Study on preparation technology of Qi Yu constitution Yangsheng granules

1. Introduction

The qi-stagnation constitution health-preserving granule is based on a clinical empirical formula, and clinical research shows that the formula has a good curative effect on the symptoms of emotional discomfort and spleen and stomach disharmony caused by liver depression and spleen deficiency [1, 2]. In traditional Chinese medicine (TCM), liver qi stagnation is treated with the health-preserving granule. It regulates qi dynamics by using herbs and sophisticated extraction processes. Clinical research demonstrates its effectiveness in enhancing mental health and reducing symptoms like irritation and stomach problems. This granule promotes general well-being and is beneficial for those with a qi-stagnation constitution. The composition comprises the following components: fingered citron, mint, rose, Chinese date, tuckahoe, and honey-fried licorice root [3, 4, 5]. The monarch drug is rose and jujube, the ministerial drug is finger citron, the adjuvant drug is tuckahoe and mint, and the guide drug is honey-fried licorice root. According to the Measures for the Administration of the Catalogue of Traditional Chinese Medicinal Materials, these TCM belong to 101 kinds of drugs with the exact origin of medicine and food [6]. Consistency between batches is ensured by quality control procedures in extraction operations. These consist of reference standards, batch-to-batch monitoring, sampling methods, analytical techniques, documentation, staff training, statistical process control, standardized processes, calibration, validation, and continuous improvement. For the preparation of medicinal products, a strong quality control process entails assessing raw materials, adhering to standard operating procedures, performing in-process inspections, rigorous testing, stability assessments, documentation, validation studies, quality assurance supervision, and ongoing efforts to improve [7, 8]. Drugs known as TCM have the same roots as food and medicine and support a holistic approach to healthcare. Research, standardization, and public education are all necessary to incorporate TCM into healthcare procedures and foster an awareness of its nutritional and therapeutic qualities.

This study intends to preliminarily determine the reasonable preparation process of the Qi Yu granule by network pharmacology analysis and the analysis of existing preparation methods.

2. Analysis of efficacy and chemical composition of monarch drug

Rose (meiguihua) tastes slightly bitter, sweet, and warm. The liver, spleen, and qi-regulating drugs can regulate liver-qi stagnation, qi, and blood pain [9]. Doctors from historical periods assessed its healing properties. According to “Materia Medica Justice”, roses were believed to have the ability to soothe the liver and stimulate the stomach, facilitate the flow of qi, and improve blood circulation. The medicine can be used for treating hypochondriac pain and epigastric distention caused by liver-qi disharmony, epigastric distention and pain caused by liver-stomach disharmony, gynecological menoxenia, premenstrual breast distention and pain caused by liver channel obstruction, and the like, and has sound effects of regulating qi and treating traumatic swelling. Treatment for epigastric distention resulting from an imbalance in liver qi and hypochondriac pain is a comprehensive treatment in TCM [10]. This covers acupuncture, herbal medicine, dietary modifications, stress reduction, and mind-body activities. Patients are encouraged to speak with healthcare professionals while the treatment attempts to relieve symptoms and bring the liver system back into balance. It is reported that there are many chemical components in rose, up to more than 100 kinds [3], and the main bioactive components include volatile oil, flavone, polysaccharide, phenolic acid, etc. The rose polysaccharide mainly comprises the following components: galacturonic acid (45.5%), galactose (5.5%), and arabinose (4.7%). Most of the polysaccharides were methyl esterified (62%) and acetylated (10%). Rose pigment also has specific medicinal value. Some studies have found that the main components of rose pigment are cyanidin-3,5-diglucoside, cyanidin-3-glucoside, delphinidin-3-glucose-rhamnoside, and paeoniflorin-3-glucoside, etc. In addition, roses are rich in fatty acid components. Pengyu et al. [11] found that wild roses contain unsaturated fatty acids, and the main components are as follows: linoleic acid, methyl docosaenoate, methyl triaconitahexaenoate, ethyl nonadecenoate, methyl eicosenate, and the like.

Volatile oil components mainly include six categories: Alcohols, esters, terpenes, aldehydes, ketones, and others. Alcohols such as alpha-terpineol, leaf alcohol, benzyl alcohol, beta-phenyl ethyl alcohol, 1-hexanol, etc.; terpenoids such as alpha. -pinene, beta. -pinene, beta. -myrcene, limonene, beta. -caryophyllene, isoterpinene, cis-. Beta. -ocimene and the like; aldehydes such as hexanal, 2-hexanal, nonanal, neral, geranial, etc.; esters such as ethyl octanoate, citronellyl formate, ethyl decanoate, citronellyl acetate, and the like. Other examples include 2-nonanone, methyl nonyl ketone, 2-tridecanone, beta-damascone, geranyl acetone, etc.; such as rose oxide, methyl eugenol, eugenol and the like [3]. Treatment strategies are influenced by the volatile oil content of medicinal ingredients; a high amount necessitates steam distillation for efficacy and preservation, while a low content calls for gentle extraction techniques. Considerations include dose form, shelf life, and stability.

Doctors from historical periods often used rose as decoction or tea for treating liver and spleen symptoms, such as compendium in the treatment of liver and stomach pain: Roses are dried in the shade, made into soup, and served as tea. The “Ming Food Materia Medica” describes rose food as having a fragrant sweetness and being refreshing. Regarding the treatment for liver wind headache, according to the “Quanzhou Materia Medica”, a remedy involves using 9 to 12 grams of broad bean flower and 4 to 5 roses, which are brewed as a tea to be consumed regularly. Shandong Manual of Chinese Herbal Medicine records that “white peony root 9 grams, rose 9 grams, nutgrass galingale rhizome 12 grams, Szechwan Chinaberry fruit 9 grams” can be used to treat stomachaches [4].

Dazao (Jujubae Fructus), sweet and warm in taste, enters the heart meridian of hand shaoyin, spleen meridian of foot taiyin and stomach meridian of foot yangming. It belongs to a qi-tonifying medicine and has the effects of tonifying qi, strengthening the middle warmer, calming the nerves, and nourishing the blood. It can treat fatigue, loose stool, and anorexia caused by spleen deficiency, as well as deficiency of qi and blood caused by spleen deficiency [12]. Health analyses concentrate on blood lipids, thyroid function, renal function, and ultrasonic cardiac indicators for a thorough evaluation of cardiovascular, endocrine, and renal health. Monitoring these indicators helps with understanding the physiological system, early danger identification, and preventative measures.

Zhong et al. [13] conducted a study on the protective effect of jujube polysaccharide at different doses on the liver using a rabbit liver injury model. Ancient literature, such as the Waitai, contains reports on using jujube as a medicine. For example, it mentions proportions such as one and a half dried jujube meat, along with one and a half each of fried licorice, almond and dark plum. The results showed that jujube polysaccharide significantly reduced alanine aminotransferase activity in model rabbits. After smashing, mix with honey to make pills, the size of which is like a jujube pit. The potential of jujube’s antioxidant and anti-inflammatory qualities to mitigate oxidative stress and inflammation has been investigated in rats; however, the degree to which these characteristics may effectively cure acute liver damage caused by drugs such as CCl4 and paracetamol differs, necessitating more studies. It can be used for dry mouth. Modern pharmacological research shows that jujube protects the liver, resists fatigue, increases white blood cells, and enhances immunity. In addition, some experiments [14] showed that jujube had a protective effect on acute liver injury caused by paracetamol and CCl4 in mice. Increased blood levels of alanine aminotransferase (ALT), which are a marker of liver damage, can be caused by liver illnesses, drugs, alcohol, obesity, metabolic disorders, viral infections, gallbladder problems, and liver trauma. The chemical composition of jujube mainly includes amino acids, saccharides, vitamins, insoluble dietary fiber, and trace elements such as Ca, Fe, K, Mg, Mn, Al, and there are also a large number of nucleotide derivatives in jujube. In the preparation process, the processes related to the above substances mainly focus on whether to extract with water and precipitate with alcohol, the specific indicators of the extraction process, and the treatment mode of the rose volatile oil.

To further understand the pharmacological components of jujube rose, BAT-MAN (Bioinformatics Analysis Tool for Molecular mechANism) was utilized to study the components of Ganpishuangli granules. This involved a comprehensive analysis of the molecular mechanisms and bioactive compounds present in Ganpishuangli granules, particularly focusing on how these components interact and contribute to the pharmacological effects associated with jujube rose. The use of BAT-MAN allowed for a detailed examination of the chemical composition and potential therapeutic properties of Ganpishuangli granules, shedding light on the intricate relationships between its constituents and their biological activities. The methods are outlined below.

Firstly, the pharmacological tool database is put in to investigate the formula. After the formula data is put in, the ion channel of the whole component acting on the organism is obtained. A critical first step towards comprehending how a pharmaceutical formula targets a particular illness is to enrich ion channel data. This entails determining the effective illness spectrum, translating effects to the organism level, and analyzing the regulation of ion channels by the formula’s constituent parts. This thorough study directs further investigation and validation of focused therapy strategies. The effective disease spectrum of the formula substance is obtained through the enrichment of the ion channel. It is found that the granule drug combination has a potential curative effect on liver and spleen system diseases such as depression and breast cancer, especially the jujube containing various components with curative effects on breast cancer [15]. Data of the top 40 effective disease spectrum of prescription substances are shown in the Table 1.

According to the theory of TCM, the diseases related to the liver and gallbladder, liver depression, and spleen deficiency in the above conclusions are:

Breast cancer (No. 4 stagnation of liver-qi) [16], Parkinson’s disease (No. 10 internal movement of wind yang in TCM), hypertension (No. 8 is closely related to spleen dysfunction, “Origin of TCM Etiology and Pathogenesis Theory of Prehypertension”), schizophrenia (No. 14), anxiety disorder (No. 15), depression (No. 17), migraine (No. 22), and epilepsy (No. 25). Epilepsy is closely related to phlegm and wind, and the disease location is mainly in liver, spleen and other viscera [4].

The following information is obtained by backtracking (Fig. 1): disease (square in the figure), target gene protein (five-pointed star part), and drug composition. In rose, the substances highly related to liver depression are isoamyl alcohol and isoamyl amine. The proteins against which the above substances act is e.g. DRD1.

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According to the analysis of existing pharmacological materials, saccharides are effective components for liver and spleen symptoms.

There are three categories of sugars: monosaccharides, oligosaccharides, and polysaccharides, commonly known as carbohydrates. Monosaccharides are readily soluble in water. Weifang’s study [9] showed that the pharmacological experiment on rose polysaccharides showed that the spleen index of mice taking rose polysaccharides was superior to that of the control group, and it was confirmed that rose polysaccharides had antitumor activity. El-Sayed et al. [17] showed that the water extract of taif rose had a clear inhibitory effect on the growth of hepatoma cell line HepG2 in vitro, and IC50 was less than 20 μ g/ml. In addition, the literature shows that polysaccharides in other TCM in the formula also have liver protection effects [4], such as fingered citron polysaccharides having specific immune enhancement effects. Chun et al. showed that pachymaran has a noticeable anti-jaundice and hepatoprotective effect.

Response surface methodology (RSM) is a statistical approach that modifies temperature and duration in tests in a systematic way to find the parameters for water extraction. A mathematical model represented as a response surface is constructed using the gathered data to help optimize circumstances to maximize or minimize desired results. Take the times of decoction, extraction time (start timing after slight boiling), and the multiple water additions as the investigation factors, and each factor is set with three levels. According to the clinical decoction habits of tea drinks, the factor levels are shown in Table 5. Weigh two parts of medicinal materials in the prescription proportion, put them into a 500 ml flask, experiment according to Table 6, extract the dry extract and weigh the weight, and select the best extraction conditions according to the dry extract rate by response surface methodology. In developing pharmaceutical products in factories, scalability, efficiency, and dependability are critical factors in experimental design. Maximizing production includes scale-up studies, realistic operating conditions, robust processes, sensitive parameters, appropriate equipment, environmental control, FMEA, regulatory compliance, QbD principles, and continuous improvement.

Based on the dry extract weight and the obtained hesperidin content, a response surface experiment was performed:

The weight of dry extract powder after water extraction 1, 2, and 3 times was compared by extracting for 30 minutes and adding water nine times. The weight of dry extract powder was reached when the extraction time was 30 minutes, and the times of adding water were 6, 9, and 12. Comparing the weight of dry extract powder after adding nine times of war and extracting for 20 minutes, 30 minutes, and 4,0 minutes, it was found that the above parameters greatly influenced the weight of dry extract powder. The main factors affecting water extraction were decocting times, extracting time, and adding water and removing times. Various factors influence decocting times, including plant variability, economics, standardization, efficiency optimization, traditional practices, temperature sensitivity, bioactive constituent extraction, and material complexity. The ultimate goal is to maximize the desired compounds. According to the pre-experiment results, each factor was coded with - 1, 0, and 1, and the dry extract rate (Y) was used as the index (Tables 6–8). Fill the above experimental results into the practical record form to obtain the ranking.

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After obtaining the above experimental results, the analysis model was established using the Design-Expert V. 10 software to analyze the response surface methodology (Fig. 3).

Comparing the significance of the fitted equations, the results were all “significant”. Observe the normal distribution of the residuals (i.e., the difference between the actual and estimated values) as shown in Figs 4 and 5.

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See Fig. 6 for the plot of residuals versus equation predictions (the more scattered and irregular the distribution, the better).

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Multiple linear regression and equation fitting were carried out on the three factors (independent variables) A, B, and C, respectively, and the regression equation was as follows:

According to the determined equation as the model, the corresponding dry extract weight and hesperidin content as the dependent variables, draw the three-dimensional map of the response surface. In hesperidin extraction processes, a three-dimensional map of the response surface facilitates the identification of ideal conditions, the visualization of variable interactions, the validation of models, the performance of sensitivity analyses, the effective optimization of processes, the definition of perfect varying ranges, the disclosure of nonlinear relationships, and the evaluation of risk and robustness. The decoction times are discontinuous variables, hence C can only be taken as one time, two times, and three times. The corresponding contour lines and three-dimensional map are as outlined in the following figures: Decocting once (Figs 7–10); decocting ptwice(Supplementary Figs 1–4); decocting thrice (Supplementary Figs 5-7); equation Predictions vs. actual (Supplementary Fig. 8).

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According to Figs 7–10 and Supplementary Figs 1-8, the following conclusions can be drawn:

Each part of the experimental medicinal materials is four parts of Chinese restorative materials, and the weight of each part is 18 g, performing the water extraction and concentration processes.

(1) Under one extraction, the dry extract rate increased with extraction time and water content.

The reason for the two high points in the chart is that at the beginning of extraction, because the liquid medicine has been thoroughly soaked, many substances are dissolved in the aqueous solution. Hence, the software simulates the rising trend according to the data in the image under the condition of short extraction time and less water addition. The trend is confirmed by experimental verification. Water-soluble components of plant materials, such as extractives, phenolic compounds, vitamins, alkaloids, amino acids, and pigments, dissolve in an aqueous solution during extraction. Other solvents can collect more bioactive substances. Thorough experimental settings, such as reproducing parameters, managing the environment, robust sampling, real-time monitoring, sensitivity testing, comparison analysis, feedback loops, documentation, validation criteria, and peer review, are essential to validate the increasing trend in simulations. In addition, by observing the dry paste trend of the second and third extractions, it can be seen that the trend is gradually not evident with the increase of extraction times.

The hesperidin content decreased first and then increased with water content, but the trend was not obvious. There are several reasons why the concentration of hesperidin decreases as the water content increases, including the influence of temperature and extraction time, matrix interactions, dilution effects, hitting solubility limitations, and possible degradation. You may lessen these impacts and increase hesperidin output by optimizing the extraction conditions. With the increase in heating time, the hesperidin content increased first and decreased slightly when the heating time was close to 40 minutes.

Under these conditions, the highest dry extract weight of a sample of experimental medicinal materials was 10.98 g, and the hesperidin content was about 185.99 μ g. The conditions for the highest hesperidin content are: Add 12 times water for approximately 30 minutes.

(2) The dry extract rate increased with the increase of the two conditions when the times of adding water were more than nine and the time was more than 30 minutes, and the maximum dry extract rate was obtained when the conditions reached the upper limit of the interval. According to the contour map, it can be clearly seen that the effect of the water addition ratio on dry extract rate is greater than that of extraction time.

The content of hesperidin decreased with the increase of the water addition ratio. The content of hesperidin increased at first and then reduced with heating time.

Under these conditions, the maximum amount of hesperidin extracted from a sample of experimental medicinal materials was 271.53 μ g, and the weight of the dry extract was 7.06 g. The optimum conditions were adding six times of water and extracting for 33.94 min. When the highest dry extract weight was 13.78 g, the hesperidin content was 158.71 μ g, and the conditions were as follows: the amount of water was 10.75 times and the heating time was 40 min.

(3) The weight of the dry extract increased with the ratio of water and heating time when the extraction time was fixed three times. The weight of the dry extract was higher than 14 g when the water ratio was 12 times and the extraction time was 20 minutes or the water ratio was 8 ∼ 10 times and the heating time was 40 minutes.

The hesperidin content is more than 300 μ g when the amount of water is six times and the heating time is about 27.5 ∼ 27.5 min. The content of hesperidin decreased with the increase of water addition times.

Under these conditions, the maximum hesperidin extracted from a sample of experimental medicinal materials was 309.60 μ g, and the dry extract weighed 7.88 g. The conditions were as follows: adding six times water and extracting for 32.325 min. To obtain the highest dry paste weight, 15. The content of hesperidin was 201.97 μ g when the amount of water was 8.697 times and the heating time was 40 min.

Therefore, if hesperidin is taken as the only index, the best index is extraction three times, the condition of adding six times, and the time of 32.325 min. If that weight of dry extract was taken as the only index, the optimum condition was as follows: extract three times, adding 8.697 times of wat, heating for 40 min.

If those two parameters are higher than 95% of the high value, i.e., higher than 14.74 g, 294.12 μ g, available unconditionally. If calculated as higher than 80% respectively, i.e., higher than 12.42 g and 247.68 μ g respectively, the range of values is shown in Supplementary Fig. 9.

According to the 20 optimal solutions given by the system, if according to the dry paste weight. The weight of hesperidin content is 6:4, and the two solutions with the highest scores are selected as follows(Supplementary Fig. 10).

The screen solution can be determined if the weight ratio of the dry extract weight to the hesperidin content is 5:5. The final parameters were determined to facilitate the actual operation: extraction three times, extraction time for 40 min, and water addition multiple approaching six times.

Response surface validation experiment: Three validation experiments were carried out according to the optimized process conditions, and the results of sample 18 19 and 20 showed that the average content of dry extract was 14.80 g and the moderate content of hesperidin was 254.34 μ g, which had little deviation from the predicted value, indicating that the established mathematical model was reliable and had good predictability.

Through the literature analysis, network pharmacology analysis, and experiments, the feasible scheme of producing Qi Yu constitution Yangsheng granules in the factory is determined, and the optimal process is determined according to the mathematical model established by the content of hesperidin measured by HPLC and the weight of dry extract. For a factory to achieve its operational excellence and product quality goals, it is essential to optimize the manufacturing process of Qi Yu constitution Yangsheng granules manufacturing process. This includes optimizing formulation, efficient resource utilization, consistent product quality, yield maximization, cost reduction, and compliance with quality standards. By comparison, response surface methodology was chosen as the analysis tool instead of the orthogonal experiment.

This choice is based on the following considerations:

The response surface model is more realistic: The mathematical model based on the orthogonal experiment is linear, which is inconsistent with the fact that multiple factors cross-affect the experimental results. When the experimental conditions change, the results are often not linear (for example, the increase in water volume is beneficial to the dissolution of effective substances but also increases the difficulty of concentration. If the heating method is used for concentration, the material loss caused by heating will also increase). The response surface method based on a multi-dimensional high-precision regression equation makes up for this shortcoming.

The premise of the orthogonal experiment was that the extraction time was the same. If we want to analyze the optimal solution under fixed conditions, we need to redesign the experiment, and the results of the past orthogonal experiment need to be more competent. The response surface experiment provides the possibility of predicting the experimental results under fixed conditions, which can ensure the stability of product quality according to the formula of response surface simulation.

Author contributions

All authors reviewed the results, approved the final version of the manuscript and agreed to its publication.

Data availability

The experimental data used to support the findings of this study are available from the corresponding author upon request.

Funding

This study is supported by the National Key R&D Plan Fund (No. 2019YFC1711700); Chinese Sleep Quality Assessment Technology (No. JBGS2021009); and the Science and Technology Innovation Engineering Project of the China Academy of Chinese Medical Sciences (No. CI2021A05407).

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/THC-232034.