Comparison of in vitro dissolution and pharmacodynamics of different forms of Chuanxiong

Liu Li?, Xu Xingang?, Gao Peng?, Dai Long?

( 1. Shandong University of Traditional Chinese Medicine, Jinan 250014, China; 2. Shandong Institute of Traditional Chinese Medicine, Jinan 250014, China)

Key words : Chuanxiong; ultrafine powder; ferulic acid; analgesia

OBJECTIVE: To compare the in vitro ferric acid dissolution and analgesic effect of Chuanxiong ultrafine powder, fine powder and water extract, alcohol extract and double extract.

METHODS : The in vitro dissolution of ferulic acid in the various forms of Chuanxiong was determined by in-situ dissolution test and thin-layer scanning method . The analgesic effect was compared by writhing method and hot plate method.

RESULTS : The dissolution of ferulic acid in each drug form ( 3g) was ultrafine powder (0.645±0.008) mg, fine powder (0.359±0.005) mg, water extract (0.220±0.004) mg, and alcohol extract ( 0.526±0.007) mg, double extract (0.061±0.003) mg; the analgesic effect of Chuanxiong ultrafine powder is good.

Conclusion : Chuanxiong ultrafine powder can increase the dissolution of active ingredients and improve the efficacy of fine powder.

CLC number: R284.2      Document identification code: A     Article ID : 1001-1528(2002)04-0246-03

Comparison on dissolution rates in vitropharmacodynamics of Rhizoma Chuanxiong by different forms used as intermediate

LIU Li?, XU Xin-gang ?, GAO Peng?, DAI Long?

(1.Shandong University of Traditional Chinese Medicine, Jinan250014, China; 2. Shandong Institute of Traditonal Chinese Medicine, Jinan250014, China)

Key words : Chuanxiong; supper-micronized powder ferulic acid; analgesic effect

Abstact: Objective: To compare the dissolution quantities in vitro of ferulic acid the analgesic effect of Rhizoma Chuanxiong by supper-micronized powder, fine powder, water extraction, alcohol extractiondouble extraction. Methods: Their dissolution rates in vitro were determined by paddle stirring method, the Dissolution quantities of ferulic acid in 60 min were determined by TLC-scaning, the analgesic effects were determined by torsionhot-plate methods. Results: The dissolution quantities of ferulic acid were respectively: supper-micronized powder (0.645±0.008) mg,fine powder (0.359 ± 0.005) mg, water extraction (0.220 ± 0.004) mg, alcohol extraction (0.526 ± 0.007) mg, double extraction (0.061 ± 0.003) mg. The analgesic effect of supper-micronized powder of Rhizoma Chuanxiong was the best. Conclusion : The supper-micronized powder of Chuanxiong improved the dissolution quantity of active componentsimproved the potency.

Chuanxiong is a commonly used traditional Chinese medicine. It has the effect of activating blood circulation, relieving phlegm and relieving pain. It is mainly used for irregular menstruation, menstrual dysmenorrhea, headache, rheumatism and other symptoms ^{[ 1 ]} . Its prescription preparations are more effective in treating pain. At present, the application of all powders into medicine or water, alcohol and other extraction drugs. In order to explore its scientific and rational form of drug use, we compared the in vitro dissolution of ferulic acid in two kinds of pulverization processes of Sichuan-Yunnan ultrafine powder and fine powder and three extraction processes of water extraction, alcohol extraction and double extraction by in vitro dissolution test. The pharmacodynamic comparison test of analgesic effect was carried out, and as a result, the ultrafine powder and the alcohol extract ferulic acid were eluted in a large amount, and the analgesic effect was strong. Explain that the correct form of Chuanxiong is: If the whole powder is used as a medicine, the ultrafine powder can increase the dissolution of the active ingredient and improve the efficacy. In order to reduce the dose of medication, it is better to use the Chuanxiong alcohol extract as a medicine.

1 experimental material

1.1 Instrument

939 Thin Layer Automatic Planer (Chongqing Nan'an Baird Instrument Technology Factory); Intelligent Dissolution Tester (Tianjin University Radio Factory); CS-9301

Thin layer scanner (Shimadzu, Japan); 82-1 centrifuge (Shanghai Medical Instrument Factory).

1.2    Medicinal herbs

Chuanxiong medicinal materials were purchased from Jinan Jianlian Chinese Medicine Store, and were identified as dried roots of the umbrella family Ligus-ticum Chuanxiong Hoort. by the Chinese Medicine Laboratory of Shandong Institute of Traditional Chinese Medicine.

1.3    Reagent

The reagents used were analytically pure; ferulic acid reference substance (China National Institute for the Control of Pharmaceutical and Biological Products).

1.4    animal

Kunming mice, shared by sputum, weigh 18~22g, 24~26g, provided by Animal Experimental Center of Shandong University of Traditional Chinese Medicine.

2 methods and results

2.1 Sample preparation

2.1.1 Chuanxiong fine powder Chuanxiong is crushed into powder that passes through the No. 6 sieve.

2.1.2 Chuanxiong micropowder Ultrafine pulverization of Chuanxiong into fine powder with a center particle size of 10 μm or less and not less than 95%.

2.1.3 Chuanxiong water extract Take Chuanxiong medicinal material and crush it into coarse powder. Weigh 50g, add water to cook twice, the first 10 times the amount of water for 2h, the second 8 times the amount of water for 1h, filtered, the filtrate was combined, concentrated under reduced pressure to a thick paste, dried in vacuo, to obtain a dry paste 18.1g.

2.1.4 Chuanxiong alcohol extract The Chuanxiong medicinal material is pulverized into coarse powder, weighed 50g, extracted with 80% ethanol twice, each time 8 times, each time 2h, filtered, the filtrate is combined, the ethanol is recovered under reduced pressure and concentrated to The thick paste was vacuum dried to obtain 12.0 g of a dry paste.

2.1.5 Chuanxiong double extracts were prepared by crushing the Chuanxiong medicinal materials into coarse powder, weighing 50g, adding water to extract the volatile oil to the end, collecting the volatile oil, filtering the extract, and boiling the slag with water for 1 hour, filtering, combining the filtrate, and concentrating under reduced pressure. Thick paste, vacuum dried, 20.2 g dry paste.

2.2     Eluate preparation

Adjust the dissolution bath water bath to a constant temperature of 37 ° C, accurately measure 1000 mL of artificial gastric juice in a beaker of the dissolution apparatus, and keep warm in a constant temperature water bath, and the stirring speed is set to 100 r·min ^-1 . Take the pulverized sample, accurately weigh the weight (fine powder, ultrafine powder weighed about 3g, extract the amount of dry paste equivalent to 3g of the original medicinal material), 3 parts per group, placed in the beaker of the analyzer, the artificial gastric juice is contacted The sample was started, and after 1 h, the liquid was taken out and centrifuged at 2000 r·min ^-1 for 20 min. The supernatant was taken and filtered through a filter to obtain a test solution.

2.3    Determination of ferulic acid content

2.3.1 Thin layer chromatography and scanning conditions. Take silica gel G, add 0.2% CMC-Na, and lay a thin layer plate of 10cm×20 cm×0.03cm by automatic plater. After activation at 105°C for 30min, store it in a desiccator. Standby after cooling. N-hexane-chloroform-glacial acetic acid (8:8:1) unfolded, 8cm spread, dual-wavelength reflective sawtooth scan, λ _s =328nm, λ _R =370nm, chromatogram shown in Figure 1.

1 2 3 4 5 6

Figure 1 TLC map of ferulic acid in Chuanxiong

1 , ferulic acid reference substance 4 , Chuanxiong water extract

2 , Chuanxiong micropowder 5 , Chuanxiong alcohol extract

3 , Chuanxiong fine powder 6 , Sichuan and Chongqing double extract

2.3.2    Drawing of standard curves    Accurately weigh about 1mg of ferulic acid reference substance, dissolve it in methanol, transfer it to a 2mL volumetric flask, add methanol and dilute to the mark and shake it to use as a reference solution. Accurately draw 1, 2, 3, 4, 5? L points on the same silica gel G thin-layer plate, unfold according to the above conditions, take out, air dry, view at 365nm under ultraviolet light, locate, determine, the results show that ferulic acid The loading amount between 0.5 and 2.5?g is linear with the integral value of the spot peak area. The regression equation is Y = 1124.68X + 273.21, r = 0.9992.

2.3.3    Sample measurement Precision extraction of 50 mL of eluate, extraction of ether 3 times, each time 50 mL of diethyl ether was combined, evaporated, and the residue was dissolved in methanol, and the volume was taken up in a 2 mL volumetric flask as a test solution. Accurately draw the test solution 10?L, the reference solution 1?L and 5?L point on the same silica gel G thin-layer plate, determined according to the law, the measurement results are shown in Table 1. (The content of ferulic acid in the original medicinal materials of Chuanxiong was 0.0314%).

Table 1 Ferulic acid yield in Chuanxiong (mg/3g medicinal materials) (n=3)

Sample set                             Ferulic acid dissolution x ± s

Chuanxiong ultrafine powder 0.645 ± 0.008

Chuanxiong fine powder 0.359 ± 0.005

Chuanxiong water extract 0.220 ± 0.004

Chuanxiong alcohol extract 0.526 ± 0.007

Chuanxiong double extract 0.061 ± 0.012

2.4    Analgesic effect of different comminuted degree of Chuanxiong

2.4.1 Analgesic effect of 0.6% acetic acid writhing method Compared with 60 mice, weighing 24~26g, they were randomly divided into 3 groups according to body weight, respectively, given to each drug group, 1 time/day, continuous 3 On the day, the control group was given an equal volume of distilled water. At the end of 30 minutes after the last administration, 0.6% acetic acid (0.1 mL/10g rat weight) was injected intraperitoneally. The time of * writhing reaction and the twist within 15 min were observed after injection. The number of body, and statistical processing between groups, the results are shown in Table 2.

2.4.2 Comparison of the analgesic effect of hot plate method on mice, 60 female mice, weighing 18~20g, pre-screening pain threshold before the experiment, the pain threshold is greater than 5s, less than 30s is qualified, and then randomly divided into 3 groups. The same as 2.4.1, the pain threshold was measured according to the hot plate method ^{[ 2 ]} , and the t-test between groups was performed. The results are shown in Table 3.

Table 2     Comparison of analgesic effects of different comminuted writhing methods in Sichuan and Yunnan (x ± s )

Number of animals    The first writhing reaction within 15min writhing

Group    

                            (only)     Latency (min)        (frequency)

Distilled water 20 2.51 ±0.45 30.68 ±3.48

Chuanxiong ultrafine powder 20 3.97 ±1.52 21.56 ±5.19 ²⁾

Chuanxiong fine powder 20 3.08 ±0.57 24.65 ±5.49 ¹⁾

Water extract of Chuanxiong 20 2.65 ±0.77 28.72 ±4.21

Chuanxiong alcohol extract 20 2.96 ±0.59 25.23 ±3.76 ¹⁾

Chuanxiong double extract 20 2.81 ±1.34 27.54 ±4.53

                      Compared with distilled water group, P<0.05, ?P<0.01. (the same below)

Table 3       Different comminuted degree of Chuanxiong on the hot plate method of mice

Comparison of analgesic effects ( x ± s )

Group            Number of animals (only)           Pain threshold (s)

Distilled water 15 16.48 ±4.73

Chuanxiong ultrafine powder 14 26.57 ±7.01 ²⁾

Chuanxiong fine powder 14 20.19 ±5.44 ¹⁾

Chuanxiong water extract 15 17.21 ±3.54

Chuanxiong alcohol extract 14 20.11 ±4.12 ¹⁾

Chuanxiong double extract 14 18.65 ± 2.47

3 Summary and discussion

The experimental results show that the fertilization of the active ingredient ferulic acid in the ultrafine powder and alcohol extract is higher and the analgesic effect is stronger in the different pulverization and extraction processes of Chuanxiong. According to reports in the literature, ligustilide, the main component of the volatile oil of Rhizoma Chuanxiong, has an antagonistic effect on uterine contractions ^{[ 3 ]} . The organic acid component ferulic acid has anti-inflammatory and analgesic effects ^{[ 4 ]} , but it is easily destroyed by heat. In the water extraction and double-extraction process of Chuanxiong, ferulic acid is lost in large amount due to high temperature and long-time heating. Chuanxiong ultrafine powder has broken the wall of the medicinal materials, which is more conducive to the dissolution of the active ingredients. Therefore, the ultrafine powder of Sichuan-Yunnan and the alcohol extract ferulic acid have more dissolution and the analgesic effect is stronger. In addition, its alkaloid ligustrazine can pass through the blood-brain barrier, which is one of the medicinal ingredients ^{[ 5 ]} . However, this component is extremely susceptible to heat damage and has been lost through the process of heating extraction and concentration ^{[ 6 ]} . This part of the work needs to be supplemented because there is no reference substance for the purchase of ligustrazine.

It can be seen from this experiment that the dissolution of ferulic acid in the extraction of Ligustrazine is second only to the ultrafine powder, and the analgesic experiment also confirms that the alcohol extraction method is stronger than the water extraction method and the double extraction method, second only to the ultrafine powder and fine powder. Comprehensive consideration, we believe that in the case of the dosage form allowing the original medicinal material powder to be used as a medicine, such as powder, honey pill, etc., the form of Chuanxiong is better than ultrafine powder, but if it is a liquid preparation or a dosage form that needs to reduce the dosage, such as dropping pills, etc. The form of Chuanxiong is still better with alcohol extract.

Ultrafine pulverization technology has been increasingly reported in recent years for the application of traditional Chinese medicine. It is a compression pulverization of materials by vibration, and the particle size of the original drug powder obtained by the conventional pulverization process can be from 150 to 200 mesh (75 μm or less). Increase to the center particle size of 5 ~ 10μm or less, in order to achieve the purpose of cell wall breaking. This new technology not only retains the active ingredient in its entirety, but also exposes the active ingredients in the drug directly, so that the drug is absorbed more quickly and completely ^{[ 7 ]} . There is a certain correlation between the dissolution of the active ingredients of traditional Chinese medicines and the bioavailability of drugs in the body ^{[ 8,9 ]} . Based on the results of this study, it can be seen that the research on the preparation process of Chuanxiong ultrafine micronized medicine or prescription preparation is relatively reasonable and scientific. This study also provides a new idea for the study of ultrafine micronization of traditional Chinese medicine.

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[4] Liu Yingxiang. Anti-inflammatory effects of tetramethylpyridazine and ferulic acid [J]. Foreign Medicine·Plant Medicine, 1993, 8(2): 85.

[5] Xiao Jing. New Progress in the Pharmacological Study of Ligustrazine[J]. West China Journal of Pharmacy, 1993, 8(3): 170.

[6] Beijing Institute of Pharmaceutical Industry. Study on active constituents of Chuanxiong: extraction, isolation and structural identification of Chuanthazine [J]. Chinese Medical Journal. 1977, 57(8): 420-421.

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