Extraction of honokiol from Artemisia argyi and in vitro and in vivo investigation of its antifungal activity

Abstract Extracts from plants used in Chinese medicine can be good sources of fungicides for agricultural applications. In this study, we separated and identified antifungal compounds from four traditional Chinese medicine extracts and evaluated their antifungal activities in vitro and in vivo. In vitro, honokiol extracted from Artemisia argyi showed broad-spectrum antimicrobial and mycelial inhibitory activity with EC50 in the range 3.56 − 33.85 μg/mL against eight plant pathogens. q-PCR indicated that honokiol might induce cell cancerisation and inhibit cellular respiration, which provided significant insights into honokiol function in tobacco resistance to molecular mechanisms of the phytopathogenic fungus Phytophthora nicotianae. In vivo, honokiol significantly decreased the rate of fungal infection in eggplants, potatoes, grapes, cherry tomatoes, and cucumbers, and enhanced disease resistance in tobacco. Overall, our results indicate that honokiol has the potential to control a variety of fungal and oomycete diseases, and A. argyi could be a source of honokiol. Graphical Abstract


Introduction
The prevention and control of fungal diseases using chemical approaches constitute an important part of modern agricultural practice. However, chemical pesticides pollute the environment, endanger human health, and affect the balance of ecosystems because of long-term and large-scale use (Xie et al. 2015). It is therefore imperative to search for natural fungicides from biological sources to effectively control phytopathogenic fungi. Essential oils extracted from plants such as peppermint, eucalyptus, and cloves are reported to contain active components that exhibit potent antifungal activity against a broad spectrum of fungi (Jing et al. 2018;Xue et al. 2021). In our previous study, we reported the antifungal properties against Phytophthora nicotianae (P. nicotianae) of 40 Laoshan coastal plants (Gou et al. 2017). However, the major components with high antifungal activity are unclear. In this context, here we extracted honokiol from Artemisia argyi and investigated its antifungal activity in vitro and in vivo. The results from this study provide significant insights into the function of honokiol in tobacco resistance and exemplify its potential for application potential to control a variety of fungal and oomycete diseases in agricultural crops. TRCT-2020), and 53.26% (Cynanchum paniculatum (Bunge) Kitag. (C. paniculatum), No. TRCP-2020), respectively. There were 19 fractions showed in Figure S2a separated by silica gel column chromatography. Showed in Figure S2b, fractions from different plants exhibited significantly different inhibition activities against P. nicotianae. Among different fractions of the same plant, the third fraction from C. paniculatum, the first fraction from C. tora, the second fraction from S. flavescens, and the second fraction from A. argyi reached maximum mycelial growth inhibition of 57.55%, 70.40%, 84.01%, and 79.75%, respectively. The results indicated that the activity of the extract depended on the solvent polarity, and the ethyl acetate-soluble extract exhibited higher antifungal activities than the other soluble extracts. Similar to the results of this study, previous studies also reported that most antifungal compounds were separated from A. argyi (Zhang et al. 2021).

Characterisation of ethyl acetate fractions with the highest antifungal activity
The high-resolution mass spectrometry (UPLC-MS) chromatogram is shown in Figure  S3. In this study, 881 compounds were detected based on molecular mass, retention time, fragmentation pattern, and literature data, comprising 256 phenolic acids, 248 flavonoids, 115 alkaloids, 78 lignans and coumarins, 59 quinones, 42 terpenoids, 26 tannins, and 56 other compounds. From the pool of detected compounds, 14 compounds with high content and antifungal activity were selected (Table S2). Previous studies have suggested that flavonoids, phenolic acids, lignans, coumarins, alkaloids, and quinones have been verified for their antimicrobial activity against Fusarium oxysporum, Fusarium solani, Cylindrocarpon, and other fungal or bacterial diseases (Xie et al. 2017).

Antifungal activity of isolated components against P. nicotianae
As shown in Table S3, honokiol (EC 50 ¼ 26.95 mg/mL) exhibited the most potent antifungal activity compared with other compounds. The activity profile of other compounds against P. nicotianae showed EC 50 values ranging from 91.18 to 3803.15 mg/ mL. The results of this study suggested that antifungal properties against P. nicotianae of the four medicinal plants may be attributed to their antifungal components, and that A. argyi could be a source plant for honokiol ( Figure S4a).
2.5. Honokiol acts against P. nicotianae by regulating the expression of some genes As shown in Figure S6a, some important growth-related genes were detected by q-PCR, which might inhibit the growth of P. nicotianae. Genes 702 and 3594 might be important for subcellular lipid and cholesterol transport (Ioannou 2001). Genes 3907, 5535, and 3594 were downregulated, which might influence the Ca 2þ cycling system in mitochondria to inhibit cellular respiration (Emery et al. 2012). The downregulation of genes 2240, 796, 10228, 1198, and 1573 might cause cell cancer (Roskoski 2004;Qu et al. 2013). The actin-fragmin kinase encoded by gene 1756 is a highly conserved, pleiotropic, protein serine/threonine kinase that is essential for life in eukaryotes. Increase in honokiol concentration has been shown to potentially disrupt the balance of morphology and cell polarity, resulting in cell damage (Canton and Litchfield 2006). In this study, the upregulated and downregulated genes showed a dose-dependent efficiency and honokiol might induce cell cancerisation and inhibit cellular respiration.

In vivo antifungal efficiency of honokiol
In vivo results also showed that honokiol was effective in exerting antifungal effects in a variety of detached fruits. As shown in Figure S5a,b, the extent of lesion inhibition ranged from 24.32% to 50.45% for potato and 49.65% to 76.39% for eggplant. Honokiol showed variable lesion inhibition rates against B. cinerea, B. cinerea Pers., and C. anthracnose; however, a dose-dependent decrease in lesion size was observed with increasing honokiol concentration ( Figure S5c,d). Other studies have shown that extracts from purple sweet potato (Ipomoea batatas L.) leaves, Artemisia, and Eucalyptus could keep chili and potato fresh (Saputri and Utami 2020). These results signify those natural products, such as plant extracts and components like honokiol, might provide effective, sustainable, and environmentally friendly alternatives for the control of fungal pathogens. Thus, honokiol can be considered a promising agent to keep fruits and vegetables fresh.
With tobacco under field conditions, honokiol exhibited a disease suppressive effect 15 days after inoculation ( Figure S6b). Compared with the control, disease index and incidence rate in the group treated with different concentrations of honokiol (27 and 54 lg/mL) were 5.33-25.67 and 8.89%-24.44%, respectively. However, both samples showed higher the control efficiency than that of the positive control mancozeb. These results indicated the efficacy of honokiol application in controlling diseases such as tobacco black shank, a soil-borne disease caused by P. nicotianae, which can help improve tobacco production yield.
Administration of honokiol did not cause any significant differences in the tobacco chlorophyll content and total sugar ( Figure S7). And no significant differences were observed in roots vigour, indicating that honokiol did not influence plant roots. Superoxide dismutase (SOD) and peroxidase (POD) activities increased till reaching a maximum and then declined with further incubation, and malondialdehyde (MDA) content declined significantly after honokiol treatment, which indicated that honokiol could inhibit membrane lipid peroxidation. Thus, a low level of oxidative stress was maintained in honokiol treatments, suggesting that honokiol might prevent tobacco from membrane lipid peroxidation and enhance disease resistance.

Conclusion
In this study, we identified honokiol from A. argyi as the most effective antifungal compound against P. nicotianae using bioassay-guided isolation and UPLC-QTOF-MS/ MS analysis. q-PCR indicated that honokiol might induce cell cancerisation and inhibit cellular respiration in fungi. The in vivo test verified the antifungal activity of honokiol against P. nicotianae on potatoes and eggplants. In addition, honokiol significantly protected and prevented the efficiency of tobacco. Furthermore, honokiol also exhibited broad-spectrum inhibitory activity against eight plants pathogens. Additionally, the in vivo results indicated that honokiol has a significant protective effect on grapes, cherry tomatoes, and cucumber. Field tests suggested that honokiol enhanced disease resistance in tobacco. Our study demonstrated that honokiol has excellent potential for the development of natural fungicides.