CN115894735B - Eagle tea acidic polysaccharide, extraction and purification method and anticancer application thereof - Google Patents
Eagle tea acidic polysaccharide, extraction and purification method and anticancer application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The application discloses hawk tea acidic polysaccharide HTPA3 in the technical field of medicines, which mainly comprises mannose: rhamnose: glucose: galactose: xylose: the arabinose composition comprises 1% -5%:2% -6%:5% -10%:15% -20%:3% -8%:55% -65%. Proved by researches, the hawk tea total polysaccharide and the hawk tea acidic polysaccharide HTPA3 have the activities of resisting lung cancer and colon cancer, and the effect of the hawk tea acidic polysaccharide HTPA3 is more remarkable.
Description
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to an hawk tea acidic polysaccharide, an extraction and purification method and an anticancer application thereof.
Background
The development of lung cancer is a serious threat to human health worldwide.
Among treatments of cancers at various stages, chemotherapy plays an important role and has become a main treatment mode for cancer. However, the chemotherapeutic drugs have stronger toxic and side effects, which affects the life quality of patients, so that it is necessary to develop new anticancer drugs with good curative effect and low toxic and side effects or drugs with attenuation and synergy effects on traditional chemotherapeutic drugs. Therefore, the separation and screening of nontoxic or low-toxicity natural anti-tumor active ingredients in the traditional Chinese herbal medicines has important significance.
Hawk tea is prepared from leaves or branches and leaves of Cinnamomum camphora of Litsea of Lauraceae, and plant name Mao Baopi. Is very common in Sichuan, chongqing, yunnan, guizhou, anhui and the like, has long drinking history and no toxic or side effect, and is a medicinal and edible plant. At present, the research and utilization of the active ingredients of the hawk tea are mainly focused on flavonoids. The main pharmacological activities include antioxidation, anti-inflammatory, liver protection, blood sugar reduction, antibiosis and the like. Polysaccharides are widely found in animals, plants and microorganisms. As one of the constituent components of living matters, it is widely involved in the regulation of various life phenomena and physiological processes of cells, such as the transmission and feeling of information among immune cells, the transformation, division and regeneration of cells. The polysaccharide reported so far has more than 100 kinds of physiological activities such as antivirus, anti-infection, anti-tumor, antioxidation, anti-radiation, blood sugar reduction, liver protection, immunity regulation and the like, and has small side effect. At present, the report of the hawk tea polysaccharide is mainly focused on the research of the antioxidant function, and the report of the anti-cancer activity of the hawk tea polysaccharide is rarely reported. Therefore, it is very necessary to develop products for anticancer which can fully exert the effect of hawk tea polysaccharide.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides hawk tea acidic polysaccharide HTPA3 with better activity on colon cancer and lung cancer.
The invention aims at providing hawk tea acidic polysaccharide HTPA3, which mainly comprises mannose: rhamnose: glucose: galactose: xylose: the arabinose composition comprises 1% -5%:2% -6%:5% -10%:15% -20%:3% -8%:55% -65%.
The second purpose of the invention is to provide an extraction and purification method of hawk tea acidic polysaccharide HTPA3, which comprises the following steps:
Step S1, preparing crude polysaccharide: weighing hawk tea, adding water, heating for extraction, filtering, concentrating the filtrate under reduced pressure, centrifuging, collecting supernatant, dialyzing the supernatant, adding absolute ethyl alcohol, precipitating with ethanol overnight, centrifuging, adding distilled water for dissolving precipitate, concentrating under reduced pressure for volatilizing ethanol, and lyophilizing the concentrated solution to obtain total polysaccharides of hawk tea;
Step S2, grading: weighing total polysaccharides of Litsea coreana, adding appropriate amount of distilled water for dissolving to obtain crude polysaccharide solution with mass concentration of 30mg/ml, centrifuging at 10000r/min for 10min, collecting supernatant, eluting with DEAE sepharose fast flow anion exchange chromatography column, sequentially eluting with distilled water, 0.1mol/L, 0.22mol/L and 0.32mol/L NaCl solution, collecting 0.32mol/LNaCl solution eluate, dialyzing, and lyophilizing to obtain acidic polysaccharide HTPA3 of Litsea coreana.
Further, the specific operation of heating and extracting in the step S1 is as follows: weighing hawk tea, adding 15 times of water for decocting in boiling water for 3.5 hours, collecting filtrate, adding 10 times of water for decocting in boiling water for 3.5 hours for the third time, adding 10 times of water for decocting in boiling water for 3.5 hours for the fourth time, and decocting in boiling water for 3.5 hours.
Further, the filtrate obtained in the step S1 was heated, extracted and filtered, and the concentrated solution was concentrated under reduced pressure, and dialyzed with a 3000D dialysis bag for 48 hours.
Further, in step S2, naCl solutions with different concentrations are eluted for 3 times of column volume.
Further, the lyophilization time of each fraction of the hawk tea polysaccharide in step S2 was 94h.
The invention further aims to provide an application of the hawk tea acidic polysaccharide HTPA3 in preparing medicines for resisting lung cancer and colon cancer.
The hawk tea total polysaccharide is obtained by taking hawk tea dry leaves as raw materials and adopting a water extraction and alcohol precipitation method, and is purified by adopting DEAE sepharose fast flow anion exchange chromatography, and 4 fractions of neutral hawk tea polysaccharide HTPN, hawk tea acidic polysaccharide HTPA1, hawk tea acidic polysaccharide HTPA2 and hawk tea acidic polysaccharide HTPA3 are obtained after dialysis purification and freeze drying.
The influence of the hawk tea acid polysaccharide HTPA3 on the in-vitro proliferation of human lung cancer A549 cells and human colon cancer cells HCT-116 is measured, and the hawk tea acid polysaccharide HTPA3 can obviously inhibit the proliferation of human lung cancer A549 cells and human colon cancer HCT-116 and has small toxic and side effects, so that the hawk tea acid polysaccharide HTPA3 can be used as an important drug intermediate for developing new drugs for resisting lung cancer and colon cancer and a natural raw material of functional health food.
Drawings
FIG. 1 is a schematic diagram of a gradient elution curve of total polysaccharides of hawk tea;
FIG. 2 is a schematic diagram of elution curve of the fixed concentration of total polysaccharides of hawk tea;
FIG. 3 is a schematic diagram of the composition of the hawk tea acid polysaccharide HTPA3 monosaccharide;
FIG. 4 is a schematic diagram showing the effect of hawk tea total polysaccharide on human lung cancer A549 cell proliferation in vitro;
FIG. 5 is a schematic diagram showing the effect of various polysaccharides of hawk tea on the proliferation of human lung cancer A549 cells in vitro;
FIG. 6 is a schematic representation of the effect of hawk tea total polysaccharide on human colon cancer HCT-116 cell proliferation in vitro;
FIG. 7 is a schematic representation of the effect of various polysaccharides from Litsea coreana on the in vitro proliferation of human colon cancer HCT-116 cells.
Detailed Description
The following is a further detailed description of the embodiments:
example 1 extraction method of Litsea coreana acidic polysaccharide HTPA3
1.1 Extraction of total polysaccharides of hawk tea:
Preparation of hawk tea fractionated polysaccharide
Extracting total polysaccharides of hawk tea: 1kg of hawk tea is weighed, 15 times of water is added for decoction for 3.5 hours in the first time, filtrate is collected, 10 times of water is added for decoction for 3.5 hours in the second time, 10 times of water is added for decoction for 3.5 hours in the third time, 10 times of water is added for decoction for 3.5 hours in the fourth time, and 3.5 hours is added for decoction in the boiling water. Combining four filtrates, concentrating under reduced pressure at 80deg.C to 1.5L, dialyzing with 3000D dialysis bag for 48 hr, centrifuging at 4000rpm for 15min, and collecting supernatant. Slowly adding four times of absolute ethyl alcohol, stirring while adding, standing at room temperature overnight, and centrifuging at 4000rpm for 15min to obtain precipitate. Re-dissolving and precipitating with distilled water, concentrating and volatilizing ethanol at 50deg.C under reduced pressure, packaging, and lyophilizing to obtain 10g of total polysaccharides of Litsea coreana.
1.2DEAE sepharose fast flow purification of crude polysaccharide by anion exchange chromatography:
Weighing 3g of total polysaccharide of hawk tea, adding 100mL of distilled water for dissolution to prepare a total polysaccharide solution with the mass concentration of 30mg/mL, centrifuging at 10000rpm for 10min, taking supernatant, grading by DEAE sepharose fast flow column chromatography (column volume of 2.5L), eluting with distilled water, 0.1mol/L, 0.22mol/L and 0.32mol/L NaCl solution for 3 times of column volume in sequence, respectively collecting eluent of NaCl solutions with different concentrations, dialyzing, and freeze-drying to obtain the hawk tea fractionated polysaccharide. Wherein, the fraction eluted with 0.32mol/L NaCl solution was HTPA3, and 159mg was obtained after lyophilization.
Example 2 monosaccharide composition analysis of the eagle tea acid polysaccharide HTPA3
HPLC conditions: the mobile phase composition was acetonitrile: phosphate buffer = 18.2:81.8. Detector model: varian Prostar325, detection wavelength: 245nm. Chromatographic column model: thermo ScientificODS-2 (c 18) Hypersil dim (mm) 150×4.6; column temperature: 35 ℃. Sample injection amount: 20 μl, flow rate: 1mL/min.
Sample pretreatment:
(1) Acid hydrolysis: a4 mg sample of the hawk tea acid polysaccharide HTPA3 was weighed into a brown vial and dissolved in 2mL4M trifluoroacetic acid (TFA) and capped and sealed. The reaction mixture was heated to hydrolyze at 110℃for 4 hours, taken out and cooled to room temperature, concentrated under reduced pressure using methanol, and the operation was repeated until TFA was removed. After evaporation to dryness, the residue was dissolved in 200. Mu.L of distilled water.
(2) PMP derivatization: 100. Mu.L of the completely hydrolyzed solution of (1) was added with 100. Mu.L of 0.6M NaOH solution, mixed well, and 200. Mu.L of the now prepared 0.5M PMP methanol solution was added, sealed and vortexed well. After reacting for 100min at 70 ℃ in water bath, cooling to room temperature, neutralizing with 200 mu L of HCl with the concentration of 0.3M, and adding distilled water to make up to 1mL. And adding 1mL of chloroform for extraction, taking out an upper water phase, repeating the extraction for a plurality of times, and removing PMP as much as possible. After vortexing 15 μl of supernatant was sampled for analysis.
(3) 100. Mu.L of a 1mg/mL mixture of monosaccharide standards (Man, rha, gal, galA, glc, glcA, xyl, ara, fuc) were taken, treated in the same manner as the PMP derivatization of the sample, and then analyzed by sample injection.
The results show that eagle tea acid polysaccharide HTPA3 consists essentially of mannose: rhamnose: glucose: galactose: xylose: the arabinose composition comprises 1% -5%:2% -6%:5% -10%:15% -20%:3% -8%:55% -65%.
Example 3 experiment of the Activity of various fractions of Litsea Coreana polysaccharide against lung cancer and colon cancer
Human lung cancer A549 cells and human colon cancer HCT-116 cells are cultivated in DMEM complete medium, are cultivated in an adherence way in an incubator with 37 ℃ and 5% CO2 saturated humidity, are changed in liquid every other day, and are digested with 0.25% trypsin for passage after the degree of fusion reaches 90% after observation under a microscope.
After 3 passages of human lung cancer A549 cells and human colon cancer HCT-116 cells, the cells in logarithmic phase are digested by trypsin, counted, inoculated into 96 well plates at the density of 5 multiplied by 10 3 cells per hole, cultivated for 16 hours, after the cells are completely adhered, the total polysaccharides of hawk tea and the classified polysaccharides of hawk tea with different concentrations are respectively added, a DMEM culture medium is added into a control group, 5 compound holes are arranged in parallel, cultivated for 48 hours, 5g/L MTT solution is added into each hole, 20 μl is added into the culture box at 37 ℃, incubated for 4 hours, 150 μl of DMSO is added, and the upper chamber of an oscillator is shaken for 20 minutes, so that the crystals are fully dissolved, and the absorbance value of each hole is measured at the wavelength of 490nm of an automatic enzyme-labeling instrument. And calculating the survival rate of the A549 cell strain and the HCT-116 cell strain after different medicines and different concentrations of medicines are added. Survival (%) = (control absorbance value-experimental absorbance value)/control absorbance value x 100%.
All experiments used 5 replicates as statistical results. One-way anova with GRAPHPAD PRISM.0.1 software was used to evaluate group differences. Differences were statistically significant at P <0.05, < P <0.01, < P < 0.0001.
According to the MTT result, the hawk tea total polysaccharide has remarkable inhibition effect on lung cancer A549 cells and colon cancer HCT-116 cells, and the concentration is increased. As shown in fig. 4 and 6.
The hawk tea acidic polysaccharide HTPA3 has remarkable inhibition effect on lung cancer A549 cells and colon cancer HCT-116 cells, and the concentration is increased. And the activity is more obvious than the high inhibition effect of total sugar. As shown in fig. 5 and 7.
The hawk tea acidic polysaccharide HTPA3 is a natural plant extract, has small toxic and side effects on human bodies, and has remarkable inhibition effect on proliferation of lung cancer A549 cells and colon cancer HCT-116 cells. The hawk tea acidic HTPA3 can be used as an important drug intermediate for developing new drugs, and a natural raw material of functional health-care food.
The foregoing is merely exemplary embodiments of the present application, and specific structures and features that are well known in the art are not described in detail herein. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present application, and these should also be considered as the scope of the present application, which does not affect the effect of the implementation of the present application and the utility of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (7)
1. Hawk tea acid polysaccharide HTPA3 is characterized by mainly comprising mannose: rhamnose: glucose: galactose: xylose: the arabinose composition comprises 1% -5%:2% -6%:5% -10%:15% -20%:3% -8%:55% -65%, and the preparation method comprises the following steps:
Step S1, preparing crude polysaccharide: weighing hawk tea, adding water, heating for extraction, filtering, concentrating the filtrate under reduced pressure, centrifuging, collecting supernatant, dialyzing the supernatant, adding absolute ethyl alcohol, precipitating with ethanol overnight, centrifuging, adding distilled water for dissolving precipitate, concentrating under reduced pressure for volatilizing ethanol, and lyophilizing the concentrated solution to obtain total polysaccharides of hawk tea;
Step S2, grading: weighing total polysaccharides of Litsea coreana, adding appropriate amount of distilled water for dissolving to obtain crude polysaccharide solution with mass concentration of 30 mg/ml, centrifuging at 10000 r/min for 10min, collecting supernatant, subjecting to DEAE sepharose fast flow anion exchange chromatography column, sequentially eluting with distilled water, 0.1 mol/L, 0.22 mol/L and 0.32 mol/L NaCl solution, collecting 0.32 mol/L NaCl solution eluate, dialyzing, and lyophilizing to obtain acidic polysaccharide HTPA3 of Litsea coreana.
2. The hawk tea acid polysaccharide HTPA3 according to claim 1, wherein the specific heating and extracting operation in the step S1 is as follows: weighing hawk tea, adding 15 times of water for the first time, decocting with boiling water to 3.5 h, collecting filtrate, adding 10 times of water for the second time, decocting with boiling water to 3.5 h, adding 10 times of water for the third time, decocting with boiling water to 3.5 h, adding 10 times of water for the fourth time, and decocting with boiling water to 3.5 h.
3. The hawk tea acid polysaccharide HTPA3 according to claim 2, wherein the concentrated solution obtained by heating, extracting and filtering the filtrate in the step S1 and concentrating the filtrate under reduced pressure is dialyzed 48h by a 3000D dialysis bag.
4. An eagle tea acid polysaccharide HTPA3 according to claim 3 wherein in step S2, the NaCl solutions of different concentrations are eluted 3 column volumes.
5. The hawk tea acid polysaccharide HTPA3 of claim 4, wherein the lyophilization time of each fraction of hawk tea polysaccharide in step S2 is 94 h.
6. The use of the hawk tea acid polysaccharide HTPA3 according to claim 1 in the preparation of an anti-lung cancer medicament.
7. The use of hawk tea acid polysaccharide HTPA3 according to claim 1 in the preparation of an anti-colon cancer medicament.
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| CN1522753A (en) * | 2003-09-11 | 2004-08-25 | 安徽医科大学 | Use of glede tea aqueous extract in preparation of diabetes preventing and curing drug |
| KR100602841B1 (en) * | 2005-02-28 | 2006-07-19 | 고려대학교 산학협력단 | Acidic polysaccharide with cell-binding inhibition activity isolated from green tea and its composition for preventing and treating gastrointestinal diseases |
| CN104982540A (en) * | 2015-07-20 | 2015-10-21 | 刘冬 | Maturative agent for hawk tea golden flower cheese and preparation method of maturative agent |
| CN113717296A (en) * | 2021-09-28 | 2021-11-30 | 遵义医科大学 | Eucommia acidic polysaccharide, extraction method and application of eucommia acidic polysaccharide in preparation of anti-colon cancer drugs |
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| CN1522753A (en) * | 2003-09-11 | 2004-08-25 | 安徽医科大学 | Use of glede tea aqueous extract in preparation of diabetes preventing and curing drug |
| KR100602841B1 (en) * | 2005-02-28 | 2006-07-19 | 고려대학교 산학협력단 | Acidic polysaccharide with cell-binding inhibition activity isolated from green tea and its composition for preventing and treating gastrointestinal diseases |
| CN104982540A (en) * | 2015-07-20 | 2015-10-21 | 刘冬 | Maturative agent for hawk tea golden flower cheese and preparation method of maturative agent |
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