Ke Zhao, Xiu-Min Li, Jun-Qing Zhang,2,3, Hai-Long Li,2,3?

1.School of Pharmaceutical Sciences, Hainan Medical University, Haikou 571199, China

2.Hainan provincial key laboratory of R＆D on tropical herbs, Haikou 571199,China

3.Key Laboratory of Tropical Translational Medicine of Ministry of Education ,Hainan Key Laboratory for Research and Development of Tropical Herbs,School of Pharmacy, Hainan Medical University, Haikou 571199, China

Keywords:

ABSTRAC T

Callicarpa nudiflora (C. nudiflora) Hook. et Arn., also known as "hemostatic grass", is a large amount of authentic medicinal materials in Hainan Province, belonging to the genus of Verbenaceae. It is mainly used in local folk for the treatment of traumatic bleeding, burns, scalds and suppurative inflammation [1-2]. Clinical research shows that it has a significant effect in the treatment of burn and scald [3-4]. Pharmacology research shows that the total flavonoids and triterpenoids in C. nudiflora can significantly reduce blood viscosity, promote platelet aggregation, shorten bleeding and clotting time and anti-inflammatory activity [5-7]. It can be seen that C. nudiflora has a broad clinical prospect in the treatment of burn, scald and hemostasis. At present, there are mainly C. nudiflora tablets, C. nudiflora capsules, C. nudiflora granules [8-11], all of which are oral drugs. For acute hemorrhage, burn and scald and other acute bleeding, oral hemostatic drugs obviously can not meet the clinical needs, while traditional Chinese medicine coating agent is a kind of transdermal drug delivery agent, which is easy to use and carry, can prevent drug falling off, inhibit water evaporation on the skin surface Hair, promote hydration, especially in the protection of wound surface, prevention of bacterial infection, is the first choice of dosage form for traumatology and dermatology [12-13], so it is necessary to make the C. nudiflora into a coating agent. In this paper, the best prescription and technology of the coating agent of C. nudiflora was selected by orthogonal test, and the quality standard of the film agent was established, which laid a foundation for the further development and utilization of the coating agent of C. nudiflora.

1. Materials and Instruments

1.1 Materials and reagents

Methanol (analytical grade), ethanol (analytical grade), and formic acid (analytical grade) were purchased from Guangdong Chemical Reagent Factory; chromatography acetonitrile (Tedia); polyvinyl alcohol (Sino Pharmaceutical Group Chemical Reagent Co., Ltd.); glycerin (Tianjin Fuyu Fine Chemical Co., Ltd.); Tween 80 (Xilong Chemical Co., Ltd.); Ethylparaben (Tianjin Comeo Chemical Reagent Co., Ltd.); Acteoside Standard Reference Material (Lot No. 120804, purchased from Sichuan Vickey Biotechnology Co., Ltd.); Cynaroside standard reference (batch number AF806205, purchased from Sichuan Weikeqi Biotechnology Co., Ltd.).

1.2 Instruments and equipment

SHZ-D (III) circulating water vacuum pump (Henan Yuhua Instrument Co., Ltd.); RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); XS105DV electronic analytical balance (METTLER TOLEDO); Waters-e2695- Model 2998 high performance liquid chromatograph (Waters).

2. Methods and results

2.1 Preparation of C. nudiflora film agent

2.1.1 Preparation of 50% alcohol eluting site

Preparation of the loading solution: Weigh about 300 g of the leaves of the C. nudiflora, put it in a 10 L round bottom flask, add 6 L of distilled water for 2 hours, heat and reflux for 1 hour, filter, and add 4.5 L of distilled water to the filter residue to repeat the extraction 2 Combine the filtrate three times, concentrate to about 600 mL, add clarifying agent to flocculate and clarify (addition amount of ZTC 1 + 1 II is 8% B + 4% A, temperature is 60 ° C, stirring speed is 100 r / min) Then, dilute the clear solution to make a solution containing about 0.25 g crude drug per 1 ml as a macroporous resin loading solution.

Sample loading and elution: Weigh 3 parts of SP-207 macroporous resin, 25 g per column, and load the sample according to the ratio of resin to medicinal material 1: 0.8 (g / g). First elute with 2 times the volume (BV) of water, then elute with 2 BV 30% ethanol, continue to elute with 4 BV 50% ethanol, the flow rate is 2 BV / h. The 50% ethanol eluent was collected, and concentrated to dryness under reduced pressure to obtain the 50% alcohol elution site of C. nudiflora.

2.1.2 Research on film forming process

Preparation of coating agent base: Weigh a certain amount of polyvinyl alcohol (PVA-124) into a beaker, add an appropriate amount of 95% ethanol solution to swell for 24-48 hours, wait for the ethanol to evaporate, add an appropriate amount of ultrapure water Stir at 80 ℃ in a constant temperature water bath until gellike. Then add a certain amount of glycerin, Tween 80, and ethyl paraben, and stir well to obtain.

Evaluation index: The film-forming time and apparent traits were used as evaluation indexes for evaluation and evaluation, with a full score of 10 for each item. The evaluation criteria are shown in Table 1.

Investigation of film-forming time: Measure 1 ml of coating agent and apply evenly on a 5 cm x 5 cm glass plate, and record the filmforming time.

Observation of apparent traits: Apparent traits mainly include viscosity, uniformity and flexibility.

Table 1 scoring criteria

2.1.2.1 Influence of the amount of polyvinyl alcohol on the film-forming process

Pharmaceutical grade polyvinyl alcohol is a safe polymer organic substance, widely used in wound dressings, ophthalmology, etc. It is a commonly used safe film-forming agent. See Table 2 for the comprehensive score of polyvinyl alcohol dosage.

Table 2 polyvinyl alcohol dosage inspection

2.1.2.2 The effect of glycerin dosage on film-forming process

Glycerin has strong hydrophilicity, which can enhance the skinfriendly and wettability of the coating agent. The proper amount of glycerin can enhance the transdermal absorption rate of the drug, making the drug easy to apply on the skin. The coating agent made with low glycerin concentration has poor skin affinity and low drug penetration rate; too high solubility will cause the coating agent to be too fluid and not easy to apply externally. See Table 3 for the comprehensive score of glycerin dosage.

Table 3 investigation of glycerin dosage

2.1.2.3 Investigation of the ratio of ethanol to water

Proper ratio of water and ethanol can accelerate the film-forming speed of the C. nudiflora hemostatic coating agent, and play a great role in improving the spreadability and uniformity of the coating agent. The ratio of water to ethanol has an effect on various indicators of the coating agent, of which the film-forming time has the greatest effect. This article selects the ratio of four different levels of water and ethanol for comprehensive scoring, see Table 4.

Table 4 proportion of ethanol and water

2.1.3 Screening of C. nudiflora Hemostatic Coating Agent

The orthogonal test design was used to further optimize the preparation process of the C. nudiflora hemostatic coating agent. The film formation time, uniformity, stability and apparent properties were used as comprehensive evaluation indicators to examine the three main factors that affect the moldability: For the amount of polyvinyl alcohol (A), the amount of glycerol (B), the ratio of ethanol and water (C), select 3 levels for each factor, and design the orthogonal test according to L9(34). The levels of orthogonal design factors are shown in Table 5.

Table 5 factor levels

Table 6 comprehensive score

2.2 Process verification

Weigh 6g of polyvinyl alcohol (PVA), after pretreatment, add an appropriate amount of ultrapure water, put it in a constant temperature water bath at 80 ℃ and stir until it dissolves into phase A. Dissolve a certain amount of the powder of the effective part of the C. nudiflora with an appropriate amount of ethanol-water solution (1: 1), add phase A, then add 2 ml of glycerin, appropriate amount of Tween 80 and ethyl paraben, and mix well, that is, get.

2.3 Establishment of the quality standard of C. nudiflora coating agent

Using the best preparation process selected, 3 batches of C. nudiflora coating agent were prepared continuously, the appearance, stability, heat resistance and cold resistance of the coating agent were checked. The content determination method of main flavonoids (cynaroside and acteoside) in the coating agent was established. The method was verified and the quality standard of C. nudiflora coating agent was preliminarily established.

2.3.1 General inspection method

Apparent property inspection: Take three batches of coating agent and apply them to the same glass plate at the same time, and observe the coating agent's application: the uniformity of the coating agent, whether it is fine and shiny, whether there are obvious particles After the film is formed, observe the flexibility and thickness of the formed film.

Stability check: Take three batches of prepared coating agent, add each batch to the centrifuge tube, centrifuge at 5000 r / min, and take it out after 30 minutes, check whether the coating agent has obvious delamination.

Heat resistance check: Take 3 batches of coating agent, add the same amount to each glass test tube, place in a constant temperature water bath at 55 ℃ water bath for 24 h, check whether the coating agent has obvious layering and color appearance Variety.

Cold resistance check: Take 3 batches of coating agent, add an equal amount of each batch to a glass test tube, place in a refrigerator at -20 ℃ for 72 h, check the coating agent for obvious changes in appearance such as delamination and color.

Inspection conclusion: After inspection, the appearance of the three batches of C. nudiflora coating agent is good, the coating is uniform, fine and shiny, without obvious particles, after the film formation, the flexibility is good and the thickness is uniform.

2.3.2 Determination of the content of flavonoids in the C. nudiflora coating agent

2.3.2.1 Solution preparation and chromatographic conditions

Preparation of the mixed reference substance solution: the main flavonoids cynaroside and acteoside in the C. nudiflora coating agent were selected as the reference substance, precision weighed 1.30 mg of cynaroside and 1.44 mg of acteoside, placed in a 25 mL measuring bottle In, add 50% methanol to dissolve and dilute to the mark, shake well, filter through a 0.45 μm microporous filter membrane, and take the subsequent filtrate as the reference solution.Preparation of sample solution: Weigh 1.0 g of C. nudiflora coating agent, place in a 10 mL volumetric flask, add 50% methanol to dissolve and dilute to the mark, filter through a 0.45 μm microporous filter membrane, and take the subsequent filtrate as For test solution.Chromatographic conditions: Using octadecyl bonded silica as packing (Luna C18 column, 150 mm × 4.6 mm, 5 μm); using acetonitrile-0.1% acetic acid solution as mobile phase; flow rate is 1.0ml per minute; detection wavelength is 350 nm; column temperature is 30 ℃; injection volume is 10 μL. The chromatograms of reference solution and test solution are shown in Figures 1 and 2.

Figures 1 Chromatogram of mixed reference substance

Figures 2 sample chromatogram

2.3.2.2 Investigation of content determination methodology

Linearity: Pipette 2, 4, 6, 8 ml of the mixed reference solution accurately, place them in 10 ml measuring flasks respectively, add 50% methanol to dilute to the mark, prepare linear solutions of different concentration levels, then take the mixed control solution as At a linear high concentration point, the solutions of the abovementioned different concentration levels were injected into the high-performance liquid chromatograph, the injection volume was 10 μl, and the chromatogram was recorded. Using the injection concentration (mg / ml) as the abscissa (X) and the peak area as the ordinate (Y), draw a standard curve and calculate the regression equation. The results are shown in Table 7.

Table 7 regression equation and linear range of cynaroside and acteoside

Precision test: take the reference solution repeatedly and inject 6 times, record the peak areas of cynaroside and acteoside, and calculate RSD. The peak area RSD of 6-pin cynaroside is 0.5%, and the peak area RSD of 6-pin acteoside is 0.7%, indicating that the instrument is of good precision.

Repeatability test: Weigh 1.0 g of coating agent (batch number 20190517), weigh 6 parts in sequence, prepare the test solution, measure according to the above chromatographic conditions, record the peak area of the chromatogram, and calculate the RSD. In the 6-point test product, the RSD of the cynaroside peak area was 0.5%, and the RSD of the acteoside peak area was 0.9%, indicating that the method has good repeatability.

Solution stability: Take 1 sample solution under repeatability and inject at 0, 2, 4, 8, 12, and 24 h, record the peak areas of cynaroside and acteoside, calculate RSD, The peak area RSD of cynaroside was 0.3% within 24 hours, and the peak area RSD of acteoside within 0.4 hours was 0.4%, indicating that the test solution had good stability within 24 hours.

Accuracy test: Weigh 1.0g of the coating agent with a batch number of 20190517 in a 10ml volumetric flask, weigh 9 parts in sequence, add a mixed reference of 80%, 100% and 120% of the sample content precisely, and dilute with 50% methanol To the scale, according to the above chromatographic conditions for measurement, calculate the recovery rate, the results are shown in Table 8 and Table 9.

Table 8 recovery test results of cynaroside

Table 9 recovery test results of acteoside

2.3.2.3 Sample content determination and limit formulation

Take 3 batches of C. nudiflora coating agent of different batches, prepare test samples according to the method of preparation of test sample solution, prepare 2 batches per batch, measure 3 times for each batch, and take the average value. The results are shown in Table 10. Taking the average content of 80% as the lower limit, it is stipulated that the cynaroside content in the C. nudiflora coating agent should not be less than 0.011%, and the acteoside content should not be less than 0.018%.

Table 10 sample content determination

3. Discussion

3.1 Optimization of prescription process

The optimization of the prescription process was investigated by orthogonal experiments. The main factors affecting the formability of the coating agent are: the amount of polyvinyl alcohol (A), the amount of glycerin (B), the ratio of ethanol and water (C), each factor chooses 3 levels , Design orthogonal test according to L9(34), and evaluate by comprehensive score of coating agent. According to the experimental results, the optimal prescription process is 6.0g of polyvinyl alcohol, 2.0ml of glycerin, and a 1: 1 ratio of ethanol and water.

3.2 Quality standard of C. nudiflora coating agent

First, a general inspection method for C. nudiflora coating agent was established, and the quality of theC. nudiflora coating agent was preliminarily visually evaluated by examining the appearance, stability, heat resistance and cold resistance of the coating agent. Convenient and fast, highly compatible with the quality attributes of the coating agent. Secondly, a method for determining the content of cynaroside and acteoside in C. nudiflora coating agent was established by high performance liquid chromatography, and the method was validated. The results show that the method is linear, precise, repeatable and recoverable The stability and stability are in line with the expected requirements, which further shows that this method can accurately determine the content of cynaroside and acteoside in the C. nudiflora coating agent; After testing, the results of cynaroside and acteoside in many batches of C. nudiflora coating agent were 0.1408 mg / g and 0.2190 mg / g, respectively, and the RSD was less than 2.0%, indicating that the method can stably and reliably detect the content of cynaroside and acteoside in C. nudiflora coating agent; The lower limit of 80% of the average content of the two indicator components establishes the quality control limits of cynaroside and acteoside in the C. nudiflora coating agent. The limit stipulates that the content of cynaroside in each 1g of coating agent should not be less than 0.011%, acteoside content should not be less than 0.018%.

In summary, this article has optimized the best prescription process of the C. nudiflora coating agent, established the detection method of cynaroside and acteoside in the C. nudiflora coating agent, and established the C. nudiflora coating agent quality standards. The general inspection method in this quality standard is convenient and quick to operate, and the quality determination method has specificity, accuracy, precision and stability, and can be used for the quality control of the C. nudiflora coating agent.