Screening of effective ingredients in fennel root bark for anti liver fibrosis based on spectral effect relationship and exploration of its mechanism
Liver fibrosis is the main cause of liver cirrhosis and even liver cancer, and about 90% of liver cancer develops on the basis of liver fibrosis or cirrhosis. Therefore, the treatment of liver fibrosis is particularly important. The clinical incidence of liver fibrosis is increasing year by year, which seriously endangers people’s life safety. The main causes of liver fibrosis include chronic hepatitis virus infection, alcohol abuse, and non-alcoholic fatty liver disease. The characteristic of liver fibrosis is the excessive accumulation of extracellular matrix, which is caused by increased synthesis and deposition of extracellular matrix, as well as decreased or no degradation of extracellular matrix. Activation of hepatic stellate cells is considered a key link in liver fibrosis, and it is the main cause of excessive accumulation of extracellular matrix proteins in the liver.
Fennel root bark is the dried root bark of the Umbelliferae plant Foeniculum vulgare Mill. It is included in the standard Uyghur medicinal book and is used to treat wet cold or mucinous diseases, such as various inflammations and swelling caused by wet cold, bilateral cold pain, lower back pain, amenorrhea, urinary obstruction, and old intestinal obstruction. Hugan Buzu Re Granules is a classic formula for protecting the liver in traditional Chinese medicine. It is composed of celery seeds, celery roots, chrysanthemum seeds, dodder seeds, chicory roots, fennel root bark, and fennel, and has the effects of nourishing the liver and promoting bile flow. The preliminary research of the research group found that Hugan BuZuRe granules can protect against CCI4 induced liver fibrosis in rats. Medina Saifudin et al. found that the traditional Chinese medicine fennel root bark has a certain degree of promoting liver tissue repair and protecting liver cell membranes. Therefore, fennel root bark may have anti liver fibrosis effects.
The study of drug spectrum effect relationship is a method that combines chemical composition and pharmacological research to analyze effective chemical components. Based on the study of spectra, it maximizes the acquisition of useful chemical information, links spectra with pharmacological results, establishes a “spectrum effect” mathematical model through linear or nonlinear mathematical processing, and determines compound groups related to pharmacological effects. By analyzing known and unknown components, the types and contents of chemical components contained in complex mixed systems of traditional Chinese medicine can be maximally reflected, thereby providing an overall description and evaluation of the quality of traditional Chinese medicine. Therefore, in this study, ultra-high performance liquid chromatography-mass spectrometry was used to investigate the chemical components of the root bark of the Chinese medicinal herb fennel. The effects of ethanol extraction, petroleum ether extraction, ethyl acetate extraction, n-butanol extraction, and water extraction on HSC-T6 cell proliferation were combined with UPLC-Orbitrap-MS/MS to screen the chemical components of fennel root bark using partial least squares and grey relational analysis. The in vitro experiments were conducted to verify the anti liver fibrosis effect and mechanism of the effective components of fennel root bark, providing scientific basis for the research and development of ethnic medicine.

The synergistic effects of multiple targets and components in traditional Chinese medicine have become a hot topic in research. The study of active ingredients is the foundation of drug research, and their active ingredients and modes of action are characterized by complexity. In 2002, Li Rong proposed the theory of spectral effect relationship, which pointed out a new research direction for the study of traditional Chinese medicine efficacy. Based on the quantification of the common peaks in the UPLC-Orbitrap-MS/MS spectra of the alcohol extract and different extracts of fennel root bark and their anti liver fibrosis effects, this study used partial least squares and grey correlation analysis to investigate the correlation between the common peaks in the total ion flow spectra of fennel root bark and their anti liver fibrosis effects. This can largely reflect the contribution of the components to the pharmacological effect.
This study focuses on the alcohol extract, petroleum ether, ethyl acetate, n-butanol, and water parts of fennel root bark. High performance liquid chromatography-mass spectrometry analysis was conducted to construct a liquid chromatography-mass spectrometry (LC-MS) spectrum of fennel root bark. A total of 58 common peaks were calibrated in positive and negative ion modes, and the spectra were analyzed. It can be seen that the common peak areas of each sample are different, which can be used for spectral efficiency relationship research. The spectral effect relationship analysis showed that 11 components contributed significantly to the anti liver fibrosis effect (VIP>1 and regression coefficient>0). Among them, compounds that have been reported to have anti liver fibrosis effects in literature include gallic acid, apigenin, kaempferol, quercetin, scopoletin, and rhein, reflecting the characteristics of fennel root bark’s multi-component and multi-target anti liver fibrosis effects. Dihydrocamptothecin, dihydrocapsaicin, and scopolamine are compounds with relatively high content and good anti liver fibrosis activity found for the first time in fennel root bark, which may be potential quality markers. Among them, dehydrocamelbine may improve mouse lung tissue fibrosis caused by secondary infection of Echinococcus granulosus by reducing the expression of TGF – β 1, Smad3, and NF kB p65 in lung tissue. There have been no reports on the anti liver fibrosis effects of dihydrocapsaicin and scopolamine.
Bcl-2 belongs to the anti apoptotic gene and is a key factor in coordinating cell life and death, while Bax belongs to the pro apoptotic gene and is a pro apoptotic protein of the Bcl-2 family. As an important effector factor of cell apoptosis, caspase3 is a necessary pathway for the cascade reaction of apoptotic proteases. Its activation indicates that apoptosis has entered an irreversible stage. In apoptosis, the ratio of Bax/Bcl-2 is an important indicator for measuring cell apoptosis through the mitochondrial pathway. When the ratio of Bax/Bcl-2 increases, apoptosis increases; On the contrary, apoptosis decreases. When cells sense apoptotic signals, they can activate caspase-9 (Caspase9) by regulating the expression of apoptotic factors such as Bcl-2 and Bax, thereby activating Caspase3 and triggering a cascade reaction of the Caspase family, thereby inducing cell apoptosis. This study confirmed through experiments that dehydrocamptothecin and dihydrocapsaicin can reduce mitochondrial function and disrupt mitochondrial membrane potential balance in activated hepatic stellate cells, leading to a large release of pro apoptotic factor (Bax) and ultimately causing cell apoptosis, reducing the proliferation of hepatic stellate cells, inhibiting the expression of extracellular matrix and α – smooth muscle activator protein, and thereby alleviating liver fibrosis. In subsequent experiments, it is necessary to conduct in vivo experiments to validate the selected monomeric compounds and further investigate the anti liver fibrosis mechanism of fennel root bark monomeric compounds at the mRNA and protein levels.
In summary, this study used UPLC Orbitrap MS/MS technology combined with partial least squares and grey correlation analysis to elucidate the possible components of fennel root bark in anti liver fibrosis, reflecting the characteristics of the multi-component and multi-target anti liver fibrosis effects of fennel root bark. This is of great significance for clarifying the material basis of traditional Chinese medicine efficacy and improving the quality standards of traditional Chinese medicine, providing reference for further understanding its mechanism of action and clinical application.