Saponin compound, preparation method for same, and application thereof in immunologic adjuvant preparation

An immune adjuvant and compound technology, applied in the field of saponin compounds and their preparation, can solve the problems of crystal precipitation, high salt content, and insufficient antigen effectiveness after long-term storage, and achieve great clinical application value, simple preparation method, high quality easily controlled effects

Inactive Publication Date: 2012-03-21
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are some problems with the aluminum salt adjuvant, mainly as follows: (1) Due to its physical and chemical properties, the colloidal state of the aluminum salt vaccine is destroyed after freezing, so it cannot be freeze-dried and transported at low temperature
(2) Due to the high salt content, crystallization precipitates after long-term storage, and it is difficult to prepare the same vaccine in batches
(3) Although whether aluminum is related to the induction of senile dementia is still inconclusive, it may have an impact on the nervous system of humans and animals
(6) Because it mainly activates type 2 T hel

Method used

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  • Saponin compound, preparation method for same, and application thereof in immunologic adjuvant preparation
  • Saponin compound, preparation method for same, and application thereof in immunologic adjuvant preparation
  • Saponin compound, preparation method for same, and application thereof in immunologic adjuvant preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of Saponin Compounds

[0049] (1) Dried root of Jintiesuo (commercially available, place of origin, Nujiang, Yunnan) (41kg), crushed and extracted three times under reflux with 80% ethanol (150L) (extraction intervals were 4 hours, 3 hours, 3 hours), combined extracts, and reduced Concentrate under pressure to obtain extract (no alcohol smell), after 0.86kg of extract is diluted with 8.6L of pure water, pass through a macroporous resin column (HP-20), use pure water (400L), 70% ethanol (300L) respectively, Acetone (200 L) eluted sequentially. The 70% ethanol elution part is mainly the total saponin, and the total saponin part is passed through a C-18 reversed-phase bonded silica gel column (ODS), and then eluted with pure water, 30% ethanol, 70% ethanol, and 90% ethanol in sequence (5 L each), divided into four elution fractions (S1-S4). Segment S4 was passed through a silica gel column and eluted with methanol-ethyl acetate 1:1 (the total amount...

Embodiment 2

[0066] Example 2 Hemolytic Determination

[0067] Blood was collected from rabbit ear veins with a vacuum blood collection tube, added with normal saline, mixed evenly, centrifuged at 2000r for 10 min, washed with normal saline for 3 times, red blood cells were collected, and diluted with normal saline to make 0.5% red blood cell suspension. Take an appropriate amount of auroside A, B, and C prepared in Example 1, and dilute them with physiological saline to prepare a 4 mg / mL solution. After filtering through a 0.22 μL microporous membrane, it was diluted with normal saline to obtain dilutions with concentrations of 1000, 500, 250, 100, 50, 25, 10, and 5 μg / mL. In a 96-well plate, add 100 μL of 0.5% erythrocyte suspension to each well, then add 100 μL of saponin dilution solution of different concentrations, mix well, and repeat for 3 wells with one concentration. Then set the maximum and minimum hemolysis controls, which are physiological saline and distilled water respectiv...

Embodiment 3

[0073] Example 3 Acute Toxicology

[0074] Dilute samples Quil A, aureoside B, and aureoside C with PBS to 5 mg / kg, 10 mg / kg, 20 mg / kg, 40 mg / kg, and 80 mg / kg respectively, 5 dose groups, and use PBS as a blank control group, There were 16 groups in total, and the ICR mice were randomly divided into 16 groups, with 5 mice in each group. The mice in each group were subcutaneously injected with the corresponding dose in the neck, and were reared for 72 days, and the toxic reactions (hair loss, pain, swelling, etc.) and death of the mice were observed.

[0075] The results of acute toxicology of auroside B and auroside C are shown in Table 2

[0076]

[0077]

[0078] As can be seen from Table 2-1, when Quil A was at 100 μg, 2 mice had died, showing strong toxicity, and the median lethal dose (LD 50 ) is 125 μg. And observed obvious toxic reactions within 72 hours, such as severe hair loss at the injection site, accompanied by pain and swelling, lethargy and loss of appe...

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Abstract

The invention provides a saponin compound, which comprises psammosilene tunicoides saponin B and psammosilene tunicoides saponin C which are extracted and separated from traditional Chinese medicine psammosilene tunicoides. The saponin compound is capable of inducing organism to generate Th1 type and Th2 type immune response and inducing the organism to generate stronger cellular immunity and humoral immunity reaction on vaccines as compared with alumina gel adjuvant, thereby being capable of serving as immunologic adjuvant of various vaccines to achieve higher immunity effect. The saponin compound is low in haemolysis, fine in safety, simple and convenient in preparation method, easily controllable in quality, convenient in use, capable of realizing cryopreservation, thereby having higher clinical application value.

Description

technical field [0001] The invention relates to medicinal chemistry, in particular to a saponin compound isolated from the traditional Chinese medicine Jintiesuo, a preparation method thereof and an application in an immune adjuvant. Background technique [0002] With the development of molecular biology, the second-generation vaccines mainly use purified recombinant proteins, synthetic peptide antigens and plasmid DNA antigens to replace the safety hazards caused by the live weak virus antigens of the first-generation vaccines. However, purified antigens usually have weak immunogenicity. In order to overcome this weakness, antigens need to be mixed with appropriate adjuvants. However, it is required that the adjuvant has a long stability period, is not prone to degradation and is easy to manufacture. In addition, the most important thing is that the adjuvant can stimulate the body's strong cellular immune response and antibody production ability, so that the body can produ...

Claims

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Application Information

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IPC IPC(8): C07H15/256C07H1/08C07H1/00C07J63/00A61K31/704A61P37/04
Inventor 张卫东俞飚单磊孙建松苏娟曹文杰沈云亨田均勉
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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