Study on the spectral effect relationship between UPLC fingerprint of bone fragment patch and alkaline phosphatase activity in human osteosarcoma cell MG63
Gu Duan Bu is the dried rhizome of Drynaria fortunei (Kunze) J. Sm., a water dragon orthopedic plant. It is warm in nature, bitter in taste, and belongs to the liver and kidney meridians. It has the effects of healing and relieving pain, tonifying the kidneys and strengthening bones, and dispelling wind and spots. Modern pharmacological and clinical studies have shown that bone fragment tonics have effects such as anti osteoporosis, promoting fracture healing, kidney protection, anti-inflammatory, and promoting tooth growth. Chemical composition studies have shown that the main active ingredients in bone fragment tonics are flavonoids, triterpenoids, phenylpropanoids, and phenols. A large number of studies have shown that bone marrow tonics have significant therapeutic effects in treating bone related diseases, and in vitro experiments have shown that bone marrow tonics can promote the osteogenic differentiation ability of osteoblasts. However, there have been no reports on the specific active ingredients in bone marrow tonics. Alkaline phosphatase (ALP) is a biomarker for the differentiation and maturation of osteoblasts, and changes in ALP activity levels can be used to determine the differentiation ability of osteoblasts. Therefore, this experiment analyzed the relationship between UPLC fingerprint and ALP activity spectrum of human osteosarcoma MG63 cells, and explored the main components of bone fragment tonics used for osteoblasts.
The evaluation mode of traditional Chinese medicine spectrum efficacy is to combine the chemical component fingerprint with its pharmacological effects, so that the chemical components in the fingerprint reflect the corresponding pharmacological effects, and clarify the relationship between the characteristic peaks of the fingerprint and the pharmacological effects, determine the corresponding pharmacological substance basis, and thus carry out quality control and pharmacological evaluation of traditional Chinese medicine.
This study established UPLC fingerprint spectra of raw and hot bone fragment supplements and identified their chemical components using UPLC-Q-TOF-MS technology. Grey correlation analysis, bivariate correlation analysis, and partial least squares method were used to construct the fingerprint spectra of bone fragment supplements and the spectral effect relationship of ALP activity, providing a reference for elucidating the pharmacological substance basis and quality control of bone fragment supplements.

This study used UPLC method combined with multi wavelength detection to establish fingerprint spectra of bone fragment supplements and hot products. There were 12 common peaks between different batches of bone fragment supplements and hot products, and the similarity was greater than 0.9, indicating high similarity and unclear differentiation between the two. ALP is a biomarker of osteoblast differentiation and maturation, and is one of the biochemical indicators of bone formation. The differentiation ability of osteoblasts can be determined by changes in ALP activity levels. Previous studies have shown that bone fragment supplementation can promote osteoblast differentiation. In this study, bone fragment supplements and scalds were applied to MG63 cells to analyze their effects on ALP activity. The results indicate that the same batch of bone fragments can significantly promote the secretion of ALP by MG63 cells after sand scalding. To investigate the active ingredients in bone fragmentation and repair that promote MG63 osteogenic differentiation, a spectral correlation analysis was conducted between ALP activity and characteristic peaks calibrated by fingerprint spectra. In order to comprehensively analyze the spectral effect relationship between the characteristic peaks of bone fragment patch fingerprint spectrum and ALP activity, this experiment comprehensively used three methods: grey correlation analysis, bivariate correlation analysis, and least squares regression analysis. It was found that peak 4, peak 5, and peak 12 have a high correlation with ALP activity and are positively correlated, contributing the most to ALP activity and being important variables affecting ALP activity.
UPLC-Q-TOF/MS technology has the characteristics of strong selectivity, high sensitivity, simple sample pretreatment, high accuracy, and high resolution, which can more comprehensively and quickly identify the chemical components of bone fragments. This study used UPLC-Q-TOF/MS in negative ion mode to identify 40 components in bone fragment tonics. The identified component that contributes significantly to ALP activity is peak 4 caffeic acid-4-O – β – D-glucopyranoside; Peak 5 catechins 7-glucoside; Peak 12 naringin and its activity were validated. Naringin is a flavonoid compound, and modern pharmacological research has shown that naringin can be used to treat or prevent various diseases, such as osteoporosis, antioxidant, anti-tumor, and improvement of myocardial injury. Existing research also indicates that naringin is an important active ingredient in bone fragment tonics. The pharmacological activities of caffeic acid-4-O – β – D-glucopyranoside and catechin 7-glucoside have not been reported yet, and further research is warranted. The results of this study have certain reference significance for the quality control and pharmacological research of bone fragment tonics.