Correlation Analysis of Processing Technology, Physical Parameters and Chemical

Xinhong ZHAO, Ruiying LI, Chao SUN, Zhenhua LIU, Xu XIAO, Tianchao CHEN

1.Department of Pharmacy, the Affiliated Hospital of Chengde Medical College, Chengde 067000, China; 2.Department of Pharmacy, Xuchang City Hospital of Traditional Chinese Medicine, Xuchang 461000, China; 3.Department of Pharmacy, the First Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhengzhou 450000, China

Abstract [Objectives]To explore the correlation of processing technology, physical parameters and chemical components during plain stir-baking of Trichosanthis Radix.[Methods]Based on mixture uniform experiment design, the Trichosanthis Radix was prepared by plain stir-bake method.Delphi method was used to evaluate and select the highest-scoring processed product for measuring physical parameters.UV spectrophotometry was used to determine the contents of starch and polysaccharide.The correlation and linear regression model of processing technology, physical parameters and chemical components were established with the aid of SPSS 26.0[Results]After processing by plain stir-bake method, the relative density and chromaticity showed a decreasing trend in the processed products of Trichosanthis Radix, the oxidation value, hydroscopic rate and swelling decreased firstly and then increased, and pH increased firstly and then decreased.The content of total starch decreased, the content of polysaccharide increased, and there was a negative correlation between them.There was a significant positive correlation between temperature and oxidation value, swelling and hydroscopic rate, hydroscopic rate and polysaccharide, and there was a significant negative correlation between relative density and hydroscopic rate or polysaccharide, total starch and hydroscopic rate or swelling.The linear relation model between processing technology and physical parameters and chemical components was r2>0.9.[Conclusions]After processing by plain stir-bake method, the physical parameters of Trichosanthis Radix changed, and there may be mutual conversion between total starch and polysaccharides.To a certain extent, physical parameters can be used to evaluate the quality of processed products of Trichosanthis Radix.This study is expected to provide a reference for research on quality evaluation of processed products of traditional Chinese medicine.

Key words Trichosanthis Radix, Processing technology, Physical parameters, Chemical components, Correlation

1 Introduction

Trichosanthis Radix is the dried root ofTrichosantheskirilowiiMaxim.orTrichosanthesrosthoriniiHarms in Cucurbitaceae family.It is slightly cold in nature, sweet and slightly bitter in taste, and enters the lungs and stomach meridians.It has functions of clearing the heat, purging pathogenic fire, engendering fluid and quenching the thirst, dispelling swelling and eliminating abscess, and is often used for treating febrile disease excessive thirst, dry cough due to lung heat, wasting thirst due to internal heat, and sore and ulcer, swelling and toxin[1].Shennong’sClassicofMateriaMedicainitially recorded Trichosanthis Radix, called it Gualou root and considered it medium grade drug, and theIllustratedClassicofMateriaMedicain the Song Dynasty first called it Trichosanthis Radix, and another name was while drug[2].Modern research has found that Trichosanthis Radix mainly contains starch, polysaccharides, saponins, trichosanthin(TCS), amino acids, and enzymes[3].It has anti-tumor, anti-fungal, anti-viral effects, and is often used clinically in gynecological induction of labor, diabetic syndrome, AIDS treatment, and topical treatment of skin syndromes such as swelling, heat and pain[4].

Traditional Chinese medicine processing is a unique pharmaceutical technology adopted in accordance with the theory of Chinese medicine, according to the nature of the medicinal materials, as well as the needs of adjustments, preparations and clinical applications[5].The processing of traditional Chinese medicine can change the properties of the medicine, and the physical properties and chemical components of the decoction pieces are often changed during the processing.According to findings of some scholars[6], after cold water soaking of Trichosanthis Radix, the water-soluble extracts, soluble proteins and water-soluble polysaccharides show a declining trend.On the basis of the previous study on effect of plain stir-bake method on the physical parameters and chemical components of traditional Chinese medicine processed products[7-12], we determined the physical parameters and chemical components of stir-bake to yellow, stir-bake to brown, and stir-bake to scorch processed products of Trichosanthis Radix, and explored the correlation between processing technology, physical parameters and chemical components, to explore the effect of plain stir-bake method on the quality of processed products of Trichosanthis Radix from multiple angles, all aspects, and at multiple levels, and provide a reference for the research on the quality evaluation of processed products of traditional Chinese medicine.

2 Instruments and materials

2.1 ReagentsTrichosanthis Radix(Henan Zhongyi Medicine Management Co., Ltd., China, batch no.1701021, place of origin: Hebei), identified by chief pharmacist Chen Tianchao from Department of Pharmacy, the First Affiliated Hospital of Henan University of Traditional Chinese Medicine as the Trichosanthis Radix included inChinesePharmacopoeia(2020 edition); D(+)-Anhydrous glucose reference substance(batch No.S08J6G1), amylose standard substance(batch No.J11D8N50350), and amylopectin standard substance(batch No.K01DY49573)purchased from Shanghai Yuanye Biotechnology Co., Ltd.(China)with mass fraction ≥ 98%.Other reagents were of analytical grade.

2.2 InstrumentsThermo Evolution 201 ultraviolet-visible spectrophotometer(Thermo Fisher Scientific Incorporation, USA); DT-300 electronic balance(Changshu Branch of Shanghai Medical Laser Instrument Factory, China); BSA224S-CW electronic balance(Sartorius AG, Germany); Canon DS126321 digital camera(Canon Incorporation, Japan); SJ-3F digital pH meter(Shanghai Precision & Scientific Instrument Co., Ltd., China).

3 Methods and results

3.1 Preparation and optimization of plain stir-bake processed products of Trichosanthis RadixIn accordance with the stir-bake to yellow, stir-bake to brown, and stir-bake to scorch requirements specified in General Rule 0213 of 2020ChinesePharmacopoeia(Volume 4)[5], combining the preliminary experiments in the early stage, we took the processing method, temperature and time as the influencing factors, and adopting the uniform design experiment to prepare the stir-bake to yellow, stir-bake to brown, and stir-bake to scorch processed products of Trichosanthis Radix, and we selected the best processed products using Delphi evaluation method.First, weighed about 300 g of Trichosanthis Radix decoction pieces, evenly spread them in a tray, placed them in an oven that has reached the set temperature, and heated and processed them in the oven according to the processing time in the uniform design experiment.Each group of experiments was processed in parallel for 3 batches.Finally, placed the processed products in an area with a light intensity of 300 lx, and invited several processing experts to conduct Delphi evaluations, and selected the highest-scoring processed products and processing technology, as shown in Fig.1 and Table 1.

Fig.1 Processed products of Trichosanthis Radix with the highest scores in Delphi evaluation

Table 1 Optimal processing technology of Trichosanthis Radix

3.2 Determination of physical parameters of Trichosanthis Radix decoction pieces

3.2.1Relative Density.Precisely weighed 5.0 g(M)of Trichosanthis Radix decoction pieces, placed in a 100 mL measuring cylinder containing 50 mL of light liquid paraffin, and let it stand for 5 min.ρ=M/(V-50)to calculate the relative density(g/mL).

3.2.2Oxidation value.Precisely weighed 5.0 g(M)of Trichosanthis Radix decoction pieces, and measured the oxidation value with reference to the method in reference[10].

3.2.3Hydroscopic rate.Precisely weighed 5.0 g(M)of Trichosanthis Radix decoction pieces, placed in a 100 mL measuring cylinder containing 250 mL of distilled water, filtered after 10, 20, 40, 60, 120, 240, 480, 720, and 1 440 min, and made a record of the filtrate volumeV(mL).When the filtrate volume did not change, recorded as the saturated water absorption volumeVsaturated(mL), the corresponding time was the water absorption saturation timetsaturated(min), and calculated the water absorption rate using the formula: Hydroscopic rate(mL/g)=(100-Vsaturated)/M.

3.2.4Swelling.Precisely weighed 5.0 g(M)of Trichosanthis Radix decoction pieces, placed in a 100 mL measuring cylinder containing 50 mL of distilled water, soaked according to the water absorption saturation time determined in Section3.2.3, made a record of the volumeV(mL), and calculate the swelling degree(mL/g)using the formulaS=(V-50)/M.

3.2.5pH.Took the filtrate of the Trichosanthis Radix decoction pieces under the water saturation time, and used the digital acid meter to measure the pH.

3.2.7Determination results of physical parameters.Using the above method, measured the physical parameters of the Trichosanthis Radix decoction pieces 3 times in parallel.Due to the large range of measurement results(0.07-414.54), in order to facilitate the intuitive comparison of physical property parameters, when drawing the histogram of physical parameters in GraphPad 8.0 software, we selected the scale of the vertical axis to be presented in a non-linear logarithmic form, as shown in Fig.2.From the results that the relative density and chromaticity of Trichosanthis Radix plain stir-baking processed products showed a downward trend, the oxidation value, hydroscopic rate, and swelling first decreased and then increased, and the pH first increased and then decreased.

Fig.2 Determination results of physical parameters of Trichosanthis Radix decoction pieces(n=3)

3.3 Determination of starch and polysaccharide content of Trichosanthis Radix decoction pieces

3.3.1Preparation reference substance solution.Precisely weighed 100.00 mg each of the amylose standard product and the amylopectin standard product, put them in a 100 mL beaker, added 10 mL of 1.0 mol/L potassium hydroxide solution, and placed in a boiling water bath to stir to dissolve, cooled down, and used the distilled to adjust the volume to 100 mL and prepared the amylose standard solution and the amylopectin standard solution with a mass concentration of 1.00 mg/mL.

Precisely weighed 5.57 mg of anhydrous glucose reference substance, used distilled water to adjust the volume to 50 mL, and prepared a reference substance solution with a mass concentration of 111.40 μg/mL.

3.3.2Preparation test solution.Precisely weighed about 2 g of the fine powder of Trichosanthis Radix decoction pieces dried to constant weight, and placed it in a Soxhlet fat extractor, added petroleum ether, heated and refluxed for 4 h for degreasing, filter, and put the filter residue in an oven for drying, added 80% ethanol, heated and refluxed for 2 h for desugaring treatment, filtered, and dried the filter residue to a constant weight for use.Precisely weighed 100.00 mg of the sample after degreasing and desugaring treatment, added 10 mL of 1.0 mol/L potassium hydroxide solution, stirred to dissolve in a boiling water bath, cooled down, used the distilled water to adjust the volume to 50 mL, filtered, accurately absorbed 4.0 mL of the filtrate, added 25 mL of distilled water and 0.1 mol/L hydrochloric acid solution to adjust the pH to 3.0, added 0.5 mL of iodine reagent, and used the distilled water to adjust the volume to 50 mL, and shook well to obtain the test solution required for the determination of starch content.

Precisely weighed about 1 g of the fine powder of Trichosanthis Radix decoction pieces, added 16 times the amount of distilled water, extracted at 78 ℃ for 48 min, centrifuged, extracted the supernatant with chloroform-n-butanol(4:1)twice, and added to the water layer 4 times the amount of 95% ethanol, let stand for 12 h, filtered, washed the residue twice with 95% ethanol, acetone, ether, 10 mL each time, dried, added the distilled water to make the volume up to 50 mL, accurately absorbed 1.0 mL, used the distilled water to adjust the volume to 25 mL, to obtain the test solution required for the determination of polysaccharide content.

3.3.3Plotting of standard curve.Precisely absorbed 1.00 mg/mL amylose standard solution 1.0, 2.0, 2.5, 3.5, 4.5 mL into a beaker, added 25 mL of distilled water, 0.1 mol/L hydrochloric acid solution to adjust the pH to 3.0, added 0.5 mL of iodine reagent, used the distilled water to adjust the volume to 50 mL, shook well, used the corresponding reagent as a blank control, and measured the absorbance at the wavelength of 604 nm.Took 468 nm as the reference wavelength, the concentration of the reference substanceCas the abscissa, the absorbance difference △A=A604-A468as the ordinate, and plotted the amylose standard curve equationY=0.004 5X-0.008 7(r2=0.999 2), indicating that the amylose concentration has a good linear relationship in the range of 20.00-90.00 μg/mL.

Precisely absorbed 1.00 mg/mL amylopectin standard solution 2.0, 4.0, 6.0, 8.0, 10.0 mL and placed them in a beaker.Other treatment methods are the same as amylose.Then, measured the absorbance at the wavelength of 537 nm, and took 718 nm as the reference wavelength to plot amylopectin standard curve equationY=0.002 8X-0.005 5(r2=0.998 8), indicating that amylopectin has a good linear relationship in the concentration range of 40.00-200.00 μg/mL.

Precisely absorbed 0.1, 0.4, 0.6, 0.8, 1.0 mL of 111.40 μg/mL glucose reference solution and placed them in a test tube with a stopper, made up to 1 mL with distilled water, added 1 mL of 6% phenol solution, shook well, and slowly added concentrated sulfuric acid 5 mL, shook well, placed in a boiling water bath for 25 min, cooled down, and took the corresponding reagent as a blank control, measured the absorbance at the wavelength of 490 nm using ultraviolet spectrophotometer.Taking the reference concentration C as the abscissa and absorbance A as the ordinate, plotted the glucose standard curve equationY=79.18X-0.016 3(r2=0.998 7), indicating that the glucose concentration has a good linear relationship in the range of 2.90-11.50 μg/mL.

3.3.4Methodology investigation.Took 6 pieces of Trichosanthis Radix decoction pieces in parallel, precisely weighed, prepared the test solution using the method in Section3.3.2, and carried out the precision, stability, repeatability and sample recovery rate using the method in Section3.3.3.The results indicate that in the precision test, theRSDvalues of the absorbance of amylose, amylopectin, and polysaccharide were 1.71%, 1.63%, and 1.33%, respectively; in the stability test, theRSDvalues of amylose, amylopectin, and polysaccharide were 1.66%, 1.82%, and 1.65%, respectively.The test solution was stable within 60 min; in the repeatability test, theRSDvalues of the absorbance of amylose, amylopectin, and polysaccharide were 1.97%, 1.84%, and 1.64% respectively; the average recovery rate of amylose was 100.46%, theRSDvalue was 1.37%, the average sample recovery rate of amylopectin was101.24%, theRSDvalue was 1.33%, the average sample recovery rate of polysaccharides was 100.03%, and theRSDvalue was 1.96%.

3.3.5Determination of starch and polysaccharide content in test solution.Took 3 pieces of Trichosanthis Radix decoction pieces in parallel, prepared the test solution using the method in Section3.3.2, and measured the absorbance using the method in Section3.3.3.Calculated the content of amylose and amylopectin(the sum of the two was the total starch content)and polysaccharide content in accordance with the standard curve equation.The results are shown in Table 2.

Table 2 Determination of starch and polysaccharide content of crude and processed Trichosanthis Radix products(n=3)

3.4 Correlation between processing technology, physical parameters, and chemical components of Trichosanthis RadixWe imported the processing parameters, physical parameters and chemical component measurement results of the Trichosanthis Radix into the SPSS 26.0 software, and performed the bivariate Pearson correlation analysis on the processing technology, physical parameters, and chemical components, as shown in Table 3.

3.4.1Correlation between processing technology and physical parameters.From Table 3, it can be known that there is a significant positive correlation between temperature(X)and oxidation value(Y).With the aid of SPSS 26.0 software, we established the linear equation:Y=0.035X+0.049(r2=0.977 2).

3.4.2Correlation physical parameters.From Table 3, it can be known that there is a significant negative correlation between relative density(X1)and hydroscopic rate(Y), and there is a significant positive correlation between swelling(X2)and hydroscopic rate(Y).With the aid of SPSS 26.0 software, we established the linear equation:Y=-0.387X1+0.258X2+0.924(r2=0.991 0).

3.4.3Correlation between chemical components.From Table 3, it can be known that there is a significant correlation between total starch(X1)and polysaccharides(Y).With the aid of SPSS 26.0 software, we established the linear equation:Y=-0.054X1+3.431(r2=0.908 9).

3.4.4Correlation between physical parameters and chemical components.From Table 3, it can be known that there is a significant negative correlation between relative density(X1)and polysaccharides(Y1), a significant positive correlation between hydroscopic rate(X2)and polysaccharides(Y1), and a significant negative correlation between hydroscopic rate(X2), swelling(X3)and total starch(Y2).With the aid of SPSS 26.0 software, we established the following linear equations:Y1=-3.916X1+0.784X2+3.940(r2=0.991 5),Y2=-14.153X2-32.610X3+120.265(r2=0.986 8).

Table 3 Correlation between plain stir-bake processing technology, physical parameters, and chemical components of Trichosanthis Radix

4 Discussion

4.1 Effect of processing technology on physical parameters

In the process of plain stir-baking of Trichosanthis Radix, the processing technology will change the appearance of the decoction pieces, and then affect the physical parameters.As the degree of processing deepens, the appearance color of the decoction pieces gradually deepens until the scorch becomes black, which will cause the lightness(L), red(R), green(G), blue(U)and other values that characterize the chromaticity value gradually decrease, accordingly leading to decline of the chromaticity; with the continuous volatilization of water, more cavities will be formed in the decoction piece[7], which changes the texture of the decoction piece.High temperature(>100 ℃)destroys the cell wall tissue structure[13], causing a decrease in relative density and increase in hydroscopic rate and swelling(except for the stir-bake to brown).In the process of plain stir-baking of Trichosanthis Radix, due to the participation of temperature, hydrogen bonds will break and more hydrogen ions will be released, causing the pH to drop(except for the stir-bake to brown).In addition, the processing process may cause the decomposition of large molecules inside the decoction pieces and the random combination and transformation of small molecules, resulting in more free radicals, increasing anti-oxidation degree, and thermal decomposition of volatile components in the processing process[13], thus causing the oxidation value of processed products is generally higher than that of crude products, but the specific mechanism still needs to be further studied.

4.2 Effects of processing technology on chemical components

During the processing of Trichosanthis Radix, not only the appearance of the decoction pieces change, but also the internal chemical components will change.In the process of plain stir-baking of Trichosanthis Radix, α-glycosidic bond within starch molecules continuously breaks, the starch macromolecules are converted into small molecular substances, then the starch content of processed products shows a decreasing trend(except for the stir-bake to brown), until the starch content of stir-bake to scorch processed products becomes zero.At the same time, the small-molecule carbohydrate components produced in this process will recombine into polysaccharide molecules, causing the polysaccharide content to increase continuously, and the stir-bake to scorch processed products reach the highest level(3.66 mg/g).In addition, correlation analysis shows that there is a significant negative correlation between total starch and polysaccharides(r=-0.953,P=0.047<0.05), further verifying that there may be mutual conversion between starch and polysaccharides.Trichosanthis Radix polysaccharide is a polysaccharide composed of glucose.As a main component of Trichosanthis Radix, it is processed through the plain stir-bake method to convert starch into polysaccharides, which enhances the pharmacological effects of Trichosanthis Radix polysaccharides in regulating immunity and resisting tumor[14], which is basically consistent with the enhancement of clinical efficacy by traditional Chinese medicine processing[15].

4.3 Correlation between physical parameters and chemical componentsFrom the correlation analysis results of physical parameters and chemical components, it can be seen that the differences between relative density, hydroscopic rate and polysaccharides of Trichosanthis Radix are statistically significant(P<0.05); there is a significant correlation between hydroscopic rate, swelling, and total starch, and the linear regression model established between the physical parameters and the chemical components is highly reliable(r2>0.98), indicating that the dynamic change process of the physical parameters and chemical components before and after the processing of Trichosanthis Radix can be interpreted by data.Therefore, to a certain extent, relative density and hydroscopic rate can be used to indicate polysaccharide content, and hydroscopic rate and swelling can be used to indicate total starch content, so as to realize the change trend of chemical components by measuring the physical parameters of Trichosanthis Radix, and finally realize the quality evaluation of processed products of Trichosanthis Radix.

Taking Trichosanthis Radix decoction pieces as the research object, we conducted Delphi evaluations and selected the highest scores from stir-bake to yellow, stir-bake to brown, and stir-bake to scorch processed products of Trichosanthis Radix.By measuring the relative density of decoction pieces and other six physical parameters, as well as the total starch and polysaccharide content, we clarified the correlation between the processing technology, physical parameters, and chemical components, and established a linear regression model to provide a new way and new method for the quality evaluation of processed products of Trichosanthis Radix.However, from the measurement results of physical parameters and chemical components, we can know that compared with crude products, the swelling, pH, and total starch content of stir-bake to brown and processed products of Trichosanthis Radix are inconsistent with the changes of stir-bake to yellow and stir-bake to scorch processed products.The reason may be that the interaction between processing temperature and time affects the changes of physical parameters and chemical components, but the specific mechanism needs to be further studied.Furthermore, due to the small experimental sample size in this study, and the research results need to be further verified.In future, it is necessary to increase the number of test indicators for physical parameters and chemical components by collecting Trichosanthis Radix from different origins, to expand the sample size of the experiment, and to deeply explore the influence of the two materials of Trichosanthis Radix decoction and fine powder on the physical parameters and chemical components, establish a quantitative relationship model between the processing technology, physical parameters, and chemical components, and conduct model verification to improve the credibility of the research results, and finally realize the objective, systematic, and scientific evaluation of the quality of processed Trichosanthis Radix, so as to provide a reference basis for the quality evaluation of traditional Chinese medicine processed products.