicinal plant, separation, purifying Root or leaf of Falcate dolichos flavones I, and it is easy, quick particularly to adopt high speed adverse current chromatogram that the Chinese medicine crude extract is carried out separation and purification, has also avoided sample loss simultaneously.Overcome shortcomings such as traditional preparation process method complex operation, separation cycle be long, and have that preparation amount is big, separation efficiency is high, good product purity, advantage such as simple and easy to do.Simultaneously, its chemical structural formula is determined in test by analysis, proves that this is a new compound that does not appear in the newspapers.In addition, also provide new variety with applications well prospect for antitumor drug.
Description of drawings
Fig. 1 is the color atlas (chromatographic peak I is the Root or leaf of Falcate dolichos flavone A) of half countercurrent chromatography (HSCCC) of macroporous adsorption resin chromatography thing
Fig. 2 is the color atlas (Fig. 2 A is the HPLC color atlas of crude extract, and Fig. 2 B is preparation stream part purity detecting HPLC color atlas) of the high performance liquid chromatography (HPLC) of macroporous adsorbent resin crude extract and separate part
Fig. 3 is the mass spectrum and the ultraviolet figure of compound Root or leaf of Falcate dolichos flavone A.
Fig. 4 tests the pathological section of each administration group sarcoma for anti-S180 sarcoma in the Root or leaf of Falcate dolichos flavone A body
The present invention is further described below in conjunction with specific embodiment and accompanying drawing.
Embodiment
Embodiment 1:
The extraction of compound Root or leaf of Falcate dolichos flavone A (dolichnin A) and structure are identified.The steps include:
1. take by weighing Root or leaf of Falcate dolichos meal 5.0kg, particle diameter 0.8～20.0mm, with 8 times of amount 80% alcohol reflux twice, each 1.5h filters, and merging filtrate is evaporated to the about 2000ml of medicinal extract.
2. take by weighing 4000g D101 macroporous adsorbent resin, after spending the night with 95% alcohol immersion, be soaked in water again and be added to after spending the night on the chromatography column (10.0 * 120cm), wash repeatedly with big water gaging.Full formula extraction is added to the resin upper end, open piston, flow velocity is 25ml/min, after the completion of the sample, closure piston, about 1 hour of static absorption, then with the water (30L) of 6 times of medicinal material amounts wash resin to elutriant almost colourless till, then with 40% ethanol (15L) wash-out of 3 times of medicinal material amounts, use 85% ethanol (20L) wash-out of 4 times of medicinal material amounts again, every 1000ml is a reception stream part, merge 5～9 stream parts wherein, decompression recycling ethanol final vacuum drying gets buff powder, as further separation and purification sample.
3. use high speed adverse current chromatogram (Shenzhen is with the biochemical company limited in field) separation and purification macroporous adsorbent resin and receive stream part.
Solvent system and consumption volume ratio are normal hexane: ethyl acetate: methyl alcohol: water=1: 2: 2: 1, the adverse current chromatogram column volume is 300ml, applied sample amount 400mg, rotating speed 850rpm, on be stationary phase mutually, is moving phase mutually down, flow velocity 1.8ml/min, stationary phase retention rate 58.5% detects wavelength 280nm, and Ji Lu color atlas is seen Fig. 1 in the case.
Concrete operation steps is: than the preparation solvent system, static layering behind the shake well in separating funnel is divided and is got phase up and down by above-mentioned solvent volume, on be stationary phase mutually, under be moving phase mutually.Make earlier in the counter current chromatograph pillar to be full of stationary phase, main frame is rotated, pump into moving phase again, by the sampling valve sample introduction, according to detector spectrogram receiving target composition, result's separation obtains 1 part, i.e. stream part " I ".
This part is carried out high-efficient liquid phase analysis show that " I " is single chromatographic peak, peak purity is 98.6%.
HPLC analysis condition: chromatographic column Lichrospher C
18(150mm * 4.6i.d., 5 μ m), moving phase is CH
3CN: H
2O: HAC=60: 40: 2, flow velocity 1.0ml/min detected wavelength 280mn, and macroporous adsorption resin chromatography thing and HSCCC separate part color atlas are seen Fig. 2 with this understanding.Peak 1 is the Root or leaf of Falcate dolichos flavone A.
Structure is identified: the Root or leaf of Falcate dolichos flavone A that separation is obtained carries out on Varian INOVA-500 type nuclear magnetic resonance analyser and Varian MAT-212 type mass spectrograph
1H NMR,
13C NMR and MS analyze, and the gained data are as follows.
White powder, mp213～214 ℃, [α]
D 25-120 (c.0.80, CH
3OH), and UV λ max (nm, MeOH): 340,294, the 220nm (see figure 3); ESI-MSm/z:583.2[M-H]
-(see figure 3).HR-ESI-MSm/z:583.2148 (calcd.583.2142), molecular formula is C
36H
40O
74 13C NMR and
1H NMR data see the following form 1.
Table 1 gained compound
13C NMR and
1H NMR data
| Position | δ c | δ H | Position | δ c | δ H |
| 2 | 71.3 | 5.81dd(14.0，4.0) | 3″ | 129.8 | - |
| 3 | 39.4 | 2.47dd(17.0，4.0) 3.91dd(17.0，14.0) | 4″ | 18.5 | 1.57s |
| 4 | 198.1 | - | 5″ | 24.8 | 1.42s |
| 5 | 157.1 | 12.27brs | 2
 |
77.6 | - |
| 6 | 103.5 | - | 3 | 139.1 | - |
| 7 | 159.4 | - | 4 | 112.5 | 6.15brs |
| 8 | 104.6 | - | 2
 |
25.8?26.4 | 1.36s?1.38s |
| 9 | 157.2 | - | 1″″ | 28.2 | 3.17d(7.0) |
| 10 | 102.1 | - | 2″″ | 122.6 | 5.38brs |
| 1′ | 114.2 | - | 3″″ | 130.2 | - |
| 2′ | 159.4 | - | 4″″ | 18.8 | 1.50s |
| 3′ | 107.5 | - | 5″″ | 25.6 | 1.64s |
| 4′ | 138.7 | - | 1″″′ | 199.4 | - |
| 5′ | 104.3 | 6.87brs | 4″″′ | 119.3 | 6.11d (10.0) |
| 6′ | 160.5 | - | 5″″′ | 131.6 | 6.35d (10.0) |
| 1″ | 21.5 | 3.13d(8.0) | 6″″′ | 45.8 | - |
| 2″ | 122.8 | 5.14m | 6″″′-Me×2 | 21.4?23.7 | 1.41s?1.47s |
Embodiment 2:
1. take by weighing Root or leaf of Falcate dolichos meal 5.0kg, particle diameter 0.5～20.0mm, with 6 times of amount 90% alcohol reflux twice, each 2h filters, and merging filtrate is evaporated to the about 2000ml of medicinal extract.
2. take by weighing 5000g AB-8 macroporous adsorbent resin, after spending the night with 95% alcohol immersion, be soaked in water again and be added to after spending the night on the chromatography column (10.0 * 120cm), wash repeatedly with big water gaging.Full formula extraction is added to the resin upper end, open piston, flow velocity is 25ml/min, after the completion of the sample, closure piston, about 1 hour of static absorption, then with the water (30L) of 6 times of medicinal material amounts wash resin to elutriant almost colourless till, then with 50% ethanol (20L) wash-out of 4 times of medicinal material amounts, use 95% ethanol (25L) wash-out of 5 times of medicinal material amounts again, every 2500ml is a reception stream part, merge 3～7 stream parts wherein, decompression recycling ethanol final vacuum drying gets buff powder, as further separation and purification sample.
3. use high speed adverse current chromatogram (Shenzhen is with the biochemical company limited in field) separation and purification macroporous adsorbent resin and receive stream part.
Solvent system and consumption volume ratio are normal hexane: ethyl acetate: methyl alcohol: water=1: 2.2: 2.2: 1, the adverse current chromatogram column volume is 300ml, applied sample amount 400mg, rotating speed 900rpm, on be stationary phase mutually, is moving phase mutually down, flow velocity 1.6ml/min, stationary phase retention rate 61.3% detects wavelength 280nm.
Concrete operation steps is carried out HPLC to separating obtained stream part and is analyzed and structural analysis with embodiment 1, and analytical results is all with embodiment 1.
Embodiment 3:
The body of compound Root or leaf of Falcate dolichos flavone A is interior, anticancer experiment in vitro
Select 5 kinds of human tumor cell lines, BEL-7402, SGC-7901, MCF-7, HT-29 and A498 contain the RPMI-1640 nutrient solution of 10% calf serum, 37 ℃, that the 5%CO2 incubator grows to cell is vigorous, add 0.02%EDTA digestion through 0.25% pancreatin, make cell suspension, (cell concn is 5 * 10 in 96 hole microtest plates with cell suspension inoculation
5Individual/ml), the administration group adds the compound of different concns respectively, control group adds corresponding solvent, cultivated 72 hours after the administration, press mtt assay and measure absorbancy (A) value at 550nm wavelength place with microplate reader, the inhibiting rate of computerized compound is drawn the corresponding curve that suppresses by inhibiting rate and sample concentration, calculates half-inhibition concentration (IC again
50).This compound is to the IC of each tumor cell line
50Value sees the following form 2.
Table 2 Root or leaf of Falcate dolichos flavone A is to the IC of 5 kinds of human tumor cell lines
50Value
Get the S180 mouse ascites that oneself goes down to posterity and be logarithmic phase in this chamber, be diluted to 2 * 10 with physiological saline
7/ ml, getting 0.2ml, to be inoculated in the right armpit of mouse subcutaneous.Second day gastric infusion behind the mouse inoculation, blank group gives physiological saline 0.2ml, positive controls is given abdominal injection CTX0.2ml (20mg/kg), each treatment group gives compound compound Root or leaf of Falcate dolichos flavone A 2.5mg/kg, 5.0mg/kg, 10.0mg/kg oil for injection dissolving pneumoretroperitoneum drug administration by injection respectively), continuous 10 days, put to death next day.Eye socket was got blood before mouse was put to death, and made white corpuscle (WBC) counting, and mouse is put to death in row dislocation afterwards.
Strip sarcoma and organize and get mouse spleen and thymus gland simultaneously, tumour inhibiting rate is calculated according to following formula in the back of weighing, and calculates spleen index and thymus index.After mouse was taken CTX, loose by hair, spirit was relatively poor, and drinking water diet reduces, and to compare weight increase slow with control group, but very outstanding to the tumor-inhibiting action of tumour, tumour inhibiting rate is higher than 61%.Mouse diet behind this compound of abdominal injection is good, freedom of movement, and body weight is compared there was no significant difference with model group, and the tumour rise time is delayed, knurl piece poor growth.The gained result shows that the compound that this prepared obtains has the effect that the S180 sarcoma is grown in the very strong inhibition mouse body, has compared significant difference (P＜0.01) with control group.Simultaneously, after this compound and the administration of CTX compatibility, can obviously see the rising of reduction mouse spleen index and the reduction of thymus index, the effect during high dosage is (P＜0.01) particularly significantly.White blood cell count(WBC) shows the murine interleukin reduction (P＜0.01) that this prepared product can obviously raise and be caused by CTX.
Simultaneously, we have also carried out routine paraffin wax section and microscopic analysis to mouse S180 sarcoma, as shown in Figure 4.The model group growth of tumour cell is vigorous as seen from the figure, each administration group tumour cell all has death in various degree, especially CTX and administration high dose group, the death of neoplastic cells large percentage, pathological section shows that CTX and this compound can both kill a certain amount of tumour cell, thereby suppress the growth of sarcoma,, specifically see Table 3,4 and 5 with above-mentioned experiment gained data consistent.
Table 3 Root or leaf of Falcate dolichos flavone A is to the restraining effect of S180 sarcoma (mean ± S)
Annotate: compare with model group
ΔP＜0.05; Compare with model group
The Δ ΔP＜0.01; Compare with the CTX group
#P＜0.05
The restraining effect that table 4 Root or leaf of Falcate dolichos flavone A and CTX compatibility are grown to transplantability mouse S180 sarcoma (mean ± S)
| Group | Dosage | Number of animals (only) | Body weight (g) | Sarcoma heavy (g) | Tumour inhibiting rate (%) |
| Model group | NS0.2ml/ only | 11 | 32.4±3.1 | 2.14±0.53 | - |
| CTX | 10mg/kg | 10 | 26.1±2.2 | 1.29±0.32 ΔΔ | 39.7 |
| Compound | 1.0mg/kg+10mg/kgCTX | 10 | ?29.6±2.1 | 1.28±0.46 Δ | 40.2 |
| Compound | 2.5mg/kg+10mg/kgCTX | 9 | ?30.6±2.6 | 0.96±0.38 ΔΔ | 55.1 |
| Compound | 5.0mg/kg+10mg/kgCTX | 10 | ?31.5±2.2 | 0.67±0.29 ΔΔ | 68.7 |
Annotate: compare with model group
ΔP＜0.05,
The Δ ΔP＜0.01
Table 5 Root or leaf of Falcate dolichos flavone A and CTX compatibility are to the influence of tumor-bearing mice spleen index, thymus index and leukocyte count (mean ± S)
| Group | Dosage | Number of animals (only) | Spleen index | Thymus index | WBC(×10 9/L) |
| Model group | NS0.2ml/ only | 11 | 9.7±1.6 Δ | 3.0±0.6 Δ | 9.6±0.9 ΔΔ |
| CTX | 10mg/kg | 10 | 11.3±2.5 | 2.5±0.5 | 5.2±0.8 |
| Compound | 1.0mg/kg+10mg/kgCTX | 10 | 10.4±1.8 | 2.8±0.6 | 8.6±1.0 ΔΔ |
| Compound | 2.5mg/kg+10mg/kgCTX | 9 | 9.3±1.6 Δ | 3.0±0.4 Δ | 9.8±0.9 ΔΔ |
| Compound | 5.0mg/kg+10mg/kgCTX | 10 | 7.6±2.0 Δ | 3.2±0.6 Δ | 10.5±1.2 ΔΔ |
Annotate: compare with the CTX group
ΔP＜0.05,
The Δ ΔP＜0.01
Claims (6)
1. the extracting method of antitumor component Root or leaf of Falcate dolichos flavone A in the Root or leaf of Falcate dolichos comprises: medicinal material extract, crude extract preparation and three steps of separation and purification, its technology is:
(1) adopt 6～8 times of medicinal material amount 75～95% ethanol heat to extract 2～3 times, each 1.0～2.0h, united extraction liquid is evaporated to medicinal extract;
(2) adopting the low-pole macroporous adsorbent resin is that sorbent material carries out column chromatography, and the weight ratio of resin and medicinal material is 0.5～1.1; Gained medicinal extract is gone into the absorption of macropore chromatography column, the absorption back is earlier with impurity such as abundant sweetening off of big water gaging and inorganic salt, use 3～5 times of medicinal material amount 30～50% ethanol and 4～6 times of medicinal material amount 75～95% ethanolic soln wash-outs more successively, the elutriant break into portions, wherein composition merges for close part, and underpressure distillation is also reclaimed the dry target component that gets of ethanol final vacuum;
(3) adopt high-speed countercurrent chromatography that gained macroporous adsorption resin chromatography thing is carried out separation and purification, two phase solvent system and consumption volume ratio used in the high-speed countercurrent chromatography are normal hexane: ethyl acetate: methyl alcohol: water=1: 1.8～2.2: 1.8～2.2: 1, static layering behind the shake well in separating funnel, on be stationary phase mutually, be moving phase mutually down, make earlier in the counter current chromatograph pillar during operation and be full of stationary phase, main frame is rotated, pump into moving phase again, by the sampling valve sample introduction, according to detector spectrogram receiving target composition.
2. according to the extracting method of antitumor component Root or leaf of Falcate dolichos flavone A in the described Root or leaf of Falcate dolichos of claim 1, it is characterized in that medicinal material in the described medicinal material extract is for being 0.5～20.0mm through broken particle diameter; Described heat is extracted as under the reflux state and extracts.
3. according to the extracting method of antitumor component Root or leaf of Falcate dolichos flavone A in the described Root or leaf of Falcate dolichos of claim 1, it is characterized in that in the described crude extract preparation a kind of as among low-pole resin 1300, D101, the AB-8 of the macroporous resin that uses.
4. according to the extracting method of antitumor component Root or leaf of Falcate dolichos flavone A in the described Root or leaf of Falcate dolichos of claim 1, it is characterized in that absorption back in the described crude extract preparation earlier with impurity such as abundant sweetening off of big water gaging and inorganic salt be water rinse resin with 6～7 times of medicinal material amounts to elutriant almost colourless till.
5. according to the extracting method of antitumor component Root or leaf of Falcate dolichos flavone A in the described Root or leaf of Falcate dolichos of claim 1, it is characterized in that accessing of ethanol elution and elutriant in the preparation of described crude extract to using 3～4 times of medicinal material amount 40～50% ethanol and 4～5 times of medicinal material amount 85～95% ethanolic soln wash-outs, be divided into 35～45 equal portions when ethanol eluate accesses, the 3rd～10 part of merging removes ethanol under reduced pressure in the middle of getting.
6. according to the extracting method of antitumor component Root or leaf of Falcate dolichos flavone A in the described Root or leaf of Falcate dolichos of claim 1, it is characterized in that in the described purification procedures, solvent system and consumption volume ratio are normal hexane: ethyl acetate: methyl alcohol: water=1: 2: 2: 1, and the adverse current chromatogram column volume is 300ml, applied sample amount 400mg, rotating speed 850rpm, on be stationary phase mutually, is moving phase mutually down, flow velocity 1.8ml/min, stationary phase retention rate 58.5% detects wavelength 280nm.
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| US10189762B1 (en) * | 2017-07-07 | 2019-01-29 | Orochem Technologies, Inc. | Process for purification and separation of cannabinoids, from dried hemp and cannabis leaves |
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