Establishment of fingerprint spectrum and determination of multi index component content of Chaihu Baiyaoyao formula granules
Chaihu, first recorded in the Shennong Bencao Jing, is the whole plant or dried root of Bupleurum scorzonerifolium Willd. or Bupleurum chinense DC., a plant in the Umbelliferae family. It has a bitter and pungent taste, a slight coldness, and belongs to the liver, gallbladder, and lung meridians. It has the effects of soothing the liver, relieving depression, dispersing fever, and promoting yang qi. With the development of modern natural medicinal chemistry, various valuable and important effects such as anti-inflammatory, anti-tumor, antidepressant, antiviral, immune regulation, and neural regulation have been discovered in Bupleurum chinense. Paeonia lactiflora Pall., a plant in the Ranunculaceae family, is the dried root of Paeonia lactiflora Pall. It has a bitter, sour taste and a slightly cold nature. It is mainly produced in Shandong, Hunan and other places. Currently, Bozhou has the largest production of Paeonia lactiflora, which is a famous traditional Chinese medicine in China. It has the functions of calming the liver and relieving pain, nourishing blood and regulating meridians, and reducing yin and sweating. Modern pharmacological research has shown that Paeonia lactiflora has anti-inflammatory, analgesic, anti-tumor, and antioxidant effects. The Chaihu Bai Shaoyao pair, as the basic medicine for soothing the liver and relieving depression, was first seen in the Han Dynasty’s “Treatise on Cold Damage”. Formulas such as Sini San, Xiaoyao San, Chaihu Shugan San, and Anshen Jieyu Tang all use Chaihu as the main medicine and Bai Shaoyao as the subordinate medicine. The two medicines are used in combination, with one dispersing and one converging, which conforms to the characteristics of liver medicine in traditional Chinese medicine theory.
Chinese medicine formula granules are made from single Chinese medicine decoction pieces, using modern pharmaceutical technology, and are extracted, separated, concentrated, dried, and granulated by water heating. The advantages of low dosage, easy portability, and adjustment according to symptoms endow Chinese medicine formula granules with broad development prospects. And traditional Chinese medicine formula granules are no longer limited to the development of single ingredient decoction pieces, such as honeysuckle Forsythia suspensa, ephedra almond, ephedra cassia twig, Danshen safflower, Huanglian wuzhuyu and other medicinal formula granules are gradually being widely used in clinical compatibility applications. The extraction rate of Chaihu single ingredient formula granules is relatively low. At the same time, the combination of white peony and Chaihu can increase the decoction amount of paeoniflorin and reduce the decoction amount of toxic component benzoic acid, which can enhance the solubility of active ingredients, eliminate or reduce toxic side effects, and ensure the accuracy of clinical medication dosage. At present, there are no reports of Chaihu Baiyaoyao formula granules. Therefore, this study establishes an HPLC fingerprint, chemical pattern recognition technology, and simultaneous determination of the content of four components for Chaihu Baiyaoyao formula granules, in order to improve the quality evaluation system of Chaihu Baiyaoyao formula granules and provide some reference for clinical use of traditional Chinese medicine.

This study used HPLC to establish the fingerprint spectrum of Chaihu Baiyaoyao formula particles. Different mobile phase systems (acetonitrile water, acetonitrile-0.1% phosphoric acid water, acetonitrile-0.1% formic acid water, and methanol-0.1% phosphoric acid water) were investigated, and it was found that acetonitrile-0.1% phosphoric acid water had better kurtosis and higher response values when washed with water; The extraction solvents were screened for water, methanol, ethanol, 5% concentrated ammonia methanol, and 5% concentrated ammonia ethanol solution. It was found that when using a 5% concentrated ammonia methanol solution, the chromatographic peaks were intact; Scanning the test solution of Chaihu Baiyaoyao formula granules at dual wavelengths (210nm, 230nm), the results showed that at 210nm, the fingerprint spectrum had a higher number of chromatographic peaks and a stable baseline; At the same time, the chromatographic columns (COSMOSIL 5C18-MS-II column, WONDASIL C18, Amethyst C18-H (4.6mm × 250mm, 5 μ m, 120 Å)) and column temperatures (20 ℃, 25 ℃, 30 ℃) were compared. The results showed that the COSMOSIL 5C18-MS-II column had better separation efficiency when detected at 30 ℃.
Chaihu White Peony Formula Granules are granules prepared from Chaihu White Peony decoction pieces through a series of standardized processes such as extraction, separation, concentration, drying, and granulation. They share similarities in composition and pharmacology with Chaihu White Peony and are a new form of decoction piece. Chaihu saponin a, Chaihu saponin d, and paeoniflorin are the main detection indicators for controlling the quality of Chaihu and Paeonia lactiflora in the 2020 edition of the Chinese Pharmacopoeia. Research has shown that both saikosaponin a and saikosaponin d can improve depressive behavior in rats by inhibiting the p-CREB/BDNF pathway, thereby suppressing hippocampal neuronal apoptosis and microglial activation. Paeoniflorin can alleviate CFA induced inflammatory pain by inhibiting the TRPV1 and succinic acid/SUCNR1-HIF-1 α/NLPR3 pathways. Oxidized paeoniflorin can activate receptors by upregulating the expression of peroxisome proliferator alpha protein, promote the expression of ATP binding transporter A1 gene, and improve lipid distribution. The above are the main active ingredients of Chaihu Baiyaoyao formula granules, and the determination of the content of these four ingredients is of great significance for the quality control of Chaihu Baiyaoyao formula granules. In addition, fingerprint spectra can comprehensively reflect the material basis of traditional Chinese medicine. Therefore, this study combines fingerprint spectra with content determination in order to provide some reference for improving the quality standards of Chaihu Bai Shao formula granules.
Fingerprint spectrum combined with chemical pattern recognition technology is widely used in the quality evaluation of traditional Chinese medicine, Chinese herbal decoction pieces, and formula granules. The use of this method in this study can intuitively measure the similarity of fingerprint pattern changes between formula particle samples of Chaihu Baiyaoyao from different origins. The similarity evaluation results of fingerprint spectra showed that the similarity between the formula granules and the control fingerprint spectra of 15 batches of Chaihu Baiyaoyao was greater than 0.90, indicating that Chaihu Baiyaoyao from different origins had better overall performance on the formula granules, but there were differences in the content of indicator components. The analysis may be due to the processing, harvesting methods, and production processes of the medicinal materials from different origins. The results of principal component analysis and cluster analysis mutually confirm each other, showing the regularity of the origin of medicinal materials. The origin and batch of samples collected by this research institute are limited, and further improvement is needed in the later stage.
In summary, this study established 15 batches of fingerprint spectra and simultaneous determination of four indicator components of Chaihu Baiyaoyao formula granules using HPLC method. Similarity evaluation, cluster analysis, and principal component analysis were used to evaluate and classify the quality of Chaihu Baiyaoyao formula granules. This method is easy to operate, has good repeatability, accurate and reliable results, and can provide a scientific basis for the comprehensive control and evaluation of the quality of Chaihu Baiyaoyao formula granules.