Reverse tracking and isolation of anti tuberculosis components from Guijianyu and cinnamon
Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Tuberculosis caused by drug sensitive strains can be treated with first-line drugs such as rifampicin, isoniazid, ethambutol and pyrazinamide, but the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) has brought difficulties to the treatment of tuberculosis. Due to the limited protective effect of BCG vaccination, the development of anti tuberculosis drugs with new mechanisms is the main means to control the prevalence of tuberculosis.

Natural products are one of the most important sources of new candidate drug molecules. In the past century, a large number of drug candidate molecules have been discovered from natural products. Plant chemistry researchers have conducted extensive research on the chemical composition of medicinal plants and established multiple natural product or plant chemical composition databases. For example, the Chinese Natural Product Database (CNPD) comprehensively collects information on the chemical composition of Chinese herbal medicine, including more than 10000 chemical components and involving over 4500 types of Chinese herbal medicine.

In recent years, we have conducted blind screening of over 200 Chinese herbal extracts against Mycobacterium tuberculosis and found that the minimum inhibitory concentration (MIC) values of over 80 Chinese herbal extracts against Mycobacterium tuberculosis in vitro are less than 2mg/mL. The composition of Chinese herbal medicine is complex, and elucidating its active ingredients often requires repeated ingredient separation and activity verification work. In order to quickly discover anti tuberculosis active molecules from these natural medicines, we proposed the idea of “reverse tracking separation” based on the natural product ingredient database. In this experiment, Guijianyu and cinnamon were selected as research objects, and the reverse tracking separation method was used to carry out the research work: (1) using databases such as the China Natural Product Chemical Composition Database to search for the chemical components of Guijianyu and cinnamon; (2) Through literature review, it was found that the malic acid in Guijianyu and cinnamaldehyde in cinnamon have anti tuberculosis activity. Therefore, using malic acid and cinnamaldehyde as directing molecules, Guijianyu and cinnamon extracts were gradually divided into parts containing directing molecules and parts or components without directing molecules; (3) In vitro anti tuberculosis testing revealed significant differences in the activity of different parts or components of the extract from Guijianyu. The components with higher content of malic acid had stronger activity, while those with lower content had weaker activity. Therefore, it can be concluded that malic acid is the active molecule of Guijianyu against tuberculosis. Prove using the same method that cinnamaldehyde is an active molecule of cinnamon against Mycobacterium tuberculosis; (4) Using the principle of combination, structural modifications were carried out on malic acid and cinnamaldehyde to obtain more active derivatives.

This article demonstrates the advantages of reverse tracking separation method in confirming the active ingredients of Chinese herbal medicine, and provides scientific basis for the clinical application of these two herbs, providing new ideas for discovering anti tuberculosis bacteria molecules from Chinese herbal medicine.

According to literature reports, the malic acid in Guijianyu and cinnamaldehyde in cinnamon have anti tuberculosis activity. So are they the strongest anti tuberculosis activity molecules in these two medicinal herbs, respectively? This is a matter of great concern to us. In this experiment, we proposed a reverse tracking separation approach, using malic acid as a guiding molecule to track and isolate anti tuberculosis components from Guijianyu, and cinnamaldehyde as a guiding molecule to track and isolate anti tuberculosis components from cinnamon. The results showed that there was no stronger component than malic acid in the anti tuberculosis components of Guijianyu, and cinnamaldehyde was the most active molecule against tuberculosis in cinnamon. It can be seen that the reverse tracking separation method has the advantages of saving time, manpower, and funds in exploring active ingredients in Chinese herbal medicine, especially antibacterial active ingredients. Of course, this method relies on a powerful natural product ingredient database as the foundation.

The derivatives obtained by structural modification of compounds such as malic acid and cinnamaldehyde have stronger anti tuberculosis activity. Compound 1 has a MIC value of 1 μ g/mL, which is safe and non-toxic to mammalian cells. It can be used as a lead compound for further structural optimization of anti tuberculosis bacteria.

In order to explore the possible mechanism of action of compound 1, a blind screening method was used to dock compound 1 with 20 proteins of Mycobacterium tuberculosis, indicating that compound 1 has a strong affinity for the β – ketoacyl ACP synthase III (FabH) of Mycobacterium tuberculosis. Therefore, the effect of compound 1 on the expression of FabH gene in Mycobacterium tuberculosis was determined. At sub inhibitory and inhibitory concentrations, the structural gene of β – ketoacyl ACP synthase III in Mycobacterium tuberculosis could be blocked by compound 1, leading to downregulation of expression. β – ketoacyl ACP synthase III is a key enzyme in the synthesis of long-chain fatty acids in the cell wall of Mycobacterium tuberculosis. Once inhibited, it will cause changes in the morphology of Mycobacterium tuberculosis, further leading to damage to the cell wall structure and causing bacterial swelling and disintegration. Through microscopic images, it can be seen that after intervention with compound 1, the morphology of tuberculosis bacteria undergoes significant changes, indicating that compound 1 causes damage to the cell wall of tuberculosis bacteria. The impact of cell wall destruction on the vitality of Mycobacterium tuberculosis is lethal. By measuring the bactericidal curve, we found that compound 1 (4 μ g/mL) killed all Mycobacterium tuberculosis in the culture system within 72 hours. Therefore, we speculate that the anti tuberculosis mechanism of compound 1 is related to the intervention of β – ketoacyl ACP synthase III. As this experiment did not provide conclusive evidence of compound 1 binding to the target, the mechanism of action of compound 1 still needs further investigation.

The ancient book of the Eastern Han Dynasty, “The Emergency Prescription for Back up the Elbow”, first proposed the use of ghost arrow feather to cure ghost injection and corpse injection. In ancient times, tuberculosis was called tuberculosis, ghost injection, corpse injection, etc. Cinnamomum cassia has also been recorded for the treatment of tuberculosis. This fact verifies that ghost arrow feather and cinnamon have anti tuberculous activity, providing valuable clues for the treatment of tuberculosis with these two traditional Chinese medicines.