CN101508715B - Extraction and purification process for cordycepin in cordyceps militaris link - Google Patents

Abstract

The invention provides an extraction and purification method of cordycepin from Cordyceps militaris, and belongs to the deep-processing technical field of agricultural products. The method comprises the following steps: crushing Cordyceps militaris raw material, mixing the raw material with water at 1:20 raw material/water ratio, performing ultrasonic extraction, and performing centrifugal filtration on obtained extracting solution and pouring in a macroporous resin column at the adsorption flow of 1.8BV/hr; taking 15% methanol aqueous solution as an eluant to perform resin column elution, and decompressing and condensing obtained eluent at the temperature of 60 DEG C by a rotary evaporator to obtain Cordyceps militaris cordycepin concentrate. The method can help realize aqueous solution extraction of the cordycepin from the Cordyceps militaris raw material at low temperature, and the extraction rate reaches 85%; and polysaccharide and protein can be removed from the extract by macroporous resin separation and purification, thus the content of the cordycepin in the extract can reach 10%. The cordycepin in the Cordyceps militaris product has high activity, can be taken as a raw material for producing cordycepin monomer and can also be directly taken as an additive of health products and functional food, thus the cordycepin has wide market development prospect.

Description

Extraction and purification process for cordycepin in a kind of Cordyceps militaris (L.) Link.

One, technical field

The invention provides extraction and purification process for cordycepin in a kind of Cordyceps militaris (L.) Link., belong to deep-processing technical field of agricultural products.

Two, background technology

Cordyceps militaris (L.) Link. (Cordyceps militaris) has another name called Cordyceps militaris (L.) Link., with China rare traditional Chinese medicine Cordyceps sinensis (Cordyeeps sinensis) for belonging to xenogenesis together, its cordycepin (cordycepin) content is (3.089 ± 0.046) ‰, than high about 3 to 6 times of the cordycepin in the Cordyceps sinensis. cordycepin is a kind of adenosine class formation analogue in the Chinese caterpillar fungus, molecular formula C ₁₀H ₁₃O ₃N ₅. cordycepin has proved to have effects such as antitumor, antiviral, antibiotic, has cytotoxicity and immunoregulation effect.At present, the pure product price of cordycepin is 25000 dollars/g in the international market.China's Cordyceps militaris (L.) Link. cultivation is general, but how to be used as medicine with the form of mycelium or sporophore, and therefore, the extraction process of research cordycepin has important economic value and vast market application potential.

Macroporous adsorbent resin is the isolation technique of new development both at home and abroad in recent years, and in field of medicaments (particularly natural drug is refining), widely use, be fit to suitability for industrialized production, but the separation and purification aspect that this technology is applied in cordycepin yet there are no report, and the present invention adopts the macroporous adsorbent resin isolation technique to separate

Three, summary of the invention

Technical problem

The object of the present invention is to provide extraction and purification process for cordycepin in a kind of Cordyceps militaris (L.) Link., cordycepin content can reach 10%. and can make the cordycepin in the Cordyceps militaris (L.) Link. obtain enrichment by macroporous resin after the separation and purification, and it is low to have a production cost, characteristics such as technology maturation.

Extraction and purification process for cordycepin in the technical scheme Cordyceps militaris (L.) Link. provided by the invention comprises:

1) extraction of cordycepin in the Cordyceps militaris (L.) Link.: Cordyceps militaris (L.) Link. raw material pulverizing, cross 40～100 mesh sieves, adding deionized water with 1: 20 ratio of material-water ratio then mixes, under ultrasonic frequency 90kHz, carry out ultrasonic extraction, 60 ℃ of temperature, extraction time 30-60 minute, extracting solution with 4000 rev/mins of centrifugings, is made pale brown look Cordyceps militaris (L.) Link. extracting solution.

2) separation and purification of cordycepin in the Cordyceps militaris (L.) Link.: with macroporous resin column on the extracting solution, the resin model is AB-8, and pH value is 5～5.5, and the sample solution amount is 5～6 times of resin column volumes, absorption flow velocity 1.8BV/hr; Wait to adsorb saturated back with deionized water towards post, after the effluent liquid clarification, be eluent wash-out resin column with 15% methanol aqueous solution, elutriant is 2.0～2.5 times of resin column volumes, during flow velocity 1BV/hr.

3) concentrating under reduced pressure elutriant: 60 ℃ of temperature, concentrating under reduced pressure elutriant under the vacuum tightness 0.09MP makes Cordyceps militaris (L.) Link. cordycepin enriched material with rotatory evaporator.

Extraction and purification process for cordycepin in above-mentioned a kind of Cordyceps militaris (L.) Link., used resin are that Cangzhou precious grace chemical industry company limited produces, and model is AB-8; Used eluent is 15% methanol aqueous solution; With deionized water as extraction agent and washing fluid.

Beneficial effect the present invention can keep the biological activity of cordycepin to greatest extent with the cordycepin extract at low temperature in the Cordyceps militaris (L.) Link. raw material; Utilize polysaccharide and the protein ingredient that to remove after the macroporous resin separation and purification in the extract, the cordycepin content in the extract is reached more than 10%.Cordycepin enriched material impurity is few in the product Cordyceps militaris (L.) Link., and is active high, as reducing separating difficulty as separating the monomeric raw material of cordycepin, improves separation efficiency, reduces separation costs; As also can directly adding in healthcare products and the functional food, has the vast market development prospect as additive.

Major advantage of the present invention and positively effect are as follows:

1. the present invention adopts the low-temperature ultrasonic water extraction to extract cordycepin, has kept the biological activity of cordycepin to greatest extent, extraction rate reached to 85%;

2. utilize polysaccharide and the protein ingredient that to remove after the macroporous resin separation and purification in the extract, the cordycepin content in the extract is reached more than 10%.

3. cordycepin enriched material impurity is few in the product Cordyceps militaris (L.) Link., and is active high, as reducing separating difficulty as separating the monomeric raw material of cordycepin, improves separation efficiency, reduces separation costs; Cordycepin enriched material impurity is few in the product Cordyceps militaris (L.) Link., and is active high,

4. as separating the monomeric raw material of cordycepin can reduce separating difficulty, improve separation efficiency, reduce separation costs; As also can directly adding in healthcare products and the functional food, has the vast market development prospect as additive.

Four, description of drawings

The liquid chromatogram of Fig. 1 cordycepin standard specimen

Five, embodiment

The extraction of cordycepin in the Cordyceps militaris (L.) Link.: Cordyceps militaris (L.) Link. raw material Su Ke pupa No. 1 (commercially available) is pulverized, cross 40～100 mesh sieves, mixed with 1: 20 ratio of solid-liquid ratio and extracting solution deionized water then, upward carry out ultrasonic extraction at KQ5200E type Ultrasonic Cleaners (Kunshan ultrasonic instrument company limited) with ultrasonic frequency 90kHz, extract 60 ℃ of temperature, 30 minutes extraction times, 60 ℃ of temperature, extracting solution is centrifugal with 4000 rev/mins of centrifugings with TD5A-WS type whizzer (Shanghai Lu Xiang instrument whizzer instrument company limited), make pale brown look Cordyceps militaris (L.) Link. extracting solution.The present invention adopts the low-temperature ultrasonic water extraction to extract cordycepin, has kept the biological activity of cordycepin to greatest extent, extraction rate reached to 85%.

The separation and purification of cordycepin in the Cordyceps militaris (L.) Link.: with macroporous resin column on the extracting solution, the resin model is AB-8 (the precious grace chemical industry in a Cangzhou company limited), and pH value is 5～5.5, and the sample solution amount is 5～6 times of resin column volumes, absorption flow velocity 1.8BV/hr; Wait to adsorb saturated back with deionized water towards post, after the effluent liquid clarification, be eluent wash-out resin column with 15% methanol aqueous solution, elutriant is 2～2.5 times of resin column volumes, during flow velocity 1BV/hr.

Concentrating under reduced pressure elutriant: 60 ℃ of temperature, under the vacuum tightness 0.09MP elutriant is concentrated to 10ml with RE52A type rotatory evaporator (Shanghai Yarong Biochemical Instrument Plant), makes Cordyceps militaris (L.) Link. cordycepin enriched material.

Measuring method: utilize high performance liquid chromatography to carry out the mensuration of cordycepin content. measuring method is as follows:

1 reagent and material

Potassium primary phosphate: analytical pure.Dipotassium hydrogen phosphate: analytical pure.Tetrahydrofuran (THF): chromatographically pure. dehydrated alcohol: analytical pure.4 experimental waters: should meet the requirement of GB/T 6682-1992 one-level water.Cordycepin standard substance: purity 〉=98%.

2 cordycepin standard reserving solutions: accurately take by weighing cordycepin standard substance 15mg, fixed molten with the dissolving of 50% aqueous ethanolic solution ultra-sonic oscillation to 100mL, place-18 ℃ of refrigerators to preserve, preservation period is 2 months.

3 moving phases: accurately take by weighing 1.36gKH2PO4,2.28gK2HPO43H2O in the 1L volumetric flask, be dissolved in water, constant volume, add the 10mL tetrahydrofuran (THF) again, mixing is standby.

4 instrument and equipments

Liquid chromatograph has UV-detector, 0.22 μ m filter membrane.The ultrasonoscope supercentrifuge, rotating speed is not less than 10000rpm.Air dry oven.

5 test procedures

5.1 the preparation of sample

With 60 ℃ of oven drying at low temperatures 1 hour, pulverizing the back, to cross the aperture be that 60 mesh sieves are standby with sample.Precision takes by weighing powder 0.1g, puts into reagent bottle, adds 10.00mL distilled water, supersound extraction 30 minutes, and 0.22 μ m millipore filtration filters, and extracting solution is as need testing solution.

5.2 chromatographic condition

5.2.1 typical curve

The dilute with water standard reserving solution, the standard operation solution of preparation series concentration: 3 μ g/mL, 6 μ g/mL, 30 μ g/mL, 75 μ g/mL, 150 μ g/mL.0.22 μ m membrane filtration is got filtrate, draws 10 μ L sample detection (n=3) successively with microsyringe under chromatographic condition.With the peak area is ordinate zou, and corresponding standard working solution concentration is X-coordinate.The high-efficient liquid phase chromatogram of cordycepin standard specimen (Fig. 1), the present invention utilizes polysaccharide and the protein ingredient that can remove after the macroporous resin separation and purification in the extract, and the cordycepin content in the extract is reached more than 10%.

5.2.2 chromatographic column:

C18 post (3.9mm * 300mm, 4 μ m).

5.2.3 moving phase:

The KH2PO4-K2HPO4 damping fluid (pH=6.86,0.01mol/L), isocratic elution; Flow velocity: 1.00mL/min.

5.2.4 detection wavelength: 260nm.

5.3 accurate respectively reference substance solution and each the 10 μ L of need testing solution of drawing of sample introduction inject liquid chromatograph, measure.On typical curve, calculate the concentration of garlicin in the sample solution with peak area.

The result calculates:

By (1) formula calculation result.

$X=\frac{C\×V}{m\×1000}\·\·\·\left(1\right)$

In the formula:

X---cordycepin content in the sample, unit is every kilogram (mg/g) of gram;

C---the concentration of cordycepin in the sample solution, unit is every milliliter of microgram (μ g/mL);

V---sample solution volume, unit are milliliter (mL);

M---sample mass, unit is gram (g).

Calculation result keeps two position effective digitals.

5 precision

The absolute difference of the twice independent measurement result that obtains under repeated condition must not surpass 10% of arithmetical av.

The result

Utilize polysaccharide and the protein ingredient that to remove after the macroporous resin separation and purification in the extract, the cordycepin content in the extract is reached more than 10%.

Claims (1)

1. extraction and purification process for cordycepin in the Cordyceps militaris (L.) Link. comprises:

1) extraction of cordycepin in the Cordyceps militaris (L.) Link.: Cordyceps militaris (L.) Link. raw material pulverizing, cross 40～100 mesh sieves, mixed with 1: 20 ratio of feed liquid mass ratio and extracting solution deionized water then, under ultrasonic frequency 90kHz, carry out ultrasonic extraction, extract 60 ℃ of temperature, extraction time 30-60 minute, extracting solution with 4000 rev/mins of centrifugings, is made pale brown look Cordyceps militaris (L.) Link. extracting solution;

2) separation and purification of cordycepin in the Cordyceps militaris (L.) Link.: with macroporous resin column on the extracting solution, the resin model is AB-8, and pH value is 5～5.5, and the sample solution amount is 5～6 times of resin column volumes, absorption flow velocity 1.8BV/hr; Waiting to adsorb saturated back and use the washing fluid deionized water towards post, after the effluent liquid clarification, is eluent wash-out resin column with volume ratio 15% methanol aqueous solution, and elutriant is 2～2.5 times of resin column volumes, during flow velocity 1BV/hr;

3) concentrating under reduced pressure elutriant: 60 ℃ of temperature, concentrating under reduced pressure elutriant under the vacuum tightness 0.09MP makes Cordyceps militaris (L.) Link. cordycepin enriched material with rotatory evaporator.

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