Cycas revoluta seed polysaccharide, preparation method and application in neurological disease treatment

Abstract

The invention discloses cycas revoluta seed polysaccharide as well as a preparation method and application thereof in treatment of neurological diseases. The cycas revoluta seed polysaccharide is prepared by taking the seed coats of cycas revoluta seeds as a raw material through water extraction and alcohol precipitation, ethanol fractional refining, deproteinization and desalination, and the polysaccharide has the effects of resisting neuritis and glioma. Therefore, the polysaccharide has a prospect of being developed into drugs for treating neuritis and glioma. The technical scheme and the technical effect of the invention are not disclosed in the prior art, and the technical scheme of the invention has outstanding substantive features and remarkable progress.

Description

Technical Field

[0001] The invention belongs to the field of natural drug chemistry and relates to the preparation and application of natural plant polysaccharides, and specifically to a cycad seed polysaccharide, a preparation method and application in the treatment of neurological diseases. Background Art

[0002] Cycas revoluta Thunb. is a plant of the genus Cycas in the family Cycadaceae. It is native to Fujian, Guangdong, and Guangxi Zhuang Autonomous Region, and is cultivated in Yunnan, Guizhou, and eastern Sichuan. The trunk is about 2 meters high, cylindrical, with obvious spirally arranged rhombus petiole residues; the male cones are cylindrical, with narrow microsporophyll; the female megasporophyll is densely covered with light yellow or light gray hairs. The seeds are reddish brown or orange-red, obovate or ovate, and the middle seed coat is woody. The flowering period is June-July, and the seeds mature from October to December.

[0003] Many parts of Cycas can be used as medicine. According to the "Supplement to Compendium of Materia Medica", the leaves have the effects of relieving heat and toxins, cooling blood, dispersing blood stasis and stopping bleeding; the flowers are slightly toxic, and have the effects of cooling blood and removing blood stasis, benefiting the stomach, consolidating essence and regulating qi; the roots can be used to treat jaundice; the seeds can be used to treat diarrhea, dysentery, indigestion, hiccups, cough, sputum and asthma.

[0004] Inventor Pan Taowen has been engaged in the study of chemical components of cycads for a long time, and has conducted a systematic study on the chemical components of different parts of cycads, such as the seed coat, female flowers, and male flowers (Reference: Pan Taowen, Chemical Composition of Cycads and Structural Modification of Gentiopicroside, 2012). This study used various separation materials and technical means as well as modern spectroscopic methods to separate and identify 13 compounds from different parts of cycads, such as the seed coat, female flowers, and male flowers. However, previous reports were all about small molecule compounds in cycads, and there were no reports on the study of polysaccharide components in cycad seed coats.

[0005] Plant polysaccharides are widely distributed in various plant groups in nature. Different extraction methods such as temperature, pH, enzymes, and energy (microwave, ultrasound) will cause plant polysaccharide extracts to have different molecular weights, morphologies, and other characteristics, which may ultimately affect their biological activity. With the advancement of extraction, separation, purification and other technologies and the analysis of structural characteristics, it has been found that plant polysaccharides have multiple active functions such as anti-tumor, anti-viral, anti-diabetic, and improving the body's immunity.

[0006] Based on the inventor's latest research on cycad seed polysaccharides, the present invention is specially proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a cycad seed polysaccharide, a preparation method and an application thereof in the treatment of neurological diseases.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] A cycad seed polysaccharide is prepared by taking the seed coat of cycad seeds as raw material through water extraction, alcohol precipitation, ethanol graded refining, deproteinization and desalination.

[0010] Preferably, the water extraction and alcohol precipitation step uses 4 times the volume of anhydrous ethanol for alcohol precipitation.

[0011] Preferably, the ethanol graded refining first removes 40% of the alcohol-precipitated polysaccharides, and then collects 65% of the alcohol-precipitated polysaccharides.

[0012] The method for preparing any of the above-mentioned cycad seed polysaccharides comprises the steps of:

[0013] Step S1, extraction of total polysaccharides from seed coat: taking an appropriate amount of seed coat powder of cycad seeds, adding an appropriate amount of petroleum ether for hot reflux defatting, recovering the petroleum ether, and volatilizing the residue until there is no ether smell; adding an appropriate amount of defatted powder to water, heating for extraction, filtering the extract, and concentrating under reduced pressure; slowly adding 4 times the volume of anhydrous ethanol to the above concentrate, stirring evenly, standing at low temperature overnight, centrifuging, collecting the precipitate, and freeze-drying to obtain total polysaccharides from seed coat;

[0014] Step S2, fractional purification of total polysaccharides: taking seed coat total polysaccharides, adding appropriate amount of water, stirring and dissolving, preparing seed coat total polysaccharide solution, centrifuging, collecting supernatant to obtain supernatant A, slowly adding anhydrous ethanol to supernatant A to make the ethanol volume fraction reach 40%, standing at low temperature overnight, centrifuging, collecting supernatant to obtain supernatant B, slowly adding anhydrous ethanol to supernatant B to make the ethanol volume fraction reach 65%, standing at low temperature overnight, centrifuging, collecting precipitate to obtain 65% alcohol-precipitated polysaccharide, and freeze-drying;

[0015] Step S3, deproteinization and desalting

[0016] The 65% alcohol-precipitated polysaccharide was re-dissolved in water, deproteinized using the Sevag method, dialyzed for desalination, and freeze-dried to obtain the polysaccharide.

[0017] Preferably, the temperature of the heating extraction in step S1 is 85°C.

[0018] Preferably, the temperature of the low-temperature standing overnight in step S1 and step S2 is both 4°C.

[0019] Preferably, the centrifugation parameters in step S1 and step S2 are both 4000 r / min for 10 min.

[0020] Preferably, the mass fraction of total seed coat polysaccharides in the total seed coat polysaccharide solution in step S2 is 20%.

[0021] Use of any of the above cycad seed polysaccharides in the preparation of a medicine for treating neuritis.

[0022] Use of any of the above cycad seed polysaccharides in the preparation of a drug for treating glioma.

[0023] Beneficial effects:

[0024] The invention uses the seed coat of cycad seeds as raw materials, and prepares a cycad seed polysaccharide through water extraction, alcohol precipitation, ethanol classification and refining, deproteinization and desalination. The polysaccharide has the effects of anti-neuritis and anti-glioma. Therefore, the polysaccharide has the prospect of being developed into a drug for treating neuritis and glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the IL-6 release level of BV-2 in each group;

[0026] Figure 2 is the U373 cell proliferation inhibition rate in each group. DETAILED DESCRIPTION

[0027] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0028] Example 1: Polysaccharide separation and preparation

[0029] 1. Test Materials

[0030] The cycad seeds are mature cycad (Cycas revoluta Thunb.) seeds collected from Yulin, Guangxi Zhuang Autonomous Region in December 2023. Wash with water and dry naturally, cut to remove the kernel, wash the inner wall of the seed coat with water, dry in an oven at 55°C to constant weight, and crush through a 40-mesh sieve to obtain the seed coat powder of the cycad seeds for later use.

[0031] The test water was Wahaha bottled purified water, and the organic solvent was a conventional analytical grade solvent.

[0032] 2. Test methods and results

[0033] 1. Extraction of total polysaccharides from seed coat

[0034] Take 1.5kg of cycad seed coat powder, add 10L petroleum ether and heat reflux to defat for 3h, repeat defatting once, recover petroleum ether, and evaporate the residue until there is no ether smell. Add the defatted powder to 10L water, heat and extract twice at 85℃, the first extraction is 3h, the second extraction is 2h, combine the two extracts, filter, and concentrate under reduced pressure to 1 / 10 of the original volume. Slowly add 4 times the volume of anhydrous ethanol to the above concentrate, stir evenly, then move to a 4℃ refrigerator and let it stand overnight, centrifuge at 4000r / min for 10min, collect the precipitate, and freeze-dry to obtain the total polysaccharide of the seed coat.

[0035] 2. Grading and purification of total polysaccharides

[0036] Take the total polysaccharide of the seed coat, add appropriate amount of water, stir to dissolve, prepare a total polysaccharide solution of the seed coat with a mass fraction of 20%, centrifuge at 4000r / min for 10min, collect the supernatant to obtain supernatant A, slowly add anhydrous ethanol to the supernatant A to make the ethanol volume fraction reach 40%, move it into a 4°C refrigerator and let it stand overnight, centrifuge it at 4000r / min for 10min, collect the supernatant to obtain supernatant B (remove 40% alcohol-precipitated polysaccharides), slowly add anhydrous ethanol to the supernatant B to make the ethanol volume fraction reach 65%, move it into a 4°C refrigerator and let it stand overnight, centrifuge it at 4000r / min for 10min, collect the precipitate to obtain 65% alcohol-precipitated polysaccharides, and freeze-dry.

[0037] 3. Deproteinization and desalting

[0038] 65% of the alcohol-precipitated polysaccharide was re-dissolved in water, deproteinized using the Sevag method, dialyzed for desalination (molecular weight cut-off 1000D), and freeze-dried to obtain the cycad seed fractionated polysaccharide.

[0039] Example 2: Anti-neuritis activity of polysaccharides

[0040] 1. Test Materials

[0041] The fractionated polysaccharides from cycad seeds were prepared according to the method of Example 1.

[0042] Mouse microglial cells BV-2 were purchased from Shanghai Yaji Biotechnology Co., Ltd., product number YS054C.

[0043] DMEM high glucose medium and fetal bovine serum were purchased from Gibco, and MTT was purchased from Bio-Tech.

[0044] 2. Test methods and results

[0045] 1. Cell culture

[0046] BV-2 cells were inoculated in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, cultured at 37°C and 5% CO2, and the medium was replaced every 3 days.

[0047] 2. Selection of dosing concentration

[0048] When BV-2 cells were cultured to the logarithmic growth phase, the cells were digested into a single cell suspension and the cell concentration was adjusted to 1×10 4 / mL, 100 μL of diluted cell suspension was added to the wells of 96-well plate, and after culturing for 12 hours, 100 μL of complete medium containing cycad seed graded polysaccharides was added to each well of the polysaccharide group, so that the final concentration of cycad seed graded polysaccharides was 2.5 or 5 μg / mL, and 100 μL of complete medium without cycad seed graded polysaccharides was added to each well of the control group. A blank group without cells and polysaccharides was set up, and 5 duplicate wells were added to each group. Cultured for 24 hours at 37°C and 5% CO2, 20 μL of 5g / L MTT solution was added to each well, and cultured for 4 hours, the supernatant was discarded, 150 μL of DMSO was added to each well, and the absorbance value (A value) of each well at 490nm was measured by an enzyme reader, and the average value was taken, and the cell proliferation inhibition rate was calculated according to the following formula. Inhibition rate (%) = [1-(polysaccharide group A value-blank group A value)] / (control group A value-blank group A value) × 100%. The results are shown in Table 1.

[0049] Table 1 BV-2 cell proliferation inhibition rate (24h)

[0050] Polysaccharide concentration (μg / mL) Inhibition rate (%) 2.5 1.7±0.9 5 2.1±0.8

[0051] The results of this experiment showed that 2.5 and 5 μg / mL of cycad seed fractionated polysaccharides did not cause significant damage to BV-2 cells when cultured for 24 hours. The anti-neuritis activity was determined at 2.5 and 5 μg / mL.

[0052] 3. ELISA method to determine anti-neuritis activity

[0053] When BV-2 cells were cultured to the logarithmic growth phase, the cells were digested into a single cell suspension and the cell concentration was adjusted to 1×10 4 / mL, add 100 μL of the diluted cell suspension to the wells of a 96-well plate. After culturing for 12 h, add 100 μL of the corresponding culture medium to each well according to the following groups, with 3 replicates in each group.

[0054] Control group: add 100 μL complete medium;

[0055] Model group: 100 μL of complete medium containing lipopolysaccharide (LPS) was added, and the final concentration of LPS was 1 μg / mL;

[0056] Polysaccharide L group: 100 μL of complete medium containing LPS and fractionated polysaccharides from cycad seeds was added, with a final concentration of LPS of 1 μg / mL and a final concentration of fractionated polysaccharides from cycad seeds of 2.5 μg / mL;

[0057] Polysaccharide H group: 100 μL of complete culture medium containing LPS and fractionated polysaccharides from cycad seeds was added, with a final concentration of LPS of 1 μg / mL and a final concentration of fractionated polysaccharides from cycad seeds of 5 μg / mL.

[0058] After culturing for 24 h at 37°C and 5% CO2, the supernatant was aspirated and the release level of interleukin 6 (IL-6) was determined according to the instructions of the ELISA kit. The results are shown in Table 2 and Figure 1 shown.

[0059] Table 2 IL-6 release levels

[0060] Group IL-6 content (pg / mL) IL-6 release inhibition rate (%) Control group 52±26 / Model Group 3271±295 / Polysaccharide L group 1804±217 44.8％ Polysaccharide H group 558±143 82.9％

[0061] Neuroinflammation is a key factor in cognitive dysfunction and neurodegenerative diseases. Neuroinflammation is initiated when inflammatory stimuli trigger microglia in the central nervous system. In the brain, abnormal microglial activation accelerates the occurrence and development of neuroinflammatory responses. Inhibiting the inflammatory response of microglia is an important means of treating neuritis.

[0062] The test results show that the cycad seed fractionated polysaccharide prepared in Example 1 has an anti-neuritis effect and has the prospect of being developed into a drug for treating neuritis.

[0063] Example 3: Anti-glioma activity of polysaccharides

[0064] 1. Test Materials

[0065] The fractionated polysaccharides from cycad seeds were prepared according to the method of Example 1.

[0066] Human glioma cells U373 were purchased from Shanghai Yaji Biotechnology Co., Ltd., product number YS2275C.

[0067] RPMI 1640 culture medium and fetal bovine serum were purchased from Gibco, and MTT was purchased from Bio-Tech Biotechnology.

[0068] 2. Test methods

[0069] 1. Cell culture

[0070] U373 cells were inoculated in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, cultured at 37°C and 5% CO2, and the medium was replaced every 3 days.

[0071] 2. MTT assay to determine antitumor activity

[0072] When U373 cells reached the logarithmic growth phase, the cells were digested into a single cell suspension and the cell concentration was adjusted to 1×10 4 / mL, 100 μL of the diluted cell suspension was added to the wells of a 96-well plate. After culturing for 12 h, 100 μL of complete culture medium containing cycad seed fractionated polysaccharides was added to each well of the polysaccharide group, so that the final concentration of cycad seed fractionated polysaccharides was 5, 10 or 20 μg / mL, 100 μL of complete culture medium without cycad seed fractionated polysaccharides was added to each well of the control group, and a blank group without cells and polysaccharides was set up. Each group had 5 replicate wells, and the cells were cultured at 37°C and 5% CO2 for 48 h. 20 μL of 5g / L MTT solution was added to each well, and the culture was continued for 4 h. The supernatant was discarded, 150 μL DMSO was added to each well, and the cells were shaken at room temperature for 10 min. The absorbance value (A value) of each well at 490 nm was measured by an enzyme reader, and the average value was taken. The cell proliferation inhibition rate was calculated according to the following formula.

[0073] Inhibition rate (%) = [1-(A value of polysaccharide group-A value of blank group)] / (A value of control group-A value of blank group) × 100%

[0074] 3. Test results

[0075] The results are shown in Table 3 and Figure 2 As shown in the results, it can be seen that the fractionated polysaccharides from cycad seeds can effectively inhibit the proliferation of human glioma cells U373, and show an obvious dose-dependent effect.

[0076] Table 3 U373 cell proliferation inhibition rate (48h)

[0077] Polysaccharide concentration (μg / mL) Inhibition rate (%) 5 39.5±1.2 10 56.3±1.6 20 87.9±1.5

[0078] The test results show that the cycad seed fractionated polysaccharide prepared in Example 1 has a significant anti-glioma effect and has the prospect of being developed into a drug for treating glioma.

[0079] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A cycad seed polysaccharide, characterized in that: The product is prepared from the seed coat of cycad seeds through water extraction, alcohol precipitation, ethanol graded refining, deproteinization and desalination.

2. The cycad seed polysaccharide according to claim 1, characterized in that: The water extraction and alcohol precipitation step uses 4 times the volume of anhydrous ethanol for alcohol precipitation.

3. The cycad seed polysaccharide according to claim 1, characterized in that: The ethanol graded refining first removes 40% of the alcohol-precipitated polysaccharides, and then collects 65% of the alcohol-precipitated polysaccharides.

4. A method for preparing cycad seed polysaccharide according to any one of claims 1 to 3, characterized in that: Includes steps: Step S1, extraction of total polysaccharides from seed coat: taking an appropriate amount of seed coat powder of cycad seeds, adding an appropriate amount of petroleum ether for hot reflux defatting, recovering the petroleum ether, and volatilizing the residue until there is no ether smell; adding an appropriate amount of defatted powder to water, heating for extraction, filtering the extract, and concentrating under reduced pressure; slowly adding 4 times the volume of anhydrous ethanol to the above concentrate, stirring evenly, standing at low temperature overnight, centrifuging, collecting the precipitate, and freeze-drying to obtain total polysaccharides from seed coat; Step S2, fractional purification of total polysaccharides: taking seed coat total polysaccharides, adding appropriate amount of water, stirring and dissolving, preparing seed coat total polysaccharide solution, centrifuging, collecting supernatant to obtain supernatant A, slowly adding anhydrous ethanol to supernatant A to make the ethanol volume fraction reach 40%, standing at low temperature overnight, centrifuging, collecting supernatant to obtain supernatant B, slowly adding anhydrous ethanol to supernatant B to make the ethanol volume fraction reach 65%, standing at low temperature overnight, centrifuging, collecting precipitate to obtain 65% alcohol-precipitated polysaccharide, and freeze-drying; Step S3, deproteinization and desalting The 65% alcohol-precipitated polysaccharide was re-dissolved in water, deproteinized using the Sevag method, dialyzed for desalination, and freeze-dried to obtain the polysaccharide.

5. The preparation method according to claim 4, characterized in that: The temperature of the heating extraction in step S1 is 85°C.

6. The preparation method according to claim 4, characterized in that: The temperature of the low temperature overnight standing in step S1 and step S2 is 4°C.

7. The preparation method according to claim 4, characterized in that: The centrifugation parameters in step S1 and step S2 are both 4000 r / min for 10 min.

8. The preparation method according to claim 4, characterized in that: The mass fraction of the total polysaccharides in the seed coat in the total polysaccharide solution in step S2 is 20%.

9. Use of the cycad seed polysaccharide according to any one of claims 1 to 3 in the preparation of a medicament for treating neuritis.

10. Use of the cycad seed polysaccharide according to any one of claims 1 to 3 in the preparation of a medicament for treating glioma.

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