CN101250233A - Extraction method of safflower mulberry parasitic polysaccharide - Google Patents

Abstract

The invention relates to a method for extracting plant polysaccharides, in particular to a method for extracting polysaccharides from safflower mulberry. The technical scheme adopted for realizing the purpose of the present invention is: after the safflower mulberry branches and leaves are pulverized, the filter residue is filtered after extraction with ethanol, the filter residue is extracted with hot water, and the safflower mulberry polysaccharide is obtained after removing protein and small molecular components. It is characterized in that the supernatant liquid extracted by the hot water extraction method first adopts sevage method, TCA method, enzymatic method or the combination of these three methods to remove protein, and then dialyzes to remove small molecular components. By adopting the polysaccharide extraction method of the present invention, only one precipitant is used to precipitate the polysaccharide, which saves a lot of solvents; meanwhile, the yield and water solubility of the polysaccharide are improved. The obtained safflower mulberry polysaccharide extract has a good inhibitory effect on mouse sarcoma S180, and can significantly prolong the lifespan of H22 mice bearing liver cancer ascites tumor, and its tumor inhibitory effect has no dose-dependence.

Description

Extraction method of safflower mulberry parasitic polysaccharide

technical field

The invention relates to a method for extracting plant polysaccharides, in particular to a method for extracting polysaccharides from Scurrula parasitica L.

Background technique

The safflower mulberry Scurrula parasitica L. is a semi-parasitic plant of the genus Pyroparasitica of the subfamily Morusaceae. It is a unique plant in my country and is mainly distributed in southern regions such as Guangdong, Guangxi, Yunnan and Fujian. Traditionally, safflower mulberry is often used as a substitute for mulberry tree Taxillus chinensis (DC.) Danser. It has the functions of nourishing liver and kidney, dispelling rheumatism, lowering blood pressure, calming blood and nourishing fetus, etc. It is mainly used for rheumatism, joint pain, high blood pressure, sciatic nerve Pain, low back pain, postpartum milk loss embolism.

Plants of the family Morushiaceae generally contain a large amount of flavonoids. In our previous research, we found that the total flavonoids extract of Safflower mulberry has good anti-tumor effect on acute myeloid leukemia, and can sensitize important clinical anti-tumor chemotherapy drugs Efficacy of doxorubicin, mechanism studies show that it may be a good NF-κB inhibitor. However, there is no research on the parasitic polysaccharides of safflower mulberry and their functions.

Plant polysaccharides have various biological activities, can regulate the immune function of the body, and have anti-virus, anti-bacterial, anti-parasitic, anti-tumor, anti-radiation, anti-aging and other functions, so they are getting more and more attention. In recent years, a large number of studies have shown that polysaccharides of traditional Chinese medicine have anti-tumor effects, and have less toxic and side effects, so it has become a hot spot in anti-tumor research. Lentinan, angelica polysaccharide, polyporus polysaccharide, versicolor polysaccharide and other polysaccharide drugs with stable quality, definite curative effect, little toxicity and adverse reactions have been widely used in clinic as anti-tumor drugs.

Mistletoe, the same family plant of Safflower mulberry, is a kind of plant with anti-tumor effect that has been studied abroad. In Europe, the anti-tumor effect of mistletoe has been studied since 1920. Its extract has a variety of anti-tumor effects. It has a certain therapeutic effect on common tumors such as breast cancer, gastric cancer and colon cancer. The standard plant extracts of mistletoe with trade names Iscador and Isorel have been developed in the UK and Australia respectively for clinical treatment of cancer. Existing studies have shown that the effective anti-tumor components in mistletoe extracts may mainly be polysaccharides, lectins, toxic peptides, alkaloids and polyphenols.

The extraction process of plant polysaccharides mostly adopts the traditional method of first precipitation and then protein removal. In our research, we found that using the traditional method to extract polysaccharides from the safflower mulberry parasite requires multiple use of precipitating agents to precipitate polysaccharides from the solution, which consumes a large amount of solvent and has a low yield. The extracted polysaccharides have poor water solubility. Not conducive to application. The present invention adopts the extraction method of firstly removing protein and then precipitating polysaccharide, and only needs to use a precipitant once to precipitate polysaccharide, which saves a large amount of solvent; the yield is high, and the water solubility of polysaccharide is improved at the same time, which is convenient for drug research and practical application.

Contents of the invention

The object of the present invention is to provide a method for extracting the parasitic polysaccharide of safflower mulberry by first removing the protein and then precipitating the polysaccharide.

The technical scheme adopted for realizing the purpose of the present invention is: after the safflower mulberry branches and leaves are crushed, they are extracted with ethanol under reflux, filtered to get the filter residue, the filter residue is extracted with hot water, and the safflower mulberry polysaccharide extract is obtained after precipitation and drying , which is characterized in that the supernatant extracted by the hot water extraction method first adopts the sevage method, the TCA method or the enzyme method, or any two methods between them, or a combination of the three methods to remove the protein, and then Dialysis is performed using a dialysis bag to remove small molecule components.

By adopting the method for extracting polysaccharides from safflower mulberry parasites of the present invention, only one precipitant is needed to precipitate polysaccharides, which saves a large amount of solvents; the yield of polysaccharides is improved, and the water solubility of polysaccharides is improved at the same time, which is convenient for drug research and practical application . The obtained safflower mulberry polysaccharide extract has a good inhibitory effect on mouse sarcoma S180, and has a significant synergistic effect with clinically important anti-tumor chemotherapy drugs; lifespan. Studies have shown that the anti-tumor effect of the extract is not dose-dependent, but there is an optimal dose, too high or too low a dose will reduce the anti-tumor effect.

The specific extraction method comprises the following steps:

(1) Collect safflower mulberry parasitic twigs and leaves, wash with water, dry in the air, dry at 60°C, and pulverize into powder;

(2) Take the powder, add ethanol to reflux extraction, to remove the fat-soluble components therein, filter to take the filter residue, and evaporate the solvent;

(3) Take the filter residue after evaporating the solvent, carry out hot water soaking and extraction, filter residue: water=1:5～15, the temperature of hot water is 80～95 ℃ soaking and extracting, after 2 hours, centrifuge or suction filter to remove the residue; take the above The clear liquid was concentrated under reduced pressure at 60°C;

(4) Repeat step (3) three times; combine the supernatants for 3 times.

(5) Using the sevage method, TCA method, enzymatic method or a combination of these methods, repeat three times to remove the protein in the supernatant;

(6) Put the deproteinized supernatant into a dialysis bag for dialysis, dialysis with running water for 2 days, and dialysis with distilled water for 1 day to remove small molecular components and obtain a polysaccharide solution;

(7) Add a precipitating agent to the polysaccharide solution, and precipitate the polysaccharide at low temperature so that the concentration of the precipitating agent in the solution is 65% to 85% (v/v). Washing with diethyl ether in sequence and then freeze-drying can obtain the safflower mulberry polysaccharide of the present invention.

When the dialysis bag of the present invention is used for dialysis, 2 days of running water dialysis and 1 day of distilled water dialysis; the dialysis molecular weight of the dialysis bag is 7000-14000D.

The precipitant in the present invention refers to an alcoholic solvent containing 1 to 4 carbon chains, most preferably ethanol.

The washing agent adopted in the washing step of the present invention is dehydrated alcohol, acetone and ether washing successively.

Detailed ways

The following examples are used to further illustrate the present invention, but do not limit the scope of the present invention thereby.

Example 1:

Take 180g of crushed medicinal material powder, add 80% ethanol to reflux and extract for 3 times, each time for 2 hours, take the filter residue by suction filtration, evaporate the solvent, add water according to the ratio of material to liquid 1:10, extract in hot water at 95°C, each time for 2 hours, Extracted 3 times in total, combined the 3 extracts, filtered to remove the residue, concentrated the filtrate under reduced pressure at 60°C to a volume of about 1000ml, added 1/3 volume of sevage (chloroform:n-butanol=4:1), and oscillated for 20min Afterwards, centrifuge at 8000rpm for 15min to remove the protein precipitate, take the supernatant and repeat it several times until there is no protein. The deproteinized solution was put into a dialysis bag for dialysis, running water dialysis for 2 days, distilled water dialysis for 1 day. Concentrate under reduced pressure to 1/2 volume, add 3 times the amount of 95% ethanol, let it stand in the refrigerator for 24 hours to precipitate, centrifuge to collect the precipitate, wash with absolute ethanol, acetone, ether in turn, and then freeze and vacuum-dry to obtain safflower mulberry Parasitic polysaccharide extract 3.67g.

Example 2:

Take 180g of crushed medicinal material powder, add 80% ethanol to reflux and extract for 3 times, each time for 2 hours, take the filter residue by suction filtration, evaporate the solvent, add water according to the ratio of material to liquid 1:10, extract in hot water at 95°C, each time for 2 hours, Extract 3 times, combine 3 extracts, remove residue by suction filtration, add 0.5% papain (w/v) to the filtrate, enzymatically hydrolyze at 40°C for 5h, heat to 100°C for 10min to inactivate enzyme activity, concentrate under reduced pressure at 60°C To about 1000ml volume, add 1/3 volume of sevage (chloroform:n-butanol=4:1), shake for 20min, centrifuge at 8000rpm for 15min, repeat 3 times. The deproteinized solution was put into a dialysis bag for dialysis, running water dialysis for 2 days, distilled water dialysis for 1 day. Concentrate to 1/2 volume, add 3 times the amount of 95% ethanol, put it in the refrigerator for 24 hours to precipitate, centrifuge to collect the precipitate, wash with absolute ethanol, acetone, ether in turn, and then freeze and vacuum dry to obtain the parasitic polysaccharide of Safflower mulberry Extract 4.87g.

Example 3:

Take 180g of crushed medicinal material powder, add 80% ethanol to reflux and extract for 3 times, each time for 2 hours, take the filter residue by suction filtration, evaporate the solvent, add water according to the ratio of material to liquid 1:10, extract in hot water at 95°C, each time for 2 hours, Extracted 3 times in total, combined 3 extracts, filtered to remove residue, concentrated the filtrate under reduced pressure at 60°C to a volume of about 1000ml, added an equal volume of 30% trichloroacetic acid, oscillated for 20min, then stood at 4°C for 2h, 8000rpm Centrifuge for 15 minutes to remove protein precipitates. The deproteinized solution was put into a dialysis bag for dialysis, running water dialysis for 2 days, distilled water dialysis for 1 day. Concentrate to 1/2 volume, add 3 times the amount of 95% ethanol, put it in the refrigerator for 24 hours to precipitate, centrifuge to collect the precipitate, wash with absolute ethanol, acetone, ether in turn, and then freeze and vacuum dry to obtain the parasitic polysaccharide of Safflower mulberry Extract 3.42g.

Example 4:

Take 180g of crushed medicinal material powder, add 80% ethanol to reflux and extract for 3 times, each time for 2 hours, take the filter residue by suction filtration, evaporate the solvent, add water according to the ratio of material to liquid 1:10, extract in hot water at 95°C, each time for 2 hours, Extract 3 times, combine 3 extracts, remove residue by suction filtration, add 0.2% papain (w/v) to the filtrate, enzymatically hydrolyze at 60°C for 2h, heat to 100°C for 10min to inactivate enzyme activity, and concentrate under reduced pressure at 60°C To a volume of about 1000ml, add an equal volume of 30% trichloroacetic acid, shake for 20min, let stand at 4°C for 2h, and centrifuge at 8000rpm for 15min to remove protein precipitates. The deproteinized solution was put into a dialysis bag for dialysis, running water dialysis for 2 days, distilled water dialysis for 1 day. Concentrate to 1/2 volume, add 3 times the amount of 95% ethanol, put it in the refrigerator for 24 hours to precipitate, centrifuge to collect the precipitate, wash with absolute ethanol, acetone, ether in turn, and then freeze and vacuum dry to obtain the parasitic polysaccharide of Safflower mulberry Extract 4.32g.

Example 5:

Inhibition of sarcoma S180 in mice by intraperitoneal injection of the safflower mulberry polysaccharide extract extracted according to the method described in Example 4 (hereinafter referred to as SPS).

50 Kunming mice (half male and half female, weighing 18±2 g, provided by the Experimental Animal Center of Fujian Medical University), and the S180 ascites tumor strain was provided by the School of Pharmacy, Fujian Medical University. S180 sarcoma mice passaged in the abdominal cavity for 7 days were taken and killed by dislocation. The intraperitoneal tumor cells were extracted under aseptic conditions, washed three times with normal saline, centrifuged at 2500 rpm for 3 min, and the concentration of tumor cells was adjusted to 5×10 with sterile normal saline. ⁶ /ml, inoculated subcutaneously in the right armpit of mice, each mouse was inoculated with 0.2ml.

24 hours after inoculation, they were randomly divided into groups, 10 in each group, and divided into normal saline group, cyclophosphamide group (CTX, 20mg/kg.d), SPS low-dose group (50mg/kg.d), SPS middle-dose group (100mg/kg.d). kg.d), SPS high dose group (150mg/kg.d). Each of the above groups was intraperitoneally administered 0.01ml/g per day for 10 consecutive days. 24 hours after the last administration, the tumor-bearing mice were killed by dislocation, and the tumor tissues, spleen, and thymus were removed and weighed to calculate the tumor inhibition rate. , spleen index (mg spleen weight/10g body weight) and thymus index (mg thymus weight/10g body weight). The results are shown in Table 1.

Tumor inhibition rate (%)=(average tumor weight in control group-average tumor weight in experimental group)/average tumor weight in control group×100%

n＝10)Table 1 Anti-tumor effect of safflower mulberry polysaccharides on S180 tumor-bearing mice (

n=10)

Compared with the control group: *P<0.01, **P<0.001

It can be seen from Table 1 that three doses of polysaccharide extracts of safflower mulberry parasites have good tumor inhibitory effects on mice S180. Compared with the normal saline group, the difference is significant. The dose is the best, but too high a dose will lead to a decrease in the anti-tumor effect, which is consistent with most of the immunomodulatory effects of plant polysaccharides reported in the literature. Three doses of SPS groups can increase the mouse spleen weight compared with the normal saline group, only the SPS100mg/kg.d group has a significant difference (P<0.001). Positive control drug CTX significantly reduced spleen index and thymus index.

Example 6

The synergistic effect of the safflower mulberry polysaccharide extract extracted by the method described in Example 4 on cyclophosphamide (CTX).

The materials and inoculation method are the same as those in Example 5, administered in random groups 24 hours after inoculation, and divided into normal saline group, CTX low-dose group (10mg/kg.d), CTX middle-dose group (20mg/kg.d), CTX high-dose group (30mg/kg.d), SPS (100mg/kg.d) + CTX low dose group (10mg/kg.d), SPS (100mg/kg.d) + CTX medium dose group (20mg/kg.d), SPS (100mg/kg.d)+CTX high-dose group (30mg/kg.d), the above groups were administered 0.01ml/g ip every day for 10 days, and the tumor-bearing mice were killed by dislocation 24 hours after the last administration , Take the tumor and weigh it, and calculate the tumor inhibition rate. The difference in tumor inhibition rate between the CTX treatment group alone and the polysaccharide group was compared, and the results are shown in Table 2.

n＝10)Table 2 The synergistic effect of safflower mulberry polysaccharides on CTX (

n=10)

Compared with normal saline group: *P<0.001;

Compared with CTX: ^△ P<0.05; ^△△ P<0.01

It can be seen from Table 2 that the polysaccharide extract of Safflower mulberry significantly enhanced the antitumor effect of cyclophosphamide.

Example 7

The polysaccharide extract extracted according to the method described in Example 2 significantly prolongs the lifespan of mice bearing H22 liver cancer ascites tumor.

Material and source are with embodiment 5. The H22 ascites cells passaged in the peritoneal cavity of mice for 7 days were extracted under sterile conditions, counted and diluted with normal saline, and 5×10 ⁶ cells were inoculated in the peritoneal cavity of each mouse, and administered by tail vein injection from the next day for 7 consecutive days. The survival days of the mice were counted after the administration was stopped. The negative control drug was normal saline, the positive control drug was cyclophosphamide, and SPS was divided into high, medium and low dose groups. The results are shown in Table 3.

The formula for calculating life extension rate is:

Life extension rate=(average survival days of drug administration group-average survival days of control group)/average survival days of control group×100%

n＝10)Table 3 Anti-tumor effect of safflower mulberry polysaccharides on S180 tumor-bearing mice (

n=10)

Compared with the control group: *P<0.01

As can be seen from Table 3, the polysaccharide extract of safflower mulberry parasite significantly prolongs the life of tumor-bearing mice, which is similar to the inhibition result of sarcoma S180. The dose of 100mg/kg. The effect of prolonging life in mice with tumor is the best, indicating that the anti-tumor effect of safflower mulberry polysaccharide extract has an optimal dose, and there is no linear dose-effect relationship. This point is similar to the anti-tumor effect of lentinan, indicating that the polysaccharide extract of safflower mulberry polysaccharide may have a similar anti-tumor mechanism with lentinan.

Claims (4)

1, the extracting method of safflower loranthus parasiticus polysaccharides, be the safflower loranthus parasiticus branches and leaves after crushed, extract with alcohol reflux, filter the place to go fat-soluble component, filter residue obtains the safflower loranthus parasiticus polysaccharides extract through the hot water extraction after precipitation, drying, it is characterized in that the supernatant liquor after the hot water extraction extracts, at first adopt sevage method, TCA method, enzyme process or this three kinds of method couplings to remove albumen, utilize dialysis tubing to dialyse again.

2, the extracting method of safflower loranthus parasiticus polysaccharides according to claim 1, when it is characterized in that described dialysis tubing is dialysed, flowing water dialysis 2 days, distill water dialysis 1 day; The dialysis molecular weight of said dialysis tubing is 7000～14000D.

3, the extracting method of safflower loranthus parasiticus polysaccharides according to claim 1 is characterized in that described precipitation agent, is meant the alcoholic solvent that contains 1～4 carbochain, most preferably ethanol.

4, the extracting method of safflower loranthus parasiticus polysaccharides according to claim 1 is characterized in that the washing composition that adopts in the described washing step is followed successively by dehydrated alcohol, acetone and ether washing.