2. 1. 1 Calculate the moisture absorption percentage of total salvianolic acid under different humidity. Accurately weigh 6 parts of total salvianolic acid (2g each), put it in a corresponding humidity device, stand for 72h, and then accurately weigh it again after taking it out. According to the moisture absorption percentage (%) = (weight after moisture absorption-weight before moisture absorption)/weight before moisture absorption, the moisture absorption percentage of total salvianolic acid under different humidity was calculated.
2. 1.2 Draw the hygroscopic equilibrium curve of total phenolic acid of Salvia miltiorrhiza. Taking relative humidity as abscissa and moisture absorption as ordinate, the moisture absorption balance curve is drawn.
2.2 Investigation on the influence of light, heat and humidity on the stability of total phenolic acids of Salvia miltiorrhiza Bunge.
2.2. 1 light stability test: weigh 5 parts of total phenolic acid extract and irradiate it 10d (light intensity is 4500~5000lx).
2.2.2 Thermal stability test: Weigh 5 parts of total phenolic acid extracts, seal them in glass bottles, put them in constant-temperature drying boxes at 60℃ and 80℃ for 65438±00d days, sample them at 0,65,438 0,3,5,65438±00d days, and determine the content change of total phenolic acid in Salvia miltiorrhiza by colorimetry.
2.2.3 In the wet stability test, 5 parts of total phenolic acid extracts were weighed and placed in two closed containers with relative humidity of 75%(NaCl) and 92.5%(KNO3) respectively, and kept at room temperature for 65438±00d days. Samples were taken at 0, 1, 3, 5, 10, and the content changes of total phenolic acids (calculated by protocatechuic aldehyde) of Salvia miltiorrhiza were determined by colorimetry.
2.3 Colorimetric determination of total phenolic acids in Salvia miltiorrhiza Bunge and its methodology investigation.
2.3. 1 Preparation of control solution Accurately weigh protocatechuic aldehyde standard 1mg, put it in a 1ml volumetric flask, add a small amount of methanol to dissolve it, dilute it to scale, and shake it evenly for use.
2.3.2 Preparation of test solution Accurately weigh 65438±0mg of total salvianolic acid extract, put it in a 65438±0ml volumetric flask, add a small amount of methanol to dissolve and dilute to scale, and shake well for later use.
2.3.3 Preparation of standard curve Accurately absorb 10, 20, 30, 40 and 50μl of control solution, put it in a 25ml volumetric flask, add 5.0ml of absolute ethanol, and shake well; Add 2.0ml of 0.3% sodium dodecyl sulfate, 2% ferric chloride-1% potassium ferricyanide (1: 0.9) developer 1.0ml, leave it in the dark for 5 minutes, then add 0. 1mol/l hydrochloric acid to the scale, shake well. The absorption value was measured at 730 nm. Draw a standard curve.
2.3.4 Determination of sample content Accurately absorb 30μl of sample solution, and determine the content of total salvianolic acid (calculated by protocatechuic aldehyde) according to the standard curve.
2.3.5 The stability test accurately absorbs the control solution, develops color according to the standard curve, and measures 65438 0 times every 2 hours. ..
2.3.6 The recovery rate experiment can be obtained from the moisture absorption equilibrium curve, and the critical relative humidity of the total phenolic acid extract of Salvia miltiorrhiza is 45%. It is not easy to absorb moisture at room temperature. This is of certain significance for the preservation of total phenolic acids of Salvia miltiorrhiza. Study on critical relative humidity of total phenolic acids in salvia miltiorrhiza Bge. 3. 1
3. 1. 1 Calculating the moisture absorption percentage of total salvianolic acid under different humidity According to the moisture absorption percentage formula, the moisture absorption percentage of total salvianolic acid under different relative humidity (i.e. 20%, 30%, 40%, 50%, 60% and 70%) was calculated as 0.4%, 0.8% and 0.8% respectively.
3. 1.2 Draw the hygroscopic equilibrium curve of total salvianolic acid, as shown in figure 1.
Fig. 1 hygroscopic equilibrium curve of salvianolic acid (omitted)
According to the moisture absorption equilibrium curve, the critical relative humidity of the total phenolic acid extract of Salvia miltiorrhiza is 50%. It is not easy to absorb moisture at room temperature. This is of certain significance for the preservation of total phenolic acids of Salvia miltiorrhiza.
3.2 Investigation on the influence of light, heat and humidity on the stability of total phenolic acids of Salvia miltiorrhiza Bunge.
After 3.2. 1 photostability test of total salvianolic acid 1, 3,5, 10d, the content of total salvianolic acid decreased from 69.2 1% to 69. 18%, 68.45% and 68.60.
3.2.2 Thermal stability test of total salvianolic acid after constant temperature of 80℃ 1, 3, 5, 10d, the content of total salvianolic acid changed from 69.2 1% to 69.24%, 69. 12% and 68.60%.
3.2.3 Wet stability test of total salvianolic acid After constant humidity 1, 3,5, 10d at RH=75%, the content of total salvianolic acid changed from 69.2 1% to 69.05%, 69.36% and 69./kloc-.
From the above data, it can be concluded that the content of total phenolic acids in Salvia miltiorrhiza has almost no change in light, heat and moisture stability tests, and it has certain stability.
3.3 Colorimetric determination of total phenolic acids in Salvia miltiorrhiza and its methodology investigation
3.3. 1 standard curve Take absorption value x as abscissa and concentration y as ordinate, draw standard curve Y=0.3244X+7.8× 10-3, r = 0.9998. The linear range of salvianolic acid is 1 1 ~ 55ug.
3.3.2 According to the method under the standard curve, the content of total salvianolic acid was 69.265438 0%, 68.54%, 69.78%, and the RSD was 0.90%.
3.3.3 According to the method under the standard curve, the stability test shows that the absorbance values are 0.924, 0.93 1, 0.938, 0.955, 0.976, and the RSD is 2.2 1% respectively.
3.3.4 According to the method under the standard curve, the recovery rates are 95. 13%, 105.07%, 10 1. 15%,100.40 respectively. It can be seen from the above experimental data that it is feasible to determine the total phenolic acids in Salvia miltiorrhiza by colorimetry. The critical relative humidity experiment can be used to quantitatively study the influence of humidity on drugs and provide basis for formulating the technological conditions of products. The production and storage environment of drugs must be controlled below CRH.
Theoretically speaking, the stability of total phenolic acids in Salvia miltiorrhiza is not good enough, because its structure contains phenolic hydroxyl groups, so this paper designed the stability study of total phenolic acids extract. The content of total phenolic acids was determined by protocatechuic aldehyde colorimetry, and its standard curve was used. The condition of constant humidity can be achieved by placing a saturated inorganic salt solution in the lower part of a closed container such as a dryer. According to the requirements of different humidity, NaCl saturated solution [relative humidity (75 1)%] and KNO3 saturated solution (relative humidity 92.5%) can be selected. In this experiment, under the condition of RH=92.5%, the extract was hygroscopic and almost became a solution, so the content change of total phenolic acid in Salvia miltiorrhiza was only determined under the condition of CRH of 75%. The equipment for light stability test [1] must be able to control temperature, humidity and illumination. Generally, fluorescent light source is used, and the illumination is 6000lx. In the experiment, 10d makes the total illumination reach 1.445438+005lx.
In the thermal stability experiment, only the stability of total salvianolic acid at 80℃ was studied. The results show that the stability of total phenolic acids is very good, and the experiment at 60℃ is not needed.
There is a report on the stability of compound Danshen tablets [2]. Using constant temperature accelerated test method, protocatechuic aldehyde and tanshinone in Salvia miltiorrhiza were used as indicators, and their validity periods were 3.73 years and 0.53 years respectively, indicating that protocatechuic aldehyde was relatively stable, and tanshinone was the main unstable component in Salvia miltiorrhiza [3].
The preliminary stability study of salvianolic acid in this experiment shows that it is stable in solid state. As for the stability of its aqueous solution, such as the stability at different pH values, different concentrations and different temperatures, further research can be done [4]. [1] Chen Jianxing, Yan Wang. Experiment on photostability of drugs [J]. chinese pharmaceutical affairs, 2000, 14 (1): 17. [2] Ding Qinglong, Liu Rihua. Study on the stability of compound Danshen tablets [J]. Chinese patent medicine.
Su Ziren, Chen Jiannan. Variation factors and stabilization measures of tanshinone Ⅱ A in Compound Danshen Tablets [J]. China Journal of Traditional Chinese Medicine, 2000,25 (4): 247.
Zhu Zhifang, Mo Fengkui, Zhu Ning, et al. Study on the stability of aqueous dichloroacetic acid [J]. Journal of Shenyang Pharmaceutical University, 1997, 14 (3): 170.