CN107661366B - Preparation method of gynostemma pentaphylla bioactive substance and human-derived stem cell active factor composition for preventing cell canceration - Google Patents
Preparation method of gynostemma pentaphylla bioactive substance and human-derived stem cell active factor composition for preventing cell canceration Download PDFInfo
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- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 6
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- 238000004440 column chromatography Methods 0.000 claims description 5
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
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Abstract
The invention discloses a preparation method of a gynostemma pentaphylla bioactive substance and human stem cell active factor composition for preventing cell canceration, which comprises the following operation steps of S1: preparing a gynostemma pentaphylla bioactive substance, S2: preparing human stem cell active factors. The preparation method provided by the invention is simple to operate, low in cost, suitable for industrial large-scale production and stable in process, and compared with the existing composition, the prepared gynostemma pentaphylla bioactive substance and human-derived stem cell active factor composition is more stable in drug effect and higher in activity, and has an effective effect of preventing cell canceration.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of a gynostemma pentaphylla bioactive substance and human stem cell active factor composition for preventing cell canceration.
Background
The herba Gynostemmatis is grass climbing plant of Gynostemma of Cucurbitaceae; thin and weak stem, with branches, longitudinal edges and grooves, without hair or with short and soft hair. Gynostemma pentaphylla likes the climate of yin-dampness and mildness, and is mostly wild in the shade places such as under the forest and the brook, and the gynostemma pentaphylla climbs the herbaceous plants for many years. The ginseng is mainly distributed in Hunan, Hubei, Yunnan, Guangxi provinces and the like in China, is called as 'southern ginseng', and the gynostemma pentaphylla growing in the south has higher medicinal content and is called as magical 'long-life herb' in folk.
The stem cells are the original cells. Stem cells are cells with proliferative and differentiative potential, have the ability to self-renew and replicate, and are capable of producing highly differentiated functional cells. The stem cells are the key field of the current medical research, and the bioactive factors generated by the secretion of the stem cells or bioactive substances in the stem cells are utilized to activate the stem cells of the organism, so that the cells with body injury, pathological changes and aging are physiologically repaired or replaced by the stem cells, and the stem cells have wide application prospects in the fields of disease prevention and treatment, health care and beauty.
At present, the composition prepared from the gynostemma pentaphylla bioactive substances and the human stem cell active factors is applied to the field of tumor treatment, but the composition prepared by the prior art has low activity and short validity period, and the popularization and the application of the composition are greatly limited.
Disclosure of Invention
In order to solve the above-mentioned problems, the present invention provides a method for preparing a composition of a gynostemma pentaphyllum bioactive substance and a human stem cell activator for preventing cell carcinogenesis.
The invention is realized by the following technical scheme.
A preparation method of a composition of a gynostemma pentaphylla bioactive substance and a human stem cell active factor for preventing cell canceration comprises the following operation steps:
s1: preparation of bioactive substance of Gynostemma pentaphyllum
Mixing dried gynostemma pentaphylla with a soluble soybean polysaccharide solution with the mass fraction of 3-7% according to the mass-volume ratio of 10-13kg:3L, standing at room temperature for 140min, and heating and refluxing the treated gynostemma pentaphylla by using ethanol to extract to obtain a first extracting solution and filter residue;
mixing the filter residue with 3-7% soluble soybean polysaccharide solution according to the mass volume ratio of 10-13kg:1L, and extracting with ethanol under heating and refluxing to obtain a second extractive solution;
mixing the two extractive solutions, concentrating under reduced pressure to obtain herba Gynostemmatis extract in thick paste form, and spray drying to obtain bioactive substances;
s2: preparation of human Stem cell active factor
After an umbilical cord envelope and an arteriovenous blood vessel are stripped, intermediate tissues are treated in a dividing mode, the intermediate tissues are cut into fragments with the size of 1mm multiplied by 1mm, the fragments are placed into cell culture solution for culture, when cells grow to 80% confluence, culture solution rich in stem cell bioactive factors is collected, wherein the cell culture solution is prepared from the following components in parts by weight: 87-92 parts of low-sugar DMEM medium, 0.01-0.03 part of isopropyl-beta-D-thiogalactoside, 0.1-0.5 part of sodium ascorbate, 4-6 parts of fetal bovine serum and 2-4 parts of diabesin;
adjusting the pH of the collected culture solution rich in the stem cell bioactive factors to 11.2-11.4 by using a sodium hydroxide solution, immediately adjusting the pH of the mixed solution to 6.5-7.0 by using a hydrochloric acid solution after 90-100s, placing the mixture in an environment at 14-18 ℃ for 10-12 hours, crushing, and performing column chromatography purification to obtain the human stem cell active factors;
s3: preparation of the composition
Mixing the gynostemma pentaphylla bioactive substance prepared from the S1 and the human stem cell active factor prepared from the S2 according to the mass ratio of 38-48:1, and preparing powder.
Specifically, in S1, the specific steps of heating and refluxing ethanol are as follows: mixing the treated gynostemma pentaphylla with an ethanol solution which is 40 times of the weight of the gynostemma pentaphylla and has a volume fraction of 75%, heating until the ethanol solution is boiled, continuing boiling for 120min, and finishing reflux extraction.
Specifically, in the above step S1, the inlet air temperature is 170-175 ℃ and the outlet air temperature is 65-70 ℃ during spray drying.
Specifically, in S2, the temperature of the culture was 37 ℃, the volume fraction of carbon dioxide was 7%, and the relative humidity was 95% during the cell culture.
Specifically, the concentration of the sodium hydroxide solution is 0.5mol/L, and the mass fraction of the hydrochloric acid solution is 5%.
According to the technical scheme, the beneficial effects of the invention are as follows:
the preparation method provided by the invention is simple to operate, low in cost, suitable for industrial large-scale production and stable in process, and compared with the existing composition, the prepared gynostemma pentaphylla bioactive substance and human-derived stem cell active factor composition is more stable in drug effect and higher in activity, and has an effective effect of preventing cell canceration. The active ingredients in the gynostemma pentaphylla are easy to inactivate in the distillation and extraction process, and in the invention, the soluble soybean polysaccharide solution is used for soaking the gynostemma pentaphylla, so that the inactivation of some protein active substances in the gynostemma pentaphylla can not occur in the extraction process, and the activity of the prepared gynostemma pentaphylla extract is effectively ensured; according to the invention, sodium ascorbate is added into the cell culture solution, so that the hereditary stability of the cells in the subculture process can be effectively improved, and the growth speed and proliferation capacity of the cells are improved; according to the invention, the pH adjustment operation is carried out on the prepared culture solution rich in the stem cell bioactive factors, so that the spatial structure of protein substances in the culture solution rich in the stem cell bioactive factors can be effectively more stable, and the drug effect and the stability of the composition are effectively ensured.
Detailed Description
For those skilled in the art to further understand the features and technical content of the present invention, refer to the following detailed description of the present invention.
Example 1
A preparation method of a composition of a gynostemma pentaphylla bioactive substance and a human stem cell active factor for preventing cell canceration comprises the following operation steps:
s1: preparation of bioactive substance of Gynostemma pentaphyllum
Mixing dried gynostemma pentaphylla with a soluble soybean polysaccharide solution with the mass fraction of 3% according to the mass-volume ratio of 10kg to 3L, standing at room temperature for 120min, and performing heating reflux extraction on the treated gynostemma pentaphylla by using ethanol to prepare a first extracting solution and filter residue;
mixing the filter residue and a soluble soybean polysaccharide solution with the mass fraction of 3% according to the mass-volume ratio of 10kg to 1L, and heating and refluxing the mixture by adopting ethanol to extract to obtain a second extracting solution;
mixing the two extractive solutions, concentrating under reduced pressure to obtain herba Gynostemmatis extract in thick paste form, and spray drying to obtain bioactive substances;
s2: preparation of human Stem cell active factor
After an umbilical cord envelope and an arteriovenous blood vessel are stripped, intermediate tissues are treated in a dividing mode, the intermediate tissues are cut into fragments with the size of 1mm multiplied by 1mm, the fragments are placed into cell culture solution for culture, when cells grow to 80% confluence, culture solution rich in stem cell bioactive factors is collected, wherein the cell culture solution is prepared from the following components in parts by weight: 87 parts of low-sugar DMEM medium, 0.01 part of isopropyl-beta-D-thiogalactoside, 0.1 part of sodium ascorbate, 4 parts of fetal calf serum and 2 parts of diabesin;
adjusting the pH of the collected culture solution rich in the stem cell bioactive factors to 11.2 by using a sodium hydroxide solution, immediately adjusting the pH of the mixed solution to 6.5 by using a hydrochloric acid solution after 90s, placing the mixture in an environment at 14 ℃ for 10 hours, and then crushing and purifying by column chromatography to obtain the human-derived stem cell bioactive factors;
s3: preparation of the composition
Mixing the gynostemma pentaphylla bioactive substance prepared from the S1 and the human stem cell active factor prepared from the S2 according to the mass ratio of 38:1 to prepare powder.
Specifically, in S1, the specific steps of heating and refluxing ethanol are as follows: mixing the treated gynostemma pentaphylla with an ethanol solution which is 40 times the weight of the gynostemma pentaphylla and has a volume fraction of 75%, heating until the ethanol solution boils, continuing boiling for 100min, and performing reflux extraction to finish.
Specifically, in S1, the inlet air temperature is 170 ℃ and the outlet air temperature is 65 ℃ during spray drying.
Specifically, in S2, the temperature of the culture was 37 ℃, the volume fraction of carbon dioxide was 7%, and the relative humidity was 95% during the cell culture.
Specifically, the concentration of the sodium hydroxide solution is 0.5mol/L, and the mass fraction of the hydrochloric acid solution is 5%.
Example 2
A preparation method of a composition of a gynostemma pentaphylla bioactive substance and a human stem cell active factor for preventing cell canceration comprises the following operation steps:
s1: preparation of bioactive substance of Gynostemma pentaphyllum
Mixing dried gynostemma pentaphylla with a soluble soybean polysaccharide solution with the mass fraction of 5% according to the mass-volume ratio of 12kg to 3L, standing at room temperature for 130min, and performing heating reflux extraction on the treated gynostemma pentaphylla by using ethanol to prepare a first extracting solution and filter residue;
mixing the filter residue and a soluble soybean polysaccharide solution with the mass fraction of 5% according to the mass-volume ratio of 12kg to 1L, and heating and refluxing the mixture by adopting ethanol to extract to obtain a second extracting solution;
mixing the two extractive solutions, concentrating under reduced pressure to obtain herba Gynostemmatis extract in thick paste form, and spray drying to obtain bioactive substances;
s2: preparation of human Stem cell active factor
After an umbilical cord envelope and an arteriovenous blood vessel are stripped, intermediate tissues are treated in a dividing mode, the intermediate tissues are cut into fragments with the size of 1mm multiplied by 1mm, the fragments are placed into cell culture solution for culture, when cells grow to 80% confluence, culture solution rich in stem cell bioactive factors is collected, wherein the cell culture solution is prepared from the following components in parts by weight: 90 parts of low-sugar DMEM medium, 0.02 part of isopropyl-beta-D-thiogalactoside, 0.3 part of sodium ascorbate, 5 parts of fetal calf serum and 3 parts of diabesin;
adjusting the pH of the collected culture solution rich in the stem cell bioactive factors to 11.3 by using a sodium hydroxide solution, immediately adjusting the pH of the mixed solution to 6.8 by using a hydrochloric acid solution after 95s, placing the mixture in an environment at the temperature of 16 ℃ for 11 hours, and then crushing and purifying by column chromatography to obtain the human stem cell bioactive factors;
s3: preparation of the composition
Mixing the gynostemma pentaphylla bioactive substance prepared from the S1 and the human stem cell active factor prepared from the S2 according to the mass ratio of 44:1 to prepare powder.
Specifically, in S1, the specific steps of heating and refluxing ethanol are as follows: mixing the treated gynostemma pentaphylla with an ethanol solution which is 40 times the weight of the gynostemma pentaphylla and has a volume fraction of 75%, heating until the ethanol solution boils, continuing boiling for 110min, and performing reflux extraction to finish.
Specifically, in S1, the inlet air temperature is 173 ℃ and the outlet air temperature is 68 ℃ during spray drying.
Specifically, in S2, the temperature of the culture was 37 ℃, the volume fraction of carbon dioxide was 7%, and the relative humidity was 95% during the cell culture.
Specifically, the concentration of the sodium hydroxide solution is 0.5mol/L, and the mass fraction of the hydrochloric acid solution is 5%.
Example 3
A preparation method of a composition of a gynostemma pentaphylla bioactive substance and a human stem cell active factor for preventing cell canceration comprises the following operation steps:
s1: preparation of bioactive substance of Gynostemma pentaphyllum
Mixing dried gynostemma pentaphylla with a soluble soybean polysaccharide solution with the mass fraction of 7% according to the mass-volume ratio of 13kg to 3L, standing at room temperature for 140min, and performing heating reflux extraction on the treated gynostemma pentaphylla by using ethanol to prepare a first extracting solution and filter residue;
mixing the filter residue and a soluble soybean polysaccharide solution with the mass fraction of 7% according to the mass-volume ratio of 13kg to 1L, and heating and refluxing the mixture by adopting ethanol to extract to obtain a second extracting solution;
mixing the two extractive solutions, concentrating under reduced pressure to obtain herba Gynostemmatis extract in thick paste form, and spray drying to obtain bioactive substances;
s2: preparation of human Stem cell active factor
After an umbilical cord envelope and an arteriovenous blood vessel are stripped, intermediate tissues are treated in a dividing mode, the intermediate tissues are cut into fragments with the size of 1mm multiplied by 1mm, the fragments are placed into cell culture solution for culture, when cells grow to 80% confluence, culture solution rich in stem cell bioactive factors is collected, wherein the cell culture solution is prepared from the following components in parts by weight: 92 parts of low-sugar DMEM medium, 0.03 part of isopropyl-beta-D-thiogalactoside, 0.5 part of sodium ascorbate, 6 parts of fetal calf serum and 4 parts of diabesin;
adjusting the pH of the collected culture solution rich in the stem cell bioactive factors to 11.4 by using a sodium hydroxide solution, immediately adjusting the pH of the mixed solution to 7.0 by using a hydrochloric acid solution after 100s, placing the mixture in an environment at 18 ℃ for 12 hours, and then crushing and purifying by column chromatography to obtain the human-derived stem cell bioactive factors;
s3: preparation of the composition
Mixing the gynostemma pentaphylla bioactive substance prepared from the S1 and the human stem cell active factor prepared from the S2 according to the mass ratio of 48:1 to prepare powder.
Specifically, in S1, the specific steps of heating and refluxing ethanol are as follows: mixing the treated gynostemma pentaphylla with an ethanol solution which is 40 times the weight of the gynostemma pentaphylla and has a volume fraction of 75%, heating until the ethanol solution boils, continuing boiling for 120min, and performing reflux extraction to finish.
Specifically, in S1, the inlet air temperature is 175 ℃ and the outlet air temperature is 70 ℃ during spray drying.
Specifically, in S2, the temperature of the culture was 37 ℃, the volume fraction of carbon dioxide was 7%, and the relative humidity was 95% during the cell culture.
Specifically, the concentration of the sodium hydroxide solution is 0.5mol/L, and the mass fraction of the hydrochloric acid solution is 5%.
Comparative example 1
The remaining procedure was exactly the same as in example 1 except that no soluble soybean polysaccharide solution was added to S1.
Comparative example 2
In S2, the procedure was exactly the same as in example 1 except that the pH of the culture broth rich in the stem cell bioactive factor was not adjusted.
The compositions of the bioactive substances of gynostemma pentaphyllum and the human stem cell activator were prepared by the methods of the examples and comparative examples, respectively, and then the antitumor effect test was performed, 80 male Kunming mice with the same growth conditions were selected as test mice, the average body weight was 20g, the mice were randomly and averagely divided into 8 groups, and H was established for the mice in the test groups 1 to 7 according to the method of Chimengyi et al (2016) research on antitumor activity of medicinal fomes fomentarius sporophore extract22Tumor-bearing mouse model, wherein test groups 1, 2 and 3 respectively use the composition of the bioactive substance of gynostemma pentaphyllum and the human stem cell active factor prepared in examples 1, 2 and 3, mice in test groups 4 and 5 respectively use the composition of the bioactive substance of gynostemma pentaphyllum and the human stem cell active factor prepared in comparative examples 1 and 2, and test group 6 uses the commercially available bioactive substance of gynostemma pentaphyllum and the human stem cell active factorComposition, test group 7 was normal saline group, test group 8 was normal mice without molding as blank control group, 1g of composition was dissolved in 10ml of water and then administered to stomach on day 2 after molding, 10d of continuous administration was given, water was not prohibited after one night fasting before dissection, mice were sacrificed after blood was taken from the eye canthus on day 11, tumor mass was dissected, tumor mass was taken, weighed and recorded, tumor inhibition rate was (average tumor weight of test group 7 mice-average tumor weight of administered group)/average tumor weight of test group 7 mice 100%, test results are shown in table 1:
TABLE 1 antitumor Effect test of compositions
As can be seen from table 1, the composition of the bioactive substance of gynostemma pentaphylla and the human stem cell activator prepared by the present invention has strong activity and more significant effect of preventing cell canceration.
It should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and are not limited. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the present invention.
Claims (5)
1. A preparation method of a composition of a gynostemma pentaphylla bioactive substance and a human stem cell active factor for preventing cell canceration is characterized by comprising the following operation steps:
s1: preparation of bioactive substance of Gynostemma pentaphyllum
Mixing dried gynostemma pentaphylla with a soluble soybean polysaccharide solution with the mass fraction of 3-7% according to the mass-volume ratio of 10-13kg:3L, standing at room temperature for 140min, and heating and refluxing the treated gynostemma pentaphylla by using ethanol to extract to obtain a first extracting solution and filter residue;
mixing the filter residue with 3-7% soluble soybean polysaccharide solution according to the mass volume ratio of 10-13kg:1L, and extracting with ethanol under heating and refluxing to obtain a second extractive solution;
mixing the two extractive solutions, concentrating under reduced pressure to obtain herba Gynostemmatis extract in thick paste form, and spray drying to obtain bioactive substances;
s2: preparation of human Stem cell active factor
After an umbilical cord envelope and an arteriovenous blood vessel are stripped, intermediate tissues are treated in a dividing mode, the intermediate tissues are cut into fragments with the size of 1mm multiplied by 1mm, the fragments are placed into cell culture solution for culture, when cells grow to 80% confluence, culture solution rich in stem cell bioactive factors is collected, wherein the cell culture solution is prepared from the following components in parts by weight: 87-92 parts of low-sugar DMEM medium, 0.01-0.03 part of isopropyl-beta-D-thiogalactoside, 0.1-0.5 part of sodium ascorbate, 4-6 parts of fetal bovine serum and 2-4 parts of diabesin;
adjusting the pH of the collected culture solution rich in the stem cell bioactive factors to 11.2-11.4 by using a sodium hydroxide solution, immediately adjusting the pH of the mixed solution to 6.5-7.0 by using a hydrochloric acid solution after 90-100s, placing the mixture in an environment at 14-18 ℃ for 10-12 hours, crushing, and performing column chromatography purification to obtain the human stem cell active factors;
s3: preparation of the composition
Mixing the gynostemma pentaphylla bioactive substance prepared from the S1 and the human stem cell active factor prepared from the S2 according to the mass ratio of 38-48:1, and preparing powder.
2. The method for preparing a composition of a bioactive substance of gynostemma pentaphyllum and an active factor of human stem cells for preventing cell carcinogenesis according to claim 1, wherein the ethanol reflux-heating extraction in S1 comprises the following specific steps: mixing the treated gynostemma pentaphylla with an ethanol solution which is 40 times of the weight of the gynostemma pentaphylla and has a volume fraction of 75%, heating until the ethanol solution is boiled, continuing boiling for 120min, and finishing reflux extraction.
3. The method for preparing the composition of bioactive substances of gynostemma pentaphyllum and human stem cell activator for preventing cell canceration as claimed in claim 1, wherein the air inlet temperature is 170-175 ℃ and the air outlet temperature is 65-70 ℃ during the spray drying in S1.
4. The method for producing a composition of a bioactive substance derived from Gynostemma pentaphyllum and a human stem cell activator according to claim 1, wherein the culture temperature of the cells in S2 is 37 ℃, the volume fraction of carbon dioxide is 7%, and the relative humidity is 95%.
5. The method for producing a composition of a bioactive substance derived from Gynostemma pentaphyllum and a human stem cell factor (hSCF) for preventing cell carcinogenesis according to claim 1, wherein the concentration of the sodium hydroxide solution is 0.5mol/L and the mass fraction of the hydrochloric acid solution is 5%.
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