Although the current literature has recorded many reports of identifying components from herbal preparations, all of them were largely limited to target components. This paper provides a novel and generally applicable approach to identifying nontarget components from herbal preparations, based on the use of liquid chromatography ion trap time-of-flight mass spectrometry (LC/MS-IT-TOF). A simple program was originally developed for searching the common diagnostic ions from all experimentally generated ions. The components sharing the exact same ions (mass error < 5 mDa) were classified into a family. All families were then connected into a coherent network by the bridging components that are present in two or more families. With the benefit from such a network, it is feasible to sequentially characterize the structures of all diagnostic ions once a single component has been de novo identified. The structures of the diagnostic ions could then be used as "a priori" information for selecting the exact candidates containing the substructures of the corresponding diagnostic ions from the primary database hits. This strategy enables a nearly 7-fold narrowing of the database hits and thus substantially enhances the analytical efficiency and sharpness. With the use of such an approach, 43 out of 53 components incorporated into the network have been successfully identified from the test herbal preparation. For the rest, components failed to be identified using this approach; a complementary approach to screening by sequential loss of specific chemical groups, proposed from the accurate mass differences between fragments, was established to narrow the database hits. All of the 87 peaks detected have been successfully identified by combining the use of both approaches except failed to differentiate some isomers. The presently developed approach and methodology would be useful for the identifications of complicated nontarget components from various complex mixtures such as herbal preparations, biological, and environmental samples.

BackgroundThere is an urgent need for effective treatments for hepatocellular carcinoma (HCC). Immunotherapy is promising especially when combined with traditional therapies. This study aimed to investigate the immunomodulatory function of an approved Chinese medicine formula, compound kushen injection (CKI), and its anti-HCC efficiency in combination with low-dose sorafenib.MethodsGrowth of two murine HCC cells was evaluated in an orthotopic model, a subcutaneous model, two postsurgical recurrence model, and a tumor rechallenge model with CKI and low-dose sorafenib combination treatment. In vivo macrophage or CD8+T cell depletion and in vitro primary cell coculture models were used to determine the regulation of CKI on macrophages and CD8+T cells.ResultsCKI significantly enhanced the anticancer activity of sorafenib at a subclinical dose with no obvious side effects. CKI and sorafenib combination treatment prevented the postsurgical recurrence and rechallenged tumor growth. Further, we showed that CKI activated proinflammatory responses and relieved immunosuppression of tumor-associated macrophages in the HCC microenvironment by triggering tumor necrosis factor receptor superfamily member 1 (TNFR1)-mediated NF-κB and p38 MAPK signaling cascades. CKI-primed macrophages significantly promoted the proliferation and the cytotoxic ability of CD8+T cells and decreased the exhaustion, which subsequently resulted in apoptosis of HCC cells.ConclusionsCKI acts on macrophages and CD8+T cells to reshape the immune microenvironment of HCC, which improves the therapeutic outcomes of low-dose sorafenib and avoids adverse chemotherapy effects. Our study shows that traditional Chinese medicines with immunomodulatory properties can potentiate chemotherapeutic drugs and provide a promising approach for HCC treatment.

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