CN103919835B - Preparation method and pharmaceutical use of Tripterygium wilfordii extract with triterpenes as main components - Google Patents

Abstract

The invention discloses the preparation method and its pharmaceutical applications of the triperygium wilfordii extractive with triterpenes as main component.The preparation method of triperygium wilfordii extractive of the present invention with triterpenes as main component：（1）The full root herb of dry thunder godvine is crushed, 10~40 mesh sieves are crossed, 150~200g of powder is weighed；（2）Ultrasonic extraction is carried out with Extraction solvent, Extraction solvent is 8~16mL with medicinal material amount ratio：1g, extract solution filtering, filtrate decompression reclaims Extraction solvent, obtains concentrate, dries, and obtains thunder godvine extract powder crude product；（3）Step（2）Middle gained thunder godvine extract powder 5.0~15.0g of crude product, adds solvent to redissolve, and solvent is 10~15mL with the amount ratio of thunder godvine extract powder crude product:1g, filtering, is recovered under reduced pressure solvent, obtains thunder godvine extract powder.The invention also discloses the medicine that above method gained thunder godvine extract powder is used to prepare rheumatoid arthritis.Obtained thunder godvine extract powder of the invention, total triterpene contentses improve the availability of tripterygium wilfordii, and toxicity reduction 50~75%.

Description

Preparation method and pharmacy of Tripterygium wilfordii extract mainly composed of triterpenes use

technical field

The invention belongs to the technical field of extraction of traditional Chinese medicine components, and in particular relates to an extraction optimization method of Tripterygium wilfordii extract powder with total triterpenoids as the main component and its application in medicine for treating rheumatoid arthritis.

Background technique

Tripterygium wilfordii Hook.f is a plant of the genus Tripterygium wilfordii Hook.f in the family Euonymus, mainly produced in Zhejiang, Anhui, Jiangxi, Hunan, Fujian and Yunnan. Bitter in the mouth, pungent, cool in nature, very poisonous. Return liver, kidney channel. Efficacy Expelling wind and dampness, dredging collaterals and relieving pain, reducing swelling and pain, detoxifying and killing insects. It is used for damp-heat nodules and cancerous tumors, and it is used clinically to treat leprosy reactions and rheumatoid arthritis. It is often used in Chinese patent medicines such as Tripterygium wilfordii tablets, tripterygium glycosides tablets, tripterygium wilfordii polyglycosides tablets, tripterygium total terpenes tablets, triptolide tablets, and triptolide ointment.

The main chemical components of Tripterygium wilfordii include diterpenes, triterpenes, sesquiterpenes, alkaloids, tannins, organic acids and polysaccharides. Tripterygium wilfordii can fully act on lymphocytes, reduce the production of various inflammatory factors, inhibit the proliferation of immune cells, induce apoptosis, thereby inhibiting immunity, and has a certain effect on the treatment of RA. The components of Tripterygium wilfordii are complex, and the currently recognized active ingredients of Tripterygium wilfordii for the treatment of RA are mainly diterpenoids, triterpenoids and alkaloids. Among them, the content of diterpenoids in Tripterygium wilfordii is low and highly toxic; the content of triterpenoids is high and easy to extract. There are literatures (Miao Kangli, Xu Xuehua, Wei Zhaohui, Rui Chaosong. Study on the active ingredient Demethylzeylasteral of Tripterygium wilfordii. Chinese Modern Clinical Medicine, 2005, 4 (10): 7-10.) reported the activity of triterpenoids norzelamyl The median lethal dose LD50 (mice) is 1295mg/kg, showing no cytotoxicity, and within a certain dose range, the immunosuppressive effect is positively correlated with its dose.

Most of the existing patents of Tripterygium wilfordii extract are for the purpose of extracting a single compound in Tripterygium wilfordii, mainly focusing on the purification and structural modification of triptolide. For example, Chinese patent application No. 201010603887.5 introduces a preparation The method for extracting norzeralin from tripterygium wilfordii by high performance liquid phase, the purity of the obtained norzeralin is 98.5%. 200810209679.X introduced a method for extracting triptolide from triptolide to obtain pure triptolide triptolide extract powder with a purity of 1.21%. 201110139090.9 introduced a preparation method of tripterygium wilfordii effective part powder, but its total triterpenoid content is only 15%.

Patent No. 201110139090.9 introduces a method for preparing and separating the effective part powder of Tripterygium wilfordii for the treatment of chronic nephritis. The terpene content of the effective part powder obtained in this invention is greater than 50%, and the total triterpene content is about 15%. The highly toxic sesquiterpene alkaloid component is about 40%, and the indication is chronic kidney disease.

Contents of the invention

The inventor of the present invention has found through long-term experimental research that tripterygium wilfordii triterpenoids have significant curative effects in the treatment of rheumatic arthralgia. Therefore, if the tripterygium extract powder with total triterpenoids as the main component can be prepared, the utilization of tripterygium medicinal resources can be improved, the toxicity of tripterygium can be reduced, and the curative effect and safety of it for treating rheumatoid arthritis can be improved. Based on this, the present invention provides a preparation method of Tripterygium wilfordii extract mainly composed of triterpenoids, and its use in medicine for treating rheumatoid arthritis.

Compared with untreated Tripterygium wilfordii extract powder containing a high proportion of total triterpenes, the tripterygium wilfordii extract powder provided by the present invention has significantly improved drug efficacy in inhibiting the proliferation of arthritis synoviocytes and the like, and has reduced toxicity , can realize industrial production.

To achieve the above object, the present invention takes the following technical solutions:

A preparation method of Tripterygium wilfordii extract with triterpenoids as the main component, the specific steps are as follows:

(1) Grind the dried whole root of Tripterygium wilfordii, pass through a 10-40 mesh sieve, and weigh 150g-200g of the powder;

(2) Ultrasonic extraction is carried out with ethanol solution, ethyl acetate or acetone and other solvents, the ratio of extraction solvent to medicinal material is 8ml-16mL: 1g, the extract is filtered, the filtrate is decompressed and the solvent is recovered to obtain a concentrated solution, which is dried to obtain Tripterygium wilfordii Extract powder crude product;

(3) The crude extract powder obtained in step (2) is 5.0g ~ 15.0g, add ethyl acetate, ethanol, absolute ethanol or an acidic ethanol solution with hydrochloric acid to adjust the pH to redissolve, the solvent and the crude extract powder The dosage ratio is 10～15mL:1g, filter, recover the solvent under reduced pressure, and obtain Tripterygium wilfordii extract powder.

Preferably, in step (2), the concentration of the ethanol solution is 80% to 95%, and the number of ultrasonic waves is 1 to 3 times, 0.5 to 2.0 hours each time.

Preferably, in step (2), the drying is vacuum drying at 55-75°C.

Preferably, in step (2), the ultrasonic extraction power is 250W-500W, the stirring speed is 750-1250rpm, and the ultrasonic temperature is 25-50°C.

The invention also discloses that the tripterygium twig extract powder obtained by the above extraction method is used to prepare a medicine for treating rheumatoid arthritis.

In the present invention, the less toxic total triterpene component among the terpene components is used as the target object, and norzelamin is used as the effective monomer component, and the total triterpene content in the obtained extract is 50% to 75%. The content of the effective monomer component norzeratin is about 10%, and the indication is rheumatoid arthritis.

The inventors have found through research that the solubility of total triterpenes represented by norzeralin in solvents with different polarities varies greatly, and its solubility is affected by pH. Using this solubility characteristic can achieve a high proportion of total triterpenes. Preparation of Triterpenoid Tripterygium Extract Powder. Adopting the technical solution of the present invention, the total triterpene content in the treated Tripterygium wilfordii extract powder is determined to be 50% to 75% by a chromogenic method.

The inventor of the present invention has conducted long-term related experimental research on Tripterygium wilfordii and found that in the treatment of rheumatic arthralgia, both triterpenoids and diterpenoids in Tripterygium wilfordii are active ingredients, but the diterpenoid triptolide is the main one. Toxic ingredients. When the content of triptolide exceeds a certain limit, it is very easy to cause poisoning, but no obvious toxicity has been found for triterpenoids represented by norzelamyl.

Based on the research on the toxicity and medicinal components of Tripterygium wilfordii, the present invention provides a preparation method of Tripterygium wilfordii extract powder whose main component is total triterpenes, the tripterygium wilfordii extract powder prepared by this method contains higher The total triterpenoids, and can be used exclusively for the treatment of rheumatic arthralgia, effectively solve the waste of tripterygium medicinal resources and the safety of tripterygium preparations, the problem of narrow clinical application range, improve the bioavailability of the drug, and provide a new way for tripterygium. The application of rattan Chinese medicinal materials provides a new technical development direction.

The Tripterygium wilfordii extract powder containing a high proportion of total triterpenes prepared by the preparation method of the present invention found that the content of total triterpenes is 50-75% after measurement; the present invention proposes to treat rheumatoid arthritis with total triterpenes as active ingredients The total triterpenoid tripterygium extract powder prepared by the method of the present invention takes the total triterpenes in Tripterygium wilfordii as the target object, improves the utilization of tripterygium wilfordii medicinal materials, and significantly reduces toxicity.

detailed description

The technical solutions of the present invention are further illustrated below in conjunction with the examples, but these examples do not limit the protection scope of the present invention.

Embodiment 1: prepare the method for the tripterygium officinalis extract powder containing high proportion total triterpenes:

(1) Take the whole root of Tripterygium wilfordii, crush it, pass through a 40-mesh sieve, and weigh 150g;

(2) Extract once with 2400mL ethanol ultrasonic (ultrasonic power 250W, speed 750rpm, temperature 20°C), extract for 2 hours, filter the extract, recover the solvent from the filtrate under reduced pressure, obtain a concentrated solution, and dry it in vacuum at 75°C to obtain Tripterygium wilfordii extract Crude powder 10.30g;

(3) Add 103 mL of ethyl acetate to the crude extract powder to redissolve, filter, and recover the solvent from the filtrate under reduced pressure to obtain 9.28 g of Tripterygium wilfordii extract powder. The total triterpene content is 54.7% determined by UV method, and determined by HPLC Among them, the content of norzera wood aldehyde is 6.2%.

Embodiment 2: prepare the method for the tripterygium twig extract powder containing high proportion of total triterpenes:

(1) Take the whole root of Tripterygium wilfordii, crush it, pass through a 10-mesh sieve, and weigh 200g;

(2) Use 1600mL ethyl acetate ultrasonic extraction (ultrasonic power 500W, rotation speed 1250rpm, temperature 50°C) to extract 3 times, 0.5h each time, combine the extracts, filter, and recover the solvent from the filtrate under reduced pressure to obtain a concentrated solution, vacuum at 55°C Dried to obtain 6.46g of the crude product of Tripterygium wilfordii extract powder;

(3) Add 85 mL of absolute ethanol to the above-mentioned crude extract powder to redissolve, filter, and recover the solvent from the filtrate under reduced pressure to obtain 5.89 g of Tripterygium wilfordii extract powder. The total triterpene content is 72.8% determined by UV method, and determined by HPLC Among them, the content of norzera wood aldehyde is 10.5%.

Embodiment 3: the method for preparing the tripterygium twig extract powder containing high proportion of total triterpenes:

(1) Take the whole root of Tripterygium wilfordii, crush it, pass through a 24-mesh sieve, and weigh 200g;

(2) Ultrasonic extraction with 2000mL acetone (ultrasonic power 400W, rotation speed 1000rpm, temperature 40°C) was used to extract twice, each time for 1.0h, the extracts were combined, filtered, and the filtrate was decompressed to recover the solvent to obtain a concentrated solution, which was vacuum-dried at 70°C. Obtain 5.98g of the crude product of Tripterygium wilfordii extract powder;

(3) Add 55 mL of acidic ethanol (containing 0.1% hydrochloric acid) to the crude product of the above-mentioned extract powder to redissolve with hydrochloric acid, filter, and recover the solvent from the filtrate under reduced pressure to obtain 5.32 g of tripterygium twig extract powder. The content of triterpene is 68.4%, and the content of norzeralin is 8.6% measured by HPLC.

Embodiment 4: the method for preparing the tripterygium twig extract powder containing high proportion of total triterpenes:

(1) Take the whole root of Tripterygium wilfordii, crush it, pass through a 24-mesh sieve, and weigh 200g;

(2) Ultrasonic extraction with 2400mL80% ethanol (ultrasonic power 450W, rotation speed 1000rpm, temperature 40°C) was used to extract twice, each time for 0.5h, the extracts were combined, filtered, the filtrate was reduced in pressure to recover the solvent, and the concentrated solution was obtained, which was vacuum-dried at 70°C , to obtain Tripterygium wilfordii extract powder crude product 11.39g;

(3) Add 250mL of ethyl acetate to the crude extract powder to redissolve, filter, and recover the solvent under reduced pressure to obtain 9.35g of Tripterygium wilfordii extract powder. The total triterpene content is 52.5% by UV method, and 52.5% of which is determined by HPLC. The norzeralin content is 7.5%.

Embodiment 5: rat ear swelling test of tripterygium twig extract powder:

Select embodiment 1, the tripterygium wilfordii extract powder of gained is carried out preliminary animal anti-inflammatory efficacy evaluation:

1. Experimental materials

1.1 Materials and instruments: CMC‐Na (CP, batch number F20110609, Sinopharm Chemical Reagent Co., Ltd.), xylene (CP, batch number 20030501, Juhua Group Corporation Reagent Factory), indomethacin enteric-coated tablets (100 tablets/bottle , batch number 110101, Shanghai Xinyi Huanghe Pharmaceutical Co., Ltd.), norzeratin reference substance (1g, batch number FY18540201, Nantong Feiyu Biotechnology Co., Ltd.), 6mm diameter punch.

1.2 Animals: Kunming male mice (animal use license number: SCXK (Zhejiang)‐20080033)

2. Methods and Results

2.1 Sample: Weigh an appropriate amount of tripterygium twig extract powder, norzeramydal, and indomethacin enteric-coated tablets, add 15mL of 0.5% CMC-Na solution, stir and ultrasonically make the suspension uniform, as shown in Tables 1 and 2 below .

Table 1 Preparation of the drug solution of the norzeramylal efficacy test group

Table 2 Preparation of tripterygium wilfordii extract powder efficacy test group liquid

group Extract powder concentration/c (mg/mL) Dose (mg/kg/d) high dose 6.34 127.08 medium dose 1.43 28.46 low dose 0.47 9.45

2.2 Anti-inflammatory efficacy experiment of norzeramylal:

The mice were fasted without food and water the night before administration. Grouping of mice: Divide the mice into 3 groups according to body weight, and then randomly divide the mice of each body weight into 8 groups, with 10 mice in each group. The five groups are blank control group (0.5% CMC-Na solution), positive control group group (indomethacin), high-dose group, middle-dose group, and low-dose group, as shown in Tables 1 and 2. Oral administration of 0.4 mL per day for 3 consecutive days. One hour after the last administration, 0.05 mL of xylene was applied to the right ear of the mice with a syringe, and the left ear was left untreated. After 4 hours, the mice were killed by neck dislocation, and the two ears were cut off along the base line of the auricles, and round ear pieces were respectively punched in the same part of the left and right ears with a 6 mm diameter punch, and weighed. Calculate swelling.

Swelling degree (mg) = left ear piece weight (mg) - right ear piece weight (mg)

Swelling inhibition rate (%) = (average swelling degree of the control group - average swelling degree of the administration group) / average swelling degree of the control group

Table 3 The results of the swelling inhibition rate of different tripterygium wilfordii drugs and indomethacin on mouse ear swelling

group Inhibition rate% blank control 0 Indomethacin 32.92±2.665 High Dose (norzeratal) 31.43±1.949 Medium dose (norzeratal) 20.05±1.749 Low Dose (norzeratal) 5.94±1.324 High dose (tripterygium wilfordii extract powder) 68.41±1.247** Medium dose (tripterygium wilfordii extract powder) 37.76±0.851 Low dose (tripterygium wilfordii extract powder) 14.11±2.345

Note: Compared with indomethacin, *P<0.05, **P<0.01.

From the above results, it can be seen that: norzeramydal and Tripterygium officinalis extract powder have obvious anti-inflammatory activity; compared with the reference drug indomethacin, the anti-inflammatory effect of norzeramydal at high concentrations is comparable to that of indomethacin. Domethacin is similar, and the anti-inflammatory effect of tripterygium wilfordii extract obtained in the present invention is greater than or equal to that of indomethacin at high and medium concentrations.

Embodiment 6: Acute toxicity test of Tripterygium wilfordii extract powder to mice

Select embodiment 3, carry out preliminary toxicity evaluation to the extract powder of gained:

1. Experimental materials

1.1 Materials and instruments: CMC‐Na (CP, batch number F20110609, Sinopharm Chemical Reagent Co., Ltd.), norzeralin (prepared according to the method of patent 201010603887.5),

1.2 Animal: Kunming male mouse (animal use license number: SCXK (Zhejiang)‐20080033)

2. Methods and Results

2.1 Sample preparation: Weigh Tripterygium wilfordii extract powder, norzelamaldehyde plus 0.5% CMC-Na solution, stir and ultrasonically make the suspension uniform to obtain acute toxicity reagent, as shown in Table 4.

Table 4 Acute Toxicity Test Liquid of Norzeramylal and Tripterygium wilfordii Extract Powder

serial number Concentration of extract powder (mg/mL) Dissolution status blank control 0 ‐‐‐ norzeratal 1 63.56 suspend norzeratal 2 135.75 suspend norzeratal 3 260.35 The drug solution is thick Tripterygium wilfordii extract powder 1 387.44 The drug solution is thick Tripterygium twig extract powder 2 502.63 The drug solution is thick Tripterygium wilfordii extract powder 3 795.84 There is little solvent, and the extract powder is in an infiltrated state

2.2 Acute toxicity test methods and results:

Mice were fasted without food and water the night before administration. Grouping of mice: Divide mice into 3 groups according to body weight, and then randomly divide mice of each body weight into 7 groups, 10 in each group, and blank control group (0.5% CMC-Na solution), as shown in Table 4. Oral administration of 0.2mL. See Table 5 for statistical calculation of LD ₅₀ .

Table 5 Tripterygium wilfordii acute toxicity test result

group norzeratal 1357.50 Tripterygium wilfordii extract powder 5235.81

From the above results, it can be known that the LD ₅₀ of norzeratumaldehyde in this experiment is close to the 1295mg/kg reported in the literature _; The LD ₅₀ of metzeralin is less toxic than pure norzeralin.

Example 7: Tripterygium wilfordii extract powder inhibits the proliferation of arthritis synoviocytes

Selecting embodiment 3, the tripterygium wilfordii extract powder of gained is carried out preliminary efficacy evaluation:

1. Experimental materials

1.1 Samples: Weigh an appropriate amount of Tripterygium wilfordii extract powder, triptolide, and norzeramydal, and dissolve them in cell culture medium to obtain a drug-containing culture medium as shown in Table 6.

Table 6 Drug-containing culture medium of each group (mg/mL)

1.2 Reagents: Fetal bovine serum (Gibco, batch number: 623311); MTT (sigama subpackage, batch number: M2128), DMEM low-sugar culture medium (Gino Biomedical Technology Co., Ltd., batch number: 12102204).

1.3 Instruments: Inverted phase contrast microscope (Leica DMIL), cell culture incubator (Thermo Forma3111), full-wavelength microplate reader (M4 type, MD Company).

1.4 Cells: Primary HFLS‐RA cells were purchased from Cell Applications and subcultured in our laboratory.

2. Methods and Results

Take the 5th passage HFLS‐RA cells, add 100 μl of cell suspension to each well of a 96-well cell culture plate, and the number of cells is 1×10 ⁴ /well. After culturing for 24 hours to allow the cells to adhere to the wall, 200 μl of triptolide extract powder, triptolide and norzelamydral were added in four concentrations of A, B, C and D respectively (see Table 6). Three replicate wells were set up for each concentration. The DMEM culture medium containing cells without drugs was used as the standard control, and the DMEM culture medium without cells was used as the blank control. After administration and incubation for 24 hours, add 200 μl of MTT working solution to each well; after 4 hours of incubation, remove the MTT working solution, and then add 200 μl of DMSO to each well, shake at 180 rpm, avoid light, and incubate at room temperature for 10 minutes; measure with a microplate reader at a wavelength of 570 nm Absorbance values.

Calculate the cell inhibition rate, the calculation formula of the inhibition rate is as follows: inhibition rate%=[1‐(absorbance of sample well/absorbance of standard control well)]×100%, and the calculation results are shown in Table 7.

Table 7 Inhibition of different components of Tripterygium wilfordii on the proliferation of HFLS-RA cells n=3

group triptolide norzeratal Tripterygium wilfordii extract powder Group A 98.19±1.43 70.77±4.48** 72.28±3.43 Group B 93.11±3.55 50.32±2.87** 55.50±6.52 Group C 82.25±4.09 46.87±4.90** 50.22±5.31** Group D 63.37±7.21 18.85±6.34** 22.13±4.08**

Note:

Under the same concentration level group, compared with the triptolide group, *P<0.05, **P<0.01; compared with the norzeramaldehyde group, ^△ P<0.05, ^△△ P<0.01.

The above experimental results show that triptolide and norzelamyl have inhibitory effect on the proliferation of arthritic synoviocytes, and the effect is weakened with the decrease of dose. The product (i.e. Tripterygium officinalis extract powder) prepared in the present invention has obvious effect of treating rheumatic arthralgia, and is equivalent to pure norzeramydal, but the preparation process is relatively pure norzeramydal Aldehydes are simple and easy to work with.

Example 8: Inhibitory effect of Tripterygium wilfordii extract powder on RAW264.7 cell proliferation

Select embodiment 3, the tripterygium wilfordii extract powder of gained is carried out preliminary anti-inflammatory action evaluation:

1. Experimental materials

1.1 Weigh an appropriate amount of Tripterygium wilfordii extract powder, triptolide, and norzeramydal, and dissolve them in cell culture medium to obtain a drug-containing culture medium as shown in Table 8.

Table 8 Drug-containing culture solution of each group (mg/mL)

1.2 Reagents: Fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd., batch number: 121112); MTT (sigama subpackage, batch number: M2128), DMEM high-glucose culture medium (Gino Biomedical Technology Co., Ltd., batch number: 13040204).

1.3 Instruments: Inverted phase contrast microscope (Leica DMIL), cell culture incubator (Thermo Forma3111), full-wavelength microplate reader (M4 type, MD Company).

1.4 Cells: RAW264.7 cells, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, were subcultured in our laboratory.

2. Methods and results

Take RAW264.7 cells, add 100 μl of cell suspension to each well of a 96-well cell culture plate, and the number of cells is 1×104/well. After culturing for 24 hours to allow the cells to adhere to the wall, 200 μl of triptolide extract powder, triptolide and norzelamydal were added in four groups of concentrations A, B, C and D respectively (see Table 8). Three replicate wells were set up for each concentration. The DMEM culture medium containing cells without drugs was used as the standard control, and the DMEM culture medium without cells was used as the blank control. After administration and incubation for 24 hours, add 200 μl of MTT working solution to each well; after 4 hours of incubation, remove the MTT working solution, and then add 200 μl of DMSO to each well, shake at 180 rpm, avoid light, and incubate at room temperature for 10 minutes; measure with a microplate reader at a wavelength of 570 nm Absorbance values.

Calculate the cell inhibition rate, the inhibition rate calculation formula is as follows: inhibition rate%=[1‐(absorbance of sample well/absorbance of standard control well)]×100%, and the calculation results are shown in Table 9.

Table 9 Inhibition of different components of Tripterygium wilfordii on the proliferation of RAW264.7 cells n=3

group triptolide norzeratal Tripterygium wilfordii extract powder Group A 100.19±1.67 101.77±1.48 98.28±9.13 Group B 94.11±3.26 86.32±3.67 90.50±6.34 Group C 85.25±6.09 50.87±4.70** Group D 83.52±5.21 23.85±7.34**

Note:

Under the same concentration level group, compared with triptolide group, *P<0.05, **P<0.01; compared with norzeramaldehyde, ^△ P<0.05, ^△△ P<0.01.

The above experimental results show that: at high concentrations, tripterygium twig extract powder has no significant difference in proliferation inhibitory effect on RAW264. RAW264.7 cells have a stronger inhibitory effect on proliferation than pure norzeralin, and the inhibitory effect has a significant difference. The tripterygium wilfordii extract powder prepared by the invention is easy to prepare compared with pure norzelamaldehyde, and is beneficial to industrial production and clinical application.

Example 9: Inhibitory effect of Tripterygium wilfordii components on the proliferation of HaCaT cells

Selecting embodiment 2, the tripterygium wilfordii extract powder of gained is carried out preliminary toxicity evaluation:

1. Experimental materials

1.1 Sample: Weigh and weigh triptolide, Tripterygium wilfordii extract powder and total diterpenes of Tripterygium wilfordii (containing 70% of the total diterpenes) used in the examples, and dissolve them in cell culture medium to obtain the drug-containing culture medium as shown in Table 10.

Table 10 Drug-containing culture medium of each group (mg/mL)

group triptolide Total Diterpenes of Tripterygium wilfordii Tripterygium wilfordii extract powder Group A 0.0128 0.0135 0.0129 Group B 0.0064 0.0067 0.0064 Group C 0.0032 0.0034 0.0032 Group D 0.0016 0.0017 0.0016 Group E 0.0008 0.0008 0.0008

1.2 Reagents: Fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd., batch number: 121112); MTT (sigama subpackage, batch number: M2128), DMEM high-glucose culture medium (Gino Biomedical Technology Co., Ltd., batch number: 13040204).

1.3 Instruments: Inverted phase contrast microscope (Leica DMIL), cell culture incubator (Thermo Forma3111), full-wavelength microplate reader (M4 type, MD Company).

1.4 Cells: HaCaT cells, purchased from Wuhan Cell Bank Company, were subcultured in our laboratory.

2. Methods and Results

Take HaCaT cells, add 100 μl of cell suspension to each well of a 96-well cell culture plate, and the number of cells is 1×10 ⁴ /well. After culturing for 24 hours to allow the cells to adhere to the wall, 200 μl of drug-containing culture medium containing strychnine and strychnine in different proportions were added respectively (see Table 10). Three replicate wells were set up for each concentration. The DMEM culture medium containing cells without drugs was used as the standard control, and the DMEM culture medium without cells was used as the blank control. After administration and incubation for 24 hours, add 200 μl of MTT working solution to each well; after 4 hours of incubation, remove the MTT working solution, and then add 200 μl of DMSO to each well, shake at 180 rpm, avoid light, and incubate at room temperature for 10 minutes; measure with a microplate reader at a wavelength of 570 nm Absorbance values.

Calculate the cell inhibition rate, the inhibition rate calculation formula is as follows: inhibition rate%=[1-(absorbance of sample well/absorbance of standard control well)]×100%, and the calculation results are shown in Table 11.

Table 11 Inhibition of HaCaT cell proliferation by different components of Tripterygium wilfordii

group triptolide Total Diterpenes of Tripterygium wilfordii Tripterygium wilfordii extract powder Group A 73.94±3.74 75.05±4.34 68.79±4.68 Group B 70.95±5.13 69.17±5.29 69.62±1.58 Group C 76.30±3.34 73.29±3.23 Group D 71.76±5.89 70.26±6.52 Group E 76.79±1.95 64.32±5.08

Note:

1. Compared with triptolide, **P<0.01; compared with triptolide total diterpenes, ^△△ P<0.01.

The above experimental results show: triptolide has a strong proliferation inhibitory effect on HaCaT cells, and there is no significant difference as the concentration decreases, indicating that the cytotoxicity of triptolide is relatively large; , and the toxicity of triptolide group is basically equal. The toxicity of triptolide extract powder to HaCaT cells decreased with the decrease of concentration, and there was a significant difference between triptolide and total diterpenes of triptolide at low concentrations. Table 9 also proves that the products obtained by the extraction and purification method of the present invention (i.e. the "tripterygium officinalis extract powder" group) can achieve high efficiency and low toxicity in the treatment of rheumatic arthralgia, and can be used for long-term treatment of rheumatoid Rheumatoid arthritis.

Claims (4)

1. the preparation method of a kind of triperygium wilfordii extractive with triterpenes as main component, it is comprised the following steps that：

(1) the full root herb of dry thunder godvine is crushed, crosses 10 mesh~40 mesh sieves, weigh powder 150g~200g；

(2) ultrasonic extraction is carried out with Extraction solvent, Extraction solvent is 8ml~16mL with medicinal material amount ratio：1g, extract solution filtering, Filtrate decompression reclaims Extraction solvent, obtains concentrate, dries, and obtains thunder godvine extract powder crude product；

Described Extraction solvent is selected from ethanol solution, ethyl acetate or acetone；

(3) gained thunder godvine extract powder crude product 5.0g~15.0g in step (2), adds solvent to be redissolved, the solvent and Thunder God The amount ratio of rattan extract powder crude product is 10~15mL：1g, filtering, is recovered under reduced pressure the solvent, obtains thunder godvine extract powder；

The described solvent that redissolves is selected from ethyl acetate, absolute ethyl alcohol or the acid ethanol solution with salt acid for adjusting pH；

Described Extraction solvent is the different solvent of polarity with solvent is redissolved.

2. the preparation method of the triperygium wilfordii extractive with triterpenes as main component as claimed in claim 1, it is characterized in that：Step Suddenly (2), described ethanol solution concentration is 80%~95%, and ultrasonic number of times is 1~3 time, every time 0.5~2.0h.

3. the preparation method of the triperygium wilfordii extractive with triterpenes as main component as claimed in claim 1, it is characterized in that：Step Suddenly (2), described drying is 55~75 DEG C of vacuum drying.

4. the preparation method of the triperygium wilfordii extractive with triterpenes as main component as claimed in claim 1, it is characterized in that：Step Suddenly (2), described ultrasonic extraction power is 250W~500W, and 750~1250rpm of mixing speed, ultrasonic temperature is 25~50 DEG C.