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Evaluation of cholinesterase inhibitory activity of six Indonesian Cassia species

Alzheimer’s disease (AD) is a neurodegenerative disorder, which is the most common cause of dementia. The aging population means that the number of people suffering from AD is expected to increase each year if there is no effective treatment found. One of the strategies for the treatment of AD is the use of cholinesterase inhibitors. Plants have been the source of many bioactive metabolites, including cholinesterase inhibitors. The objective of this study is to investigate the potency of several plant extracts from the genus Cassia as cholinesterase inhibitors. The cholinesterase inhibitory screening was carried out against two enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), according to the modified Ellman’s method. The chemistry of the active fractions was studied by LC-MS/MS method. The results suggested that amongst six plant extracts from the genus Cassia investigated, the ethanolic extract of Cassia spectabilis showed the strongest inhibition against both AChE and BChE enzymes, with IC50 values of 39.5 and 36.9 μg/mL, respectively. Investigation on the n-hexane, ethyl acetate, and nbutanol fractions obtained from the C. spectabilis extract showed that the ethyl acetate and the n-butanol fractions gave better inhibitory activity compared to the n-hexane fraction. Based on the LC-MS/MS data, the two active fractions gave similar profile. Both fractions contained alkaloid cassine and spectaline, which may responsible for the cholinesterase inhibitory activity.

been studied thoroughly as AChE inhibitor is a sesquiterpene alkaloid, Huperzine A, obtained from Huperzia spp. The product containing extract of Huperzia spp has been commercialized as a food supplement for memory support in China [2].
Considering the two AChE inhibitors above are natural origin, research on finding either new or more effective compounds has been a target of many researchers. Recently we have reported the potency of several marine sponges as acetylcholinesterase inhibitor [7]. In the present study investigation of Cassia spp as cholinesterase inhibitor was carried out. The genus Cassia in the family Caesalpiniaceae have been known as flowering plants which comprising of more than 500 species, and are distributed widely in the tropical and subtropical regions [8][9][10]. Cassia spp are usually grown as ornamental plants, but many of these plants have great economic importance as well as its utilization in traditional medicine. The leave and the bark of Cassia fistula have been used to treat blackwater fever as well as a laxative [8]. Other traditional usages of Cassia spp have also been documented, such as for gastrointestinal problems, skin diseases, cough, bronchitis and cardiac disorders [10,11]. Metabolites reported from Cassia species include anthraquinones, terpenoids, xanthones, flavonoids as well as alkaloids. Many of these compounds posses promising biological activities, such as antioxidant, anti-inflammatory, anticancer, antiplasmodial, and hepatoprotective agents [11][12][13][14]. Anti-AD activity was reported from Cassia obtusifolia; the seeds extract of this plant can improve memory and learning in mice [15], as well as ameliorates amyloid β-induced synaptic dysfunction through anti-inflammatory and Akt/GSK-3β pathways [16]. C. obtusifolia extract was also found to inhibit AChE, BChE, and BACE1 (β-site amyloid precursor protein cleaving enzyme 1) [17]. It was suggested that the napthopyrone compounds may be responsible for these activities [18]. Freitosa et al. (2011) have reported that the extract of Cassia alata leaves has shown AChE inhibition [19]. Based on these reports, the potency of other Cassia species as cholinesterase inhibitors is worth to be investigated.

Cholinesterase inhibitory assay
The ethanolic extracts were prepared from the leaves of six species of the genus Cassia, namely  Figure 1 suggested that the highest inhibition against both enzymes was given by C. spectabilis and the lowest inhibition was shown by C. fistula extract. The inhibitory activity of C. spectabilis extract was higher against BChE (85.2%) compared to that against AChE enzyme (53.8%). The C. spectabilis extract was further partitioned with, n-hexane, ethyl acetate, and n-butanol, sequentially. Each of the fractions was then subjected to cholinesterase inhibitory assay against both AChE and BChE enzymes. The results presented in Figure 2 indicated that the ethyl acetate and the n-butanol fractions gave higher inhibition against both enzymes, which suggested that the active compounds are possibly semipolar to polar compounds.  The 50% inhibitory activities of the ethanolic extract as well as the ethyl acetate and n-butanol fractions of C. spectabilis were also investigated. The results given in Table 1 show that all samples tested except galantamine exhibited lower IC50 values against BChE compare to AChE. The lowest IC50 value was given by the ethyl acetate fraction of C. spectabilis at 10.8 and 4.7µg/mL against AChE and BChE, respectively. The ethanolic extract of C. spectabilis was subjected to enzyme inhibiting kinetics study, and a Michaelis-Menten plot was determined ( Figure 3). The Km value of the substrate ATCI (control) for AChE was 68.4 µM and Vmax was 99.1 µM/min. When the ethanolic extract of C. spectabilis leaves was added the Km and Vmax were 62.9 µM and 74.7 µM/min, respectively. There was no significant change in the Km values; indicated that the ethanolic extract of C. spectabilis inhibited AChE enzyme in a non-competitive way.

Identification of metabolites by LC-MS/MS
The chemistry of the ethyl acetate and the n-butanol fractions were studied by using LC-MS/MS instruments. The total ion chromatograms (TIC) of the two fractions were compared (Figure 4), which showed a similar TIC profile. Both fractions exhibited 2 major peaks at RT 2.57 (1) and 6.14 (2) mins, however, the ethyl acetate fraction also gave several minor peaks at RT approximately 6.9 -17.9 mins which was not seen in the n-butanol fraction. Based on the MS/MS data ( Table 2) and comparison to the literature, the two major peaks were identified as cassine (1) and spectaline (2) ( Figure 5) [20,21]. In the ethyl acetate fraction, a peak at RT 6.95 mins was observed, which was identified as 3-O-acetylspectaline (3). However, other minor peaks can not be identified unambiguously. Cassine and spectaline are two major piperidine alkaloids that have been reported from several parts of C. spectabilis, such as from the leaves and flowers [11,[22][23][24]. Piperidine alkaloids, such as iso-6-cassine and iso-6-spectaline, isolated from C. spectabilis have shown potency as depressant and anticonvulsant agents [25,26]

CONCLUSION
Based on these results, the ethanolic extract of Cassia spectabilis showed the strongest inhibition against both AChE and BChE enzymes amongst Cassia species tested. Further fractionation on C. spectabilis extract suggested that the ethyl acetate and the n-butanol fractions gave better inhibitory activities compared to the n-hexane fraction. Piperidine alkaloids, cassine, spectaline and 3-O-acetylspectaline which were identified in the active fractions, may contribute to the cholinesterase inhibitory activity of C. spectabilis.

Plant collection, extraction and fractionation
Plant materials were collected from Purwodadi Botanic Garden, East Java, Indonesia on March 9 th , 2019. Sample specimens (Voucher numbers: PWD 01 -PWD 06) were kept at the Institute of Tropical Diseases Universitas Airlangga. The identification of plant materials was conducted by Purwodadi Botanic Garden (Identification letter number: 0371/IPH.06/HM/III/2019).
The leaves were air-dried for seven days at room temperature followed by grinding to obtain powdered materials. Ten grams of each samples were extracted with ethanol by using ultrasonic extraction for 3 x 10 mins, then the residue and filtrate were separated by filtration. The residue was re-extracted with ethanol using the same procedure for 3 times. The collected filtrate was then concentrated in vacuo to yield the crude ethanolic extracts (0.5 -2.0 g). The ethanolic extract of C. spectabilis (0.5 g) was dissolved with a mixture of ethanol and H2O (1:1) (20 mL), then further separated by a liquid-liquid partition with n-hexane (15 mL), ethyl acetate (15 mL), and followed by n-butanol (15 mL). Fractionation with each solvent was conducted 3 times. Each of the fractions obtained was evaporated in vacuo, and yielded n-hexane (46.9 mg), ethyl acetate (184.4 mg), and n-butanol (97.2 mg) fractions.

Cholinesterase inhibitory assay
The assay was carried out according to the modified Ellman's method [7,[29][30][31]. Plant extracts were dissolved in methanol to make 10 mg/mL concentration, and was serially diluted with 50 mM Tris buffer to obtain a 1 mg/mL concentration containing not more than 10% of methanol. These samples were further diluted in the microplate well to a final test concentration of 100 µg/mL. Sample solutions were added to a 96-well microplate, followed by the addition of 1.5 mM ATCI or 1.5 mM BTCI (25 µL), 3 mM DTNB (125 µL), and Tris buffer (50 µL). The substrate was then hydrolyzed by the addition of 25 µL of 0.22 U/mL of either EeAChE, hrAChE or BChE. The solutions were shaken for 30 s in a microplate reader (Bio-Tek Instrument, USA) before measurement. The product, 5-thio-2-nitrobenzoate, indicated by a yellow color was measured at 405 nm every 5 s for 2 min. Every experiment was carried out in triplicates. Galantamine (100 µL) was used as a positive control, and 10% methanol was used as a negative control. For the measurement of IC50, serial concentrations of the samples were prepared. The inhibitory activity was calculated as: % Inhibition =[(Mean velocity of control -Mean velocity of sample)/ Mean velocity of control]x100.

Identification of Metabolites by LC-MS/MS
Identification of metabolites was conducted by using Agilent 1260 Infinity Series HPLC system with an auto-sampler fitted with analytical C-18 column Agilent Poroshell 120 (Agilent Technologies, Santa, Clara, CA, USA). The HPLC was connected with an Agilent 6530 UHD Accurate-Mass Q-TOF LC/MS (Agilent Technologies), equipped with dual electrospray ionization (ESI). Samples were dissolved in methanol, and 10 µL of samples were injected into the system employing gradient elution of 40-100% methanol/H2O containing 0.1% v/v formic acid for 30 mins, flowrate 0.25 mL/min. N2 was employed as drying gas at a flowrate of 10 L/min. The temperature of the nebulizer was set at 325ºC, and the potential of the capillary was 3.5 kV. Positive mode was used and the scan mass range was 100-1000. The MS/MS collision energies were set at 10, 20, and 40 eV. The mass spectrometry data was analyzed using Agilent Mass Hunter Qualitative Analysis software version B06.00.