Chemical profile, anti-hepatoma activity, anti-acetylcholinesterase and antioxidant activity of aerial part of Aconitum carmichaeli Debx

Abstract Five extracts of the aerial parts of Aconitum carmichaeli were obtained by different solvent extraction or macroporous adsorption resin purification: ethyl acetate layer extract (EAE), n-butanol layer extract (BuE), water layer extract (WE), extract eluted by 10% ethanol from macroporous resin (10%EE), extract eluted by 80% ethanol from macroporous resin (80%EE). Antioxidant activities of the five extracts were determined by ABTS, DPPH, FRAP assays, anti-AChE activities by modified Ellman’s method, in vitro anti-hepatoma activities by CCK-8 assay, and chemical constituents of 80%EE were identified by UPLC-QE-Orbitrap-MS. The results demonstrated that the 80%EE showed the best in vitro anti-hepatoma activity on Huh-7 cell line with an IC 50 of 103.91 ± 11.02 μg/mL. 10%EE and 80%EE gave the highest antioxidant activity. Furthermore, current findings demonstrated that the aerial part of Aconitum carmichaeli Debx. has high medicinal value and may be a good natural medicine. Graphical abstract


Introduction
Aconitum carmichaeli Debx. is a plant belonging to the genus Aconitum in the Ranunculaceae family.Modern pharmacological studies have shown that the lateral roots of Aconitum plants possesses cardiotonic, anti-shock, anti-arrhythmic and sedative medicinal effects (Fei et al. 2021).As the lateral roots of Aconitum carmichaeli are the main medicinal sites, the aerial parts are often abandoned, resulting in a serious waste of medicinal plant resources.In recent years, the aerial parts of Aconitum carmichaeli Debx.were found to have similarities in chemical composition with the lateral roots.Alkaloids were the main medicinal components of the lateral roots of Aconitum carmichaeli and they were also found in the aerial parts of Aconitum carmichaeli, such as mesaconitine, talatisamine, isotalatizdine (Konno et al. 1982;Liu et al. 2012;Xiong et al. 2019).However, there are few studies focus on the medicinal activity of the aerial parts of Aconitum carmichaeli Debx.Therefore, in order to improve the utilization rate of Aconitum, it is important to explore the medicinal value of the aerial parts of Aconitum carmichaeli Debx.
The present objective of our research was to investigate the chemical profile, antihepatoma activity, anti-acetylcholinesterase and antioxidant activity of aerial part of Aconitum carmichaeli Debx. to provide reliable theoretical basis for its medicinal use.

Antioxidant activity evaluation
In this study, the antioxidant activities of the ethanol extract purified by macroporous resin eluted with 10% ethanol (10%EE) and 80% ethanol (80%EE), extracts obtained by extracting ethanol extract with ethyl acetate(EAE), n-butanol (BuE) and water (WE) were measured by ABTS, DPPH and FRAP methods.Ascorbic acid(VC) was used as a positive contrast (Krakowska et al. 2017;La et al. 2021).Table 1 lists the results of ABTS and DPPH methods.The ABTS radical scavenging ability of the five extracts were dose-dependent.Among the extracts, WE exhibited a weak antioxidant activity while 10%EE and 80%EE exhibited significant antioxidant activities.EAE exhibited greater scavenging ability for ABTS than that of BuE, but all were weaker than VC (p < 0.05).The result of DPPH method was consistent with ABTS method.Fe 3þ -TPTZ could be reduced to Fe 2þ -TPTZ by reducing reaction, and the maximum absorption wavelength was 593 nm (Yu et al. 2012;Kolar et al. 2018).The result of FRAP assay was exhibited in Figure S1 (Supplementary material), it was observed that the inhibition percentage of the five extracts were dose-dependent.WE exhibited the weakest antioxidant activity while 10%EE exhibited the highest antioxidant activity.It was demonstrated that the scavenging abilities for FRAP of 10%EE and 80%EE were higher than EAE, BuE, WE.
The results of antioxidant activity experiment showed that the extracts of the aerial parts of Aconitum carmichaeli Debx.had significant antioxidant activity, and the extract purified by macroporous resin had stronger antioxidant activity.

Anti-acetylcholinesterase activity evaluation
Ellman's method (Ellman et al. 1961) was used to determined the anti-AChE activity of the five extracts.The in vitro anti-AChE activities of the five extracts were presented in Table 1.Results indicated that the anti-AChE activities of extracts were dose-dependent.Among the extracts, 10%EE and 80%EE showed significant anti-AChE activities, WE showed weak anti-AChE activity, EAE and BuE had similar anti-AChE activities.Huperinze A was used as a positive control in this study.Studies have shown that Huperinze A has strong anti-AChE activity (Gul et al. 2019).As the extracts were obtained from natural plants, the extracts of the aerial parts of Aconitum carmichaeli Debx.had lower anti-AChE activities compared with the positive control.It is concluded that 10%EE and 80%EE possessed good inhibitory activity, whereas the WE showed moderate and poor inhibitory activities, respectively at the concentration tested.Ramli et al. (2014) had proved that some alkaloids have strong acetylcholinesterase inhibition, according to the results of UPLC-QE-Orbitrap-MS, 80%EE contains a greater variety of alkaloids, which may be related to its significant anti-AChE activity.

In vitro anti-hepatoma activity evaluation by CCK-8 method
CCK-8 method was used to determine the cell survival rate after the intervention of extracts.The anti-hepatoma activities of the five extracts were presented in Table 1.It was demonstrated that the extracts of aerial parts of Aconitum carmichaeli Debx.could inhibit the growth of Huh-7 cells, and the inhibitory effects were dose-dependent.Among the five extracts, 80%EE showed the strongest anti-hepatoma activity with a IC 50 of 441.17 (±12.33)lg/mL, WE showed the weakest anti-hepatoma activity.Alkaloids, as a class of natural ingredients derived from traditional Chinese medicines, have previously been shown to exert prominent anti-hepatocarcinogenic effects, through various mechanisms including inhibition of proliferation, metastasis and angiogenesis, and so on (Liu et al. 2019).80%EE had lower anti-hepatoma activity compared with the positive control 5-Fluorouracil, but the anti-hepatoma activity of 80%EE was still significant, which may due to the rich variety of alkaloids.

UPLC-QE-Orbitrap-MS analysis
UPLC-QE-Orbitrap-MS was used to determine the chemical profile of 80%EE.The chemical profile identification of 80%EE by UPLC-QE-Orbitrap-MS in positive ion mode was shown in Figure S2 and Table S1 (Supplementary material), in negative ion mode was shown in Figure S3 and Table S2 (Supplementary material).The determination of the each peak was based on the structure and the reported fragment information of the compounds.By comparing positive and negative ion modes, more fragmentation information for the alkaloids can be appeared in positive ion mode.The result demonstrated that a total of 41 compounds were identified in 80%EE in positive ion mode and a total of 18 compounds were identified in 80%EE in negative ion mode, which contained a large number of alkaloids common to the lateral root part of Aconitum carmichaeli Debx., such as isotalatizidine, karakanine, songoramine, 14-acetyltalatisamine, beiwutine, karakoline, carmichaenine D and hypaconitine.Using negative ion mode, some non-alkaloid components were detected, such as cis-aconitic acid, 6-gingerol, linoleic acid and palmitic acid.
The traditional Chinese medicine Fuzi, has the medicinal value of analgesia, antiinflammatory and anti-tumor.Alkaloids are the main medicinal components of Fuzi, which are crucial to the efficacy of Fuzi.The current results of this paper show that the aerial parts of Aconitum carmichaeli Debx.and Fuzi contain many similar ingredients, so we speculate that the aerial parts of Aconitum carmichaeli Debx.may have medicinal value similar to the Fuzi.

Conclusion
In this study, five extracts of the aerial part of Aconitum carmichaeli were obtained by different solvent extraction, macroporous adsorption resin purification.Antioxidant activities, anti-AChE activities, in vitro anti-hepatoma activities of the five extracts were determined, chemical constituents of the 80%EE were identified by UPLC-QE-Orbitrap-MS.The results showed that 10%EE and 80%EE presented strong antioxidant activity in DPPH, ABTS and FRAP test.80%EE presented strong anti-AChE activity in Ellman's method.The result of UPLC-QE-Orbitrap-MS indicated that 80%EE contained rich variety of alkaloids.This study can provide reference for finding new antioxidant, anti-AD and anti-hepatoma drugs, and is of great significance to promote the further comprehensive development and utilization of Aconitum carmichaeli resources.
characteristic industry chain project of Shaanxi Provincial Department of science and technology [grant number 2019TSLSF02-02].

Table 1 .
Antioxidant, anti-acetylcholinesterase and anti-AChE activity of the five extracts.mean values with different letters are significantly different (p<0.05) according one-way Anova in the same test.