CN104107199A - Preparation method of peganum harmal L total alkaloids - Google Patents

Abstract

The invention relates to effective parts in total alkaloids extracted from a natural product-peganum harmal L alkaloid. A preparation method in the invention includes following steps: weighing seeds of peganum harmal L; drying the seeds in air; crushing the seeds; adding ethanol to obtain an ethanol extract; processing the ethanol extract in a rotational thin film evaporator to obtain a purple-brown crude extract paste; adding hydrochloric acid to the crude extract paste and heating the crude extract paste until the crude extract paste is dissolved; performing an extraction process for twice with CHCl3 after a hot filtering process to obtain a mother liquid; neutralizing the mother liquid with an NaOH aqueous solution to obtain a brown-yellow solid after a separating-out process; performing a heating and dissolving process with hydrochloric acid; carrying out a filtering process to obtain a crude product; adding ethanol to the crude product with heating; performing a dissolving process and a filtering process; carrying out a crystallization separating-out process to obtain peganum harmal L total alkaloids hydrocoloride; dissolving the peganum harmal L total alkaloids hydrocoloride in deionized water with heating; performing a separation process; carrying out a leaching process through water to obtain a leached liquid; separating crystals out after cooling the leaching liquid; heating the separated crystals with medical ethanol; and carrying out a dissolving process, a filtering process and a crystallization separating-out process to obtain peganum harmal L alkaloid. The peganum harmal L alkaloid can be used for treating breast cancer, melanoma, colon cancer, gastric cancer and esophageal cancer. In addition, an experimental result of the peganum harmal L alkaloid on hepatoma cell BEC-7402 proves that inhibition ratio of the peganum harmal L alkaloid on the hepatoma cell BEC-7402 can reach 60%, which establishes a basis for future popularization and application.

Description

The preparation method of Harmala alkaloids

Technical field:

The present invention is the effective site of the total alkaloids of extraction in a kind of natural plants Herba pegani harmalae grass (Peganum Harmal L), is used for the treatment of breast carcinoma, melanoma, colon cancer, gastric cancer, esophageal carcinoma.

Background technology:

Herba pegani harmalae grass Active Fractions Alkaloid A and B, molecular structure is confirmed, and this parent nucleus is also that current cancer therapy drug has ground the melanomatous report for the treatment of, but effective percentage is greatly about 10% left and right.The modification of parent nucleus and transformation, for the research and development of innovation cancer therapy drug provide the thinking of a sustainable development.

Herba pegani harmalae grass is grown in sandy beach, Gobi desert, is that one is fixed the sand crop, excessively excavates the heavy damage that can cause ecological environment, and because the low demand of alkaloid is large, quality is difficult to be controlled, and is difficult to satisfy the demands.

Summary of the invention:

The object of this invention is to provide a kind of chemosynthesis new technology, not only reduce medicinal cost, alleviate patient's financial burden, the controllability of quality improves, and as the effectiveness of a new drug, the controllability of safety, quality has had guarantee.

Preparation method of the present invention is achieved in that

Take seed of peganum harmala, air-dry, crush, dress post or be placed in three-necked bottle, adds ethanol, and drip washing to leacheate is pale red, or with Mayer reagent inspection inanimate object alkali precipitation; Ethanol extract, in rotary film evaporator, is reclaimed after ethanol, make puce and slightly carry cream;

To slightly carry cream and add water, add chemical pure concentrated hydrochloric acid, and be heated to dissolve, after heat filtering, put to room temperature, use CHCl ₃extract twice, then CHCl ₃a little washing for layer, reclaims alkaloid salt hydrochlorate; Filtrate and water lotion merge, and with the neutralization of NaOH aqueous solution, after tune pH value, separate out precipitation, sucking filtration, and wash with water, obtain the total alkaloids of brown color solid, shaped.By this total alkaloids adding distil water and chemical pure concentrated hydrochloric acid again, adjust pH value, be heated to dissolve, heat filtering, is placed to room temperature, obtains brown color precipitation, then through sucking filtration, air-dry, obtains the hydrochlorate crystal of Harmala alkaloids;

By gained Harmala alkaloids hydrochlorate deionized water heating for dissolving, adjust pH value, through resin column chromatography, the deionized water drip washing of reusable heat, collect yellow leacheate, then adjust pH value with sodium hydroxide, separate out yellow crystal after cooling, be washed till neutrality with distilled water, this solid adding distil water is adjusted to pH value, heating for dissolving, heat filtering with analytically pure hydrochloric acid, the crystallization of yellow rib shape is separated out in placement, sucking filtration, then wash three times with medical ethanol, air-dry, obtain Harmala alkaloids effective site, this Active Fractions Alkaloid is named as HRM3422.

Harmala alkaloids of the present invention is used for the treatment of gastric cancer, esophageal carcinoma, is also useful on the experiment of hepatoma carcinoma cell BEC-7402, and its suppression ratio is up to 60%, for applying and lay a good foundation from now on.

In this project development process, we contact with radiological medicine expert, HRM3422 penetrates effect to the anti-width of cultured cell in vitro and is studied, experimental result shows: HRM3422 has radiation resistance under aerobic conditions, under weary oxygen condition, nonreactive is put effect, and increase the mortality rate of cell, this biphasic effect to anticancer be significant.The extensive utilization of nuclear energy, be from now on one period irreversible development trend, but its safety is also absorbed focus.

Experience according to us to HRM3422 research and development, no matter external application for curing melanoma, breast carcinoma, enema treatment colorectal cancer, oral medication esophageal carcinoma and gastric cancer, all that medicine directly contacts with tumor body, kill or the transfer diffusion of anticancer, reduce the side effect that medicine produces through intestinal, stomach, blood, Absorption And Metabolism, improve the curative effect of medicine.

brief description of the drawings

Fig. 1 is the influence curve figure of HRM3422 to Growth of Gastric,

Fig. 2 is the influence curve figure of HRM3422 to S180 Growth of Cells.

Detailed description of the invention:

Preparation method of the present invention is achieved in that

Take 1000 grams of seed of peganum harmala, air-dry, crush, dress post or be placed in three-necked bottle of 2500cc, adds the ethanol of mass concentration 75-95%, and drip washing to leacheate is pale red, or with Mayer reagent inspection inanimate object alkali precipitation; Ethanol extract, in rotary film evaporator, is controlled to temperature and reclaimed after ethanol at 40-45 DEG C, make puce and slightly carry cream;

To slightly carry the cream 90-110cc that adds water, adding chemical pure concentrated hydrochloric acid, to be transferred to PH be 1.9-2.1, be heated to alkaloid salt hydrochlorate and dissolve, and heat filtering, filtrate is put to room temperature, uses CHCl ₃extract twice, each consumption 50cc, this is CHCl ₃layer, for removing grease, and with a little wash CHCl ₃layer, reclaims alkaloid salt hydrochlorate; Filtrate and water lotion merge, then neutralize with mass concentration 10%NaOH aqueous solution, and tune PH is 12-14, after separating out brown precipitation, carry out sucking filtration, then wash with water to PH be 7.5-8.5, obtain the total alkaloids of brown color shape, add 70cc distilled water and chemical pure concentrated hydrochloric acid, tune PH is 2.5-3.5 again, is heated to dissolve, heat filtering, be placed to room temperature, obtain brown color precipitation, sucking filtration, air-dry, obtain the hydrochlorate crystal of total alkaloids.

By gained Harmala alkaloids hydrochlorate deionized water heating for dissolving, adjusting PH is 6.5, through resin column chromatography, use again deionized water drip washing, collect yellow leacheate, then to adjust PH with 5% analytical pure sodium hydroxide be 12, after cooling, separate out yellow crystal, be washed till neutrality with distilled water, it is 3 that this solid adding distil water is adjusted to pH value with analytically pure hydrochloric acid, heating for dissolving, heat filtering, places and separates out the crystallization of yellow rib shape, sucking filtration, wash three times with medical ethanol again, air-dry, obtain alkaloid HRM3422.

Refining HRM3422, through ultraviolet spectral analysis, is the mixture being made up of banisterine (Harmaline) and two alkaloids of dehydrogenation camel alkali (Harmine), and banisterine (Harmaline) accounts for 19.35%.Yageine (Harmine) accounts for 71.30%.

The structural analysis of banisterine (Harmaline) and dehydrogenation camel alkali (Harmine) is measured:

1, banisterine

Chemical name: 1-methyl～3,4-dihydro-7-methoxyl group-β-Ka Bolin

English name: 1-Methyl-3,4-dihydro-7-Methoxy-β-Carboline

Structural formula:

Molecular formula: CH ₁₃cH ₁₄n ₂o

Molecular weight: 214.28 m.p.260 DEG C

H nuclear magnetic resonance map:

By FX-90Q type nuclear magnetic resonance analyser, taking TMS as interior mark, DMSO is as solvent.

2, dehydrogenation camel alkali

Chemical name: 7-methoxyl group-1-methyl-pyrido (3,4-b)-indole

English name: 7-Methoxy-1-Methyl-9H-ayrido (3,4-b)-indole

Structural formula:

Molecular formula: C ₁₃h ₁₂n ₂o

Molecular weight: 212.26 m.p.249.0～250 DEG C

¹h nuclear magnetic resonance map:

Instrument: by FX-90Q type nuclear magnetic resonance analyser, taking TMS as interior mark, DMSO is as solvent

Yageine (Harmine)

One, the toxicological experiment of Harmala alkaloids:

1, acute toxicity test:

Mice is oral: (kunming mice, 18-22g, male and female half and half)

LD ₅₀and letter limit (mg/kg)=352.14

95％Lower Limit＝304.47

95％upper Limit＝423.07

Mouse vein: LD ₅₀=53.53

95％Lower Limit＝46.16

95％upper Limit＝59.43

2, special toxicity test:

Micronucleus test, chromosomal aberration, Ames experiment is all negative.

3, long term toxication

Animal: wistar rat, body weight 100～150g.

Medicine: HRM3422

Instrument: SF-2 automatic biochemistry analyzer, microscope, microtome

6,12, (high dose group is up to 48mg/kg to tri-dosage groups of 24mg/kg, 20 of every group of rats, 20 of control rats test: rat oral gavage dosage:.Each treated animal male and female half and half above.

Result and discussion:

Animal blood conventional analysis: rat oral gavage does not have a significant effect to hematological indices, urine analysis of blood is all in normal range.

Animal blood biochemical indicator is observed: after rat oral gavage, its biochemical indicator, blood urea nitrogen (BUN), alanine amino transaminase (ALT), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), blood glucose (GLU), total bilirubin (T-BIL), creatinine (Crea), T-CHOL (T-CHO) etc. detect index all within normal range.

Zoopathology checks: all animals are become celestial, and stomach pylorus and the stomach bottom part of high dose group and low dose group rat are rubescent.The rat stomach of drug withdrawal after two weeks has no variation.Survey rat each dosage group organ coefficient, comprise the heart, liver, lung, spleen, stomach, kidney, adrenal gland, thyroid, testis, uterus, ovary, brain, prostate etc., learn by statistics inspection, the organ coefficient of administration group and matched group is without significant difference.

Administration group and matched group to rat have carried out histological inspection, comprise the tissues such as the heart, liver, lung, spleen, stomach, kidney, adrenal gland, duodenum, ileum, colon, pancreas, prostate, hypophysis, lymph node, bladder, testis, uterus, ovary, fallopian tube, thyroid, brain, optic nerve, the gastric mucosa of rat of observing high dose group has that part comes off, chief cell and the parietal cell of gastric tissue have atrophy, and intestinal mucosa also has part obscission.

(Beijing's clinical pharmacology institute)

Two, the test of pesticide effectiveness:

1, the impact of HRM3422 on mouse melanoma B16:

Animal: purebred mice C57BL/6J, 18-22 gram, DBA/2, kunming mice is 18-22 gram;

Medicine: 0.5% tween 80 hydrotropy for HRM3422 preparation, and it is for subsequent use to be made into suspension with distilled water;

Tumor strain: B16 melanoma is this research department conservation that goes down to posterity, for experiment tumor.

Experimental technique: get band tumor kind Mus, under aseptic condition, peel off or draw tumor, make homogenate with normal saline 1: 3 (gram/ml), through subcutaneous or abdominal cavity kind in intact animal oxter or abdominal cavity, grouping next day after inoculation, weigh, start gavage or intraperitoneal injection, once a day, give 12 days continuously, after drug withdrawal, peel off tumor, weigh, record suppression ratio, take statistics to learn and process.

Table 1-1HRM3422 oral administration is to mouse melanin cancer B ₁₆inhibitory action

Table 1-2HRM3422 intraperitoneal administration is to mouse melanin cancer B ₁₆inhibitory action

2, the inhibitory action to mouse breast cancer (EMT-6):

Medicine and reagent: HRM3422 preparation is with after 0.5% temperature-80 hydrotropy, be made into suspension with distilled water, and 4 DEG C save backup.Cyclophosphamide is fastened sea 12 pharmaceutical manufacturing, prepares with distilled water.

Animal and tumor strain: purebred mice C57BL/6J, body weight is 18-22 gram, tumor strain is that mouse breast cancer (EMT-6) is provided by Hoffman-La Roche Molecular Biology Research Lab of the U.S..

Experimental technique: get band tumor kind Mus and peel off or draw tumor liquid under aseptic condition, make homogenate with normal saline 1: 3 (g/ml), subcutaneous vaccination is in intact animal oxter according to a conventional method, and inoculation random packet, next day, weighs, respectively with LD ₅₀1/5,1/10,1/20 amount oral, intraperitoneal administration, once a day, successive administration 10-39 days, using cyclophosphamide as positive control, at inoculation tumor once daily next day; Give matched group every day corresponding solvent.

Within second day after drug withdrawal, put to death animal, weigh, peel off, claim tumor weight, calculate tumour inhibiting rate and carry out statistical procedures.

The oral impact on mouse breast cancer (EMT-6) of table 2-1HRM3422

The impact of table 2-2HRM3422 intraperitoneal administration on mouse breast cancer (EMT-6)

Result shows: HRM3422 is oral to melanin tumour b16, breast carcinoma EMT-6, lumbar injection all has strong inhibitory action, and particularly lumbar injection is more remarkable.

(institute of Materia Medica,Chinese Academy of Medical Sciences pharmacological room)

3, cell killing is tested:

Cell is adjusted to 5-10 ten thousand/ml concentration, be inoculated in sterile culture flask, add respectively the medicinal liquid having diluted, place in 37 DEG C of calorstats and cultivate.After 24 hours, 48 hours, add dyestuff counting cell number anyway, calculate suppression ratio.

(1) lethal effect to gastric cancer (823) cell:

The lethal effect of table 3-1HRM3422 to stomach cancer cell

Note: * * * P < 0.001, * * P < 0.01, * P < 0.02

Result shows:

HRM3422 small dose group does not have obvious inhibitory action to proliferation of human gastric cancer cell, and HRM3422 is heavy dose of and two dosage groups of positive control drug 5-Fu all have obvious inhibitory action to proliferation of human gastric cancer cell, and extend in time its inhibitory action and strengthen, statistical procedures has highly significant difference.

(2) lethal effect to S180 cell:

The lethal effect of table 3-2HRM3422 to S180 cell

Note: * * P < 0.01

Result shows:

HRM3422 low dose is to S-180 cell unrestraint effect.HRM3422 heavy dose and positive control drug 5-Fu all have obvious inhibitory action to the propagation of S-180 cell, and extend in time its effect enhancing, and statistical procedures all has notable difference.

(3) impact on Growth of Gastric:

Identical with said method, after 48 hours, discard medicinal liquid, add new RPMI-1640 to continue to cultivate, in 72 hours, 96 hours living cell counting numbers

Referring to Fig. 1, be the influence curve figure of HRM3422 to Growth of Gastric, in figure:

●-● matched group ▲-▲ 1 μ g/ml

Δ-Δ 10 μ g/ml mouth-mouth 5-Fu10 μ g/ml

Find out from growth curve chart, in inoculation latter 24 hours, 4 groups of cells were all in adaptive phase, after 48 hours, matched group growth curve rises gradually, and HRM34221 μ g/ml group Growth of Cells is slower, the slope of growth curve is less, relatively has obvious difference with matched group.The cell of HRM342210 μ g/ml and 5-Fu10 μ g/ml group is not grown, and after 48 hours, discards medicinal liquid, and this inhibitory action is improved not yet.

(4) impact of HRM3422 on S180 Growth of Cells:

Referring to Fig. 2, be the influence curve figure of HRM3422 to S180 Growth of Cells, in figure:

●-● matched group ▲-▲ HRM34221 μ g/ml mouth-mouth 5-Fu1 μ g/ml

○-○5-Fu10μg/ml Δ-ΔHRM342210μg/ml

Find out from growth curve chart, in inoculation latter 24 hours, 48 hour cells are all in adaptive phase, contrast and two groups of growth curves of HRM34221 μ g/ml sharply rise later, and the cell of 5-Fu1 μ g/ml, 10 μ g/ml and HRM342210 μ g/ml is not bred, after 48 hours, discard medicinal liquid, this inhibitory action is improved not yet, and has compared obvious difference.

(5) lethal effect of HRM3422 to stomach cancer cell:

Cell strain: BGC-823, is incubated at the HPMI-1640K containing 20% calf serum

Medicine: total alkali and HRM3422 are made into 100,200,300,400 μ g/ml with RPMI-1640 respectively

Experimental technique: BGC-823 cell is inoculated in 24 well culture plates to every hole 5 × 10 ⁵cell, waits after cell attachment, abandons culture fluid, adds respectively above-mentioned each concentration liquid, and not dosing of matched group, separately adds culture fluid, at 37 DEG C, and CO ₂in incubator, cultivate 24 hours, 0.02%EDTA peptic cell, centrifugal, drip sheet, expect that with platform blue repelling attack observes kill rate, count the dead cell number in 100 cells.

Result: table 4

(institute of oncology of Beijing pharmacology group) (1989)

(6) lethal effect of HRM3422 to esophageal cancer cell:

Cell strain: the EC871214 esophageal carcinoma, is incubated at containing in the RPMI-1640 of 20% calf serum;

Medicine: HRM3422 is made into the medicinal liquid of 100,200,300,400,500 μ g/ml with RPMI-1640;

Experimental technique: EC87121 cell is inoculated in 24 well culture plates to every hole 5 × 10 ⁵cell, waits after cell attachment, abandons culture fluid, adds respectively above-mentioned each concentration liquid, and not dosing of matched group, only adds culture fluid, at 37 DEG C, and CO ₂in incubator, cultivate 24 hours, 0.02%EDTA peptic cell, centrifugal, drip sheet, observe lethality by the blue repelling attack of platform, count the dead cell number in 100 cells.

Result: table 5

Result shows: when HRM3422 is during at higher concentration 400 μ g/ml, the esophageal cancer cell of In vitro culture is had to strong lethal effect.

(institute of oncology of Beijing pharmacology group) (1989)

4, observe the lethal effect of HRM3422 to cell in gastric cancer to measure the method for cell protein amount

Cell is made up of protein, adopts and makes cytolysis become the reagent of protein solution, makes cytolysis, by the uv absorption of UV-260 spectrophotometric determination protein, can understand cell quantity number.Protein is linear with trap A value within the scope of finite concentration.

The impact of table 6HRM3422 on stomach cancer cell protein content

Note: * * P < 0.01 * P < 0.02

Result shows, except HRM3422 small dose group, the optical density value of the heavy dose of group of HRM3422 and positive controls is all starkly lower than matched group, illustrates that HRM3422 has inhibitory action to Growth of Gastric.Statistical procedures all has significant difference.

5, the impact on stomach cancer cell DNA content

Stomach cancer cell is diluted into about 100,000/ml, be inoculated in and in bottle, add the medicinal liquid having diluted, 37 DEG C of constant temperature culture, after 48 hours, cell is taken out, with PBS (PH7.47) washing, drip sheet, fix, add fluorescent dye, hexagram sheet, with microspectrofluorimeter Observe and measure cell DNA relative amount.

The impact of table 7HRM3422 on stomach cancer cell DNA relative amount

***P＜0.001

Result shows, two dosage groups of HRM3422 and positive controls cell DNA content are all starkly lower than matched group, learns by statistics and processes the difference that all has highly significant.

6, the impact of HRM3422 on stomach cancer cell microfilament

Cell and medicament contact be after 24 hours, with PBS washing, fixing, phalloidin dyeing 15-20 minute, mounting, at fluorescence microscopy Microscopic observation, takes pictures.HRM34225 μ g/ml and matched group comparison, in cell, microfilament obviously reduces, and in HRM342210 μ g/ml group cell, Subfilament Structure almost disappears.

Brief summary:

The above results shows, HRM3422 has obvious inhibitory action to the growth of stomach cancer cell and S-180 cell, and along with its inhibitory action of prolongation of time constantly strengthens.When discarding after medicinal liquid, this effect changes not yet, illustrates that HRM3422 is very strong to the direct effect of gastric cancer and S-180 cell.This experiment finds that HRM3422 can make stomach cancer cell DNA content obviously decline, and this is closely related with the cytostatic effect of HRM3422, and the reason that DNA content obviously reduces needs further to study.

We have also observed the impact of HRM3422 on microfilament in stomach cancer cell simultaneously.Known microfilament is to be prevalent in eukaryotic cell, and with the form of cell, closely-related material moves.Microfilament is again the important component that forms cytoskeleton.As medicine destroys Subfilament Structure in cell, reflect to a certain extent the effect of medicine to tumor.This experiment shows, HRM3422 can obviously make microfilament in stomach cancer cell reduce and disappear.Due to depolymerization of microfilaments in cell, may hinder the transport of the material such as DNA and protein synthesis and protein because transmission of information is obstructed, thus anticancer growth and motion.

(Peking University's natural drug and bionical medicine National Key Laboratory)

Five, HRM3422 animal pharmacokinetics test:

Absorb: adopt HPLC fluoroscopic examination, measure the method for blood drug level, the pharmacokinetics of Oral Administration in Rats HRM3422.Result shows: after oral, in rat body, absorb comparatively fast, within approximately 67 minutes, blood drug level reaches summit.In rat body eliminate also fast, after 4 hours, blood drug level is 0.0254 μ g/ml.

Metabolism: adopt Isolated Rat Liver circulation perfusion method, by separation, purification in perfusate, carry out structure determination through EI-MS spectrum with HPLC, determine metabolite.

Rat oral gavage, its internal metabolism, it is consistent with liver perfusion experiment that metabolite generates situation.

Distribute: adopt tissue homogenate method to study the distribution in tissue, result shows: this medicine can be distributed to each tissue rapidly, great majority organize in 2 hours for the highest, especially with liver for the highest, be followed successively by lung, pancreas, kidney, brain, the heart, blood.

Excretion: set up urine concentration PHLC algoscopy, ultraviolet detection.Result shows: after Oral Administration in Rats HRM3422, mainly by renal excretion, 45.19% of oral dose by homaluria in 72 hours.

(analytical chemistry teaching and research room of College of Pharmacy, Beijing Univ)

Six, HRM3422 clinical observation effect:

1, case: all through proved by pathology, existing toward not using any chemotherapy or radiotherapy before Drug therapy.Late case is estimated more than one month life cycle.Inoperable case, the variation of observe the symptoms improvement situation and tumor size.

2, medicine: the effective site HRM3422 extracting from natural goods

3, dosage: HRM3422 400mg every day, points three times oral, continues one month.

(1) rectal cancer is coloclysis administration, for 600mg adds normal saline 50-100ml, once a day coloclysis.

(2) melanoma and advanced breast cancer adopt Herbal medicines for external use, and dosage is depending on wound surface size, at every turn 200-1000mg.The next day, changes dressings once.

4, result:

The pathology evaluation criteria of Drug therapy: according to the degeneration of tumor cell, degree of necrosis and ask that the extent of reaction of matter evaluates.

Seven, model case:

(1) sieve Duan Shi: female 80 years old

Left foot melanoma, local application's external application HRM3422200mg/ day, medication one month, tumor body flattens, and carries out biopsy, and under mirror, tumor cell disappears completely.

(2) Lee × ×: female 52 years old

After left breast cancer operation, extensively recur part, and local external application HRM3422600-1000mg/ day, medication is after one month, and wound surface is that crust covers, and there are the growth of part normal epithelial, stellate ganglion deliquescing in the lower right corner.

(3) × × ×: male rectal cancer patient

Focus is positioned at rectum, apart from anus 10cm place, and big or small 4cm × 3cm, sick inspection is middle differentiation adenocarcinoma, with the physiological water coloclysis of HRM3422400mg, once a day.The postoperative sick inspection of row rectal carcinoma radical after 48 days, under mirror, tumor cell disappears completely, in cancer bed, is granulation and scar tissue.

Harmala alkaloids is used for the treatment of gastric cancer, esophageal carcinoma, is also useful on the experiment of hepatoma carcinoma cell BEC-7402, and its suppression ratio is up to 60%, for applying and lay a good foundation from now on.

Claims (2)

1. the preparation method of Harmala alkaloids, realizes with following step:

Take seed of peganum harmala, air-dry, crush, dress post or be placed in three-necked bottle, adds ethanol, and drip washing to leacheate is pale red, or with Mayer reagent inspection inanimate object alkali precipitation; Ethanol extract, in rotary film evaporator, is reclaimed after ethanol, make puce and slightly carry cream;

To slightly carry cream and add water, add chemical pure concentrated hydrochloric acid, and be heated to dissolve, after heat filtering, put to room temperature, use CHCl ₃extract twice, then CHCl ₃a little washing for layer, reclaims alkaloid salt hydrochlorate; Filtrate and water lotion merge, and with the neutralization of NaOH aqueous solution, after tune pH value, separate out precipitation, sucking filtration, and wash with water, the total alkaloids of brown color solid, shaped obtained by this total alkaloids adding distil water and chemical pure concentrated hydrochloric acid again, adjust pH value, be heated to dissolve, heat filtering, is placed to room temperature, obtain brown color precipitation, through sucking filtration, air-dry again, obtain the hydrochlorate crystal of Harmala alkaloids;

By gained Harmala alkaloids hydrochlorate deionized water heating for dissolving, adjust pH value, through resin column chromatography, use again deionized water drip washing, collect yellow leacheate, then adjust pH value with sodium hydroxide, after cooling, separate out yellow crystal, be washed till neutrality with distilled water, analytically pure hydrochloric acid for this solid adding distil water is adjusted to pH value, heating for dissolving, heat filtering, places and separates out the crystallization of yellow rib shape, sucking filtration, wash three times with medical ethanol again, air-dry, obtain Herba pegani harmalae alkaloid.

2. the preparation method of Harmala alkaloids according to claim 1, with following step specific implementation:

Take 1000 grams of seed of peganum harmala, air-dry, crush, dress post or be placed in three-necked bottle of 2500cc, adds the ethanol of mass concentration 75-95%, and drip washing to leacheate is pale red, or with Mayer reagent inspection inanimate object alkali precipitation; Ethanol extract, in rotary film evaporator, is controlled to temperature and reclaimed after ethanol at 40-45 DEG C, make puce and slightly carry cream;

To slightly carry the cream 90-110cc that adds water, adding chemical pure concentrated hydrochloric acid, to be transferred to PH be 1.9-2.1, be heated to alkaloid salt hydrochlorate and dissolve, and heat filtering, filtrate is put to room temperature, uses CHCl ₃extract twice, each consumption 50cc, this is CHCl ₃layer, for removing grease, and with a little wash CHCl ₃layer, reclaims alkaloid salt hydrochlorate; Filtrate and water lotion merge, then neutralize with mass concentration 10%NaOH aqueous solution, and tune PH is 12-14, after separating out brown precipitation, carry out sucking filtration, then wash with water to PH be 7.5-8.5, obtain the total alkaloids of brown color solid, shaped, add 70cc distilled water and chemical pure concentrated hydrochloric acid, tune PH is 2.5-3.5 again, is heated to dissolve, heat filtering, be placed to room temperature, obtain brown color precipitation, sucking filtration, air-dry, obtain the hydrochlorate crystal of total alkaloids

By gained Harmala alkaloids hydrochlorate deionized water heating for dissolving, adjusting PH is 6.5, through resin column chromatography, use again deionized water drip washing, collect yellow leacheate, then to adjust PH with 5% analytical pure sodium hydroxide be 12, after cooling, separate out yellow crystal, be washed till neutrality with distilled water, it is 3 that this solid adding distil water is adjusted to pH value with analytically pure hydrochloric acid, heating for dissolving, heat filtering, places and separates out the crystallization of yellow rib shape, sucking filtration, wash three times with medical ethanol again, air-dry, obtain Herba pegani harmalae alkaloid.