Marker group and method for evaluating immune curative effect of ginseng

Abstract

The invention discloses a marker group and a method for evaluating the immune efficacy of ginseng, including the immune efficacy, and belongs to the technical field of ginseng quality evaluation. The marker group comprises at least three of Rg1, Re, Rb1, Rc, Rb2 and Rd. The invention provides a ginseng quality evaluation mode and method. The mode is based on two core parameters: one is saponin content, and the other is correlation between the sum of the saponin content and the drug effect. The method not only meets the requirements of the quality standard on the analysis method, but also reflects the strength of the pharmacological activity of the main medicinal components (saponin components) of the ginseng, and establishes a method for reflecting the advantages and disadvantages of the immune efficacy of the ginseng. According to the method, the accuracy and the robustness of a chemical determination method are continuously used, the integrity and the objectivity of evaluating the drug effect strength by the whole animal model are reflected, the defect that the traditional quality control method emphasizes chemical indexes and neglects the whole drug effect is overcome, and rapid and accurate evaluation of the quality of the ginseng is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of detecting and evaluating the immunotherapy effect of indirect reaction ginseng, and particularly relates to a marker combination and method for evaluating the advantages and disadvantages of the immunotherapy effect of ginseng. BACKGROUND

[0002] As a traditional precious Chinese medicinal material, the quality evaluation of ginseng has always been the core problem of guaranteeing the standardization of medicinal material market and clinical efficacy. At present, the quality standards of ginseng recorded in Chinese Pharmacopoeia mainly based on the identification of appearance, inspection items and content determination, which belongs to a qualified evaluation system and has significant limitations: only a few chemical markers such as ginsenoside Rg1, Re and Rb1 are used as content determination indicators, which is difficult to fully reflect the internal quality differences caused by different producing areas, growth years and processing methods, and cannot realize the advantages and disadvantages of reflecting the efficacy of medicinal materials.

[0003] In recent years, some scholars have tried to introduce in vitro biological effect evaluation methods to make up for the shortcomings of chemical analysis, aiming to establish a quality evaluation strategy reflecting the effectiveness and safety of traditional Chinese medicine. However, such methods generally have poor experimental repeatability, unclear concentration-effect linear relationship, and complex operation, which is difficult to meet the requirements of simplicity, robustness and accuracy of quality control analysis methods. In addition, pharmacological studies have shown that traditional Chinese medicine often realizes cross-organ and long-distance regulation through “intermediate mediators”, and it is difficult to fully reflect its overall efficacy by simply relying on in vitro biological effects. SUMMARY

[0004] In view of the above shortcomings in the prior art, the present application provides a marker group and method for evaluating the advantages and disadvantages of the immunotherapy effect of ginseng. The present application proposes a quality consistency evaluation mode and a medicinal material quality evaluation method. The mode is based on two core parameters: one is the content of chemical markers, and the other is the correlation strength between the main component addition content and pharmacological activity. This method not only meets the requirements of quality standards for analysis methods, but also further reflects the pharmacological activity of components, in vivo absorption characteristics and the mutual transformation relationship between components, thereby overcoming the shortcomings of traditional quality control methods that focus on chemical indicators while ignoring overall efficacy. By applying this evaluation mode, the quality consistency of different medicinal materials and products can be evaluated, and the optimal efficacy of immune promotion can also be reflected, thereby providing new ideas and method support for the quality control and standard improvement of ginseng and other traditional Chinese medicinal materials.

[0005] To achieve the above purpose, the technical scheme adopted by the present application to solve its technical problems is: The purpose of the present application is to provide a method for evaluating the advantages and disadvantages of the immunotherapy effect of ginseng, which is specifically: The marker group consisting of at least three of Rg1, Re, Rb1, Rc, Rb2 and Rd is detected in ginseng, and then the total saponin content in each marker group is determined, and the total saponin content is used to determine the immune efficacy of the ginseng to be detected.

[0006] Further, the total saponin content of the marker group is linearly correlated with the immune regulation activity of ginseng.

[0007] Further, the marker group consists of Re, Rb1, Rc and Rd four saponins; if the total content of saponins in the marker group is ≥72.97, it is determined that it has excellent immune efficacy; if the total content of saponins is ≤29.25%, it is determined that its immune efficacy is poor; if the total content of saponins is between 29.25% and 72.97%, it is determined that its immune efficacy is moderate.

[0008] Further, the marker group consists of Rg1, Re, Rb1, Rc, Rb2 and Rd six saponins; if the total content of saponins in the marker group is ≥77.60, it is determined that it has excellent immune efficacy; if the total content of saponins is ≤37.57%, it is determined that its immune efficacy is poor; if the total content of saponins is between 37.57% and 77.60%, it is determined that its immune efficacy is moderate.

[0009] Further, the marker group consists of Rg1, Re, Rb1, Rc and Rd five saponins; if the total content of saponins in the marker group is ≥75.22%, it is determined that it has excellent immune efficacy; if the total content of saponins is ≤31.57%, it is determined that its immune efficacy is poor; if the total content of saponins is between 31.57% and 75.22%, it is determined that its immune efficacy is moderate.

[0010] Further, the marker group consists of Rg1, Re and Rb1 three saponins; if the total content of saponins in the marker group is ≥54.81%, it is determined that it has excellent immune efficacy; if the total content of saponins is ≤21.11%, it is determined that its immune efficacy is poor; if the total content of saponins is between 21.11% and 54.81%, it is determined that its immune efficacy is moderate.

[0011] Further, the marker group consists of Re, Rb1 and Rd three saponins; if the total content of saponins in the marker group is ≥62.32%, it is determined that it has excellent immune efficacy; if the total content of saponins is ≤23.51%, it is determined that its immune efficacy is poor; if the total content of saponins is between 23.51% and 62.32%, it is determined that its immune efficacy is moderate.

[0012] Further, the sample of ginseng to be detected is ginseng leaf, ginseng stem, ginseng root, ginseng total saponin extract or corresponding preparation.

[0013] Another object of the present application is to provide a marker group for evaluating the pros and cons of ginseng immunotherapy, which consists of at least three saponins from Rg1, Re, Rb1, Rc, Rb2 and Rd.

[0014] Another object of the present application is to provide the use of the above marker group in the evaluation of the pros and cons of ginseng immunotherapy quality.

[0015] Another object of the present application is to provide the use of the above marker group in the preparation of a preparation for evaluating the pros and cons of ginseng immunotherapy quality.

[0016] Further, the ginseng sample for evaluating the pros and cons of immunotherapy quality is ginseng leaf, ginseng stem, ginseng root, ginseng total saponin extract or corresponding ginseng preparation.

[0017] The beneficial effects of the present application are: The present application proposes a quality consistency evaluation mode and a medicinal material quality evaluation method. The mode is based on two core parameters: the content of chemical markers and the correlation strength between the additive content of main components and pharmacological activity. The method not only meets the requirements of the quality standard for the analysis method, but also further reflects the pharmacological activity of the components, the in vivo absorption characteristics and the mutual transformation relationship between the components, thereby overcoming the shortcomings of traditional quality control methods that emphasize chemical indicators and ignore overall efficacy. The application of the evaluation mode can not only evaluate the consistency of the quality of different medicinal materials and products, but also reflect the optimal efficacy of the immune boosting effect, thereby providing new ideas and method support for the quality control and standard improvement of ginseng and other Chinese medicinal materials. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure is the clustering analysis result graph of five batches of ginseng root extracts; Figure 2 Figure is the animal experiment design flow chart Figure 3 Figure is the detection of mouse organ index and changes in serum cytokines; Figure 4 Figure is the correlation analysis of the content of multiple components and activity indicators (IgM and IFN-γ) (strong correlation); Figure 5 Figure is the correlation analysis of the content of multiple components and activity indicators (IgM and IFN-γ) (not correlated). DETAILED DESCRIPTION

[0019] The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0020] Quantitative analysis of seven saponin components and sample fractionation of Example 1 Preparation of standard solution: 1.0 mg of Rg1 reference substance was accurately weighed, dissolved in 1.0 mL of methanol, shaken uniformly, and prepared into a reference solution with a concentration of 1.0 mg·mL -1 -20°C for standby, and diluted to the appropriate concentration with methanol before sampling.

[0021] Preparation of test solution: 30.00 mg of ginseng root extract was accurately weighed, 10 mL of methanol was added, ultrasonic extraction was performed for 30 min (room temperature, 40 kHz), centrifugation was performed for 10 min (10000 r·min -1 ), and the supernatant was filtered through a 0.22 μm microporous filter to obtain the test solution.

[0022] Chromatographic conditions: Waters Acquity UPLC BEH C18 column (2.1×100 mm, 1.7 μm); mobile phase: acetonitrile (A) and water (B); gradient elution program: 0~8 min, 19% A; 8~13 min, 19%-29% A; 13~16 min, 29% A; 16~23 min, 29%-40% A; 23~26 min, 40%-19% A, flow rate 0.3 mL·min -1 , injection volume 2 µL, column temperature 30°C, detection wavelength 203 nm.

[0023] 2 μL of each of the test solution and the single standard solution (Rg1) was precisely taken and injected into the high performance liquid chromatograph for chromatographic analysis, and the content of the seven ginsenosides in the ginseng root extract was calculated by using the detection and calculation method established in the present application.

[0024] Results: The standard linear equation of Rg1 is y =1265.7 x +2.541, and the quantitative equations of the remaining components are converted according to the following formula as shown in Table 1, and the molar coefficient quantitative results are shown in Table 2;

[0025] Among them, M r ginsenoside Rg1,M 测 Molar mass of other six ginsenosides (Re, Rf, Rb1, Rc, Rb2, Rd); The peak area y measured by the instrument is brought into y =1265.7 x +2.541, the mass concentration of Rg1 is calculated x (mg / mL), and according to the preparation method of the control test solution, the volume of the final test solution 10 mL is multiplied, and the original medicinal material powder 30.00 mg is divided to obtain the percentage content of Rg1 in the root extract, that is, the data in Table 2. The quantitative results of the rest of the six ginsenosides are calculated by using the corresponding quantitative equation to calculate the sample content, see Table 2. The quality of five batches of ginseng root extracts is evaluated by combining the quantitative results with cluster analysis, and the results are shown in Figure 1 As shown in the table, the five batches of samples can be divided into three groups: high content group (YY), medium content group (LMT) and low content group, wherein the low content group DST / KLM / PS is divided into three groups, indicating that the quality consistency of DST / KLM / PS is good, and the average value of the three is finally taken as the determination standard. The content of Rb1 in YY is the highest, and the content of Re in LMT is the highest, which is independent of a group. It is worth noting that the grouping results are highly consistent with the total content gradient of the seven ginsenosides in the root extract (total content YY>LMT>DST / KLM / PS), so the above grouping results reflect the difference of the total content of the seven ginsenosides in the root extract. The quality of all the samples above meets the requirements of the pharmacopoeia.

[0026] Table 1 Linear equation for calculating five ginsenosides in Example 1

[0027] Table 2 Quantitative results of the detection method described in Example 1

[0028] Note: a Not reaching the quantitative limit; b Not detected.

[0029] Example 2 Evaluation of immune boosting activity of three groups of graded samples 1. Drug configuration scheme According to the results of Example 1, high content group (YY), medium content group (LMT) and low content group (DST) (biological drug amount 211.14 mg / kg) are selected as the administration group. In order to eliminate the potential interference of polysaccharides in ginseng root extract, the extract is further treated by alcohol precipitation. 4 g of the above ginseng root extract is weighed, 80% ethanol is added, mixed, and placed at 4°C overnight, centrifuged at 7000 rpm / min for 20 min, and the supernatant is rotary evaporated to remove alcohol, and then freeze-dried and stored.

[0030] 2. Experimental animal modeling and drug treatment 5-8 weeks old male ICR mice were selected as the research object. After the mice were purchased, they were given a week of suitability feeding to adapt to the experimental environment. According to the experimental requirements, the mice after adaptation were randomly divided into five groups, namely the control group, the model group, the YY drug group, the LMT drug group and the DST drug group, 10 in each group. The drug groups were given the above ginsenoside extract (distilled water preparation) by gavage, and the normal group and the model group were given the same dose of distilled water. On the 4th day of administration, the model group and the drug group were injected with cyclophosphamide 100 mg / kg intraperitoneally, and the reaction of the mice was observed, such as dull, loose hair, cold limbs and tail, curling and arching back, etc. The normal group was injected with the same volume of normal saline, and the administration was continued until the 7th day. The specific administration and modeling time is shown in Figure 2 .

[0031] 3. Determination of organ index (spleen and thymus), peripheral cytokine (IFN-γ) and antibody (IgM) After 0.5h of administration on the 7th day, 1% sodium pentobarbital (prepared with normal saline) was injected intraperitoneally at a dose of 50 mg / kg for anesthesia. Immediately, blood was taken from the fundus vein and centrifuged for 10 min to obtain the upper serum (3000 rpm / min), which was stored at -80℃. Then, the concentration of peripheral blood cytokine IFN-γ and antibody IgM was detected by ELISA kit. After the mice were sacrificed by spinal dislocation, the thymus and spleen were washed with pre-cooled normal saline, dried with filter paper, weighed, and the organ index was calculated. The results are shown in Figure 3 .

[0032] As shown in Figure 3 A and 3B, the spleen index ( P P<0.001) and thymus index ( P P<0.0001) of the model group were significantly lower than those of the normal group. As shown in Figure 3 C and 3D, serum detection showed that the levels of IFN-γ (Th1 type inflammatory factor) and antibody IgM (marker of excessive activation of humoral immunity) in the model group were abnormally high ( P P<0.01 vs P<0.0001), which proved that the immunocompromised model was successfully replicated.

[0033] After administration, ginseng root extract showed a significant dose-dependent immune regulation effect: the high content group (YY) significantly inhibited IFN-γ ( P P<0.01 vs model group) and IgM ( P P<0.001); the medium content group (LMT) significantly regulated IgM ( P<0.01); the low content group (DST) had no statistical difference in the recall effect. It showed that among the three samples, the DST sample was invalid, the YY sample had the best immune boosting effect, and the LMT was in the middle, with certain immune boosting effect. Therefore, the total content of saponins is closely related to the strength of its immune regulation activity.

[0034] Example 3 Correlation analysis of different saponin combinations and ginseng immunotherapy efficacy and screening of medicinal material evaluation indexes Different saponin combinations were constructed, and the correlation between different saponin combinations and ginseng efficacy was detected. The combinations are as follows: 1) Re, Rb1, Rc and Rd; 2) Rg1, Re, Rb1, Rc, Rb2 and Rd; 3) Rg1, Re, Rb1, Rc and Rd; 4) Rg1, Re and Rb1; 5) Re, Rb1 and Rd; 6) Rg1, Re, Rc, Rb2 and Rd; 7) Rg1, Re, Rb2 and Rd; 8) Rg1, Re and Rb2; Among them, the detection results of the combination with strong correlation with the immune quality of medicinal materials are shown in Table 3 and Figure 4 , and the detection results of the combination with no correlation are shown in Figure 5 .

[0035] Table 3 Different combination contents and correlation coefficients

[0036] As shown in Table 3 and Figure 4 , among the different saponin combinations constructed, the combination of 4 components (Re+Rb1+Rc+Rd) has the best prediction effect on efficacy ( r >0.98, P <0.05), and the remaining combinations with strong correlation ( r >0.95) also include 6-component combinations: Rg1+Re+Rb1+Rb2+Rc+Rd, 5-component combinations: Rg1+Re+Rb1+Rc+Rd, and 3-component combinations: Rg1+Re+Rb1, Re+Rd+Rb1.

[0037] The combinations with poor correlation and unable to predict the immune regulation ability of ginsenosides are 5-component combinations: Rg1+Re+Rc+Rb2+Rd ( r <0.4), 4-component combinations: Rg1+Re+Rb2+Rd ( r <0.2), and 3-component combinations: Rg1+Re+Rb2 ( r<0.2), while Rg1+Rb2 is the worst, which is related to the lowest content of the two. Figure 5 ).

[0038] Therefore, according to the above detection results, even if the quality of the product has met the current pharmacopoeia specified qualified standard, it still cannot reflect the pros and cons of the product in the efficacy evaluation. However, the specific saponin combination developed by the present application as a marker group can effectively evaluate the pros and cons of the ginseng immunotherapy.

[0039] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the examples, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A method for evaluating the immunotherapeutic effect of ginseng, characterized in that, Detecting the content of the marker group consisting of at least three of Rg1, Re, Rb1, Rc, Rb2 and Rd in ginseng, and then comprehensively judging the immunotherapy effect of the ginseng sample to be detected according to the total content of the saponins in each marker group.

2. The method of claim 1, wherein, The total saponin content of the marker group is linearly correlated with the immunomodulatory activity of ginseng.

3. The method of claim 1, wherein, The marker group consists of Re, Rb1, Rc and Rd, and if the total content of saponins in the marker group is ≥72.97%, it is determined that it has excellent immunotherapy effect; if the total content of saponins is ≤29.25%, it is determined that it has poor immunotherapy effect; and if the total content of saponins is between 29.25% and 72.97%, it is determined that it has moderate immunotherapy effect.

4. The method of claim 1, wherein, The marker group consists of Rg1, Re, Rb1, Rc, Rb2 and Rd, and if the total content of saponins in the marker group is ≥77.60%, it is determined that it has excellent immunotherapy effect; if the total content of saponins is ≤37.57%, it is determined that it has poor immunotherapy effect; and if the total content of saponins is between 37.57% and 77.60%, it is determined that it has moderate immunotherapy effect.

5. The method of claim 1, wherein, The marker group consists of Rg1, Re, Rb1, Rc and Rd, and if the total content of saponins in the marker group is ≥75.22%, it is determined that it has excellent immunotherapy effect; if the total content of saponins is ≤31.57%, it is determined that it has poor immunotherapy effect; and if the total content of saponins is between 31.57% and 75.22%, it is determined that it has moderate immunotherapy effect.

6. The method of claim 1, wherein, The marker group consists of Rg1, Re and Rb1, and if the total content of saponins in the marker group is ≥54.81%, it is determined that it has excellent immunotherapy effect; if the total content of saponins is ≤21.11%, it is determined that it has poor immunotherapy effect; and if the total content of saponins is between 21.11% and 54.81%, it is determined that it has moderate immunotherapy effect.

7. The method of claim 1, wherein, The marker group consists of Re, Rb1 and Rd, and if the total content of saponins in the marker group is ≥62.32%, it is determined that it has excellent immunotherapy effect; if the total content of saponins is ≤23.51%, it is determined that it has poor immunotherapy effect; and if the total content of saponins is between 23.51% and 62.32%, it is determined that it has moderate immunotherapy effect.

8. The method according to any one of claims 1 to 7, characterized in that, IFN-γ and IgM are used as indexes for judging the immunotherapy effect, wherein the sample with excellent immunotherapy effect significantly inhibits both IFN-γ and IgM; the sample with moderate immunotherapy effect adjusts IgM; and the sample with poor immunotherapy effect has no adjustment effect on IgM.

9. The method of claim 8, wherein, The sample to be detected is ginseng leaf, ginseng stem, ginseng root, ginseng total saponin extract or corresponding preparation.

10. The marker group for evaluating the immunotherapeutic effect of ginseng according to any one of claims 1 to 9, wherein the marker group comprises the following markers: The marker group consists of at least three of Rg1, Re, Rb1, Rc, Rb2 and Rd. ​ 11. Use of the marker group of claim 10 in a preparation for evaluating the quality of ginseng immunotherapy.

12. Use according to claim 10 or 11, characterized in that, The sample of ginseng for evaluating the quality of immunotherapy is ginseng leaf, ginseng stem, ginseng root, ginseng total saponin extract or corresponding preparation.

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