CN106668188B - Application of polygala tenuifolia total saponin in anti-aging and immunoregulation - Google Patents
Application of polygala tenuifolia total saponin in anti-aging and immunoregulation Download PDFInfo
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Abstract
The invention discloses application of polygala total saponins in resisting aging and regulating immunity. The polygala tenuifolia saponin can be used for preparing anti-aging products or products for enhancing the immune function. The anti-aging effect research of the invention shows that compared with a model group, the polygala total saponin is high, the thymus index and the spleen index of a medium-dose group are both obviously reduced, and the CAT activity of serum is extremely obviously increased; the SOD activity of the low-dose group is obviously improved. The difference between the MDA level of the brain tissue and the SOD activity of the high, medium and low dose groups has statistical significance. Compared with the model group, the difference between the MDA content of the brain tissue and the SOD activity of the high, medium and low dose groups has statistical significance. The research on the immune function regulation effect shows that compared with a model group, the polygala tenuifolia saponin high-dosage and medium-dosage groups remarkably increase spleen index and thymus index; the high dose group had significantly elevated levels of both serum cytokines IL-2 and IL-6.
Description
Technical Field
The invention relates to application of total saponins of polygala tenuifolia in resisting aging and regulating immunity.
Background
Polygala tenuifolia Willd is dried root of Polygala tenuifolia Willd or Polygala rigidum L. Bitter, pungent and warm in flavor. It enters heart, kidney and lung meridians. Has the effects of soothing the nerves, promoting intelligence, restoring normal coordination between the heart and the kidney, eliminating phlegm and reducing swelling. Polygalaxolide is one of main active ingredients, more than 80 different types of Polygalaxolide are separated, the main structure is pentacyclic triterpene type, and the basic mother nucleus is oleanolic acid. At present, ginsenoside, notoginsenoside and astragaloside which take triterpenoid saponin as main active ingredients have anti-inflammatory and immune effects, and the research on polygala tenuifolia total saponin is less, so that the research on the anti-aging and immune regulation effects of polygala tenuifolia total saponin is needed to be carried out, and a reference is provided for further development of the pharmacological effect of polygala tenuifolia saponin.
Disclosure of Invention
The invention aims to provide the application of polygala tenuifolia total saponins in resisting aging and regulating immunity, and researches on the anti-aging effect of different doses of polygala tenuifolia total saponins on aging mice caused by D-galactose and the regulating effect on the immunologic function of mice with low immunity caused by cyclophosphamide show that polygala tenuifolia saponins have the effects of resisting aging and strengthening the immunologic function.
The polygala total saponin can be prepared according to the existing method, for example, the following method is adopted: firstly, carrying out petroleum ether reflux degreasing treatment; extracting the degreased polygala root with ethanol under reflux; extracting the obtained extract, and purifying with macroporous adsorbent resin;
wherein, the petroleum ether reflux degreasing treatment can be carried out according to the following steps:
crushing polygala tenuifolia to 40 meshes, carrying out reflux degreasing treatment on 30-60 ℃ petroleum ether in a water bath condition at 45 ℃, degreasing the petroleum ether for 1 hour in a volume which is 5 times that of the petroleum ether for 1 time, degreasing the petroleum ether for 0.5 hour in a volume which is 3 times that of the petroleum ether for 2 times, and airing the degreased polygala tenuifolia powder for later use.
The ethanol reflux extraction can be carried out according to the following steps:
adding 8 times (by mass) of 85% ethanol water solution into the degreased polygala tenuifolia, placing in a reflux device for reflux extraction for 3 times, wherein each time is 1.5h, recovering ethanol from the extract until no alcohol smell exists, placing in a water bath to volatilize the solvent, placing in a drying oven at 35 ℃ for 24h, and preparing into extract for later use.
The macroporous adsorption resin purification can be carried out according to the following steps (Konghui, Zhengde, Zhonghonglei, and the like. the purification process research of the polygala tenuifolia total saponin macroporous adsorption resin [ J ]. Liaoning traditional Chinese medicine J.2014, 41(10): 2196-: d101 loading the macroporous resin into a column by a 95% ethanol water solution wet method, eluting by using distilled water until no alcohol smell exists; and (3) absorbing the test solution to sample on a D101 macroporous column, eluting with 2BV of distilled water and 3BV of 70% ethanol aqueous solution in sequence at an elution speed of 1BV/h, and collecting the eluent of the ethanol part. Recovering solvent with rotary evaporator, volatilizing solvent in water bath, and oven drying at 35 deg.C for 24 hr to obtain cortex et radix Polygalae total saponin.
The research on the anti-aging effect of the polygala tenuifolia saponins on the aged D-galactose mice shows that the polygala tenuifolia total saponins can be used for preparing anti-aging products.
The anti-aging product has the following functions:
1) increasing spleen and thymus indices;
2) the activity of CAT and SOD in serum is improved;
3) reducing the MDA content in brain tissue;
4) increase SOD content in brain tissue and reduce SOD content.
The application of the polygala total saponin in preparing any one of the following products 1) to 4) also belongs to the protection scope of the invention:
1) products that increase spleen and thymus indices;
2) products for improving CAT and SOD activity in serum;
3) products that reduce the amount of MDA in brain tissue;
4) a product for increasing SOD content in brain tissue.
The research on the regulation effect of the polygala total saponin on the immune function of mice with low immunity caused by cyclophosphamide shows that the polygala total saponin can be used for preparing products for enhancing the immune function.
The product for enhancing the immune function has the following functions:
the application of the polygala total saponin in preparing products for improving the content of serum cytokines IL-2 and IL-6 also belongs to the protection scope of the invention.
The research on the anti-aging effect of the invention shows that the polygala total saponin is high (400mg kg) compared with the model group-1) Middle dose group (200 mg. kg)-1) The thymus index and the spleen index of the patient are both obviously reduced (P)<0.01，P<0.05，P<0.05，P<0.05), the CAT activity of the serum is remarkably increased (P)<0.01); low dose group (100mg kg)-1) The SOD activity is obviously improved (P)<0.05). The difference between the MDA level and SOD activity of the brain tissue of the high, medium and low dose groups has statistical significance (P)<0.05). Compared with the model group, the difference between the MDA content and the SOD activity of the brain tissues of the high, medium and low dose groups has statistical significance (P)<0.05). The research on the immune function regulation effect of the invention shows that the polygala tenuifolia saponin is high (400 m) compared with a model groupg·kg-1) Middle dose group (200 mg. kg)-1) Both significantly increased spleen index and thymus index (P)<0.05); the content of serum cytokines IL-2 and IL-6 in the high-dose group is obviously increased (P)<0.01). Experiments of the invention fully prove that the polygala tenuifolia saponin has the functions of resisting aging and enhancing the immunologic function.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The material and the method are as follows:
1. animal(s) production
144 SPF-level Kunming mice, each half of male and female, 4 weeks old, with the body mass of 20 +/-2 g, are provided by the experimental animal center of the military medical academy of sciences of the Chinese people liberation army (license number: SCXK- (army) 2012-0004).
2. Drugs and reagents
Polygala tenuifolia (Hebei Quantai pharmaceutical Co., Ltd., production lot: 1601001), petroleum ether (Tianjin chemical reagent Co., Ltd., lot: 20160108), absolute ethyl alcohol (Tianjin chemical company, advanced Toyobo chemical Co., Ltd., production lot: 20101014), D-galactose (Tianjin photoreplication chemical research institute, lot: 20141212), vitamin E (Chengdu Kelong chemical reagent factory, lot: 2015828), 0.9% physiological saline (Shijiazhuang Siyao Co., Ltd., product lot: 1508103201), superoxide dismutase (SOD) determination kit (Nanjing Biochemical engineering research institute, production lot: 20160714), and Catalase (CAT) test kit (Nanjing Biochemical research Co., Ltd., lot: 20160415). Cyclophosphamide (Solarbio cat. No. c8650); levamisole hydrochloride (Solarbio cat. No. l 8230); a macroporous resin (D101); mouse IL-2ELISA Kit; mouse IL-6ELISA Kit.
3. Main instrument
Ultra-3000 Series ultraviolet-visible spectrophotometer (Beijing Puyuan Seiki technologies, Inc.), MultiskanFC type microplate reader (Shanghai Saimer Feishiel instruments, Inc.), AX224ZH/E electronic balance (Aohaus, Inc.), DE-100g universal high speed pulverizer (rhodiola Kimura industries, Inc.), RE-52AA rotary evaporator (Shanghai Yangrong Biochemical instruments, Inc.), SC-02 low speed centrifuge (Zhongkai Kao science instruments, Inc., Anhui), 02-0510PRO200Bio-Gen Series homogenizer, RB3 vortex mixer (Haiman Beijing instruments, Inc., Haiman City, Inc.).
The total saponins of polygala tenuifolia used in the following examples were prepared as follows:
pulverizing cortex et radix Polygalae to 40 mesh, defatting with 30-60 deg.C petroleum ether under 45 deg.C water bath condition for 1 time and 5 times of petroleum ether for 1 hr and 2 times of petroleum ether for 0.5 hr, and air drying the defatted cortex et radix Polygalae powder.
Adding 85% ethanol water solution (volume concentration) 8 times the mass of the degreased polygala tenuifolia, placing in a reflux device for reflux extraction for 3 times, wherein each time is 1.5h, recovering ethanol from the extracting solution until no alcohol smell exists, placing in a water bath to volatilize the solvent, placing in a 35 ℃ oven for drying for 24h, and preparing into extract for later use.
D101, filling the macroporous resin into a column by a 95% ethanol aqueous solution (volume concentration) through a wet method, eluting by using distilled water until no alcohol smell exists; and (3) absorbing the test solution to sample on a D101 macroporous column, eluting with 2BV of distilled water and 3BV of 70 percent ethanol in sequence at an elution speed of 1BV/h, and collecting the eluent of the ethanol part. Recovering solvent with rotary evaporator, volatilizing solvent in water bath, and oven drying at 35 deg.C for 24 hr to obtain cortex et radix Polygalae total saponin.
Example 1 study of Polygala tenuifolia Saponin on anti-aging Effect
72 SPF-level Kunming mice, which are half male and female, are randomly divided into a blank group, a positive group, a model group and an administration group (high, medium and low dose groups), each group comprises 12 mice, and the mice are fed adaptively for one week. Model group and each administration group injected with D-galactose at 1.25 g.kg in back and neck-1The blank group was injected subcutaneously with an equal volume of saline; positive group gavage vitamin E1.25 g.kg-1Sequentially intragastric polygala total saponin 400, 200 and 100 mg/kg in high, medium and low dose administration groups-1Equal volume of distilled water was perfused into the stomach for the blank and model groups. Body weight of mice was recorded once a week for 4 consecutive weeks, and the general state of the mice, the ratio between groups was observedThe hair gloss, the sparseness and the activity degree of the mouse are reduced. Mice were weighed in vivo after fasting for 12h at the end of experiment week 4.
After the last administration, fasting was performed for 12 hours, and the body weight of the mouse was measured, and the thymus and spleen were taken and weighed, and the thymus index and spleen index were calculated. The mouse serum and brain tissue were measured for CAT, SOD and MDA, respectively, according to the kit instructions.
Statistical methods experimental data were analyzed for one-way anova using SPSS17.0 software, and the results were expressed as mean ± standard deviation (x ± s), with t-test for comparisons between groups, with P <0.05 indicating significant differences and P <0.01 indicating very significant differences.
1. Effect of Total saponins of Polygala tenuifolia on immune organ index of mice
As shown in Table 1, spleen index and thymus index of the model mice are significantly reduced compared with those of the blank group (P <0.01, P <0.05), and spleen and thymus are observed to have signs of atrophy, which indicates that the model of aging caused by D-galactose is successful. The positive group had a significantly increased thymic index (P <0.05) compared to the model group; differences between thymus index and spleen index groups of the senegenin high dose group have statistical significance (P <0.01, P < 0.05); the spleen index and the thymus index of the dose group in the polygalasaponin are obviously increased (P <0.05), which indicates that the polygalasaponin can obviously inhibit the reduction of the spleen and thymus indexes.
TABLE 1 immune organ index comparison
Note: in comparison with the blank set, the results,*P<0.05，**P<0.01; in comparison with the set of models,#P<0.05，##P<0.01
2. effect of Total saponins of Polygala tenuifolia on CAT and SOD activities in mouse serum
As shown in Table 2, compared with the blank group, the activities of CAT and SOD in the serum of the model group are both significantly reduced (P <0.01 and P <0.05), and CAT and SOD are main antioxidant enzymes for eliminating free radicals in vivo. The enzyme activities of CAT and SOD indirectly reflect the capability of organism for eliminating oxygen free radical, which shows that the model of aging caused by D-galactose is successful. The CAT of the serum of the positive group is obviously increased compared with the model group (P < 0.05); the difference between CAT activity groups of high and medium dose group serum has statistical significance (P < 0.01); SOD activity of the serum of the low-dose group is obviously improved (P is less than 0.05), which indicates that the polygala tenuifolia saponin can obviously inhibit the reduction of the activity of two enzymes to a certain extent.
TABLE 2 comparison of serum antioxidant capacity
Note: in comparison with the blank set, the results,*P<0.05，**P<0.01; in comparison with the set of models,#P<0.05，##P<0.01
3. effect of Total saponins of Polygala tenuifolia on MDA and SOD Activity of brain tissue of mouse
As shown in table 3, the model brain tissue has higher MDA content (P <0.05) and significantly lower SOD activity (P <0.05) compared to the blank group. Compared with the model group, the positive brain tissue has obviously reduced MDA content (P < 0.05); the difference between the MDA content of the brain tissue and the SOD activity of the high, medium and low dose groups has statistical significance (P <0.01 or P < 0.05).
TABLE 3 comparison of antioxidant capacity of brain tissues
Note: in comparison with the blank set, the results,*P<0.05，**P<0.01; in comparison with the set of models,#P<0.05，##P<0.01
the injection of the D-galactose solution causes the agingA classical subacute model of aging is characterized by that the D-galactose solution is continuously injected to the back of the neck of mouse, so changing several biochemical indexes in body of mouse, such as the decrease of SOD and CAT activities in serum[13]. The activities of CAT and SOD indirectly reflect the capability of organism to eliminate oxygen free radical, which indicates that D-galactose can cause cell aging. MDA is lipid peroxide formed by oxygen radicals attacking unsaturated fatty acids in biological membranes, and the level indirectly reflects the severity of free radical attack on body cells.
The experimental result of the embodiment shows that the tenuigenin can effectively resist the senile mice caused by D-galactose and has the anti-aging effect; the anti-aging mechanism of the mouse can be to resist the damage of free radicals to cells by improving the activities of antioxidase such as CAT, SOD and the like of the organism, and the polygalasaponin of an aging mouse can obviously improve the activities of CAT and SOD in the aging mouse and reduce the content of MDA, so that the mouse aging caused by D-galactose can be relieved to a certain extent, and the antioxidant capacity of the whole organism is improved.
Example 2 immunization study of Polygala tenuifolia Saponin
Randomly dividing mice into blank group, model group, positive group, saponin high, medium and low dosage groups, wherein each group comprises 12 mice, each half of the mice is female and male, and the intraperitoneal injection dosage of each group except the blank group is 30 mg/kg-1The cyclophosphamide solution is injected into blank group with equal dosage of normal saline, and the gastric lavage dosage of positive group is 20mg kg-1Levamisole hydrochloride solution, sequentially intragastric administration of 400, 200 and 100 mg/kg of polygala total saponin in high, medium and low dose administration groups-1The blank and model groups were drenched with distilled water of equal volume and administered continuously for 14 days.
After the last administration, fasting is carried out for 12h, the weight of the mouse is weighed, the thymus and the spleen are taken, and the thymus index and the spleen index are calculated by weighing. The IL-2 and IL-6 levels in mouse sera were measured according to ELISA instructions.
Statistical methods experimental data were analyzed for one-way anova using SPSS17.0 software, and the results were expressed as mean ± standard deviation (x ± s), with t-test for comparisons between groups, with P <0.05 indicating significant differences and P <0.01 indicating very significant differences.
1. Effect of Total saponins of Polygala tenuifolia on spleen index and thymus index of mice
As shown in table 4, the thymus index and spleen index of the mice in the model group were significantly decreased compared to the blank group (P <0.05 or P <0.01), and the quality of the immune organs was able to reflect the strength of the immune regulation to some extent, which indicates the success of the mouse model with low immunity. Compared with the model group, the thymus index and the spleen index of the positive group are obviously increased (P is less than 0.05), which indicates that the positive medicine can relieve the atrophy of the organs of the mice with low immunity; from the level of spleen index, the polygala tenuifolia saponin high and medium dose groups are obviously increased (P <0.05), and from the level of thymus index, the polygala tenuifolia saponin high, medium and low dose groups are obviously increased (P <0.01 or P <0.05), which indicates that the polygala tenuifolia saponin plays a certain role in relieving the atrophy of the organs of the mice.
TABLE 4 Effect of Polygala saponins on spleen and thymus index in mice
Note: in comparison with the blank set, the results,#P<0.05；##P<0.01; in comparison with the set of models,*P<0.05；**P<0.01
2. influence of Polygala tenuifolia Total saponins on mouse serum cytokine IL-2 and IL-6 content
As shown in Table 5, compared with the blank group, the serum cytokines IL-2 and IL-6 of the model group mice are both significantly reduced (P <0.05), and the reduction of the serum cytokine reflects the reduction of the immune regulation function of the mice, which indicates that the immunosuppressive mouse model is successfully manufactured.
Compared with the model group, the content of serum cytokines IL-2 and IL-6 of the mice in the positive group is obviously increased (P <0.05 or P <0.01), which indicates that levamisole hydrochloride can relieve the reduction of the serum cytokine content of the mice so as to regulate the immune function of the mice. The content of serum cytokines IL-2 and IL-6 in the saponin high-dose group is remarkably increased compared with that in the model group (P <0.01), and the content of serum cytokines IL-6 in the medium-dose group is remarkably increased (P < 0.05). Therefore, the polygala tenuifolia saponin can inhibit the reduction of the serum cytokine content of mice caused by cyclophosphamide.
TABLE 5 Effect of Polygala tenuifolia saponins on the levels of mouse serum cytokines IL-2, IL-6
Note: in comparison with the blank set, the results,#P<0.05，##P<0.01,; in comparison with the set of models,*P<0.05，**P<0.01；
in the regulation of the whole immune system, the selective induction of mixed reaction of Th1/Th2 plays a major role, cytokines such as IFN-gamma, IL-2 and the like secreted by Th1 cells mediate cellular immune response by activating effector cells, and cytokines such as IL-4, IL-5, IL-6 and the like secreted by Th2 cells assist B cells to generate antibodies as a major role, so that humoral immune response is mediated, therefore, the content of IL-2 and IL-6 is selected as two indexes for evaluating the influence of tenuigenin on the immune regulation of mice in the embodiment.
The experimental results of the embodiment show that the polygalasaponin has certain influence on thymus index and spleen index of mice, and the content of IL-2 and IL-6, and the influence degree is increased along with the increase of the dosage.
Claims (1)
1. The application of the polygala total saponin in preparing the product for regulating immunity is characterized in that: the product can increase the content of serum cytokines IL-2 and IL-6;
the polygala total saponin is prepared by the method comprising the following steps:
firstly, petroleum ether is adopted to carry out reflux degreasing treatment on polygala tenuifolia; extracting the degreased polygala root with ethanol under reflux; extracting the obtained extract, and purifying with macroporous adsorbent resin; wherein the content of the first and second substances,
the steps of the petroleum ether reflux degreasing treatment are as follows: crushing polygala tenuifolia to 40 meshes, carrying out reflux degreasing treatment on petroleum ether at the temperature of 30-60 ℃ in a water bath condition at the temperature of 45 ℃, degreasing the petroleum ether for 1 hour in a volume which is 5 times that of the petroleum ether for 1 time, degreasing the petroleum ether for 0.5 hour in a volume which is 3 times that of the petroleum ether for 2 times, and airing the degreased polygala tenuifolia powder for later use;
the ethanol reflux extraction steps are as follows: adding 85% ethanol water solution with the mass of 8 times of that of the degreased polygala tenuifolia, placing the degreased polygala tenuifolia into a reflux device for reflux extraction for 3 times, wherein each time is 1.5h, recovering ethanol from an extracting solution until no alcohol smell exists, placing the extracting solution in a water bath pot for volatilizing the solvent, placing the extracting solution in a drying oven at the temperature of 35 ℃ for drying for 24h, and preparing an extract for macroporous adsorption resin purification;
the macroporous adsorption resin purification steps are as follows: d101 loading the macroporous resin into a column by a 95% ethanol water solution wet method, eluting by using distilled water until no alcohol smell exists; taking a sample solution to be tested, loading the sample solution into a D101 macroporous column, eluting with 2BV of distilled water and 3BV of 70% ethanol aqueous solution in sequence at the elution speed of 1BV/h, and collecting the eluent of the ethanol part; recovering solvent with rotary evaporator, volatilizing solvent in water bath, and oven drying at 35 deg.C for 24 hr.
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| CN105055248A (en) * | 2015-08-26 | 2015-11-18 | 湖北中医药大学 | Application of polygala tenuifolia total saponins to preparation of skin aging resistant daily chemical product and aging resistant daily chemical product prepared from same |
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| CN103099911A (en) * | 2012-12-28 | 2013-05-15 | 广东万年青制药有限公司 | Application of mental treasure pills in preparation of medicine for treating senile dementia |
| CN105055248A (en) * | 2015-08-26 | 2015-11-18 | 湖北中医药大学 | Application of polygala tenuifolia total saponins to preparation of skin aging resistant daily chemical product and aging resistant daily chemical product prepared from same |
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