Anti-arboviral activity and chemical characterization of hispidulin and ethanolic extracts from Millingtonia hortensis L.f. and Oroxylum indicum (L.) Kurz (Bignoniaceae)

Abstract Millingtonia hortensis L.f. and Oroxylum indicum (L.) Kurz (Bignoniaceae) are native species from the Asian continent. They are popularly used in traditional medicine and their extracts are rich in flavonoids. In this work, ethanolic extracts of stems and leaves of these species were evaluated against the Chikungunya, Zika and Mayaro virus. The extracts were subjected to analysis by ultra-efficient liquid chromatography coupled to mass spectrometry. Additionally, M. hortensis leaves extract was fractionated, leading to the isolation of hispidulin. Anti-arboviral activity against the three viruses was detected for M. hortensis leaves extract with EC50 ranging from 37.8 to 134.1 µg/mL and for O. indicum stems extract with EC50 ranging from 18.6 to 55.9 µg/mL. Hispidulin inhibited viral cytopathic effect of MAYV (EC50 value 32.2 µM) and CHIKV (EC50 value 78.8 µM). In LC-DAD-ESI-MS/MS analysis we characterized 25 flavonoids confirming once again the presence of these substances in extracts of these species. Graphical Abstract


Introduction
Millingtonia hortensis L.f. and Oroxylum indicum (L.) Kurz belong to the Oroxyleae tribe from the Bignoniaceae family and are not only known as ornamental plants, but also as plants used in traditional medicine (Anulakanapakorn et al. 1987;Dinda et al. 2015). M. hortensis is used in the treatment of asthma, tuberculosis, sinusitis and as a tonic and cholagogue (Anulakanapakorn et al. 1987). O. indicum is used for prevention and treatment of several diseases, such as gastric ulcers, tumors, jaundice, diarrhea and dysentery, diabetes, arthritic and rhematic problems, among others (Dinda et al. 2015). These Oroxyleae tribe species are rich in flavonoids, mainly flavones, including hispidulin (Chulasiri et al. 1992;Dinda et al. 2015).
A number of natural and synthetic flavonoids have been tested for antiviral activity (Zakaryan et al. 2017), mainly against arboviruses of public health importance, such as Zika virus (ZIKV), dengue virus (DENV), Chikungunya virus (CHIKV) and Mayaro virus (MAYV), due to their rapid expansion and their potential for emergence and re-emergence, but hispidulin has not. In this investigation, the in vitro antiviral activity of M. hortensis and O. indicum leaves and stems ethanolic extracts and the isolated flavone hispidulin were evaluated against the arboviruses ZIKV, CHIKV and MAYV. In addition, the chemical composition of the crude extracts was analyzed by ultra-efficient liquid chromatography coupled to diode array and mass spectrometry detectors (UHPLC-DAD-ESI-MS).

Results and discussion
The rapid expansion of arboviruses, such as ZIKV, CHIKV and MAYV, and their emerging and re-emerging potential, characterizes them as an important public health problem (Mayer et al. 2017;Weaver et al. 2018). In addition, so far, there is no specific treatment or vaccine for Zika, Chikungunya and Mayaro fever. These facts make the search for antiviral drugs urgent, being of great importance to carry out research for the discovery of specific chemotherapeutics for the treatment of these arboviruses.
Ethanolic extracts from Oroxyleae tribe species, M. hortensis and O. indicum, were screened for in vitro antiviral activity against ZIKV, CHIKV and MAYV, results are shown in Table S6 in the Supplementary Material.
M. hortensis stems extract was active against the three arboviruses exhibiting moderate antiviral activity with EC 50 values ranging from 176.30 to 99.86 mg/mL. The leaves extract showed low activity against ZIKV and CHIKV (EC 50 values 134.10 and 97.60 mg/ mL, respectively) but high against MAYV (EC 50 value 37.79 mg/mL).
O. indicum extracts inhibited the multiplication cycle of the tested viruses, with a high activity for stems extract against ZIKV and MAYV (EC 50 values 20.57 and 18.59 mg/ mL, respectively), and for leaves extract against ZIKV and CHIKV (EC 50 values 40.35 and 48.33 mg/mL, respectively). In addition, the stems extract showed moderate activity against CHIKV (EC 50 ¼ 55.85 mg/mL), as well as the leaves extract against MAYV (EC 50 ¼ 67.65 mg/mL).
This paper describes for the first time the investigation of the anti-arboviral activity against ZIKV, CHIKV and MAYV of these Oroxyleae tribe species. M. hortensis and O. indicum are traditional folk medicinal plants used in the Asian continent due to their diverse biological properties (Anulakanapakorn et al. 1987;Dinda et al. 2015). Although there are no reports on the antiviral activity of the species in the scientific literature. All extracts evaluated were active against arboviruses, of them O. indicum crude extracts were selective and promising against the three viruses, however when fractionated there was no maintenance or increase in biological activity (data not shown). On the other hand, from M. hortensis, only leaves extract showed high activity for MAYV.
Bio-guided fractionation of M. hortensis leaves extract (65.0 g) was performed by liquid-liquid partition and led to three fractions: CH 2 Cl 2 fraction (6.8 g), EtOAc fraction (5.5 g) and H 2 O fraction (26.9 g). H 2 O fraction was not active against the arboviruses. EtOAc fraction showed only low activity against CHIKV (EC 50 ¼ 183.9 mg/mL and SI ¼ 1.2) and high activity anti-MAYV (EC 50 ¼ 47.9 mg/mL and SI ¼ 4.6), while CH 2 Cl 2 fraction was active against the three arboviruses, showing high activity with EC 50 values ranging from 39.6 to 14.8 mg/mL.
During the liquid-liquid partition there was the formation of a precipitate and it was isolated by vacuum filtration as a yellow solid, called MH01 (934 mg). Furthermore, the active CH 2 Cl 2 fraction (6.7 g) was chosen to be fractionated by silica column chromatography allowing isolation of more MH01 (92 mg). This substance MH01 was identified by 13 C and 1 H NMR as hispidulin (6-methyl-scutellarein or 4 0 ,5,7trihidroxy-6-methoxyflavone) (Supplementary Material, Figures S1-S7) and was confirmed using previously reported NMR data. It was previously isolated from flowers and leaves of M. hortensis (Chulasiri et al. 1992;Hase et al. 1995). Structure of hispidulin obtained from dichloromethane fraction is shown in Figure 1. This specie is a good source of obtaining hispidulin, about 2.2 g of hispidulin/Kg of plant.
This in vitro anti-MAYV and anti-CHIKV activities of hispidulin observed is reported for the first time. Furthermore, from these results, it is observed that hispidulin is 2.5 times more active against MAYV when compared to activity against CHIKV.
In order to confirm the antiviral activity observed in the MTT assay, extracts and isolated hispidulin compound were subjected to an in vitro cytopathic effect inhibition assay. It was observed that hispidulin inhibited MAYV cytopathic effect. It has shown antiviral activity with monolayer cell protection greater than 80% for all tested substances when compared to viral control (Supplemental material, Figures S8-S14).
According to this study, the flavone hispidulin antiviral activity shows that it is a potential substance with in vitro anti-CHIKV and anti-MAYV activity and it presents better activity than the origin crude extract, CH 2 Cl 2 fraction and positive controls. It was observed that treatment with hispidulin protected the cell monolayer whereas cells without inhibitor were thoroughly destroyed during MAYV multiplication cycle.
In the literature, regarding the antiviral activity investigation of hispidulin, only the in vitro evaluation of anti-HIV (Osei-Safo et al. 2009) and anti-H1N1 (Bang et al. 2016) activities are reported. Therefore, the unprecedented anti-MAYV and anti-CHIKV activity observed in this study for hispidulin demonstrates that this flavone is a promising substance with antiviral potential against MAYV and CHIKV, and further studies should be carried out in the investigation of this biological activity.
The screening, identification, and further confirmation of several components in the M. hortensis leaves extract was performed by UHPLC-DAD-ESI-MS/MS. The M. hortensis stems extract and O. indicum leaves and stems extracts were analyzed by UHPLC-DAD-ESI-MS and the data are shown in the Supplemental material (Table S7 and Figures S16-S37. The UV spectrum of these compounds are characteristic of flavonoids or phenylethanoids. The UV spectral data and the mass/charge relation (m/z) were compared with the published literature data, allowing the characterization of thirteen flavonoids and two phenylethanoids glycosides in M. hortensis leaves (Table S6 and Figure S15), of which six flavonoids (apigenin, luteolin, nepetin, nepetrin, apigenin-7-O-glucoside and nepetin-7-O-glucuronide) and the phenylethanoid glycoside isoverbascoside were reported for the first time in M. hortensis.
It has been reported in the literature that polyphenols, such as flavonoids, are one of the main secondary metabolites classes that have antiviral activity against several viruses (Kamboj et al. 2012;Zakaryan et al. 2017). Furthermore, the antiviral activity may be associated with the synergistic effect between flavonoids and other natural constituents (Mor an-Santibañez et al. 2018).

Experimental
See supplementary material.

Conclusion
Millingtonia hortensis L.f. and Oroxylum indicum (L.) Kurz (Bignoniaceae) are potential plants with anti-arboviral activity and source of phenolic compounds, such as flavonoids. Furthermore, M. hortensis leaves can be a source of obtaining hispidulin, a flavone that inhibits the cytopathic effect of MAYV and CHIKV viruses in cell cultures.