CN105044300A - Method for adjusting dosage of medicinal material coptidis rhizoma on basis of anti-dysentery bacillus effect equivalency factor - Google Patents

Abstract

The invention provides a method for adjusting dosage of a medicinal material coptidis rhizoma on the basis of an anti-dysentery bacillus effect equivalency factor. The method comprises the following steps: (1), measuring the contents of active ingredients in the medicinal material coptidis rhizoma; (2), calculating an anti-dysentery bacillus effect equivalency factor of the coptidis rhizoma; (3), adjusting the dosage of the medicinal material coptidis rhizoma according to the anti-dysentery bacillus effect equivalency factor. According to the invention, a clinical function is used as guidance, the anti-dysentery bacillus effect equivalency factor can represent the coptidis rhizoma anti-dysentery bacillus pharmacological activity, and guide establishment and improvement of a clinical individualized medication standard; the problem of contribution difference of multiple component indexes to the efficacy of the coptidis rhizoma is solved, and comprehensiveness, quantization and integration of the coptidis rhizoma quality evaluation are realized; the efficacy of the coptidis rhizoma can be represented by detecting the contents of the coptidis rhizoma components directly, implementation is simple, easy and repeatable and the operability is high.

Description

A method for adjusting the dosage of Coptidis rhizome based on anti-shigella effect equivalent factor

technical field

The invention relates to a method for adjusting the dosage of coptis chinensis based on the anti-shigella effect equivalent factor.

Background technique

Coptis Rhizoma (Coptidis Rhizoma) is the dry rhizome of Coptis chinensis Franch., Coptis deltoidea C.Y. Chenget Hsiao and Coptisteeta Wall. of Ranunculaceae plants. It is often used for clearing away heat and dampness, purging fire and detoxifying. The ingredients related to its medicinal activity are alkaloids, including berberine, epiberberine, coptisine, palmatine, jatrorrhizine, etc. Modern pharmacological studies have shown that Coptis chinensis has antibacterial, antitumor, hypoglycemic, antiviral, and anti-inflammatory effects, among which the inhibitory effect of Coptis chinensis on Shigella has an important correlation with its traditional effect of "stopping dysentery".

As a bulk medicinal material in clinical practice, how to effectively evaluate and control the quality of Coptis chinensis will be of great significance to ensure its clinical efficacy and promote its product development. At present, the quantitative analysis of multiple index components is mainly used to realize the evaluation and control of the quality of Coptis chinensis, but this method does not reflect the difference in the contribution of different components to the overall efficacy of the medicinal material, and ignores the difference in the efficacy of different components. , it is difficult to evaluate the quality of different medicinal materials in practical application. For Coptis chinensis, the current standards for its quality control index components include four alkaloid components, berberine, coptisine, epiberberine, and palmatine. The content of berberine in one batch is 8.2%, coptisine is 2.7%, and the content of berberine in another batch is 9.5%, and coptisine is 1.8%, how to judge the quality of these two batches of medicinal materials by observing the content of ingredients What about the difference?

In addition, unlike chemical drugs, its component structure is clear and its structure-activity relationship is clear. Identification, inspection, and content determination can be directly used as indicators for efficacy evaluation. For traditional Chinese medicine, its material connotation is complex, and only testing the content of ingredients will lead to many limitations. It is necessary to correlate the quality control indicators with the efficacy and activity of the medicine. However, if these pharmacodynamic and active indicators are simply added on the basis of the current component content test, it will increase the difficulty and complexity of implementing quality control standards in the future. Because the addition of each drug efficacy index will lead to more standards to be met for each medicinal material, more cumbersome operations, and more experimental materials and experimental animals will be consumed, which will inevitably result in a waste of energy resources. Moreover, the problem of difference in contribution caused by this multi-indicator quality control model is still unresolved, and it is impossible to explain which indicator has a more important relationship with the quality of medicinal materials.

Therefore, if we can establish a comprehensive quality assessment and control method for Coptis chinensis that can directly concern "controllable and effective" without increasing the difficulty of implementing the quality control model, and can guide clinical medication, it will be of great importance to the quality control standards of Coptis chinensis and other traditional Chinese medicines. perfection is of great significance.

Contents of the invention

The object of the present invention is to provide a method for adjusting the dosage of Coptidis Rhizoma based on the anti-shigella effect equivalent factor (Effectequivalencyfactor, EEF).

The method for regulating the dosage of Coptidis Rhizoma based on the anti-Shigella effect equivalent factor of Coptidis Rhizoma of the present invention comprises the following steps:

(1) measure the content of berberine (BER), epiberberine (EPI), coptisine (COP), palmatine (PAL) and jatrorrhizine (JAT) in the Coptidis Rhizoma medicinal material, measure respectively X _BER , X _EPI , X _COP , X _PAL , X _JAT ;

(2) Utilize formula 1 to calculate the anti-shigella effect equivalent factor of Coptidis rhizome medicinal material to be tested:

Formula I: EEF＝3.352*X _BER +1.293*X _EPI +6.635*X _COP +0.140*X _PAL +0.419*X _JAT -0.111;

Described EEF is the anti-Shigella effect equivalent factor of Coptis Rhizoma medicinal material to be tested;

(3) Utilize formula II to calculate the actual consumption of Coptidis medicinal material:

Formula II is _Dmeasure =D _target /EEF; wherein Dmeasure is the actual dosage of the Coptidis Rhizoma to be _tested , and D _target is the target dosage of the Coptidis Rhizoma.

The method of adjusting the dosage of Coptis rhizome based on the anti-shigella effect equivalent factor of the present invention can not only reflect its "efficacy" by measuring the content of ingredients, but will not increase the difficulty of implementing quality standards, and is accurate, quantifiable, integrated and reproducible , comprehensively operable, effective and applicable, and can correlate with the clinical efficacy of medicinal materials, adjust the dosage of Coptidis Rhizoma according to the equivalent factor of anti-Shigella effect, to ensure the consistency of effect equivalent, innovative integration research on the quality evaluation and control standards of Coptidis medicinal materials and Clinical medication guidance is of great significance.

Detailed ways

The content of the invention will be further described below in conjunction with specific examples.

The method for adjusting the dosage of Coptis Rhizoma based on the anti-Shigella effect equivalent factor of the present invention can carry out activity evaluation and clinical dosage adjustment of Coptis Rhizoma samples.

Instruments used in the test: pulverizer; analytical balance (JM-B type, Yuyao Jiming Weighing Calibration Equipment Co., Ltd.); rotary evaporator (R205B, Shanghai Shensheng Technology Co., Ltd.); electric blast drying oven (DHG9070A, Shanghai Yiheng Scientific Instrument Co., Ltd.); circulating water multipurpose vacuum pump (SHB-B95, Zhengzhou Great Wall Technology Industry and Trade Co., Ltd.); Agilent1100 high performance liquid chromatography (DAD detector), Chemstations chromatographic workstation (Agilent Co., Ltd., USA ); BS210S electronic balance (Beijing Sartorious Co., Ltd.); water is distilled water.

(1) measure the content of berberine, epiberberine, coptisine, palmatine, jatrorrhizine in the collected Coptidis medicinal material:

Coptidis medicinal materials collected in Table 1

(i) Firstly prepare the water extract of Coptidis rhizome: take Coptidis rhizome medicinal material to remove impurities, wash and dry, then pulverize into coarse powder. Weigh 50g of different batches of Coptidis rhizome medicinal powder, soak in water for 30 minutes, decoct and extract 3 times (the amount of water added is 10, 10, and 8 times the weight of the medicinal material, and the extraction is 1.5, 1.0, and 0.5 hours respectively), and filter while hot. After drying, the filtrates were combined, concentrated under reduced pressure, the extract was transferred to an oven, and dried at 75°C to obtain the dry powder of the water-extracted sample of Coptis chinensis.

(ii) Preparation of reference solution: Accurately weigh appropriate amounts of berberine, epiberberine, coptisine, palmatine, and jatrorrhizine, place them in brown measuring bottles, add hydrochloric acid-methanol (1:100) Prepare each reference substance stock solution respectively. Precisely measure the appropriate amount of each reference substance stock solution, put it in the same brown measuring bottle, and prepare it to contain 0.4337mg of berberine, 0.1052mg of epiberberine, 0.0606mg of coptis, 0.1160mg of palmatine, and jatrorrhizine in each 1ml. 0.0721 mg of the mixed reference substance stock solution is set aside (sealed storage below 10°C).

(iii) Preparation of the test solution: Weigh about 50 mg of Coptis chinensis extract powder sample, accurately weigh it, put it in a stoppered conical flask, add 50 ml of hydrochloric acid-methanol (1:100), weigh it, and set the weight at 60°C Heat in a water bath for 15 minutes, take it out, ultrasonically treat it for 20 minutes, let it cool down, weigh it again, make up the lost weight with hydrochloric acid-methanol (1:100), shake well, and filter through a microporous membrane (0.45 μm) , that is.

(iv) liquid chromatography measurement:

Chromatographic conditions: Chromatographic column: Kromasil100-8C ₁₈ (250mm×4.6mm); mobile phase A: acetonitrile (1.5g sodium dodecyl sulfate/500ml)-water (2:1) (phosphoric acid adjusted to pH 6.0), mobile Phase B: water (3.0g potassium dihydrogen phosphate/500ml), linear gradient elution program (0min: 38%B, 55min: 38%B, 65min: 15%B, 70min: 0%B); detection wavelength: 345nm ; Flow rate: 1.0ml/min; Column temperature: 30°C; Injection volume: 10μl.

The measurement results are shown in Table 2.

Table 2: Contents of active ingredients in Coptidis Rhizoma

(2) Construction of Coptidis rhizome anti-shigella effect equivalent factor:

① Shigella inoculation: extract 1.1mL of Shigella original bacteria liquid (fourth generation), dilute it with LB medium to form an inoculation medium with a total volume of 44mL, that is, the original bacteria liquid is enlarged by 40 times, and the Shigella concentration is 1×10 ⁶ CFU/mL. ②In a clean and sterile environment, accurately add 5mL of LB culture medium inoculated with Shigella dysenteriae into each glass ampoule bottle with a volume of 20mL, and then add different volumes of active ingredient stock solution to each bottle, and constant volume to 10mL, so that the final concentration of the active ingredient is 0.00mg/mL, 0.05mg/mL, 0.10mg/mL, 0.15mg/mL, 0.20mg/mL, 0.25mg/mL, cap the bottle stopper, seal it, and put it into a micropipette In the calorimeter, the "power-time (Pt)" curve changes of the growth and metabolism of Shigella in each ampoule were detected by the recording and tracking software that comes with the instrument. The temperature of the instrument was controlled at 37°C, and the experiment was terminated when all the curves returned to the baseline. The corresponding data were exported by software, and the data on the curve were imported into Origin8.5 software (OriginLab, USA) for drawing. ③ Calculate the bacteriostatic rate I% (Inhibitionratio), the formula is as follows: I%=(t ₂ -t ₀ )/t ₀ × 100%, wherein, t ₀ is the second index of Shigella growth metabolism without drug intervention The peak time of the growth phase, _t2 is the peak time of the second exponential growth phase of the Shigella growth metabolism during drug intervention. According to the peak time t ₂ of the second exponential growth phase of the growth and metabolism of Shigella with the action of the active ingredients of Coptidis Rhizoma, the inhibitory rate I% of each ingredient at different concentrations on the growth of Shigella was calculated. Bacteriostatic rate I% (y) and concentration C (x) are carried out linear relationship analysis, calculate the dose-effect relationship equation of the two, the results are shown in Table 3.

table 3

The sum of the dose-effect relationship equations of the five active ingredients was used to obtain the sum of the anti-Shigella effect of Coptis chinensis, and the ratio of the sum of the anti-Shigella effect of the control medicinal ingredients 0.9583 was the equivalent factor of the anti-Shigella effect of this batch of Coptis chinensis. Equation, which is Formula I:

EEF=3.352* _XBER +1.293* _XEPI +6.635* _XCOP +0.140* _XPAL +0.419* _XJAT -0.111.

(3) To verify the representativeness of Coptidis rhizome anti-Shigella effect equivalent factor to its medicinal activity:

① Shigella inoculation: extract 1.1mL of Shigella original bacteria liquid (fourth generation), dilute it with LB medium to form an inoculation medium with a total volume of 44mL, that is, the original bacteria liquid is enlarged by 40 times, and the Shigella concentration is 1×10 ⁶ CFU/mL. ②Using the ampoule method, in a clean and sterile environment, accurately add 5 mL of LB culture medium inoculated with Shigella dysenteriae into each glass ampoule bottle with a volume of 20 mL, and then add different volumes of Coptis rhizome sample reserves to each bottle Solution, dilute to 10mL, so that the final concentration of Coptis chinensis water extract is 0.00mg/mL, 0.20mg/mL, 0.40mg/mL, 0.60mg/mL, 0.80mg/mL, 1.00mg/mL, and cap the bottle , sealed, put into a microcalorimeter, and the Pt curve changes of Shigella growth and metabolism in each ampoule were detected by the recording and tracking software that comes with the instrument. The temperature of the instrument was controlled at 37°C, and the experiment was terminated when all the curves returned to the baseline. ③With the same concentration of berberine as the positive control, 0.20 mg/mL was used as the concentration to investigate the inhibitory effect of different batches of Coptis chinensis water extracts on Shigella, and according to t ₂ , calculate the inhibition rate of different batches of Coptis chinensis samples against Shigella I _m %; and take the berberine inhibition rate I _BER % of the same concentration as a contrast, calculate the relative bacteriostatic rate RI _m % of the water extract of Coptidis rhizome of each batch, the formula is RI _m %=I _m /I _BER × 100 %. 4. Bring the active ingredient content value of each batch of Coptis chinensis water extract into formula I, calculate their EEF, carry out correlation analysis with the relative inhibition rate result of each batch of samples and EEF, the result is as shown in table 4, illustrates that both There is a significant correlation, and EEF can better represent the antibacterial activity of Coptidis Rhizoma.

Table 4

Application of Coptidis rhizome anti-shigella effect equivalent factor EEF:

The content of the active ingredients of each batch of Coptidis Rhizoma measured according to the method of the aforementioned step (1)

Quantitative value is substituted in the formula I, obtains the EEF of Coptidis rhizome medicinal material, and the result is as shown in table 5:

table 5

Then, according to the formula II: D=D _target /EEF, assuming that the target dose D _target is 1 gram, then

The actual dosage of Coptidis Rhizoma to be tested is shown in Table 6:

Table 6

The method established by the present invention for adjusting the dosage of Coptidis Rhizoma based on the equivalent factor of anti-Shigella effect factor can better represent the anti-Shigella activity of Coptidis Rhizome. As a comprehensive weighted index of chemical composition analysis and biological activity, it solves the problem of inconsistency between indexes when evaluating the quality of Coptis chinensis by multiple indexes in the form of integrated quantification, and it is difficult to evaluate the quality difference of medicinal materials. Since each active ingredient is directly assigned a pharmacodynamic weight coefficient, the larger the coefficient, the stronger the representativeness of the ingredient on the anti-Shigella effect of Coptis chinensis. When evaluating and controlling the quality of Coptis chinensis in clinical use, the "efficacy" of the batch of Coptis chinensis can be directly reflected by detecting the content of ingredients, without increasing the difficulty of implementing quality standards, and realizing a simple, scientific and effective mode of quality assessment and control of traditional Chinese medicine. At the same time, the effect equivalence factor can guide clinical medication to a certain extent. The medicinal materials with high index can be appropriately reduced in clinical use, and the clinical dosage of medicinal materials with low index can be increased to ensure the consistency of effect equivalence. In conclusion, the dosage adjustment method of Coptidis rhizome based on the consistency of effect equivalence makes it possible to evaluate and control the quality of traditional Chinese medicine comprehensively and quantitatively and guide clinical medication.

The method of the present invention can also carry out effect equivalent factor construction to different active components in Coptidis Rhizoma, also can adopt the consumption adjustment of different target doses, the above is only the example enumerated for explaining the content of the invention, all the Coptidis Rhizoma made based on the essential content of the present invention Simple changes such as batches, ingredients, and target dosages should all fall within the protection scope of the present invention.

Claims (3)

1. regulate a method for Rhizoma Coptidis consumption based on anti-shigella dysenteriae effect equivalence factor, said method comprising the steps of:

(1) measure the content of the jamaicin (BER) in described Rhizoma Coptidis, epiberberine (EPI), coptisine (COP), palmatine (PAL) and jateorrhizine (JAT), namely measure X respectively _bER, X _ePI, X _cOP, X _pAL, X _jAT;

(2) formula I is utilized to calculate the anti-shigella dysenteriae effect equivalence factor of Rhizoma Coptidis to be measured

Formula I:EEF _survey=3.352*X _bER+ 1.293*X _ePI+ 6.635*X _cOP+ 0.140*X _pAL+ 0.419*X _jAT-0.111

Described EEF _surveyfor the anti-shigella dysenteriae effect equivalence factor of Rhizoma Coptidis to be measured;

(3) formula II is utilized to calculate the actual amount of Rhizoma Coptidis

Formula II:D _survey=D _target/ EEF _survey; Wherein said D _surveyfor the actual amount of Rhizoma Coptidis to be measured, D _targetfor the target amount of Rhizoma Coptidis.

2. the method for claim 1, the described measurement wherein in step (1) adopts high performance liquid chromatography.

3. the method for claim 1, wherein said Rhizoma Coptidis is selected from ranunculaceae plant coptis CoptischinensisFranch., triangle leaf coptis CoptisdeltoideaC.Y.ChengetHsiao and Yun Lian CoptisteetaWall.