Comparative study on the quality of Paeonia lactiflora and its processed products using HPLC characteristic spectrum combined with content determination
White peony is the dry root of Paeonia lactiflora Pall, a plant in the Ranunculaceae family. It is bitter, sour, and slightly cold in nature. Return to the liver and spleen meridians. It has the effects of nourishing blood and regulating meridians, tonifying yin and stopping sweating, softening the liver and relieving pain, and calming liver yang. Used for blood deficiency and yellowing, menstrual disorders, spontaneous sweating, night sweats, rib pain, abdominal pain, limb spasms, headache and dizziness. The main active ingredient of Paeonia lactiflora is total glucosides of Paeonia lactiflora (TGP). TGP is a mixture of paeoniflorin, paeoniflorin, benzoyl paeoniflorin, and other ingredients, which has anti-inflammatory, immune regulating, and hepatoprotective effects. Stir fried white peony and wine white peony are both processed products of white peony. Modern pharmacological research has shown that the pharmacological effects of processed white peony are generally enhanced, and the analgesic effect of processed products is more significant than that of raw products, fully demonstrating the significance of white peony processing. The National Chinese Medicine Processing Standards contain the processing methods for wine white peony, stir fried white peony, and earth stir fried white peony; The “Jingyue Quanshu” and Jiao Shude’s “Ten Lectures on Medication Experience” both record the use of stir fried white peony. After stir frying white peony, its cooling properties are alleviated, and it has the effects of nourishing blood and collaterals, relieving spleen pain, and can be used to treat diarrhea and abdominal pain caused by spleen deficiency or liver spleen disharmony. The book “Bian Que Xin Shu” pioneered the method of “wine stir frying” white peony, while “Tang Ye Ben Cao” records that soaking white peony wine in the meridian can relieve central abdominal pain; The Compendium of Materia Medica states that “those who avoid the middle cold should stir fry with wine” and “stir fry with wine to nourish yin”. After stir frying with white peony wine, its sour and cold nature can be reduced, and it is good at harmonizing the middle, slow and urgent. It is often used for rib pain, abdominal pain, especially postpartum abdominal pain.

Sulfur fumigation is a commonly used method for preventing insect infestation, mold growth, and prolonging the storage time of traditional Chinese medicinal materials. However, sulfur fumigation can cause excessive SO2 residue in some traditional Chinese medicinal materials, resulting in changes in the effective ingredients and their content, reducing their efficacy and even turning them into “poison”, seriously affecting the quality and safety of traditional Chinese medicinal materials. Modern processing studies of Paeonia lactiflora have shown that sulfur fumigation can lead to a decrease in the content of paeoniflorin in Paeonia lactiflora. It is speculated that sulfur fumigation can transform some paeoniflorin into a new component, paeoniflorin sulfite, thereby reducing the content of paeoniflorin.

On the basis of the 2020 edition of the Chinese Pharmacopoeia standard for Paeonia lactiflora medicinal materials and processed products, this study established characteristic spectra of Paeonia lactiflora, Paeonia lactiflora wine, Paeonia lactiflora stir fried, and Paeonia lactiflora sulfur smoked using HPLC high-performance liquid chromatography, and identified six active ingredients: gallic acid, catechins, paeoniflorin, paeoniflorin, 1,2,3,4,6-pentagalloyl glucose, and benzoyl paeoniflorin; Simultaneously measure the content of the six active ingredients mentioned above in white peony, wine white peony, stir fried white peony, and sulfur smoked white peony, and evaluate the quality of white peony, processed products, and sulfur smoked products using similarity calculation of characteristic spectra and chemometric analysis methods. This provides a reference for the identification and quality evaluation of white peony, processed products, and sulfur smoked products.

Preparation of test solution. This study investigated the extraction efficiency of different concentrations of methanol and ethanol through single factor analysis. The chromatographic peak “total peak area/sample size” was used as the evaluation index for the characteristic spectrum, and the total content of gallic acid, catechins, paeoniflorin, paeoniflorin, 1,2,3,4,6-pentagalloyl glucose, and benzoyl paeoniflorin was used as the content determination index. Finally, dilute ethanol was determined as the extraction solvent; A comparative study was conducted on two extraction methods, heating reflux and ultrasonic treatment, and it was found that different extraction methods had little effect on the extraction efficiency of Paeonia lactiflora. Therefore, considering the simplicity of operation, ultrasonic treatment was chosen; At the same time, the ultrasonic treatment time (15, 30, 45 minutes) of the samples was investigated, and it was found that 30 minutes of ultrasonic treatment could completely extract various components. Based on the above investigation, it was determined that the best preparation method for the white peony test solution was to treat 50mL of dilute ethanol with ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes.

Establishment and analysis of feature maps. The research results of characteristic spectra show that there are 6 common peaks in the HPLC characteristic spectra of white peony, fried white peony, and wine white peony, and 7 common peaks in the HPLC characteristic spectra of sulfur smoked white peony. Due to the use of Agilent, Waters, and Thermo instruments from different manufacturers for sample detection to shorten working time and improve work efficiency, even if the same chromatographic column is used, the retention time of chromatographic peaks in the characteristic spectrum may vary due to differences in machine dead volume among different manufacturers. If peak 4 is taken as the S peak and the relative retention time is calculated, the relative retention time of each characteristic peak will be relatively stable. Peak 7 is generated after sulfur fumigation of Paeonia lactiflora, which is speculated to be paeoniflorin sulfite. Six characteristic components of Paeonia lactiflora, fried Paeonia lactiflora, and wine Paeonia lactiflora were identified by reference standards, namely gallic acid, catechins, paeoniflorin, paeoniflorin, 1,2,3,4,6-pentagalloyl glucose, and benzoyl paeoniflorin. OPLS-DA analysis identified three differential biomarkers, indicating significant changes in peak 7, peak 4 (paeoniflorin), and peak 5 (1,2,3,4,6-pentagalloyl glucose) after processing and sulfur fumigation of Paeonia lactiflora.

Changes in chemical composition of Paeonia lactiflora after stir frying, wine roasting, and sulfur fumigation. The content determination results of six known components in white peony, fried white peony, wine white peony, and sulfur smoked white peony showed that, except for gallic acid, the other five components had varying degrees of changes. After frying and wine roasting, the content of paeoniflorin in white peony decreased, while paeoniflorin lactone and benzoyl paeoniflorin both increased. This may be due to the transformation of paeoniflorin into paeoniflorin lactone and benzoyl paeoniflorin during the processing of white peony; After sulfur fumigation, the content of paeoniflorin decreased, which is speculated to be caused by the conversion of white peony to paeoniflorin sulfite after sulfur fumigation. Catechins are unstable when exposed to heat. Therefore, the frying and wine roasting processes significantly reduced the catechin content in white peony, while the sulfur fumigation process did not show significant changes in catechin content due to the lack of obvious heat exposure. In addition, after wine roasting and sulfur fumigation, the content of 1,2,3,4,6-pentagalloylglucose increased significantly, which is inferred to be due to the catalytic effect of heat and some chemical components in the auxiliary materials during the processing of Paeonia lactiflora, which promotes the conversion of 1,2,3,4,6-pentagalloylglucose. The specific mechanism needs further research.

According to traditional Chinese medicine theory, there should be differences in the clinical application of different processed products of traditional Chinese medicine, which are related to the differences in their internal chemical composition. Paeonia lactiflora has the effects of nourishing blood, nourishing yin, protecting the liver, and relieving spasms. After stir frying, its cooling properties are alleviated, and its spleen relieving and pain relieving effects are increased. After alcohol roasting, its sour and cold properties are reduced, and its moderate and urgent effects are enhanced. This may be related to the decrease in the content of paeoniflorin in Paeonia lactiflora, the increase in the content of glycoside lactones and benzoyl paeoniflorin. Although sulfur fumigation of Paeonia lactiflora can prevent insects, prevent mold and rot, excessive sulfur fumigation leads to a significant decrease in the content of paeoniflorin, which may be due to the conversion of some paeoniflorin into paeoniflorin sulfite, thus seriously affecting the medicinal materials. The quality of the decoction pieces should be strictly controlled, therefore, the sulfur fumigation process should be strictly controlled. In the later stage, further investigation will be conducted on the sulfur fumigation process of Paeonia lactiflora, exploring the effects of different degrees of sulfur fumigation on the effective components of Paeonia lactiflora and determining the optimal sulfur fumigation process.