阅读说明：本技术 一种蕨菜中原蕨苷的分离纯化制备方法 (Method for separating, purifying and preparing pteridophytin in fiddlehead ) 是由 陈乃东 陈乃富 郝经文 朱安玲 徐惠敏 刘孝全 李强 杨维瀚 于 2021-01-18 设计创作，主要内容包括：本发明公开了一种蕨菜中原蕨苷的分离纯化制备方法,属于医药生物技术领域。操作步骤如下：(1)将新鲜蕨菜,-50℃以下冷冻干燥,粉碎,获得冻干粉；(2)将冻干粉进行正丁醇冷凝回流提取2遍,合并滤液,减压回收正丁醇得浸膏,冷冻干燥,获得正丁醇提取物；(3)将正丁醇提取物正向硅胶柱层析分离,使用氯仿-甲醇梯度洗脱,减压浓缩,获得原蕨苷粗品；(4)将原蕨苷粗品进行C18反向柱纯化精制,30％甲醇除杂,45％甲醇洗脱,低温冷冻干燥除去溶剂,获得纯度大于98％的原蕨苷。本发明原蕨苷的提取率可达65％,为药物开发提供了可能；本发明具有分离样品量大、目标物质损失少、提取率高、装置简单、操作简便、适合工业化生产的优点。(The invention discloses a separation and purification preparation method of pteridium aquilinum glycoside in pteridium aquilinum, belonging to the technical field of medical biology. The operation steps are as follows: (1) freeze drying fresh herba Fimbristylis Dichotomae at-50 deg.C, and pulverizing to obtain lyophilized powder; (2) condensing and refluxing the freeze-dried powder with n-butanol for 2 times, mixing filtrates, recovering n-butanol under reduced pressure to obtain extract, and freeze-drying to obtain n-butanol extract; (3) separating n-butanol extract by forward silica gel column chromatography, gradient eluting with chloroform-methanol, and concentrating under reduced pressure to obtain crude pteridonin; (4) and (3) refining the crude product of the crude pteridophytin by C18 reverse column purification, removing impurities by 30% methanol, eluting by 45% methanol, and removing the solvent by low-temperature freeze drying to obtain the pteridophytin with the purity of more than 98%. The extraction rate of the pteridophytin can reach 65 percent, and the possibility is provided for drug development; the method has the advantages of large sample separation amount, less target substance loss, high extraction rate, simple device, simple and convenient operation and suitability for industrial production.)

1. A separation and purification preparation method of pteridium aquilinum glycoside in pteridium aquilinum is characterized by comprising the following operation steps:

(1) preparation of lyophilized powder

Freeze drying fresh herba Fimbristylis Dichotomae at a temperature below-50 deg.C, and pulverizing to obtain lyophilized powder;

(2) preparation of n-butanol extract

Carrying out n-butanol condensation reflux extraction on the freeze-dried powder, filtering, carrying out n-butanol condensation reflux extraction on filter residues, combining filtrates, recovering n-butanol under reduced pressure to obtain an extract, and carrying out 50 freeze drying to constant weight to obtain an n-butanol extract;

(3) preparation of crude product of crude Proteosine

And (3) carrying out forward silica gel column chromatography separation on the n-butanol extract by adopting a method comprising the following steps of: 1, chloroform-methanol, in a volume ratio of 15: 1 and 10: 1, sequentially carrying out gradient elution by chloroform-methanol, and collecting the eluate with the volume ratio of 10: 1, concentrating the chloroform-methanol eluent under reduced pressure to obtain a crude product of the protopterosin;

(4) preparation of Proteosine

And C18 reverse column purification is carried out on the crude product of the crude pteridophytin, the impurities are removed by using 30% methanol solution, the methanol solution with the concentration of 45% is used for elution, the eluted part of the methanol solution with the concentration of 45% is collected, the solvent is removed by low-temperature freeze drying, and the methanol is recrystallized to obtain the pteridophytin with the purity of more than 98%, wherein the pteridophytin is white powder.

2. The method for separating, purifying and preparing pteridoidin from fiddlehead according to claim 1, wherein the method comprises the following steps: in the step (1), the mixture is crushed and sieved by a 40-mesh sieve.

3. The method for separating, purifying and preparing pteridoidin from fiddlehead according to claim 1, wherein the method comprises the following steps: in the step (2), n-butanol is added into the freeze-dried powder according to the feed-liquid ratio of 1g to 15mL, and the reflux extraction is carried out for 1.5h at the temperature of 85 ℃.

4. The method for separating, purifying and preparing pteridoidin from fiddlehead according to claim 1, wherein the method comprises the following steps: in the step (3), dissolving the n-butanol extract in methanol, adding 200-mesh 300-mesh forward silica gel, uniformly mixing, concentrating under reduced pressure until the mixture is completely dried to obtain sample-mixed silica gel, and sieving with a 200-mesh sieve; loading 200-plus-300-mesh forward silica gel into a chromatographic column and compacting, vibrating and flattening the surface of the separated silica gel, and sequentially adding sample-mixing silica gel and protective silica gel; the volume ratio of 30: 1, eluting with chloroform-methanol to remove part of impurities in the extract, which have lower polarity than the original pterosin; followed by a volume ratio of 15: 1, eluting with chloroform-methanol to remove small polar impurities with polarity closer to that of the protopanaxadin in the extract; and finally, mixing the components in a volume ratio of 10: 1, eluting with chloroform-methanol, and collecting the eluate at a volume ratio of 10: 1, concentrating the chloroform-methanol eluent at 45 ℃ under reduced pressure to obtain crude pteridonin.

5. The method for separating, purifying and preparing pteridoidin from fiddlehead according to claim 1, wherein the method comprises the following steps: in the step (4), the C18 reverse silica gel filler is filled into a glass chromatographic column, and after activation, the glass chromatographic column is sequentially eluted by double distilled water and balanced by a methanol solution with the volume concentration of 10 percent; dissolving crude pteridophytin in methanol solution with volume concentration of 10%, filtering with 0.22 μm filter membrane, slowly dripping filtrate onto C18 reverse column which is eluted by double distilled water and balanced by methanol solution with concentration of 10%, sequentially eluting with methanol solution with volume concentration of 10% and methanol solution with volume concentration of 30%, removing part of impurities with polarity larger than that of the pteridophytin, eluting with methanol solution with volume concentration of 45%, collecting the eluted part of methanol solution with volume concentration of 45%, pre-freezing at-20 deg.C for 24h, freeze-drying the eluate at-105 deg.C with a freeze dryer, and recrystallizing with methanol for 3 times to obtain pteridophytin with purity of 98%.

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to a separation and purification preparation method of pteridoidin in fiddlehead.

Background

Fern (with the scientific name of Pteridium aquilinum (L.) Kuhn var. latiuscumum (Desv.) Underw.ex Heller) is a variety of European fern belonging to the genus fern of the family fern, and the plant height can reach 1 m. The rootstock grows and walks transversely, is densely rusted, yellow and soft, and then gradually falls off; the leaves are far-growing, the stem length is 20-80 cm, the base part is 3-6 mm thick, and the color is brown or brown straw; the blade is in a wide triangle or long triangle shape, the length is 30-60 cm, the width is 20-45 cm, the tip is tapered, the base is in a circular wedge shape, and the blade is in a three-feather pinnate shape. Fern is abundant in resources and widely distributed in China and all over the world.

Fiddlehead is derived from seedlings and tender shoots of fern (Pteridium aquilinum (L.) Kuhn), namely fist vegetable, Ruyi vegetable, Longpaw vegetable, etc., according to the record of compendium of materia Medica: fern is sweet and cold in flavor and has the effects of clearing heat, smoothing intestines, descending qi, dispelling wind, reducing phlegm and the like. The fiddlehead is less polluted because the fiddlehead grows in forests, mountain wild and pine forests, is rich in various nutrient components beneficial to human bodies, is a mountain wild vegetable deeply favored by people, is eaten by people for a long time, and has the reputation of the king of the mountain vegetable.

In 19 years, excessive consumption of fiddlehead can cause poisoning and even death of cattle, in 1960, the fiddlehead is discovered to have carcinogenic effect by researches on the poisoning symptoms of the fiddlehead of cattle by Rosenberger, Heeschen and the like, and in 1965, various intestinal canceration phenomena are discovered to occur in mice by experiments that Evans and Mason feed the fiddlehead to the mice, which indicates that the fiddlehead may contain certain carcinogenic components. In 1983-1984, Niwa, a Japanese scientist, and Hoeven, a Netherlands scientist, isolated from fern, a substance capable of causing carcinogenesis in experimental animals, namely, protoporphyrin (Ptaquiloside, PTA for short, CAS: 87625-62-5, FIG. 1), and the content of the protoporphyrin in fern and fern is reported to be 0.0-12945 μ g/g, and the average content is about 0.64%.

Pharmacological activity tracking studies have shown that protoporphyrin can induce tumors in the lesions of the urethra and the gastrointestinal tract of experimental animals, and according to this, protoporphyrin is ranked as a class 3 carcinogen by the cancer organization in the world. The research on the preparation method of the protopterosin can obtain the high-purity protopterosin, and has important significance for the safety evaluation and quality control of the fiddlehead and the products thereof.

Studies by Shahin et al have found that protoporphyrin has a relevant immunomodulatory effect, and that prolonged use of the active substance, fidadienone, of protoporphyrin increases monocytes and increases the number of tumor necrosis factors, studies by latore et al have found that the carcinogenic effect of protoporphyrin can be reversed when selenium is used for simultaneous or post-treatment, and that the combination of selenium and protoporphyrin can have a therapeutic effect on cancer. Recent research shows that pterosin sesquiterpenoids containing 1-indanone structure, such as primordin, have biological activities of resisting inflammation, rheumatism, Alzheimer disease/senile dementia, virus and the like, and have wide application prospects in the field of drug test research. Fern and fern resources are extremely abundant in China and even all over the world, and a method for quickly preparing high-purity protoporphyrin from the fern is established, so that the method has important significance for research and development of new drugs and deep development of the fern resources.

Protopanabasine is stable in organic reagents, but is unstable in aqueous solution, is extremely unstable in hot water, acidic and alkaline solvents, and is easily subjected to desugarization and aromatization to generate Pterosin B (Pterosin B, CAS:34175-96-7, FIG. 1).

Since the structure of the protoporphyrin is complex and the artificial synthesis is difficult, the substance is mainly obtained from pteridium aquilinum, for example, the azalea and the like are reported in Chinese patent document CN106083593 to prepare and separate the protoporphyrin from the pteridium aquilinum by water extraction, polyamide resin and macroporous resin purification, and the extraction rate of the protoporphyrin is 25%; haruki Niwa and Makoto Oika of Japan, extracting with boiling water, adsorbing with resin XAD-2, eluting with methanol, extracting with n-butanol, purifying with silica gel column, and refining to obtain protopterosin, with extraction rate of 0.01% and extraction rate of about 16.7%; yoshida et al extract with 0.5% acetic acid-methanol, dissolve the extract in 0.1% acetic acid, then extract with n-hexane-ether, dissolve the extract with methanol-ethanol, concentrate the filtrate to get crude product of crude pteridonin, separate with reversed phase high performance liquid chromatography to get the crude pteridonin, the extraction yield is 0.006%, the extraction rate is only 1%.

According to the reports in the literature, the main steps for separating the protopterosin from the bracken are as follows: (1) the extraction method is mainly hot water extraction or acid-containing organic reagent extraction; (2) the separation method comprises the following steps: dissolving the extract in water, purifying with polyamide resin column and macroporous resin column, and separating with C18 preparative column.

However, hot water extraction or acid-containing organic reagent extraction can cause substantial decomposition of the protopanaxadin, reducing the yield of the protopanaxadin; before C18 column separation, the extract is dissolved in water and then is enriched by polyamide resin column, macroporous resin column, etc., and water is used as solvent and eluent for many times, so as to further increase the decomposition of the crude pteridophytin and further reduce the extraction rate of the crude pteridophytin. It is urgently needed to construct an efficient method for preparing and separating the protopanaxadin.

Disclosure of Invention

In order to solve the problems existing in the prior separation and preparation of the protoporphyrin glycoside, the invention provides a separation and purification preparation method of the protoporphyrin glycoside in fiddlehead.

The operation steps for separating, purifying and preparing the pteridophytin in the fiddlehead are as follows:

(1) preparation of lyophilized powder

Freeze drying fresh herba Fimbristylis Dichotomae at a temperature below-50 deg.C, and pulverizing to obtain lyophilized powder;

(2) preparation of n-butanol extract

Carrying out n-butanol condensation reflux extraction on the freeze-dried powder, filtering, carrying out n-butanol condensation reflux extraction on filter residues, combining filtrates, recovering n-butanol under reduced pressure to obtain an extract, and carrying out 50 freeze drying to constant weight to obtain an n-butanol extract;

(3) preparation of crude product of crude Proteosine

And (3) carrying out forward silica gel column chromatography separation on the n-butanol extract by adopting a method comprising the following steps of: 1, chloroform-methanol, in a volume ratio of 15: 1 and 10: 1, sequentially carrying out gradient elution by chloroform-methanol, and collecting the eluate with the volume ratio of 10: 1, concentrating the chloroform-methanol eluent under reduced pressure to obtain crude pteridonin;

(4) preparation of Proteosine

And C18 reverse column purification is carried out on the crude product of the crude pteridophytin, the impurities are removed by using 30% methanol solution, the methanol solution with the concentration of 45% is used for elution, the eluted part of the methanol solution with the concentration of 45% is collected, the solvent is removed by low-temperature freeze drying, and the methanol is recrystallized to obtain the pteridophytin with the purity of more than 98%, wherein the pteridophytin is white powder.

The technical scheme for further limiting is as follows:

in the step (1), the mixture is crushed and sieved by a 40-mesh sieve.

In the step (2), n-butanol is added into the freeze-dried powder according to the feed-liquid ratio of 1g to 15mL, and the reflux extraction is carried out for 1.5h at the temperature of 85 ℃.

In the step (3), dissolving the n-butanol extract in methanol, adding 200-mesh 300-mesh forward silica gel, uniformly mixing, concentrating under reduced pressure until the mixture is completely dried to obtain sample-mixed silica gel, and sieving with a 200-mesh sieve; loading 200-plus-300-mesh forward silica gel into a chromatographic column and compacting, vibrating and flattening the surface of the separated silica gel, and sequentially adding sample-mixing silica gel and protective silica gel; the volume ratio of 30: 1, eluting with chloroform-methanol to remove part of impurities with polarity lower than that of the protopterosin in the extract; followed by a volume ratio of 15: 1, eluting with chloroform-methanol to remove small polar impurities with polarity closer to that of the protopanaxadin in the extract; and finally, mixing the components in a volume ratio of 10: 1, eluting with chloroform-methanol, and collecting the eluate at a volume ratio of 10: 1, concentrating the chloroform-methanol eluent at 45 ℃ under reduced pressure to obtain crude pteridonin.

In the step (4), the C18 reverse silica gel filler is filled into a glass chromatographic column, and after activation, the glass chromatographic column is sequentially eluted by double distilled water and balanced by a methanol solution with the volume concentration of 10 percent; dissolving crude pteridophytin in methanol solution with volume concentration of 10%, filtering with 0.22 μm filter membrane, slowly dripping filtrate onto C18 reverse column which is eluted by double distilled water and balanced by methanol solution with concentration of 10%, eluting with methanol solution with volume concentration of 10% and methanol solution with volume concentration of 30% in sequence, removing part of impurities with polarity larger than that of the pteridophytin, eluting with methanol solution with volume concentration of 45%, collecting the eluted part of methanol solution with volume concentration of 45%, pre-freezing at-20 deg.C for 24h, freeze-drying the eluate at-105 deg.C with a freeze dryer, and recrystallizing with methanol for 3 times to obtain the pteridophytin with purity of 98%.

The beneficial technical effects of the invention are embodied in the following aspects:

(1) the innovation of the invention lies in the almost anhydrous operation of the whole process, because the crude pteridonin is easy to decompose in water, the existing method is carried out in the environment of water or even acid water. Fresh fern and bracken raw materials are subjected to freeze drying dehydration at the temperature of below 50 ℃ below zero, compared with the drying dehydration reported in documents, the decomposition of the protoporphyrin caused in the drying dehydration process is reduced, and the structures of plant tissues and cells of the fresh fern and the bracken can be damaged by freezing and expanding of water at low temperature, so that the dissolution of the protoporphyrin in the subsequent extraction process is facilitated, and the extraction rate of the protoporphyrin is improved.

(2) The method adopts the n-butanol for extraction, can reduce the decomposition of the protopanaxadin in the water extraction or acid-containing organic reagent extraction process, improves the extraction rate of the protopanaxadin, can be repeatedly used after the extraction solvent is recovered, reduces the cost, and is suitable for industrial production.

(3) The method adopts silica gel column chromatography for separation, and uses an organic reagent chloroform-methanol as an eluent, so as to reduce the decomposition of the pteridopsis latifolia glycoside in the separation process.

(4) In the refining process of the crude pteridophytin, the methanol solution elution part with the volume concentration of 45 percent is collected, and the solvent is removed by adopting low-temperature freeze drying, so that compared with the traditional decompression concentration, the decomposition of the crude pteridophytin is effectively reduced, and the yield of the crude pteridophytin is improved.

(5) The invention provides a common C18 reversed phase column refined raw pteridipenum glycoside, which is suitable for industrial production.

(6) The extraction rate of the protopanaxadin of the method can reach 65 percent, which is obviously higher than that reported in the literature by 1 to 25 percent.

Drawings

FIG. 1 is a chemical transformation diagram of protopanaxadin;

FIG. 2 is a first order mass spectrum of protopanaxadin prepared by the present invention;

FIG. 3 is a second-order mass spectrum of protopanaxadin prepared by the present invention;

FIG. 4 is an HPLC chromatogram of prepared protopanaxadin.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments, and it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.

Example 1

The operation steps for separating, purifying and preparing the pteridophytin in the fiddlehead are as follows:

(1) preparation of lyophilized powder

Cleaning fresh herba Fimbristylis Dichotomae with tap water to remove surface floating dust, cutting into small segments of about 0.5cm, pre-freezing at-20 deg.C for 24 hr, transferring to-50 deg.C freeze-drying machine, freeze-drying for 48 hr, pulverizing, and sieving with 40 mesh sieve to obtain lyophilized powder.

(2) Preparation of n-butanol extract

Weighing 200g of freeze-dried powder, adding n-butyl alcohol according to a material-liquid ratio of 1g:15mL, carrying out reflux extraction at 85 ℃ for 1.5h, filtering, collecting filtrate, extracting the filtrate from filter residue according to the same method, combining the filtrates collected twice, recycling n-butyl alcohol under reduced pressure at 70 ℃ by using a rotary evaporator to obtain extract, and carrying out freeze drying on the extract to constant weight by using 50 ℃ to obtain 19.2g of n-butyl alcohol extract.

(3) Preparation of crude product of crude Proteosine

Weighing 18.0g of n-butanol extract, dissolving in 120mL of methanol, adding 26g of 200-mesh 300-mesh forward silica gel, mixing uniformly, concentrating under reduced pressure until the mixture is completely dried to obtain sample-mixed silica gel, and sieving with a 200-mesh sieve; weighing 360g of 200-mesh 300-mesh forward silica gel, filling the forward silica gel into a chromatographic column, compacting, vibrating and flattening the surface of the separated silica gel, and sequentially adding sample-mixing silica gel and protective silica gel with the thickness of 2 cm; the volume ratio of 7.6L to 30: 1, eluting with chloroform-methanol to remove part of impurities in the extract, which have lower polarity than the original pterosin; followed by 4.0L volume ratio 15: 1, eluting with chloroform-methanol to remove small polar impurities with polarity closer to that of the protopanaxadin in the extract; finally, 4.5L of the mixture with the volume ratio of 10: 1, eluting with chloroform-methanol, and collecting the eluate at a volume ratio of 10: 1, and concentrating under reduced pressure at 45 ℃ to obtain 120.0mg of crude pteridonin.

(4) Preparation of Proteosine

Weighing 60g of C18 reverse silica gel filler, loading into a glass chromatographic column, and sequentially eluting with double distilled water and balancing with 10% methanol after activation; dissolving 120.0mg crude protopterosin in 3.0mL 10% methanol aqueous solution, filtering with 0.22 μm filter membrane, slowly dripping the filtrate on a C18 reverse column which is eluted by double distilled water and balanced by 10% methanol aqueous solution, eluting with 10% methanol aqueous solution 180mL, eluting with 30% methanol aqueous solution 300mL, removing part of impurities with polarity higher than protopterosin, and eluting with 45% methanol aqueous solution 450 mL. Collecting 45% methanol aqueous solution eluate, pre-freezing at-20 deg.C for 24 hr, lyophilizing the eluate at-105 deg.C with lyophilizer, recrystallizing with methanol for 3 times to obtain white powder 36.2mg, and performing primary mass spectrometry to obtain molecular ion peak M/Z of 421.1839([ M + Na) ([ M + Na ])]⁺) The secondary mass spectrometric results are shown in fig. 3, which shows that the ion fragments m/Z201.1278, m/Z219.1380 and m/Z241.1208 are completely the same as the primary mass spectrometric and secondary mass spectrometric results of the standard protopterosin, and the separated white powder is identified as the protopterosin. The purity of the obtained pteridopsis latifolia white powder was 98% by HPLC.

The structural formula of the protopanaxadin is as follows:

accurately weighing the crude pteridophytin standard substance, dissolving in methanol to obtain crude pteridophytin standard solutions with different concentration gradients, and measuring the crude pteridophytin standard curve equation by using an HPLC method as follows: y4.44 x-1.86 (R)²0.999). Liquid phase conditions: c₁₈Reverse phase chromatography column (250X 4.6mm, 5 μm); mobile phase: the mobile phase A is water, the mobile phase B is methanol, and gradient elution is carried out; the detection wavelength is 214 nm; the column temperature is 30 ℃; flow rate: 1.0 mL/min^-1(ii) a A chromatographic column: waters Atlantis C₁₈Reverse directionChromatography column (250X 4.6mm, 5 μm); mobile phase: the mobile phase A is water, the mobile phase B is methanol, and the detection wavelength is 214 nm; the column temperature is 30 ℃; flow rate: 1.0 mL/min^-1(ii) a The sample volume is 10 mu L; gradient elution: 0-5min, 20-40% B; 5-10min, 40-45% B; 10-20min, 45-50% B; 20-25min, 50-57% B; 25-27min, 57-100% B; 27-35min, 100% B. The HPLC chromatogram of the isolated protoporphyrin glycoside prepared in this example is shown in FIG. 4, and the content of the protoporphyrin glycoside is determined to be 98%.

Accurately weighing n-butanol extract, dissolving in methanol to obtain 0.5mg/mL, and measuring the content of protoporphyrin in the n-butanol extract by HPLC method with the same standard curve. Further calculations showed that the extraction yield of orthopterosin was 64.6% and 0.197% using the materials and methods described in example 1.