The main chemical compounds and protein targets of the LCTE soup formula
It is well held that Chinese herbal plants contain bioactive compounds and that the therapeutic effects of herbal treatments are achieved via compound/target interaction. We started our work by researching LCTE-contained chemical compounds and their protein targets. The LCTE formula consists of 20 herbal ingredients and one mineral material (raw gypsum, Rudis Gypsi Miscueris (Table 1). The main component of raw gypsum is inorganic CaSO42H2O, and the inclusion of gypsum in the formula is explainable by findings that it reduces body temperature15 as well as attenuates heat-induced hypothalamic inflammation via down-regulating IL–1β16. As such, we focused our study on the 20 herbal plants used to formulate LCTE. We found a complete chemical compound list for each plant as recorded in three Chinese herbal databases6–8 (detailed in the Methods section). The listed compounds were then filtered using ADME (absorption, distribution, metabolism, and excretion) indices to find and keep which might be absorbable via oral administration17. ADME filtration for oral bioavailability is necessary in that Chinese herbal plants are boiled with water and the obtained soup then orally administered. The passing rates ranged from 0.026 to 0.37 with an average of 0.11 (Table 1), indicating that the majority of the compounds present in the related plants would not be absorbed. After finding which compounds were most likely to be absorbed, we again checked the three Chinese herbal databases to find which proteins would be affected by each of these compounds. Each herbal ingredient, their total number of compounds, number of orally absorbable compounds, and their number of protein targets are listed in Table 1. More detailed information on plants and their compounds that passing filtration, and the proteins targeted by each compound screened out are available in the supplementary Table S1 and S2.
The potentials to directly inhibit SARS-CoV–2 and the anti-inflammatory properties of the main LCTE-contained compounds
In all, the 20 herbal ingredients in the LCTE formula contain 207 different chemical components that may be absorbed through oral administration. 27 of these components were present in at least 2 of the plant sources. 5 components were of high concurrence, existing in 6 or more plant sources (supplementary Table S3). Stigmasterol was present in 8 plants, quercetin was present in 7 plants, luteolin and beta-sitosterol and kaempferol were present in 6 plants (Fig. 1). Notably, we recently found that two of the five most prevalent compounds, kaempferol and quercetin, have the potential to directly inhibit papain-like protease (PLpro) and 3C-like protease (3CLpro), two enzymes which are critical to the replication of COVID–19-causing pathogen SARS-CoV–218.
COVID–19’s basic pathology is viral-caused inflammation. Based on publications in PubMed, we found that 19 of the 27 compounds present in 2 or more of the LCTE herbal plants possess anti-inflammatory properties (supplementary Table S3). The indication is that the main compounds of LCTE have the potential to directly inhibit SARS-CoV–2 and down-regulate inflammation (Fig. 1).
Enrichment of proteins targeted by LCTE in protein sets related to common COVID–19 symptoms
Fifteen main symptoms have been reported in COVID–19 patients19,20. These include fever, cough, myalgia, fatigue, and dyspnea and etc., which could be catalogued as general, respiratory and digestive symptoms. The LCTE formula’s clinical effectiveness in reliving these symptoms has been confirmed in China. To understand the molecular basis for LCTE’s relief of COVID–19 symptoms, we used DisGeNET21 to download the proteins related to each symptom and then studied the correspondence between these and the protein targets of LCTE’s orally-absorbable compounds. The exact proteins related to each symptom and the results for the DisGeNet search ‘viral respiratory infection’ are available in supplementary Table S4.
Hypergeometric distribution probability computation showed that the component-targeted proteins were significantly enriched in 11 of the symptoms-related proteins (P < 0.05) (Fig.2 and supplementary Table S5), including 5 of the highest occurrence symptoms, fever, coughing, myalgia, fatigue, and dyspnea. The protein sets for anoxia, diarrhea, nausea, headache, vomiting, and abdominal pain also significantly correlated to proteins targeted by LCTE. Computational enrichment for dry cough, pharyngalgia, dizziness, and sputum proteins did not yield significant results, which might be due to the low number of proteins recorded for these symptoms in the DisGeNET database. The proteins related to the search query ‘viral respiratory infection’ in DisGeNET also correlated significantly with LCTE-targeted proteins. Since COVID–19 is a viral respiratory disease, such results further support the effectiveness of LCTE in treating COVID–19.
Key compounds and proteins for LCTE’s symptoms relief
The field of network pharmacology suggests that the effects of a drug containing multiple components can be predicted by a network constructed of the linkages between the multiple components and their targets9. Inspired by this methodology, we built a network using the following linkages: herbal plants and their corresponding chemical compounds, chemical compounds and their affected proteins, and proteins related to each symptom. By calculating the degree for each vertex, we were able to derive which plants, compounds, and proteins are key to LCTE’s symptom alleviation. For example, in LCTE there are two herbal ingredients, Gan Cao and Huang Qin, and 6 compounds (quercetin, luetolin, stigmasterol, kaempferol, beta-sitosterol, and wogonin) useful in treating fever, and the key proteins affected by these components are PTGS2 (prostaglandin-endoperoxide synthase 2) and PRSS1 (Serine Protease
1) (Fig. 3). While for treating cough, three LCTE’s plants (Gan Cao, Huang Qin, and Zhi Shi) and five compounds (quercetin, luetolin, stigmasterol, kaempferol, and beta-sitostero) were involved, targeting the proteins TOP2B (DNA topoisomerase II beta) and ADRB2 (adrenoceptor beta 2) (supplementary Fig S1). The network data for the key herbal plants, their compounds, the proteins involved in LCTE’s effects on other COVID–19 symptoms, and information related to LCTE treatment of ‘viral respiratory infection’ are available in supplementary Fig S1 to Fig S15 and Table S6.
The general effects of LCTE soup
To predict the general in vivo effects of LCTE soup, we mapped all the proteins targeted by the orally-absorbable compounds to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Disease Ontology (DO) database and to find which pathways or diseases were enriched. The top 30 enriched KEGG pathways were mainly related to viral and other microbial infections, and inflammation/cytokine responses (Fig. 4). The full list of enriched KEGG pathways and the genes involved are given in supplementary Table S7. The top 2 diseases enriched in DO were ‘chronic obstructive pulmonary disease’ and ‘lung disease’ (the full list of enrichment is in supplementary Table S8). Taken as a whole, our findings suggest that that this formula’s general effects are well suited to treating viral infection in the respiratory system.