Normal phase HPLC-based activity profiling of non-polar crude plant extracts – acetylcholinesterase inhibiting guttiferones from Montrouziera cauliflora as a case study

Abstract The study describes bioactive compounds as inhibitors of acetylcholinesterase (AChE), from the stem bark extract of Montrouziera cauliflora, selected among 19 dichloromethane extracts from Clusiaceae species. Our work focused on the development of an original normal phase HPLC microfractionation strategy to rapidly assess highly active zones from this crude active non-polar plant extract. Two different microfraction collection methods were evaluated for the assessment of the AChE inhibition. Two guttiferones and a tocotrienol were directly isolated among five compounds identified off-line by NMR after upscaling the fractionation and their AChE inhibition was evaluated. The strengths and weaknesses of the two microfractionation collection methods for HPLC-AChE activity-based profiling are discussed.


Introduction
Alzheimer's disease (AD) is one of the major causes of dementia among old people and it has been estimated that 10% of the world's population exceeding 65 years is affected (Di Giovanni et al. 2008). Associated with other observations, this disease is combined with a very strong decrease of the acetylcholine, the main neurotransmitter of the cortex. Since acetylcholinesterase (AChE) is involved in the hydrolysis of the acetylcholine, its inhibition would restore cognitive functions (Cummings 2004). According to the Committee of Transparency of the High Authority of Health (HAS, France) (Haute Autorité de Santé 2012), only four drugs have a marketing authorisation (AMM) in the symptomatic treatment of AD. Three of them are AChE inhibitors and two are from natural source: galanthamine (Reminyl®) a natural product and rivastigmine (Exelon®) a synthetic analogue of the natural drug physostigmine. The Clusiaceae family is known to biosynthesise polyphenolic compounds sometimes linked to AChE inhibitory activity, like phenylcoumarins (Awang et al. 2010) and benzophenones (Lenta et al. 2007;Costa Júnior et al. 2013). Considering the interest for natural AChE inhibitors (Jiang et al. 2015) and our previous phytochemical studies on Clusiaceae and Callophyllaceae (Hay et al. 2003(Hay et al. , 2008Ferchichi et al. 2012), 49 crude extracts from both families were evaluated for their AChE inhibitory activity using the microdilution Ellman's assay. Among them, three showed an inhibition higher than 50% at 50 μg/ml, all from Clusiaceae: the methanolic roots extract of Calophyllum caledonicum Vieill. ex Planch. & Triana, the methanolic bark extract of Garcinia virgata Vieill. ex Guillaumin and the dichloromethane stem-bark extract of Montrouziera cauliflora Planch. & Triana. Only this later extract exhibited 80% of inhibition at 200 μg/ml, suggesting a possible dose-response effect. Therefore, it was selected for further study by high-resolution activity-based profiling through an HPLC microfractionation strategy (Bertrand et al. 2014;Potterat & Hamburger 2014).

Results and discussion
M. cauliflora, namely 'houp' in French, is an endemic tree of New Caledonia growing in an environment of wet evergreen dense forests. The genus Montrouziera is known to produce xanthones and phenylcoumarins. Nevertheless, no phytochemical investigation of the species cauliflora has been reported so far (Ito et al. 2000).
The thin-layer chromatography (TLC) AChE assay (bioautography) is usually considered as a method of choice for non-polar extracts (Marston et al. 2002) and was initially tested to screen the selected plant extracts. However, the colour decay upon the Fast Blue B salt reaction was difficult to observe due to highly coloured zones, leading to poor identification of the bioactive fractions (data not shown). Furthermore, the TLC resolution was not sufficient for a complete separation of bioactive constituents. Thus, we evaluate an at-line HPLC activity-based profiling based on microfractionation to get through these issues and enhance chromatographic resolution for a better deconvolution of bioactive principles. Indeed, the use of new strategies combining HPLC microfractionation with spectroscopic and bioactivity data enables prioritisation of hits and identification of bioactive constituents at an early stage (Bertrand et al. 2014;Potterat & Hamburger 2014).
Because of the non-polar nature of the bioactive extracts, optimal metabolite profiling of the stem bark of M. cauliflora had been developed based on analytical normal phase (NP) HPLC and detection was ensured by both uV and ELSD ( Figure S1). To obtain microfractions containing sufficient amounts of metabolites for a satisfactory readout with the AChE bioassay, the analytical conditions were upscaled by geometric chromatographic transfer to semipreparative NP HPLC. This allowed the injection of 50 mg of crude extract and the recovery of 88 fractions (Figure 1(A)). Two different microfractionation methods were evaluated: (1) collection into separate vials, allowing fractions to be dried and weighed prior to bioactivity evaluation; and (2) collection into a 96-well reservoir allowing fractions to be dried and re-suspended in a fixed volume. Method 1 provides microfractions containing all the same metabolites' concentrations, while method 2 gives fraction of concentrations that were varying according to the proportion of the extract constituents. The AChE inhibitory activity was measured for each microfraction using the microdilution Ellman's assay and different concentrations were tested to obtain a satisfactory readout (Figure 1(B)-(E)). Finally, the AChE inhibitory activity profiles were generated based on all the individual assay results and both collection methods were compared to check for convergent results. The fractions weighed into individual vials (collection method 1) were tested at 1, 10 and 50 μg/ml after dilution (Figure 1(B)-(D)). Alternatively, method 2 (collection of fixed-volume fractions) was also used. Here, an average concentration of 1 μg/ml corresponded to a 0.09% aliquot of the wells (considering a mean fraction quantity of 300 μg -0.6% of the extract - Figure 1(A)). When tested at 10 and 50 μg/ml, the microfractions from collection method 1 showed inhibition values too high to be discriminant (Figure 1(B) and (C)). However, at 1 μg/ml, a satisfactory discrimination between active and inactive fractions was observed (Figure 1(D)). Method 2, (Figure 1(E)) also discriminated active zones in the chromatogram. The activity levels were in accordance with the calculated dilution, and also with the weighed quantity, showing a dose-response relationship. For example, the highest amount (0.8-1.2 mg, Figure 1(A)) was weighed between 10 and 13 min of elution, corresponding to one of the active zones and to some of the isolated compounds (Figure 1(D)): gutifferone I and M, respectively 12 and 13 min, δ-tocotrienol, lupeol, globuloxanthone, 6, 7 and 8 min). The activity profile at 1 μg/ml (method 1: fixed concentration) (Figure 1(D)) was compared to that of method 2 (concentration varying according to extract composition) (Figure 1(E)). With method 2, only two zones were found active: 4-8 min and 10-13 min, while three zones were active with method 1: 4-6 min, 7-10 min and 10-13 min. With method 2, a residual activity (~20%) was found after 15 min. This might be related to the higher amounts of metabolites in the corresponding microfractions compared to method 1.
A second microfractionation with three times higher loading (150 mg) provided enough material for isolation and purification of five compounds from the actives zones by collection method 1 (4-6, 7-10 and 10-13 min). Based on NMR and MS data, these were identified as two benzophenones (guttiferones M (Masullo et al. 2008) and I (Nguyen et al. 2005), a mixture of globuxanthone (Iinuma et al. 1995) and lupeol (Mouffok et al. 2012) (0.7:1.0 mol/mol ratio estimated by 1 H NMR) and the δ-tocotrienol (Ohnmacht et al. 2008) ( Figure S2). Their NMR spectra were in accordance with previously reported data (Marston et al. 2002). The amounts isolated indicated that the guttiferones M and I represent roughly 13 and 9% of the crude extract, respectively. All these compounds and fractions were evaluated for their AChE inhibition at 10 −3 M or 500 μg/ml, respectively (Table S1). The AChE inhibitory activity of the isolated compounds was considered as moderate relative to galanthamine. Nevertheless, only the guttiferones A and F have been previously described for their anticholinesterase properties (Lenta et al. 2007) and synthetic benzophenone derivatives have been recently described as a novel potent and selective inhibitors of AChE (Belluti et al. 2011). Tocotrienols are well documented as dietary neuroprotective agents in AD and have been able to decrease induced AChE activity in vivo (Frank et al. 2012).

Conclusion
We herein evaluated the efficiency of NP HPLC for rapidly assessing the AChE inhibitory activity of the constituents of a representative non-polar medicinal plant extract. Two microfraction collection methods were evaluated to establish the best way to highlight zones of bioactivity in the NP HPLC profiles. There are very few papers describing non-polar HPLC microfractionation using NP HPLC chromatography. The two collection methods compared (fixed concentration and concentration dependent on extract composition) led to HPLC high-resolution inhibition profiles and allowed the rapid evaluation of the presence of bioactive compounds within the plant crude extract. The amount of extract injected was found to considerably influence the bioactivity results and variation of concentration in the extract also impact the bioactivity profiles. From a bioactivity point of view, since the higher value appeared in the same zone for both methods, the detection of major active compounds could be independent of collection methods. Likewise, the use of 0.09% fixed-volume fractions (collection method 2) clearly showed additional zones of activity not detected with the first method. This suggests that minor active compounds in the zone 7-10 min were too diluted to be detected as active compounds in method 2. On the other hand, the compounds eluting after 15 min showing activity in method 2 have been considered as inactive compounds at 1 μg/ml (method 1). Both methods have advantages and limitations, but the usage of method 2 was time saving and allows a satisfactory assessment of the AChE inhibitory activity in solution using the Ellmann's microdilution method. Simple NP HPLC microfractionation with at-line coupling to bioassay in 96 well-plates provided an easy way to localise AChE inhibitors in crude non-polar plant extracts and represents an easy strategy, which can be readily implemented in a screening protocol for large number of extract. However, as shown, the appropriate dilution needs to be adapted in order to discriminate bioactive fractions. The identification of previously reported AChE inhibitors further validates the efficiency of the approach.

Supplementary material
The experimental section can be access as supplementary material.