Chinese medicine extract and preparation method and application thereof

Abstract

A Chinese medicine extract and preparation method and application thereof. Raw materials of the extract are Artemisia carvifolia and propolis. The extract has an excellent effect of inhibiting CaN-NF-AT pathway and a lower relative toxicity for cells.

Claims

1. A method for preparing a Traditional Chinese medicine extract, comprising extracting the raw materials consisting of Artemisia carvifolia and propolis, wherein the mass ratio of Artemisia carvifolia to propolis is 3:5 to 5:3.

2. The method according to claim 1, wherein a solvent for extraction is one of the group consisting of alcohols, alkanes, chloroform, acetone, water and ethyl acetate, or a mixture thereof.

3. The method according to claim 1, wherein the extraction method is percolation, impregnation, decoction, reflux extraction, continuous reflux extraction, supercritical fluid extraction or ultrasound extraction.

Description

DETAILED DESCRIPTION OF EMBODIMENTS

(1) The present invention provides a Chinese medicine extract, preparation method and use thereof. One of ordinary skill in the art can use this disclosure for reference and improve the technological parameter to reach the same result. In particular, all similar replacements and changes are obvious for those of ordinary skill in the art, so they will be considered within the scope of the present invention. The method and use of the present invention have been described in preferred embodiment. It is apparent that others can achieve and apply the technology in the present invention through reasonable changes and combinations within the scope of the present invention, to implement and apply the techniques of the present invention.

(2) The instruments and reagents used in the present invention are regular market products and commercial available.

(3) Hereinafter, the present invention will be further described in conjunction with the examples:

Example 1

(4) 30 g Artemisia carvifolia and 50 g propolis were added to 20-fold mass of 95% ethanol and percolation extraction was performed for 48 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Example 2

(5) 30 g Artemisia carvifolia and 50 g propolis were added to 25-fold mass of 95% ethanol and percolation extraction was performed for 48 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Example 3

(6) 50 g Artemisia carvifolia and 30 g propolis were added to 30-fold mass of 95% ethanol and percolation extraction was performed for 48 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Example 4

(7) 50 g Artemisia carvifolia and 30 g propolis were added to 30-fold mass of methanol and percolation extraction was performed for 48 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Example 5

(8) 40 g Artemisia carvifolia and 40 g propolis were added to 10-fold mass of chloroform and continuous reflux extraction (Soxhlet extraction) was performed at room temperature for 24 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Example 6

(9) 35 g Artemisia carvifolia and 45 g propolis were added to 10-fold mass of ethyl acetate and reflux extraction was performed at room temperature for 36 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Example 7

(10) 50 g Artemisia carvifolia and 30 g propolis were added to the same mass of butanediol and ultrasound extraction was performed at room temperature for 2 h. The extract was obtained by collecting the filtered solution and adjusting the mass to 80 g in butanediol.

Example 8

(11) 50 g Artemisia carvifolia and 30 g propolis were added to the same amount of propanedione and ultrasound extraction was performed at 50 C. for 2 h. The extract was obtained by collecting the filtered solution and adjusting the mass to 80 g in propanedione.

Example 9

(12) 50 g Artemisia carvifolia and 30 g propolis were added to 30-fold mass of petroleum ether and impregnation extraction was performed at room temperature for 48 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Example 10

(13) 45 g Artemisia carvifolia and 35 g propolis were added to 30-fold mass of water and decoction extraction was performed for 48 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Example 11

(14) 30 g Artemisia carvifolia and 50 g propolis were used for supercritical fluid extraction under the conditions: pressure 33-35 MPa, temperature 50 C., time 3.5 h, CO.sub.2 flow rate 15-25 L/h, ethanol volume rate in the cosolvent 15%. The extract was obtained by collecting and concentrating the solution to 80 g under low temperature and reduced pressure.

Comparative Example 1

(15) 80 g Artemisia carvifolia was added to 25-fold mass of 95% ethanol and percolation extraction was performed for 48 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Comparative Example 2

(16) 80 g propolis was added to 25-fold mass of 95% ethanol and percolation extraction was performed for 48 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Comparative Example 3

(17) 60 g Artemisia carvifolia and 20 g propolis were added to 25-fold mass of 95% ethanol and percolation extraction was performed for 48 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Comparative Example 4

(18) 20 g Artemisia carvifolia and 60 g propolis were added to 25-fold mass of 95% ethanol and percolation extraction was performed for 48 h. The extract was obtained by collecting and concentrating the filtered solution to 80 g under low temperature and reduced pressure.

Example 12

(19) Test sample: all extracts were dissolved in 70% ethanol and diluted in deionized water to a working solution of 0.2 mg/ml. For detection, 10 l sample, in which the final concentration of test extract is 0.2 mg/ml, was added to the test tube.

(20) CaN enzyme activity test: CaN activity test kit from Enzo Life Science was used and the test was performed according to the manufacture's instruction on the kit. 25 l CaM working solution, 5 l CaN working solution and 10 l 50 M test sample working solution were added to a small centrifuge tube, mixed well and incubated at 30 C. for 10 minutes. In the control tube, 10 l 0.5% DMSO water solution was added instead of test sample. After incubation, 10 l substrate working solution was added to each tube and mixed well. After incubation at 30 C. for 50-60 minutes, 100 l chromogenic reagent was added and mixed well. After standing for 20-30 minutes, 135 l solution from each tube was transferred to a 96-well plate and subjected to absorbance measurement at 620 nm.

(21) The determination of the optimum concentration of activator PMA and A23187: to determine the optimum concentrations of activator PMA and A23187 which activate the CaN-NF-AT pathway, PMA was added at concentration: 2.5 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml and 40 ng/ml. A23187 was added at concentration: 0.125 M, 0.25 M, 0.5 M, 1 M and 2 M. Each concentration has 4 repeat wells. After activation for 18 h, fluorescence intensity from all experiment groups was detected at 1 m.

(22) Test method: NFAT K562 reporter stable cell line, in which the NFAT reporter gene was expressed constantly, was purchased from Affimatrix (U.S.A.). After K562 cells were treated with PMA (CPKC kinase activator) and A23187 (Calcium channel activator), the luciferase gene expression level, which was controlled by transcription factor NFAT, was detected. The expression level of luciferase reporter gene reflects the activation level of CaN pathway. Results were shown in table 1:

(23) TABLE-US-00001 TABLE 1 The effects of PMA and A23187 to the expression of K562 NF-AT reporter gene at different concentrations Activator concentration Value at 1 M PMA A23187 Well Well Well Well ng/ml M 1 2 3 4 Average 0 0 261 197 179 148 196 2.5 0.125 1165 1608 1128 1228 1282 5 0.25 1859 1708 1918 2163 1912 10 0.5 3228 2970 2368 2518 2771 20 1 2317 2963 2527 3278 2771 40 2 3332 2530 3780 3239 3220

(24) As shown in table 1, PMA and A23187 can activate the expression of reporter gene at a dose-dependant manner. The difference of fluorescence readings between repeat wells can be controlled in a reasonable range. The optimum concentrations determined by above experiment for CaN pathway activators were: PMA 10 ng/ml, A23187 0.5 M.

(25) K562 cells in which the NFAT reporter gene was expressed constantly were conventional subcultured in RPMI-1640 medium with 10% fetal bovine serum. Healthy and logarithmic growth phase cells were used in the experiment. Cells were cultured in 24-well plate at a density of 210.sup.5/well and treated with the extracts from example 1-3 and comparative example 1-5. The wells without extract were set as control group and the wells without PMA and A23187 were set as blank group. After one hour incubation, PMA (10 ng/ml) and A23187 (0.5 M) (channel stimulants) were added to stimulate 18 h, then cells were collected and the fluorescence intensity (reflecting the activation level of CaN pathway in the cells) was detected at 1 m by using the luciferase test kit according to the protocol.

(26) The formula to calculate the inhibition ratio is: inhibition ratio (%) W=[1(XA)/(BA)]100.

(27) Wherein, A is the average of blank at 1 m, B is the average of control at 1 m, X is the average of extract at 1 m. Results were shown in table 2:

(28) TABLE-US-00002 TABLE 2 The inhibition ratio of different samples to CaN-NF-AT pathway in K562 cells Inhibition ratio (%) Example 1 66.58 Example 2 68.97 Example 3 69.43 Comparative example 1 36.69 Comparative example 2 42.37 Comparative example 3 39.21 Comparative example 4 45.46

(29) As shown in table 2, both Artemisia carvifolia and propolis total flavonoids can inhibit the CaN-NF-AT pathway quite well. Using SPSS 19.0 version statistics software for analysis:

(30) Example averageSED: 68.32%1.53%;

(31) Comparative example averageSED: 40.93%3.81%.

(32) By one-way ANOVA, the difference between examples and comparative examples are significant (P<0.01).

(33) The result of statistic analysis indicated that the combination of Artemisia carvifolia and propolis has a much better effect than Artemisia carvifolia or propolis alone. Therefore, the formulas provided in the present invention have a better result on CaN-NF-AT pathway inhibition and can be used to treat diseases targeting CaN-NF-AT pathway, such as skin inflammation (eczema) and autoimmune diseases (RA, CD or psoriasis).

(34) The extracts from other examples in the present invention have a similar inhibition ratio as the extracts from examples 1-3 and a significant difference compared to comparative examples 1-4.

Example 13

(35) Test the cell toxicity of extracts from examples 1-3 and comparative examples 1-5 by regular CCK8 assay, comprising the steps of: (1) 100 l K562 cells suspension (110.sup.5/well) was placed into 96-well plate and preincubated in the incubator for 24 h (37 C., 5% CO.sub.2); (2) 10 l extract from example 1-3 and comparative example 1-5 was added to the wells respectively except 6 control wells; (3) 96-well plate was incubated in the incubator for 24 h; (4) 10 l CCK8 solution was added to each well; (5) 96-well plate was incubated in the incubator for 4 h; (6) The absorbance was measured in a microplate reader at 450 nm.

(36) The formula to calculate the cell toxicity is: relative toxicity (%) W=[(AX)/A]100

(37) Wherein, A is the average of blank OD.sub.450, X is the average of extract OD.sub.450. Results were shown in table 3:

(38) TABLE-US-00003 TABLE 3 The relative cell toxicity of the extracts provided by the present invention Relative toxicity (%) Example 1 2.9 Example 2 3.1 Example 3 3.3 Comparative example 1 3.0 Comparative example 2 3.2 Comparative example 3 3.1 Comparative example 4 3.2

(39) The result indicated that there is no statistic difference between the cell toxicity of test samples. The relative cell toxicities were all quite low. Because the extract provided in the present invention was made from natural raw Chinese medicine, it overcomed the hidden dangers of Western medicine or hormone drug which has a pretty big side-effect. This extract provided a material base for the anti-eczema and skin relax cosmetics.

(40) The extracts from other examples in the present invention have a similar relative cell toxicity as the extracts from examples 1-3 or comparative examples 1-4.

(41) Above embodiments are only the preferred embodiments in the present invention. What needs to be pointed out is: one of ordinary skill in the art can make modification and improvement within the scope of the present invention; these modification and improvement shall be under the protection scope of the present invention.