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Rheumatoid arthritis (RA) is a worldwide intractable disease with complex etiology and diverse symptoms. At present, the pathogenesis of RA is still not fully understood, and no curative drugs have been developed for the cause. Although the anti-rheumatoid arthritis drugs commonly used in clinics can alleviate the acute inflammation and pain of patients, they cannot prevent the course of rheumatoid arthritis and have severe adverse reactions. Phthalides are a class of important active ingredients in Angelica. Modern research has found that they have a wide range of biological activities such as analgesia, anti-tumour, and neuroprotection. In recent years, active ingredients with anti-RA effects from natural medicines have become a research hotspot. Many pharmacological studies have shown that glycosides, alkaloids, flavonoids, and terpenoids have anti-inflammatory, analgesic, and immunomodulatory activities, which provide rich resources for discovering rheumatoid arthritis drugs. The previous results of the research group showed that the main compounds in the active fraction (fraction F) from Diol-SPE with the NF-κB pathway inhibition activity were phthalide compounds. Therefore, this project intends to isolate different phthalide compounds from Angelica Sinensis and test their activities through the Jur/NF-κB/GFP reporter cell line to verify whether phthalide compounds are the main active compounds. 

Except for the bioassay-guided isolation methods, reaction-guided purification is another valuable tool to find more electrophilic natural products from the crude extract by forming a covalent bond with the target proteins. In this study, two kinds of thiol probes are chosen and aiming to find more electrophilic small molecules with Michael acceptor (e.g., α, β-unsaturated γ-lactones, α, β-unsaturated δ-lactones, etc.) or ring-strained scaffolds from Saussurea lappa and other five plants, namely Paeonia lactiflora, Piper kadsura, Notopterygium incisum, Rehmannia glutinosa, and Cinnamomum cassia Presl.

Method

DCM/MeOH (1:1) was selected as the solvent to extract Phthalides from Angelica Sinensis, and the crude extract was further isolated and purified by semi-preparative HPLC or preparative-HPLC. The structure elucidation was performed mainly by1H-NMR, HSQC, HMBC, and NOESY. 

Reaction-guided isolation was established in an inert atmosphere using a nitrogen gas source with DMF, triethylamine, and tris-HCl mixed with the extract. LC-UV test was performed to monitor the whole reaction process, which was subsequently confirmed by LC-MS analysis.

Result 

Six phthalide compounds and one phenolic acid compound were isolated from the extract of Angelica Sinensis, which were (E)-Ligustilide, (3Z)-3-Butylidene-6,7-dihydroxy-1(3H)- isobenzofuranone, (R)-Chuangxiol, Senkyunolide C, Senkyunolide H, Senkyunolide I, and Ferulic acid. Among them, Senkyunolide C showed an inhibitory effect on NF-κB, while other compounds did not show good activity. It is worth doing more research on the mechanisms of Senkyunolide C related to the NF-κB pathway. Inactive compounds may act on target(s) other than the NF-KB pathway.

A proof-of-concept study of Saussurea lappa indicates that dehydrocostus lactone and costunolide have the structure of α, β-unsaturated γ-lactones, thus allowing them to react with the probes in the crude extract. None of the other five plants showed typical signs that the probes reacted with any of the compounds in the extract.  It might be due to the reason that no target compounds exist in these plants, or the target compound is too low in content in these plants.