CN114224936B - Extraction and purification process of effective part of white ginseng prescription for increasing leucocyte - Google Patents
Extraction and purification process of effective part of white ginseng prescription for increasing leucocyte Download PDFInfo
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Abstract
The invention relates to an extraction and purification process of an effective part of a white ginseng formula for increasing leucocytes, in particular to a white ginseng formula consisting of 4 medicinal materials of codonopsis pilosula, tremella, astragalus and gynostemma pentaphylla according to a certain dosage proportion, wherein the codonopsis pilosula, tremella, astragalus and gynostemma pentaphylla are weighed according to the prescription amount, soaked for 30min, added with 20 times of water for 3 times, each time for 4h, filtered, combined with filtrate, concentrated until each 1ml of liquid medicine contains 1g of crude medicinal material, added with 4 times of 95% ethanol, kept stand overnight, filtered, filter cake dried to obtain purified total polysaccharide, the ethanol filtrate is combined with gynostemma pentaphylla, supplemented with ethanol for adjusting to 8 times of 80% ethanol, soaked for 30min, extracted for 2h under reflux, filtered, residue is extracted for 1 time again by the same method, combined with filtrate, concentrated until each 1ml of liquid medicine contains 1g of crude medicinal material, added with 3 times of n-butanol for 3 times of extraction, a n-butanol layer is taken, n-butanol is recovered, and dried to obtain total saponins; the total polysaccharide and the total saponin are combined to obtain the effective part extract of the ginseng and baifang for increasing the white blood cells, which lays a foundation for the research of the ginseng and baifang preparation.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, and relates to an extraction and purification process of an effective part of a white ginseng formula for increasing leucocytes.
Background
With the increasing deterioration of the environment, together with the increase in the number of people with lifestyle and eating disorders, cancer has become one of the major diseases that endanger the health of people. According to the statistics of the world health organization Globocan, about 1929 thousands of cancer cases occur globally in 2020, and the number of deaths reaches 995 thousands, wherein about 24% and 30% of China are respectively higher than the global average level. The treatment of cancer mainly comprises radiotherapy, chemotherapy and tumor surgery, but no matter which treatment means is adopted, the cancer can cause damage to normal parts or cells of an organism, and local or systemic adverse reactions such as nausea, vomiting, alopecia, bone marrow suppression and the like occur, and the most common phenomenon is the leucopenia shown in the bone marrow suppression.
The traditional Chinese medicine classifies the leucopenia into 'consumptive disease' and 'blood deficiency', patients treated by radiotherapy and chemotherapy mostly show spleen and kidney deficiency symptoms such as gloomy complexion, lassitude and hypodynamia, inappetence and the like, the treatment mainly aims at tonifying the kidney and strengthening the spleen, and is assisted by replenishing qi, nourishing yin, removing stasis and detoxifying. At present, the western medicines for treating the leukopenia clinically at home and abroad mostly adopt oral or injection leukogenic medicines, mainly comprise granulocyte colony stimulating factors, leucogen, filgrastim, palbociclib and the like, but once the medicines are stopped, the level of leucocytes is reduced again, and the leucocytes are also accompanied by severe adverse reactions such as bone pain, allergy, hypodynamia, transaminase rise, even dyspnea, shock and the like. The application of traditional Chinese medicines in relieving bone marrow suppression is more and more emphasized, and for example, chinese patent medicines such as raw white oral liquid, astragalus membranaceus and raw white capsules, raw blood mixture, sanguisorba officinalis Sheng Bai tablets and the like, and classic formulas such as eight-treasure decoction, angelica sinensis blood replenishing decoction, qi-tonifying Sheng Bai decoction and the like are good choices for treating leukopenia, and the traditional Chinese medicine treatment process is often supplemented with rehabilitation means such as moxibustion, scraping therapy, massage and the like.
The ginseng and white prescription mainly comprises codonopsis pilosula, tremella, astragalus and gynostemma pentaphylla and is used for treating leukopenia after cancer radiotherapy and chemotherapy, wherein the codonopsis pilosula is a common traditional tonifying medicine in China and has the effects of tonifying spleen, benefiting lung, nourishing blood and promoting the secretion of saliva or body fluid; tremella is also called Tremella (together with radix Codonopsis is called the basis of SHENBAI prescription), and has effects of invigorating spleen, promoting appetite, nourishing yin and moistening lung; radix astragali has the effects of invigorating spleen and replenishing qi, consolidating superficial resistance and promoting diuresis, suppurative sepsis and promoting granulation, and is a holy drug for invigorating qi; gynostemma pentaphyllum has effects of invigorating qi, invigorating spleen, eliminating phlegm, relieving cough, and clearing away heat and toxic materials. The ginseng and rhizoma atractylodis macrocephalae is used as a qi-tonifying and spleen-invigorating formula, has the advantages of relieving dizziness, headache, nausea, emesis, spleen and stomach weakness, etc. after radiotherapy and chemotherapy, improving immunity and promoting increase of leukocyte.
In the formula of the ginseng white, tremella, codonopsis pilosula and astragalus root take polysaccharide as main components for treating leukopenia, and saponin in gynostemma pentaphylla is a main active component for enhancing the immune system of an organism. Therefore, the invention develops extraction and purification processes of total polysaccharide and total saponin aiming at traditional Chinese medicinal effective substances in the ginseng white formula, the extraction of the traditional Chinese medicinal compound is mostly single mixed extraction, the solubility of the polysaccharide and the saponin is greatly different based on the consideration of physicochemical properties of the polysaccharide and the saponin, the experiment adopts a method of firstly extracting water and precipitating alcohol to obtain the total polysaccharide, and then adding ethanol filtrate generated in the process of extracting the total polysaccharide into later-stage medicinal materials to extract the total saponin, so that the polysaccharide and saponin components in the ginseng white formula can be effectively and maximally extracted. The extraction of the polysaccharide components mainly comprises tremella, codonopsis pilosula and astragalus, wherein the astragalus and the codonopsis pilosula contain partial saponin, so the polysaccharide components are mixed with gynostemma pentaphylla and extracted by ethanol reflux, and the selected extraction and purification process has good novelty, creativity and practicability, and lays a solid foundation for further research of a ginseng and astragalus preparation.
Disclosure of Invention
An extraction and purification process of an effective part of a white ginseng formula for increasing white blood cells specifically relates to a white ginseng formula composed of 1-5 parts of codonopsis pilosula, 1-3 parts of tremella, 1-5 parts of astragalus and 1-10 parts of gynostemma pentaphylla, the codonopsis pilosula, tremella and astragalus are weighed according to the prescription amount, soaked for 30min, added with 20 times of water and decocted for 3 times, each time is 4h, filtered, filtrates are combined, concentrated until each 1ml of liquid medicine contains 1g of crude drug, then added with 4 times of 95% ethanol, kept stand overnight, filtered, a filter cake is dried to obtain purified total polysaccharide, the ethanol filtrate is combined with gynostemma pentaphylla, added with ethanol and adjusted to 8 times of 80% ethanol, soaked for 30min, extracted under reflux for 2h, filtered, the filter residue is extracted for 1 time again by the same method, the filtrate is combined, concentrated until each 1ml of liquid medicine contains 1g of crude drug, added with 3 times of n-butanol for 3 times of extraction, a n-butanol layer is taken, recovered and dried to obtain total saponins; mixing the total polysaccharides and total saponins to obtain the effective fraction extract of the ginseng and rhizoma anemarrhenae for increasing leucocytes.
The process for extracting and purifying the effective fraction of the white ginseng root for increasing the white blood cells according to claim 1, wherein the effective fraction of the white ginseng root for increasing the white blood cells comprises the following steps: the optimal dosage ratio of the 4 medicinal materials of the codonopsis pilosula, the tremella, the astragalus and the gynostemma pentaphylla is 3: 2: 3: 5.
The process for extracting and purifying the effective components of the leucocyte-increasing formula according to claim 1, wherein the total polysaccharides and total saponins are mixed to ensure the effect of increasing leucocytes.
For a better understanding of the present invention, the effect of the present invention and the determination of the extraction and purification process are further illustrated by animal experiments and orthogonal experiments as follows.
1. Effect test of the present invention for increasing leukocytes
1. Experimental animals: 30 male ICR mice of SPF grade 3 months old, weighing 20 +/-2 g, were purchased and bred by the Experimental animals center of Fujian Chinese medicinal university.
2. Modeling and grouping intervention: after 1 week of adaptive feeding, 30 mice were randomly divided into a blank group of 10 mice and a model group of 20 mice was constructed. The modeling module is used for performing intraperitoneal injection on 100 mg/kg cyclophosphamide solution (CTX) 1 time per day to establish a leukopenia model; the blank group was injected intraperitoneally with an equal volume of physiological saline. 3 days after molding, the mice in the molding group are randomly divided into a model group and a ginseng white square group, and each group contains 10 mice. According to the equivalent dose conversion method of human body surface area and mouse body surface area, the ginseng and white formula group uses the concentrated solution of the water decoction of the ginseng and white formula, the blank group and the model group are irrigated with equal dose of normal saline for 1 time every day, and the continuous intervention is carried out for 15 days.
3. Sample collection and index detection: on days 7 and 15, 30min after administration, blood was collected from the orbit of the mouse, and a whole blood cell analysis was performed to detect the number of leukocytes.
4. The experimental results are as follows: compared with the model group, the white blood cell level of the mice in the ginseng and white formula group is obviously increased, and the difference has statistical significance (P <0.05 See table 1).
TABLE 1 comparison of leukocyte levels in groups of mice after drug intervention: (
± s，10 -3 μL）
Note: day 7, model group and blank group comparison, model group a PLess than 0.05; comparing the Shenbai formula with the model group, the Shenbai formula group b PIs less than 0.05. Day 15, model group was compared to blank group, model group d PIs less than 0.05; comparing the Shenbai formula with the model group, the Shenbai formula group e P＜0.05。
2. Determination experiment of optimal extraction and purification process
1. Orthogonal optimization experiment of total polysaccharide extraction process
Weighing 2g of tremella fuciformis and 5g of codonopsis pilosula and astragalus root respectively, soaking for 30min, taking extraction time (A), extraction times (B) and material-liquid ratio (C) as investigation factors, taking extraction rate and content of total polysaccharide as evaluation indexes, and operating in parallel according to the formulaL 9 (3 4 ) And (2) performing orthogonal table (shown in table 2), decocting, filtering, combining filtrates, concentrating to 1g/mL of crude drug, adding 4 times of 95% ethanol, standing overnight, filtering, taking a filter cake, and drying to obtain the total polysaccharide.
Precisely measuring 1mL of 0.1mg/mL total polysaccharide solution, adding 1mL of 5% phenol solution and 5mL of concentrated sulfuric acid, boiling in a water bath for 15min, carrying out ice water bath for 10min, measuring the absorbance value at 490nm, carrying out blank, measuring the absorbance value of the total polysaccharide, and calculating the content of the total polysaccharide. The results are shown in tables 3 and 4.
TABLE 2 orthogonal factor horizon for total polysaccharide extraction
TABLE 3 orthogonal visual analysis of total polysaccharide extraction Process
TABLE 4 Quadrature variance analysis of Total polysaccharide extraction Process
From the analysis of variance shown in table 4, the influence of the factors A (extraction time), B (extraction times) and C (feed-liquid ratio) on the total saponin content is not significant: (p>0.05 The influence of the selected 3 factors on the experimental result is that A is more than B is more than C from large to small, and the preferred extraction process condition is that A is 3 B 3 C 2 Namely, the feed-liquid ratio is 1.
2. Orthogonal optimization test of total saponin extraction process
Respectively weighing 7.5G of herba Gynostemmatis 9 parts, mixing ethanol filtrates of polysaccharide extraction, soaking for 30min, taking extraction time (D), extraction frequency (E), ethanol concentration (F) and material-liquid ratio (G) as investigation factors, taking extraction rate and content of total saponin as evaluation indexes, and operating under parallel conditions according to the following formulaL 9 (3 4 ) Performing reflux extraction, filtering, mixing filtrates, concentrating to 1g/mL containing crude drug, adding 3 times of n-butanol, extracting for 3 times, recovering n-butanol, and drying to obtain total saponin. And (4) designing an experiment.
Placing the obtained total saponins in a 25mL measuring flask, and diluting to scale for later use. 0.1mL is precisely measured in a 5mL measuring flask and diluted to the scale. Precisely measuring 3mL of diluent, volatilizing in a 100 ℃ water bath, cooling, adding 0.2mL of newly-prepared 5% vanillin solution, 0.8mL of perchloric acid solution, heating in a 60 ℃ water bath for 15min, taking out, immediately cooling with ice water, precisely adding 5mL of glacial acetic acid, measuring the absorbance value at the wavelength of 550nm, and carrying out blank. And calculating the content of the total saponins. The results are shown in tables 5, 6 and 7.
TABLE 5 orthogonal factor horizon table of total saponin extraction
TABLE 6 orthogonal visual analysis of Total Saponin extraction Process
| Test No. | Time/h | Number of times/times | Alcohol concentration/%) | Ratio/times of material to liquid | Total saponins content/g | Yield/%) | Absorbance of the solution | Content/%) | Composite score |
| 1 | 1 | 1 | 70 | 8 | 1.55 | 7.945 | 0.143 | 24.848 | 60.988 |
| 2 | 1 | 2 | 80 | 10 | 2.36 | 12.102 | 0.294 | 32.157 | 83.441 |
| 3 | 1 | 3 | 90 | 12 | 2.49 | 12.766 | 0.233 | 24.414 | 72.219 |
| 4 | 1.5 | 1 | 80 | 12 | 1.55 | 7.949 | 0.181 | 30.917 | 71.085 |
| 5 | 1.5 | 2 | 90 | 8 | 2.26 | 11.592 | 0.348 | 39.494 | 94.369 |
| 6 | 1.5 | 3 | 70 | 10 | 2.13 | 10.914 | 0.224 | 27.495 | 72.747 |
| 7 | 2 | 1 | 90 | 10 | 1.91 | 9.798 | 0.193 | 26.645 | 68.567 |
| 8 | 2 | 2 | 70 | 12 | 2.24 | 11.489 | 0.280 | 32.333 | 82.213 |
| 9 | 2 | 3 | 80 | 8 | 2.36 | 12.098 | 0.389 | 42.121 | 99.989 |
| K 1 | 216.65 | 200.64 | 215.95 | 255.35 | |||||
| K 2 | 238.20 | 260.02 | 254.52 | 224.76 | |||||
| K 3 | 250.77 | 244.96 | 235.16 | 225.52 | |||||
| R | 34.12 | 59.38 | 38.57 | 30.59 |
Note: and calculating the total polysaccharide by comprehensive scoring.
TABLE 7 Quadrature variance analysis of Total Saponin extraction Process
| Factors of the fact | Sum of squares of deviations | Degree of freedom | Ratio of F | Critical value of F | Significance of |
| A extraction time | 595.57 | 2.00 | 1.00 | 19.00 | ＞0.05 |
| Number of B extractions | 1905.81 | 2.00 | 3.20 | 19.00 | ＞0.05 |
| C alcohol concentration | 743.68 | 2.00 | 1.25 | 19.00 | ＞0.05 |
| D ratio of material to liquid | 608.74 | 2.00 | 1.02 | 19.00 | ＞0.05 |
| Error of | 595.57 |
From the analysis of variance shown in table 6, the influence of factors D (extraction time), E (extraction times), F (ethanol concentration) and G (feed-liquid ratio) on the total saponin content is not significant: (p>0.05 The influence of the selected 4 factors on the experimental result is B from large to small>C>D>A, the optimal extraction process condition is A 3 B 2 C 2 D 1 The material-liquid ratio is 1h。
4. Orthogonal verification experiment of total polysaccharide and total saponin extraction process
3 parts of medicinal materials in the same batch as the orthogonal experiment are respectively weighed, and the extraction rate and the content of the total polysaccharide and the total saponin are respectively calculated according to the methods under the items of 1 orthogonal optimization experiment of the total polysaccharide extraction process and 2 orthogonal optimization experiment of the total saponin extraction process. The results are shown in tables 8 and 9.
TABLE 8 validation results of the optimal extraction process for total polysaccharides
TABLE 9 validation results of the optimal extraction process of total saponins
The result is comparable to the maximum of the quadrature result with little difference. Therefore, the preferable process is feasible and stable.
Detailed Description
The extraction and purification process of the effective part of the present invention will be further illustrated by the following examples of specific extraction and purification aspects, which are intended to be illustrative, but not limiting, of the present invention.
Example 1:
6g of codonopsis pilosula, 4g of tremella, 6g of astragalus membranaceus and 10g of gynostemma pentaphylla.
Weighing codonopsis pilosula, tremella and astragalus membranaceus according to the prescription amount, adding 520ml of 20 times of water, soaking for 30min, decocting for 4h, filtering, and collecting filtrate; decocting the filter residue for 2 times by the same method, each time for 4h, respectively adding 520ml of water, combining the filtrates extracted for 3 times, concentrating to 1g/ml containing crude drug, adding 4 times of 95% ethanol, standing overnight, filtering, drying the filter cake to obtain total polysaccharide, combining the remaining ethanol filtrate with herba Gynostemmatis, adjusting the concentration of ethanol to 8 times of 80%, soaking for 30min, reflux-extracting for 2h, filtering, extracting the filter residue for 1 time by the same method, combining the filtrates, concentrating to 1g/ml containing crude drug, adding 3 times of n-butanol, extracting for 3 times, collecting n-butanol layer, recovering n-butanol, and drying to obtain total saponin; mixing the total polysaccharides and total saponins to obtain the effective component extract of the ginseng white formula for increasing the white blood cells.
Claims (2)
1. An extraction and purification process of an effective part of a white ginseng formula for increasing leucocytes, which specifically relates to a white ginseng formula consisting of 1-5 parts of codonopsis pilosula, 1-3 parts of tremella, 1-5 parts of astragalus and 1-10 parts of gynostemma pentaphylla, wherein the codonopsis pilosula, tremella and astragalus are weighed according to the prescription amount, soaked for 30min, added with 20 times of water and decocted for 3 times, and each time is 4h, filtered, the filtrates are combined, concentrated until each 1ml of liquid medicine contains 1g of crude drug, then added with 4 times of 95% ethanol, kept stand overnight, filtered, and filter cakes are dried to obtain purified total polysaccharides, the ethanol filtrate is combined with gynostemma pentaphylla, then added with ethanol to adjust to 8 times of 80% ethanol, soaked for 30min, extracted under reflux for 2h, filtered, the filter residues are extracted by the same method for 1 time again, the filtrates are combined, concentrated until each 1ml of liquid medicine contains 1g of crude drug, respectively added with 3 times of n-butanol for 3 times of extraction, n-butanol layers are combined, n-butanol layers are recovered and dried to obtain total saponins; mixing the total polysaccharides and total saponins to obtain the effective fraction extract of the ginseng and rhizoma anemarrhenae for increasing leucocytes.
2. The process for extracting and purifying the effective fraction of the leucocyte-increasing ginseng according to claim 1, wherein the total polysaccharides and the total saponins are mixed to ensure the effect of increasing the leucocyte.
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