Inhibitory and Enzyme-Kinetic Investigation of Chelerythrine and Lupeol Isolated from Zanthoxylum rhoifolium Against Krait Snake Venom Acetylcholinesterase

A atividade in vitro de queleritrina e lupeol, dois metabólitos isolados de Zanthoxylum rhoifolium, foram estudados contra o veneno da cobra Bungarus sindanus (Elapidae). O veneno, que é altamente tóxico para os seres humanos, é constituído principalmente pela enzima acetilcolinesterase (AChE). Ambos os compostos apresentaram atividade contra o veneno, e o alcalóide queleritrina apresentou maior atividade do que triterpeno lupeol.


Introduction
Natural phenomena such as insects, microbes and mutation can cause human illness.However, nature itself remains our ally, providing remedies to treat various diseases that are more economically viable than industrialized pharmaceuticals.Death from snake poisoning is an important public health problem in rural areas, especially in countries in Africa, Asia, Oceania and South America. 1 The disadvantaged populations of these countries and those who work in agriculture or near forests, have the greatest exposure to this danger.These populations may not have access to anti-venoms, which are usually the only effective therapeutic agent.In this context, there has been much scientific interest in finding faster and cheaper solutions to mitigate or delay the ailments caused by poisonous snakes' bites.3][4][5][6] Except for Mamba snakes, Elapidae venom contains large amounts of acetylcholinesterase. 7 The serine hydrolase, acetylcholinesterase (AChE -acetylcholine acetylhydrolase, E.C.3.1.1.7),is a non proteolytic enzyme mainly found in the synaptic tissue of muscles, brain, erythrocytes and cholinergic neurons. 80] ACh is stored in the form of vesicles in the nerve terminals and when depolarization occurs, ACh is released from the vesicles and enters the synapse, binding to the receptor.
There are two types of receptors in which ACh binding takes place: muscarinic and nicotinic.Muscarine receptors are mainly found in the peripheral nervous system with smooth and cardiac muscles, while nicotinic receptors are chiefly associated with the central nervous system. 11Ch binding with muscarinic receptors is generally associated with stimulation of the parasympathetic nervous system which entails decreased blood pressure, heart rate, constriction of bronchi, increased intestinal peristalsis and salivation.In the central nervous system, ACh binding causes stimulation of nicotinic receptors which is associated with cognitive processes and memory. 12n skeletal muscles, binding of ACh causes contraction.Vol. 25, No. 1, 2014   Recent investigation has revealed that this enzyme is also present in non-synaptic tissue, i.e., snake venom. 7However, the snake venom acetylcholinesterase is different from other vertebrate tissues in that it is present in soluble non-amphiphilic monomeric form, 13,14 while from all other sources it occurs in multimeric form, having two or more subunits.Furthermore, in snake venom it is more stable than any other source. 15In snake venom, this enzyme is mainly present in family Elapidae which is well known due to highly toxic components in its venom.Furthermore, slight amount of AChE is also present in non-poisonous snake, 16 where its function is unknown.Generally, peptide sequence study shows that snake venom enzymes have 65% homology with other acetylcholinesterases, and have the same catalytic triad for substrate hydrolysis. 14edicinal plants with anti-snake venom AChE activities might be useful as first aid treatment for victims of krait (Bungarus sindanus) snake bite, as well as those from other species of the Elapidae family common in South America, such as the coral snake (Micrurus carallinus).This is of particular importance especially in local areas where antivenom is not readily available.An example is the species Zanthoxylum rhoifolium, found in Brazil and in countries of the African and Asian continents.Species of the genus Zanthoxylum, that are used as anti-snake venom, along with other medicinal uses, 17 are rich in metabolites belonging to the class of alkaloids, mainly benzophenanthridines, lignans and terpenoids.Although plant extracts containing alkaloids have been proven to possess anti-snake venom activity, 18 there is no scientific proof for this type of activity for Z. rhoifolium.

Results and Discussion
In a preliminary test of the anti-AChE activity with the crude methanol extract of Zanthoxylum rhoifolium using the bioautographic assay in thin layer chromatography (TLC) plates gave promising results. 19These results prompted the study of isolated metabolites, benzophenanthridine alkaloids and triterpenoids from this extract.Due to the small amount of enzyme available, the two most abundant compounds, chelerythrine, representing the class of benzophenanthridine alkaloids, and lupeol, a triterpenoid, were selected for the study (Scheme 1).
Chelerythrine and lupeol were found to modify the activity of the krait (Bungarus sindanus) venom acetylcholinesterase (AChE) in a dose-dependent response with the addition of 0.5 mmol L -1 acetylthiocholine (AcSCh) fixed substrate (Figure 1 A and B).
The enzymatic behavior of krait venom AChE was measured under increasing concentrations of chelerythrine (3.86-11.58µmol L -1 ) and lupeol (124-372 µmol L -1 ).The Lineweaver-Burk plot 20 and double reciprocal plot indicated that chelerythrine caused linear mixedtype inhibition, which is considered to be a partial competitive and pure noncompetitive mixture.Lupeol had a similar pattern of inhibition with krait venom AChE.The double reciprocal plot indicated that K m (Michaelis-Menten constant = substrate concentration at which the reaction is half of V max increased) and V max (the maximum rate achieved by the system at maximum substrate concentrations) decreased with increased concentrations of chelerythrine/ lupeol (Figure 2 A and B).
The K m values increased from 75 to 236.6% while V max decreased from 15 to 42% with increased of chelerythrine concentration (3.86 to 11.58 µmol L -1 , see Table 1) while for lupeol the K m values increased from 25 to 106.6% and V max decreased from 15.5 to 50.5% with concentrations ranging from 124 to 372 µmol L -1 (Table 2).
The IC 50 value for chelerythrine that inhibited 50% of enzymatic activity of venom AChE was found to be 6.7 µmol L -1 (Figure 3 A) while for lupeol it is found to be 323 µmol L -1 (Figure 3 B).
Comparing the data shown above (K m , V max and IC 50 ), we could find that the alkaloid chelerythrine presented   more potent inhibitory effect than the triterpenoid lupeol.In addition, the K i (Inhibition Constant = concentration of inhibition which is required to decrease the maximal rate of the reaction to half of the uninhibited value, in the presence of a low substrate concentration) value for chelerythrine was found to be 4.3 µmol L -1 , whereas for lupeol was found to be 223 µmol L -1 obtained using Cornish-Bowden plots of S/V vs.

Conclusions
The results of the present study indicate that chelerythrine, a benzophenanthridine alkaloid, isolated from Z. rhoifolium, has potential anti-AChE activity.This activity is less pronounced for lupeol, a triterpenoid commonly found in various plants, including Z. rhoifolium.In correlation, there is 65% amino acid sequence homology between venom and human AChE, therefore these compounds, mainly the benzophenanthridine alkaloids, need further studies for the treatment of Alzheimer's disease.In conclusion, the results support one of the popular uses of species of the genus Zanthoxylum as anti-snake venom. 15,17

General experimental procedures
Melting points were determined in an MQAPF-301 melting point apparatus and are uncorrected.NMR spectra were acquired on a Bruker DPX-400 operating at 400 and 100 MHz, for 1 H and 13 C, respectively.Chemical shifts are given in δ (ppm) using TMS as internal standard.Thin layer chromatography was performed on pre-coated TLC plates (Merck, silica 60 F-254).The spots were detected using one or more of the following methods: UV (254 nm), spraying with Dragendorff's reagent or 10% H 2 SO 4 /EtOH, followed by heating.DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)], acetylthiocholine iodide, ethopropazine, arachidonic acid and bovine serum albumin were purchased from Sigma (St. Louis, MO, USA).Sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Neon Commercial LTDA (São Paulo, Brazil).All other reagents used were of analytical grade.

Plant material
Bark of Z. rhoifolium was collected in Santana do Livramento, Rio Grande do Sul, Brazil, in November 2008, and authenticated by Prof Gilberto D. Zanetti, Department of Pharmacy, Universidade Federal de Santa Maria, RS, Brazil, where a specimen sample (HDFI 134) is retained.

Extraction and isolation
Isolation and purification of lupeol and chelerythrine from Zanthoxylum rhoifolium The dried stem bark of Z. rhoifolium (1 kg) was powdered and extracted four times with MeOH at room temperature.At this stage, we observed the formation of an insoluble precipitate, which was recovered by filtration (2.5 g) and saved for later analysis.The MeOH extract was filtered and concentrated in vacuum to obtain a crude extract (150 g).
Part of the MeOH extract (70 g) was dissolved in H 2 O (100 mL) and acidified with 2 mol L -1 HCl to pH 2-3.After exhaustive extraction with Et 2 O, the acidic solution was made basic with NH 4 OH to pH 8-9 and extracted with dichloromethane (DCM) (5 × 100 mL) to yield the basic extract (4.5 g).The DCM basic fraction (4.0 g) was chromatographed on a silica gel column (230-400 mesh, 320 g) and eluted with CH 2 Cl 2 : MeOH mixture to prepare fractions 1-90.Fractions 35-38 obtained from CH 2 Cl 2 : MeOH, 80:20, and consisting of one alkaloid (TLC), were combined and concentrated in vacuum to give chelerythrine after crystallization from Et 2 O:MeOH (120 mg).] Part of the MeOH extract (10.0 g) was dissolved in H 2 O (50 mL) and exhaustively extracted with n-hexane.The n-hexane fraction (3.0 g) was chromatographed on a silica gel column (230-400 mesh, 240 g) and eluted with n-hexane:CH 2 Cl 2 mixture to prepare fractions 1-20.Fractions 10-12, obtained from n-hexane:CH 2 Cl 2 , 80:20, were combined and concentrated in vacuum to give lupeol (150.0 mg).Lupeol was identified by direct comparison of TLC with authentic samples, and by comparison of spectral data ( 1 H and 13 C NMR).

Venom
Venom from krait (Bungarus sindanus) snakes was milked manually, mixed, lyophilized immediately and stored at -20 °C for further use.

Protein determination
Protein was assayed by the method of Bradford 24 using bovine serum albumin as standard.

Figure 1 .Scheme 1 .
Figure 1.Krait venom acetylcholinesterase activity in the presence and absence of different concentrations of chelerythrine (A) and lupeol (B).The compounds were pre-incubated at 37 o C for 10 min before addition of fixed substrate 0.5 mmol L -1 acetylthiocholine (AcSCh).The results represent the mean of three different experiments down in duplicate and significantly different from control.*P < 0.05.

Figure 2 .
Figure 2. Kinetic analysis of the inhibition of krait venom AChE by chelerythrine (A) and lupeol (B).

Figure 3 .
Figure 3. IC 50 value of chelerythrine (A) and lupeol (B).The compounds were pre-incubated at 37 o C for 10 min before addition of fixed substrate 0.5 mmol L -1 acetylthiocholine (AcSCh).The results presented are the means of three different experiments.

Table 2 .
Effect of lupeol of Km and V max of the krait (Bungarus sindanus) venom acetylcholinesterase (AChE)