Chemical Profiles and In Vitro Cholinesterase Inhibitory Activities of the Flower Extracts of Cassia spectabilis

Background Cassia spectabilis is a flowering plant containing various metabolites that provide potential for pharmacological activities. The current study aimed to investigate the ethanolic and water extracts of C. spectabilis as cholinesterase inhibitor as one of the target treatments for Alzheimer's disease. The chemical composition of the extracts was also studied to determine which components are responsible for the bioactivity. Methods The cholinesterase inhibitory activity assay was carried out by the modified Ellman's method against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). LC-MS/MS analysis was carried out to investigate the chemical profiles of the extracts, followed by a molecular networking study by GNPS. Results Both extracts showed inhibition against AChE and BChE in a dose-dependent manner, with the higher potency exhibited by the ethanolic extract with IC50 values of 7.88 and 3.78 μg/mL. The chemical analysis and molecular networking study of the flower extracts revealed similarity between the ethanolic and water extracts. Piperidine alkaloids were identified in both extracts, while the sphingolipid compounds were found in the ethanolic extract. Conclusion The water and ethanolic extracts of C. spectabilis flowers displayed potency for Alzheimer's disease treatment. The presence of piperidine alkaloids in the extract may be responsible for the cholinesterase inhibitory activity. The higher potency of the ethanolic extract compared to the water extract is possibly due to the higher amount of piperidine alkaloids in the ethanolic extract. Further study is needed to quantify the concentration of alkaloids in the extracts.


Introduction
Alzheimer's disease (AD) is a progressive neurologic disorder that mainly afects the elderly population. Te early sign of AD is mainly characterized by mild memory declines, such as forgetting recent events or conversations. As the disease progress, it will develop into severe memory loss and language impairment, as well as behavioral and social changes that will eventually afect the person's ability to carry out daily tasks [1,2]. It is estimated that more than 50 million people are sufering from AD, and the number is expected to increase to 115.4 million in 2050 [3,4].
Multiple factors are involved in the pathogenesis of AD, including the defciency of the neurotransmitter acetylcholine (ACh). Two cholinesterase enzymes are involved in regulating ACh in the brain, namely acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Both enzymes hydrolyze ACh into choline and acetate [5]. In the healthy brain, the regulation of ACh is mainly controlled by AChE compared to BChE. However, in a patient with AD, the BChE level increases from 0.6 in the normal brain to as high as 11 in the cortical areas afected by the disease [6,7]. As a consequence, the regulation of ACh is dependent on the activity of the BChE enzyme. Terefore, cholinesterase enzyme inhibition becomes one of the target therapies for AD. Te use of cholinesterase inhibitors has shown positive efects in improving the cognitive function of AD patients [8,9].
Cassia spectabilis, synonym Senna spectabilis (family Fabaceae) is a fowering plant distributed in tropical and sub-tropical regions [10]. Te fowers with unsymmetrical golden-yellow corolla located terminal. Various parts of this plant have been used as folk medicine in Indonesia, Brazil, and Tailand. Te leaves are used as laxatives and purgatives. Other traditional uses are for treating skin diseases, edema, as well as poisoning, and protozoic infection of the gut [11]. Te infusion of the leaves is traditionally used for insomnia and anxiety [12]. Chemical investigation revealed the presence of alkaloids, terpenoids, favonoids, anthraquinones, and steroids from the leaves, fowers, fruits, seeds, stems, and roots of C. spectabilis [11,13,14]. Te plant has been reported as anticonvulsant, antimicrobial, antinociceptive, and anti-infammatory agents [15][16][17][18][19][20]. In the previous study, we investigated the potency of the ethanolic and water extracts of C. spectabilis leaves and stems as antioxidant and cholinesterase inhibitors. Te leaves and stems extract of C. spectabilis exhibited signifcant cholinesterase inhibitory activities against AChE and BChE as well as moderate antioxidant activities [21,22]. In the development of herbal medicinal product for the treatment of Alzheimer's disease from C. spectabilis, information on the extraction solvent that gives the optimum potency as a cholinesterase inhibitor is needed. Traditional use of the plant as an infusion and decoction has been documented [12]. Meanwhile, studies on the methanolic extract of C. spectabilis fower as a cholinesterase inhibitor has been reported [23]. Terefore, it is interesting to know which extraction solvents that will give the best cholinesterase inhibitory activity. Te current study evaluated the cholinesterase inhibitory activities and the phytochemical profle of the water and ethanolic extracts of C. spectabilis fowers. We hypothesize that diferent extraction solvents will have an efect on the chemical profles and the cholinesterase inhibitory activities. To the best of our knowledge, there is no report on the efect of diferent extraction solvents on the cholinesterase inhibitory activity and the chemical profle of C. spectabilis fowers.

Extraction.
Te fowers of C. spectabilis were dried in the shades for approximately seven days and then grounded using an electrical blender. Te ethanolic extract of the fower was prepared by adding ethanol (500 mL) to the dried fower powdered (88.2 g), and the mixture was allowed to stand for 24 h, followed by vacuum fltration. Ten the residue was re-extracted with ethanol using the same procedure twice. Te collected fltrate was evaporated under vacuum to obtain a dark brown extract (7.0 g). For water extract preparation, the dried fowers (80.3 g) were soaked in water (500 mL) in an ultrasonic chamber for 30 minutes, followed by fltration. Ten the residue was re-extracted with water using the same procedure twice. Te fltrates were evaporated in a rotary evaporator to yield a dark brown water extract (15.0 g).

Cholinesterase Assay.
Te assay was carried out according to the modifed Ellman's method, as described in the previous publications [22,24]. Te extracts were dissolved in methanol to make 10 mg/mL and diluted with water to obtain 1 mg/mL. Tese samples were serially diluted to get a range of sample concentrations of 0.01-300 μg/mL. Sample solutions were added to a 96-well microplate (25 μL), followed by the addition of 1.5 mM ATCI (25 μL) as a substrate for the AChE enzyme, and BTCI (25 μL) as a substrate for the BChE enzyme. Te substrate was then hydrolyzed using 3 mM DTNB (125 μL), tris-bufer (50 μL), and AChE or BChE enzymes (25 μL) 0.22 μ/mL. Before measurement, the solutions were shaken for 30 s in a microplate reader (Termo Scientifc Multiskan FC). Te yellow color from the product, 5-thio-2-nitrobenzoate, was measured at 405 nm every 5 s for 2 min. Every experiment was carried out in triplicates. Methanol 10% was used as a control. Te enzyme activity was calculated as a percentage of the velocity of the sample compared with the negative control. Te inhibitory activity was calculated based on the equation.
where V: mean velocity.

LC-MS/MS and Global Natural Product Social (GNPS) Molecular
Networking. Te extracts were dissolved in methanol (10 mg/mL), sonicated, and then fltered with a 0.22 μm membrane. Chromatographic separation was performed using an Agilent 1260 Infnity Series HPLC system with an auto-sampler ftted with an analytical C-18 column (Phenomenex Luna C18 (2), 150 × 4.6 mm, 5 μm, USA). A 10 μL sample was injected into the LC system and eluted with a combination of water containing 0.1% formic acid (A) and 5% v/v acetonitrile with 0.1% formic acid (B). A linear gradient from 5 to 95% B in 30 mins, and hold on at this ratio for 10 mins. Te postrun was set for 5 mins before starting a new injection. Te solvent fow rate was set to 0.5 mL/min. Te mass analysis was performed using a QTOF 6540 UHD accurate mass spectrometer. Te analysis parameter was carried out in a positive mode with spectra acquired over a mass of m/z 100-1,000 amu. Te ESI-MS condition parameters were as follows: capillary voltage +3,500 V; drying gas (N 2 ) 7 L/min; dry gas temperature at 350°C; and nebulizer pressure at 30 psig. Fragmentations were performed using auto MS/MS experiments with collision energies at 10 V, 20 V, and 40 V. Compounds identifcation was carried out based on the MS data, MS/MS fragmentation profles, and molecular formula proposed by the Agilent MassHunter that were compared with the literature data and some databases, such as the Human Metabolome and MetFrag, with a maximum error of 5 ppm was accepted. A GNPS analysis was carried out on the LC-MS/MS data. Te LC-QTOF-MS/MS data from Agilent MassHunter data fles (.d) were converted to mzXML fle format using MSConvert software. Te data were then transferred to the GNPS server (gnps.ucsd.edu) to generate the chemical networking map (ID � 011c1f464d314d74bdf209c18a39f4a5) [25]. Te GNPS analysis workfow using the spectral clustering algorithm with a cosine score of 0.7 and a minimum of 4 matched peaks in the fragmentation spectrum. Te molecular networking data were visualized with Cytoscape software version 3.9.1. A ball-and-stick layout where nodes represent parent mass and cosine score was refected by edge thickness [25,26].

Results and Discussion
3.1. Cholinesterase Inhibitory Assays. Te ethanolic and water extracts of C. spectabilis fowers were screened against the AChE and BChE using a colorimetric method developed by Ellman et al. [27]. In this assay, AChE and BChE enzymes will hydrolyze the substrates acetylthiocholine iodide (ATCI) and butyrylthiocholine iodide (BTCI), respectively. As a result, a product thiocholine will form, which then reacts with the Ellman's reagent (DTNB) to form a yellowcolored product 5-thio-2-nitrobenzoate that can be monitored using a spectrophotometer at 405 nm. Te presence of the AChE or BChE inhibitors will prevent the hydrolysis of ATCI and BTCI so that the yellow-colored product will not be formed [27]. Various concentrations of C. spectabilis extracts were prepared to evaluate the dose-response mode and the ffty percent inhibitory activity (IC50) of the extract. Te results ( Inhibition of AChE and BChE enzymes prevents the hydrolysis of acetylcholine and increases the level of ACh in the brain. Te association between BChE enzyme and accumulation of neuritic plaques and fbrillar Aβ plaques as one of the pathologic hallmarks of AD has been reported. Terefore, inhibition against BChE serves a dual role, increasing acetylcholine levels and inhibiting fbrillar Aβ deposition [7].
Several plant species from the genus Cassia have been reported as cholinesterase inhibitors. Azman et al. have screened the potency of C. timorensis, C. grandis, C. fstula, C. alata, and C. spectabilis. Amongst the plants tested, extracts from the leaves, stems, and fowers of C. timorensis showed signifcant inhibition against AChE [28]. We have investigated the cholinesterase inhibitory activities of various extracts from the leaves and stems of C. spectabilis that showed the potency of the leaves and stems extracts against both AChE and BChE enzymes [22]. Franca et al. reported the AChE inhibitory potency of the methanolic extract of C. spectabilis fower using a TLC bioautography method, which however, cannot determine the activity quantitatively [23]. Studies on the efect of C. spectabilis on central nervous system disorders have been documented. Te ethanolic extract of C. spectabilis leaves showed anticonvulsant and sedative activity in mice [12]. Further study by Nkamguie Nkantchoua et al. investigated the anticonvulsant activity of C. spectabilis leaves decoction. Te oral administration of the decoction can completely inhibit seizure in mice induced by maximal electroshock, antagonizes completely seizures induced by pentylenetetrazole, and partially inhibit seizure induced by bicuculline [29]. Silva et al. reported that iso-6cassine and iso-6-spectaline isolated from C. spectabilis possess anticonvulsant and depressant efects on mice [19,30]. Oral administration of iso-6-cassine and iso-6spectaline at a dose of 1.5 mg/kg BW and 1.0 mg/kg BW, respectively, can signifcantly decrease motor activity of the animal on rota-rod apparatus. Both compounds were capable of promoting an increase in latency for the development of convulsion triggered by picrotoxin.

Chemical Profles and Molecular Networking Study.
Te chemistry of ethanolic and water extracts of C. spectabilis was explored using an LC-MS/MS instrument. Te total ion chromatograms (TIC) of the extracts were compared (Figure 2), which are very similar except at RT 19-33 minutes. Te GNPS molecular networking (Figure 3) shows the cluster of compounds present in the ethanolic and Advances in Pharmacological and Pharmaceutical Sciences 3 water extracts. Te red dots represent compounds identifed in the ethanolic extract, the green dots for compounds present in the water extract, and the blue dots for compounds identifed in both the ethanolic and water extracts. As can be seen in Figure 3, the majority of compounds can be detected in both extracts; this data is in agreement with the TIC profle of the extracts. Alkaloid has been reported as the major class of compound that shows promising activity as a cholinesterase inhibitor [2,31]. Terefore, in the present study, we focus on identifying alkaloids in the extracts. Te identifed alkaloids in the ethanolic and water extracts of C. spectabilis fower are presented in Tables 2 and 3. Tere are 22 compounds identifed in the ethanolic extract and 20 in the water extract. Fifteen compounds were detected in both water and ethanolic extracts, including the well-known piperidine alkaloids that have been reported from Cassia spp., such as leptophyllin A ( ), 3-O-acetylleptophyllin A (2), and leptophyllin B (3) that frst isolated from Cassia leptophylla [32] and later from Cassia spectabilis [13]. Other piperidine alkaloids detected in both extracts were (−) cassine (4), (−)-7hydroxycassine (5), (−)-spectaline (6), (−)-7-hydroxyspectaline (7), (−)-3-O-Acetylspectaline (8), (−)-spectalinine (9), and (−)-6-iso-carnavaline ( 0) (Figure 4). Based on the TIC profles, the diference between the water and ethanolic extracts is the peaks at 25-31 mins region, from which sphingolipids were detected.
Selegato et al. have summarized secondary metabolites reported from various parts of C. spectabilis [13]. Te fower contains alkaloids, pentacyclic triterpenes, steroids, pyrones, anthraquinones, and phenylpropenoic acid. Piperidine alkaloids have been reported as the major constituent in the   [38]. Our previous study showed that the ethanolic extract of C. spectabilis leaves contains piperidine alkaloids and displays signifcant cholinesterase inhibitory activities [21,22]. Based on the various reports on the potency of piperidine alkaloids as cholinesterase inhibitors [34][35][36][37][38], it can be suggested that the cholinesterase inhibitory activities of the ethanolic and water extracts of C. spectabilis fowers in this study possibly due to the presence of the piperidine alkaloids detected in the samples. Te higher cholinesterase inhibitory activity of the ethanolic extract compared to the water extract may occur due to the higher amount of piperidine alkaloids in the ethanolic extract. To quantify the concentration of alkaloids in the extracts, additional research is required.   Advances in Pharmacological and Pharmaceutical Sciences 3(R)-benzoyl-2-(R)-methyl-6(R)-11′-oxododecyl)-piperidine *

Conclusions
Te ethanolic extract of C. spectabilis fowers demonstrated higher potency as cholinesterase inhibitors compared to the water extract. Despite the qualitative analysis of the component in the extract, the presence of piperidine alkaloids in the extract may be responsible for the bioactivity. Based on this study, the ethanolic extract of the C. spectabilis fower can be a good candidate for the development of herbal medicine for Alzheimer's disease. Future works could focus on the quantitative analysis of the alkaloid in the extract and the in vivo study using the Alzheimer's disease animal model in the behavioral assays.

Data Availability
Te data used to support the fndings of this study are available upon reasonable request from the corresponding author.

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding the publication of this paper.

Authors' Contributions
SS and WE were involved in the conception and design of the study. SS, ERL, KK, WE, NN, and NS were involved in experiments and data analysis. SS drafted and edited the manuscript. All authors have read and approved the fnal manuscript.