Chemical composition and evaluation of prolyl oligopeptidase and acetylcholinesterase inhibitory activities of Leonurus Sibiricus L. from Brazil

Abstract Chemical investigation of the aerial parts of Leonurus sibiricus L. used in Brazilian folk medicine led to the identification of the following constituents: the labdane-type diterpenoid leojaponin, the phytosterols β-sitosterol and β-sitosterol glucoside and the alkaloid leonurine. The crude extracts obtained from methanol and methanol/1% HCl and pure compounds isolated from L. sibirius were investigated as acetylcholinesterase (AChE) and prolyl oligopeptidase (POP) inhibitors. Extracts obtained by maceration were active against POP (53–58%), but showed weak activity against AChE. The isolated leojaponin and leonurine were evaluated as POP inhibitors.


Introduction
Leonurus species (Lamiaceae) are used in a diversified manner in folk medicine to treat various diseases in the Chinese culture (Lorenzi & Matos 2002;Shang et al. 2014). Leonurus sibiricus L., a medicinal plant native of southern Siberia, China, Korea, Japan and Vietnam (Wu et al. 2011) and popularly known in Brazil as 'rubim' or 'erva-de-macaé' , is used in traditional medicine to treat digestive disorders, viral infections, hypertension, headache, diabetes and respiratory diseases (Lorenzi & Matos 2002). Major classes of phytochemicals identified in L. sibiricus are alkaloids, mono, sesqui and diterpenes, furan-lactones and flavonoids (Pan et al. 2006;Lin et al. 2007;Wu et al. 2011). Despite its widespread medicinal use, Ahmed et al. (2005) observed that the crude methanol extract from aerial parts of L. sibiricus from African origin has central nervous system (CNS) depressant action. Its effects are described in the literature as moderately narcotic and psychoactive 'cannabis-like' (Wu et al. 2011). Considering the spectrum of pharmacological effects and extensive popular use of L. sibiricus, more studies are needed to better understand the CNS effects of L. sibiricus naturalized in Brazil. Previous studies have shown that the maintaining of the correct levels of enzymes such as AChE and POP is directly related to different diseases that affect the CNS, as Alzheimer's disease (AD), bipolar disorder, depression and schizophrenia (Brandt et al. 2007;Orhan 2012). Thus, this study aimed to perform the isolation of L. sibiricus secondary metabolites as well as to evaluate the inhibitory capacity against prolyl oligopeptidase (POP) and acetylcholinesterase (AChE) of its crude extracts and isolated compounds.

Results and discussion
In this work, maceration method was used to obtain the crude extracts methanol (CE-MeOH) and methanol/HCl 1% (CE-MeOH + ). Chemical studies of these extracts resulted in the isolation of leojaponin (1), β-sitosterol (2), β-sitosterol-glucoside (3) and leonurine (4). Compound 1 has been previously identified by Romero-González et al. (2006) and Wu et al. (2015) in Leonurus japonicus extracts, while the phytosterols 2 and 3 were isolated from L. heterophyllus by Shang et al. (2014) and Liu et al. (2014), respectively. This is the first report on the isolation of these metabolites from Leonurus sibiricus. Leonurine 4 was isolated from some species of the genus Leonurus, including L. sibiricus, L. heterophyllus and L. japonicus. The structures of the isolated compounds 1-3 were elucidated by GC-MS/ HRMS-ESI + and 1 H and 13 C NMR compared with data reported in the literature (Romero-González et al. 2006;Khatun et al. 2012;Chaturvedula and Prakash 2012). Compound 4 was identified by HPLC in comparison with authentic standard and by mass spectroscopy (HRMS-ESI + ).
GC/MS chromatogram of compound 1 presented one peak at t R = 14.660 min, with a mass spectrum showing a molecular ion with m/z value of 314 [M + H] + . Its 1 H NMR spectrum exhibited the characteristic signals of a labdane-type diterpene as well as signals at δ H 7.38, 7.29 and 6.32 ppm, assigned to the furan ring protons attached to the carbons C-15, C-16 and C-14, respectively (Hammami et al. 2016), signals of four methyl groups hydrogens between δ H 1.99-1.38 ppm and a signal at δ H 6.98 ppm suggesting a hydroxyl hydrogen attached to the C-6 carbon. Through the analysis of the 13 C NMR spectrum of 1 in comparison to the literature data, it was possible to relate all spectrum signals with the carbons of structure 1. The signal observed at δ C 181.7 ppm suggests the presence of a ketone group from diterpene of leojaponin, assigned to C-7. The signals related to sp 2 carbons of the cyclohexene ring and of furan ring were identified in the region between δ C 165.8 and 110.5 ppm. TLC analysis of CE-MeOH + using the Dragendorff reagent showed the presence of one alkaloid. The alkaloid leonurine (4) was isolated in small quantity from this extract as a white amorphous solid, identified by HRMS and comparative analysis by HPLC using a commercially purchased standard (Supplementary material).
Alzheimer's disease (AD) has one of highest incidence currently (Prince et al. 2015). Inhibition of AChE proved to be the most successful means to balance the cholinergic system for the stabilization of (AD) symptoms (Hyde et al. 2013). Several studies have also indicated the involvement of the POP in AD, especially regarding memory loss observed in patients affected by the disease (Orhan 2012). Other CNS disorders such as depression, mania and schizophrenia are also related to the alterations on the levels of this enzyme in the body (Brandt et al. 2007). Thus, the importance of searching for inhibitors of these enzymes is evident, justifying the in vitro evaluation of the potential AChE and POP inhibition of extracts of L. sibiricus, a medicinal species with CNS activity, slightly narcotic and psychoactive (Ahmed et al. 2005;Wu et al. 2011;Kjellgren et al. 2013). L. sibiricus crude methanol (CE-MeOH) and methanol/HCl 1% (CE-MeOH + ) extracts were subjected to screening at a concentration of 200 μg/mL against AChE and POP (Toide et al. 1995;Rhee et al. 2001). Regarding the potential to inhibit AChE, the extracts of L. sibiricus did not show high inhibitory capacity, even at the concentration of 200 μg/mL (Table 1), thus the AChE assay with the isolated compounds was not performed. In contrast, both extracts from L. sibiricus at 200 μg/mL exhibited inhibitory capacity in the range of 53-58% against POP. It is worth noting that L. sibiricus extracts, which were investigated for the first time against AChE and POP, demonstrated the presence of potential POP inhibitors among its various secondary metabolites. Therefore, the POP inhibitory activity is likely to be related to the L. sibiricus CNS depressant action observed by Ahmed et al. (2005).
POP IC 50 of β-sitosterol-glucoside (3) isolated from Caryophylli flos was previously determined by Lee et al. (1998) as 27.5 μg/mL (46.8 μM). To establish whether leojaponin (1) and leonurine (4) could be involved in the POP activity of L. sibiricus, these compounds were tested against this enzyme. Leojaponin (1) showed weak POP inhibitory activity, with an IC 50 of 184.7 μM, while leonurine (4) was inactive against this enzyme. Although 1 is not a potent POP inhibitor, the structure of this diterpene is prone to be used as a hit or lead compound for further structural optimisation on the search for new POP inhibitors with improved potency.

Conclusions
Researches related to the identification of naturally occurring compounds have allowed the discovery of important inhibitors of enzymes-target of the pharmaceutical industry to the development of new drugs, such as AChE and POP. From Leonurus sibiricus, we isolated and identified only one compound with good ability to inhibit POP, the phytosterol β-sitosterol glucoside (3). Despite leojaponin (1) present weak inhibitory effect, this labdane-type diterpene shows potential as starting molecule for obtaining new derivatives with increased inhibitory activity of POP. Therefore, this compound could be a candidate for further studies in the development of new drugs for the treatment of neuropsychiatric disorders such as AD and bipolar disorder.

Disclosure statement
No potential conflict of interest was reported by the authors.