Isolation of Amaryllidaceae alkaloids from Nerine bowdenii W. Watson and their biological activities

Abstract

Twenty-two isoquinoline alkaloids (1–22) were isolated from fresh bulbs of Nerine bowdenii (Amaryllidaceae) by standard chromatographic methods. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic analyses, and by comparison with literature data. 6-O-Demethylbelladine (11) and 4′-O-demethylbelladine (12) are reported here for the first time. Compounds isolated in sufficient amounts were evaluated for their acetylcholinesterase, and butyrylcholinesterase inhibition activity using Ellman's method. In the prolyl oligopeptidase assay, Z-Gly-Pro-p-nitroanilide was used as substrate. Untested alkaloids were also screened for their cytotoxic activity against p53-mutated Caco-2 and HT-29 colorectal adenocarcinoma cells. At the same time, healthy small intestine cells FH-74 Int were used to determine overall toxicity against noncancerous cells. The crinine-type alkaloid buphanisine (7) demonstrated interesting cytotoxicity against both tested cancer cell lines with IC₅₀ values of 8.59 ± 0.15 μM for Caco-2 and 5.32 ± 1.70 μM for HT-29.

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Introduction

The Amaryllidaceae family consists of about seventy genera, whose species are widely distributed in the tropics and warm-temperature regions of the world.¹ The plants of the Amaryllidaceae are known to contain a specific type of compound, namely the Amaryllidaceae alkaloids, which are of great interest due to their wide range of biological activities, including antiviral, antimalarial, anticancer and anticholinesteratic.^1–3 Some species of this family contain galantamine, a long-acting, selective, reversible and competitive acetylcholinesterase inhibitor, which has been approved by the Food and Drug Administration (FDA) for the treatment of mild to moderate Alzheimer's disease under the commercial name Reminyl© (galantamine hydrobromide).

Species of Nerine Herbert (Amaryllidaceae), the second largest genus within the Amaryllidaceae with ca. 30 species, are autumn-flowering, perennial, bulbous plants confined to temperate regions of southern Africa with summer rainfall and cool, dry winters. Bulbs from Nerine species have been used in the traditional medicine of the Sotho and Zulu tribes of southern Africa, who use decoctions of bulbs to treat coughs and colds, in renal and hepatic dysfunctions, to obtain relief from back pain, and as a remedy for infertility.⁴

N. bowdenii is an endemic Amaryllidaceae species native to KwaZulu-Natal Drakensberg and Eastern Cape Provinces in South Africa and is widely used as ornamental flowers.⁵ Previous phytochemical studies led to the isolation of about 25 Amaryllidaceae alkaloids of various structural types.^5–11

Our previous phytochemical study of the alkaloid extract of this species led to the detection of 22 compounds, 19 of which were identified by GC-MS. The alkaloid extract also showed promising butyrylcholinesterase (BuChE) inhibition activity with an IC₅₀ value of 14.8 ± 1.1 μg mL⁻¹.¹¹ The important bioactivities of the Amaryllidaceae alkaloids, together with the absence of a detailed current phytochemical report on N. bowdenii, encouraged us to examine this species. In the present study we report the isolation of Amaryllidaceae alkaloids from the fresh bulbs of N. bowdenii and the inhibitory effect of these compounds on the activity of erythrocyte acetylcholinesterase (AChE), plasma BuChE and prolyloligopeptidase (POP). Prolyl oligopeptidase (POP) is a cytosolic serine peptidase that cleaves peptide bonds at the carboxyl end of proline, and is widely distributed in the organs of the body, including the brain.^12,13 In recent studies, some POP inhibitors have been found to be efficacious antidementia drugs.¹⁴ Thus, POP inhibition can represent an important supporting approach in AD treatment, and, therefore, the search for new compounds influencing more therapeutic targets connected with AD is required. So far untested alkaloids were also screened for their cytotoxic activity against p53-mutated Caco-2 and HT-29 colorectal adenocarcinoma cells. At the same time, healthy small intestine cells were used to determine overall toxicity against noncancerous cells.

Results and discussion

Extensive chromatographic purification led to the isolation of two new and twenty known Amaryllidaceae alkaloids (Fig. 1). The structures of the known alkaloids were determined by comparison of their MS and NMR spectra with literature data, and additional physical properties as: belladine⁴ (1), acetylcaranine¹⁵ (2), undulatine¹⁶ (3), caranine¹⁷ (4), 11-O-acetylambelline¹⁶ (5), buphanidrine¹⁶ (6), buphanisine¹⁶ (7), ambelline¹⁶ (8), deacetylbowdesine¹⁸ (9, synonym bulbisine), buphanamine¹⁹ (10), tazettine²⁰ (13), 1,2β-epoxyambelline²¹ (14), 6α-hydroxyundulatine²¹ (15), 1-O-acetylbulbisine²² (16), haemanthamine²³ (17), 1-O-acetyllycorine²⁴ (18), crinamidine¹⁸ (19), hamayne²⁵ (20), crinine¹⁸ (21), and powelline¹⁸ (22).

Fig. 1 Structures of isolated alkaloids from fresh bulbs of Nerine bowdenii.

The isolated alkaloids belong to the belladine (1, 11, 12), crinine (3, 5–10, 14–16, 19, 21, 22), haemanthamine (17, 20), lycorine (2, 4, 18), and tazettine (13), structure types.

The novel compounds, 6-O-demethylbelladine (11) and 4′-O-demethylbelladine (12), were obtained as a light yellow viscous mass. ESI-HRMS of 11 and 12 showed molecular ion peaks [M + H]⁺ at m/z 302.1751, and m/z 302.1750, respectively, corresponding to the formula C₁₈H₂₃NO₃ (calc. 301.1678). NMR spectra of both new belladine-derivatives are similar to those of belladine.⁴ They differ only by the absence of one methoxy group in the spectra of both compounds (the signal of this group is missing in the ¹H and ¹³C NMR spectra). Assignments of the chemical shifts in compounds 11 and 12 were made employing gHSQC and gHMBC experiments. 1D NMR experiments (¹H and ¹³C) revealed the fact that the structures contain two aromatic rings, one of these 1,2,4-trisubstituted and the second one 1,4-disubstituted. Furthermore, the compounds incorporate a nitrogen atom to which the methyl group, benzyl group and 2-phenylethyl group are attached. The correlations of the hydrogen atoms of the N-methyl group to the carbon of the benzyl group and carbon of the 2-phenylethyl group can be clearly observed in the gHMBC experiment (see Fig. 2). Thus, the NMR spectra of compounds 11 and 12 are similar to those of belladine.⁴ They differ only by the absence of one methoxy group in both of the structures. The signal of this group is missing in both the ¹H and ¹³C NMR spectra. The assignments of chemical shifts in compounds 11 and 12 were made employing gHSQC and gHMBC experiments. Important correlations obtained from the gHMBC experiment are shown in Fig. 2. The chemical shifts of both compounds are very similar (see Table 1). There are differences in the shifts of carbons C6 and C4′ and in the adjacent carbon atoms. In the case of compound 11 there is a correlation between hydrogens of the methoxy group and carbon C6 (δ = 158.3 ppm). Further correlation in the gHMBC experiment indicates the presence of a second methoxy group at carbon C5′ (δ = 146.9 ppm). Thus, carbon C4′ bears only a hydroxy group. In the case of compound 12, gHMBC experiment indicates correlations between hydrogens of the methoxy groups and carbons C4′ (δ = 149.1 ppm) and C5′ (δ = 148.7 ppm) leading to the conclusion that the methoxy group is missing at carbon C6. The chemical shifts of compounds 11 and 12 are summarized in Table 1.

Fig. 2 Key gHMBC correlation of new compounds isolated from Nerine bowdenii.

Table 1 ¹H- and ¹³C-NMR data of new belladine-derivatives 11 and 12 isolated from N. bowdenii

Position	11		12
	^1H	^13C	^1H	^13C
1	2.89–2.82 m	57.8	2.86–2.73 m	58.3
2	2.98–2.91 m	31.3	2.86–2.73 m	31.6
3	—	130.4	—	130.4
4	7.13–7.09 m	129.7	7.00–6.94 m	129.7
5	6.86–6.81 m	114.0	6.76–6.72 m	115.5
6	—	158.3	—	155.0
7	6.86–6.81 m	114.1	6.76–6.72 m	115.5
8	7.13–7.09 m	129.7	7.00–6.94 m	129.7
1′	3.80–3.77 m	60.5	3.72–3.67 m	61.1
2′	—	126.7	—	128.4
3′	7.00–6.97 m	116.3	7.00–6.94 m	112.6
4′	—	145.8	—	149.1
5′	—	146.9	—	148.7
6′	6.86–6.81 m	110.8	6.86–6.79 m	110.8
7′	6.92–6.88 m	121.9	6.86–6.79 m	122.0
6-OCH3	3.79 s	55.3	—	—
4′-OCH3	—	—	3.85 s	55.9
5′-OCH3	3.90 s	56.0	3.88 s	55.9
N-CH3	2.48 s	40.6	2.40 s	41.0

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All isolated compounds obtained in sufficient amounts were assayed for their HuAChE, HuBuChE and POP inhibition activities. Galantamine hydrobromide and huperzine A were used as positive controls in the HuAChE and HuBuChE assays, and Z-Pro-prolinal and berberine in the POP assay. The results, expressed as IC₅₀ values, are summarized in Table 2. The most active alkaloids in the HuAChE assay were two crinine-type alkaloids, undulatine and powelline, with IC₅₀ values of 23.5 ± 1.2 μM and 29.1 ± 1.6 μM, respectively. Undulatine has been reported previously as an important AChE inhibitor, which acts via a mixed inhibition mechanism, and, on the basis of the parallel artificial permeation assay (PAMPA) for the prediction of blood–brain barrier (BBB) method, is able to cross the BBB by passive permeation. Additionally, undulatine (2) has shown interesting POP inhibition activity.^3,26 Contrary to previous reports (IC₅₀ = 0.96 ± 0.04 μM – identical results in both reports),^27,28 we found 1-O-acetyllycorine to be inactive in the HuAChE inhibition assay (IC₅₀ > 1000 μM). Based on the conflicting results, we decided to perform the same experiments with commercially available acetylcholinesterase from electric eel to compare the IC₅₀ value of 1-O-acetyllycorine with reported data. In these experiments (six replications), 1-O-acetyllycorine showed moderate inhibitory activity with an IC₅₀ value of 28.4 ± 0.35 μM. A similar situation has been described with the isoquinoline alkaloid protopine; in the HuAChE inhibition assay protopine was inactive (IC₅₀ = 423 ± 10 μM),²⁹ in contrast to results in the assay with commercially available acetylcholinesterase from electric eel (IC₅₀ = 16.1 μM).³⁰ It seems that the source of enzyme is, in some cases, crucial for the determination of AChE inhibition activity of tested compounds. For determination of the inhibition mechanism of 1-O-acetyllycorine, a Lineweaver–Burk plot was used.³¹ From the results obtained it could be concluded that 1-O-acetyllycorine acts via a mixed inhibition mechanism. The kinetic analysis of AChE inhibition is shown in Fig. 3. The K_m and V_m values were calculated from the Lineweaver–Burk plot. The values of K_m and V_m for the reaction in the presence of 1-O-acetyllycorine were decreased compared with the values for the reaction in its absence (Fig. 3). In the BuChE inhibition assay we obtained similar data for each type of BuChE (IC₅₀ = 176.2 ± 14.2 μM for HuBuChE and IC₅₀ = 93.6 ± 5.2 μM for horse serum BuChE). The most potent HuBuChE inhibition activity has been demonstrated by the newly identified alkaloid 4′-O-demethylbelladine (IC₅₀ = 30.7 ± 4.0 μM). The other tested alkaloids showed only weak or no inhibition activity in the HuAChE and HuBuChE assays.

Table 2 HuAChE, HuBuChE, and POP inhibitory activity of the tested Amaryllidaceae alkaloids isolated from Nerine bowdenii expressed as IC₅₀

Compound	HuAChE	HuBuChE	POP
	IC50^a (μM)	IC50^a (μM)	IC50^a (mM)
a Results are the mean values ± standard deviations of three independent replications.b nm: not measured due to limited material.c Standard.d In higher concentrations turbidity was formed, could not be accurately determined the IC50 value.
Belladine (1)	699.2 ± 19.4	315.3 ± 10.5	>100
Acetylcaranine (2)	443.7 ± 62.4	141.2 ± 12.6	0.65 ± 0.04
Undulatine (3)	23.5 ± 1.2	>1000	1.97 ± 0.12
Caranine (4)	>1000	187.6 ± 51.3	1.99 ± 0.33
11-O-Acetylambelline (5)	>1000	>1000	>0.79^d
Buphanidrine (6)	72.6 ± 8.2	>1000	0.37 ± 0.04
Buphanisine (7)	99.2 ± 4.2	>1000	>100
Ambelline (8)	169.2 ± 7.4	985.4 ± 25.6	>100
Deacetylbowdesine (9)	427.9 ± 31.4	>1000	0.79 ± 0.06
Buphanamine (10)	236.5 ± 32.3	626.2 ± 67.9	3.11 ± 0.36
6-O-Demethylbelladine (11)	223.2 ± 23.6	115.7 ± 10.1	0.66 ± 0.09
4′-O-Demethylbelladine (12)	606.8 ± 74.2	30.7 ± 4.0	0.37 ± 0.03
Tazettine (13)	>1000	>1000	>100
1,2β-epoxyambelline (14)	nm^b	nm^b	nm^b
6α-Hydroxyundulatine (15)	>1000	624.8 ± 95.0	nm^b
1-O-Acetylbulbisine (16)	84.8 ± 11.0	481.7 ± 84.1	2.45 ± 0.21
Haemanthamine (17)	>1000	>1000	>100
1-O-Acetyllycorine (18)	>1000	176.2 ± 14.2	0.45 ± 0.05
Crinamidine (19)	230.1 ± 9.8	>1000	0.79 ± 0.06
Hamayne (20)	992.7 ± 220.7	472.0 ± 37.0	>100
Crinine (21)	>1000	770.0 ± 46.9	1.47 ± 0.12
Powelline (22)	29.1 ± 1.6	394.0 ± 4.8	0.77 ± 0.02
Galantamine^c	1.7 ± 0.1	42.3 ± 1.3	>100
Berberine^c	—	—	0.14 ± 0.02

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Fig. 3 Lineweaver–Burk plot of 1/v vs. 1/[acetylthiocholine] in the presence or absence of 1-O-acetyllycorine.

As mentioned, inhibition of POP can represent an important supporting approach in AD treatment, and, therefore, we tested all isolated compounds on their ability to inhibit POP. The most interesting inhibition activity has been demonstrated by 4′-O-demethylbelladine (belladine type), buphanidrine (crinine type) and 1-O-acetyllycorine (lycorine type), with IC₅₀ values of 0.37 ± 0.03 mM, 0.37 ± 0.04 mM, and 0.45 ± 0.05 mM, respectively (Table 2). Some of Amaryllidaceae alkaloids have been previously tested for their POP inhibition activity; the best results have been shown by the lycorine type alkaloid 9-O-demethylgalanthine (IC₅₀ = 150 ± 20 μM) isolated from Zephyranthes robusta.³² In comparison with the used standard isoquinoline alkaloid berberine, the best obtained activities are about three times weaker, but only a limited number of Amaryllidaceae alkaloids have been tested so far. The lycorine structure seems to be interesting for POP inhibition, but a wider range of compounds of either natural origin or semisynthetic analogues must be tested first.

Acetylcaranine (2), caranine (4), 11-O-acetylambelline (5), buphanisine (7), and crinine (21) were also screened for their cytotoxic activity against p53-mutated Caco-2 and HT-29 colorectal adenocarcinoma cells. At the same time, healthy small intestine cells were used to determine overall toxicity against noncancerous cells. The cytotoxicity of undulatine, haemanthamine, hamayne, ambelline, 1-O-acetylbulbisine, buphanamine, and tazettine has already been published in our previous report.² From the tested compounds, interesting cytotoxicity has been shown by the α-crinine-type alkaloid buphanisine, which showed interesting toxicity against both p53-mutated Caco-2 and HT-29 colorectal adenocarcinoma cells, while showing significantly lower toxicity against normal intestine FHs-74 Int cells (Table 3). The basic SAR of crinine-type alkaloids can be hypothesized from the study of McNulty et al.,³³ who screened a mini-library of crinane-type alkaloids for their ability to induce apoptosis in rat liver hepatoma (5123c) cells. In conformity to the results of McNulty, also in our previous study² the potent cytotoxic activity of crinine-type alkaloids is connected with the presence of an α-C2 bridge such as in haemanthamine and haemanthidine.² Alkaloids with a β-C2 bridge in their structure, such as in the tested buphanisine, showed no cytotoxicity except for a rare alkaloid, isolated from the bulb of Boophone disticha, which demonstrated significant antiproliferative activity in human acute lymphoblastic leukemia (CEM) cells³⁴ and potent cytotoxic effects against the human cervical adenocarcinoma (HeLa) cell line.³⁵

Table 3 Cytotoxicity of tested compounds against two cancer cell lines and one noncancerous gastrointestinal cell line

Compound	Cancer cells		Normal cells
	Caco-2	HT-29	FHs 74 Int
	IC50^a (μM)	IC50^a (μM)	IC50^a (μM)
a Results are the mean values ± standard deviations of three independent replications, NT – not tested.
Acetylcaranine (2)	29.5 ± 0.6	19.2 ± 1.2	66.1 ± 6.8
Caranine (4)	64.4 ± 4.5	46.6 ± 1.9	>100
11-O-Acetylambelline (5)	>100	>100	>100
Buphanisine (7)	8.6 ± 0.2	5.3 ± 1.7	22.8 ± 2.6
Crinine (21)	64.5 ± 17.8	50.8 ± 1.4	>100
Vinorelbine	0.03 ± 0.00	NT	4.0 ± 0.3

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Conclusions

In conclusion, two new compounds, 11 and 12, were isolated together with twenty known Amaryllidaceae alkaloids from fresh bulbs of Nerine bowdenii. This plant is a rich source of diverse Amaryllidaceae alkaloids, especially of the crinine type, with important biological activities. Compounds isolated in sufficient amount were screened for their biological activities connected with Alzheimer's disease and oncological diseases. Some alkaloids were isolated in amounts that will allow detailed study of their mechanism of action and also preparation of new derivatives for biological assays.

Experimental

General experimental procedures

NMR spectra were recorded for CDCl₃ and CD₃OD solutions at ambient temperature on a VNMR S500 NMR (Varian) spectrometer operating at 500 MHz for ¹H and 125 MHz for ¹³C. Chemical shifts were recorded as δ values in parts per million (ppm), and were indirectly referenced to tetramethylsilane (TMS) via the solvent signal (7.26 ppm for ¹H and 77.0 ppm for ¹³C for CDCl₃, and 3.30 ppm for ¹H and 49.0 ppm for ¹³C for CD₃OD). Coupling constants (J) are given in Hz. For unambiguous assignment of ¹H and ¹³C signals 2D NMR spectra (COSY, gHSQC, gHMBC and ROESY) were measured using standard parameter settings and pulse programs delivered by the producer of the spectrometer. ESI-HRMS were obtained with a Waters Synapt G7-Si with a hybrid mass analyzer quadrupole-time-of-flight (Q-TOF), coupled to a Waters Acquity I-Class UHPLC system. The EI-MS were obtained on an Agilent 7890A GC 5975 inert MSD operating in EI mode at 70 eV (Agilent Technologies, Santa Clara, CA, USA). A DB-5 column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, USA) was used. The temperature program was: 100–180 °C at 15 °C min⁻¹, 1 min hold at 180 °C, and 180–300 °C at 5 °C min⁻¹ and 5 min hold at 300 °C; detection range m/z 40–600. The injector temperature was 280 °C. The flow-rate of carrier gas (helium) was 0.8 mL min⁻¹. A split ratio of 1 : 15 was used. TLC was carried out on Merck precoated silica gel 60 F₂₅₄ plates, and neutral Al₂O₃ (ACROSS) was used for CC. Compounds on the plate were observed under UV light (254 and 366 nm) and visualized by spraying with Dragendorff's reagent.

The authors declare that all experimental procedures were undertaken in accordance with the Czech guidelines for the care and use of farm, experimental animals and live subjects and were performed under the supervision of Ethical Committee of the Charles University in Prague, Faculty of Pharmacy in Hradec Králové (Protection of Animals from Cruelty Act No. 246/92, Czech Republic). The informed consent was obtained from volunteer Dr Jakub Chlebek (member of authors team).

Plant materials

The fresh bulbs of Nerine bowdenii Watson were obtained from the herbal dealer Lukon Glads (Sadská, Czech Republic). Botanical identification was performed by Prof. L. Opletal. A voucher specimen is deposited in the Herbarium of the Faculty of Pharmacy in Hradec Králové under number: CUFPH-16130/AL-254.

Extraction and isolation of alkaloids

Fresh bulbs (10 kg) were minced and exhaustively extracted with ethanol (EtOH) (96%, v/v, 3×) by boiling for 30 min under reflux; the combined extract was filtered and evaporated to dryness under reduced pressure. The crude extract (425 g) was acidified to pH 1.5 with 2% hydrochloric acid (HCl; 2.5 L), filtered, the filtrate defatted with diethyl ether (Et₂O; 3 × 3.5 L), alkalized to pH 10 with a 25% solution of ammonia and exhaustively extracted with ethyl acetate (3 × 3.5 L). The organic layer was evaporated to give 51 g of fluid residue. The obtained extract, which was Dragendorff positive, was further fractionated by CC on Al₂O₃ (2500 g), eluting with light petrol gradually enriched with CHCl₃ (20 : 80–10 : 90), and then CHCl₃ enriched with EtOH (99 : 1–50 : 50). Fractions of 500 mL were collected and monitored by TLC, yielding 157 fractions, which were combined into 8 fractions, and analyzed by GC-MS.

Preparative TLC (cHx : To : DEA 48 : 48 : 4; 2×) of fraction I (2.483 g) gave belladine (1, 2.104 g). Fraction II (4.285 g) was further chromatographed by preparative TLC (To : DEA 95 : 5; 1×) to give acetylcaranine (2; 235 mg) and undulatine (3; 1.571 g); both compounds were recrystallized from an ethanol and chloroform mixture. Preparative TLC of fraction III (6.598 g) (cHx : acetone : NH₄OH 30 : 60 : 2; 1×) gave 4 sub-fractions IIIa–d. Caranine (4; 339 mg) and 11-O-acetylambelline (5; 24 mg) were obtained by subsequent TLC of sub-fraction IIIc (cHx : EtOAc : DEA 90 : 5 : 5; 1×). Sub-fraction IIId was further chromatographed by preparative TLC (cHx : EtOAc : DEA 90 : 5 : 5; 3×) to give buphanidrine (6; 784 mg) and buphanisine (7; 971 mg). Fraction IV (7.275 g) was crystallized from EtOH yielding 3.16 g of ambelline (8). The mother liquor of fraction IV was further treated by preparative TLC (cHx : To : EtOH : DEA 50 : 40 : 5 : 5; 3×) to give deacetylbowdensine (9; 14 mg) and buphanamine (10; 108 mg), which was crystallized from EtOH. Preparative TLC (cHx : To : DEA 45 : 45 : 10; 3×) of fraction V (1.387 g) led to separation of 3 sub-fractions Va–Vc. Sub-fraction Va was chromatographed to yield 4′-O-demethylbelladine (11; 12 mg) and 6-O-demethylbelladine (12; 18 mg). Sub-fraction Vb gave tazettine (13, 17 mg), epoxyambelline (14; 4.28 mg) and 6-hydroxyundulatine (15; 12 mg). Fraction VI (4.524 g) was treated by preparative TLC (cHx : acetone : NH₄OH, 30 : 60 : 2; 2×); 3 zones were isolated. Sub-fraction VIb (647 mg) was subsequently chromatographed (To : EtOAc : DEA 55 : 40 : 5; 1×) and gave 1-O-acetylbulbisine (16; 468 mg). Recrystallization of sub-fraction VIc (1.025 g) gave haemanthamine (17; 652 mg). Fraction VII (1.024 g) was treated by preparative TLC chromatography (cHx : acetone : NH₄OH, 30 : 60 : 2; 3×) to give 1-O-acetyllycorine (18; 39 mg), crinamidine (19; 65 mg) and hamayne (20; 398 mg). Preparative TLC (cHx : acetone : NH₄OH, 30 : 60 : 2; 1× and subsequently cHx : EtOAc : DEA 80 : 10 : 10; 2×) of fraction VIII (1.086 g) yielded crinine (21; 768 mg) and powelline (22; 32 mg).

Preparation of enzymes for HuAChE, HuBuChE assays

Enzymes were prepared from freshly drawn blood (taken from healthy volunteers), to which 2 mL 3.4% sodium citrate (w/v) per 18 mL blood was added, according to Steck and Kant,³⁶ with slight modification. Briefly, plasma (HuBuChE) was removed from the whole blood by centrifugation at 4000 rpm in a Boeco U-32R centrifuge fitted with a Hettich 1611 rotor. Red blood cells were transferred to 50 mL tubes and washed 3 times with 5 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride (centrifugation under same conditions). The washed erythrocytes were stirred with 5 mM phosphate buffer (pH 7.4) for 10 min to ensure lysis. The lysed cells were dispensed for subsequent measurement. Activity of each enzyme preparation was measured immediately after preparation and adjusted with 5 mM phosphate buffer (pH 7.4) to reach activity of blank sample A = 0.08–0.15 for AChE and A = 0.15–0.20 for BuChE.

HuAChE and HuBuChE assay

HuAChE and HuBuChE activities were determined using a modified method of Ellman³⁷ with ATChI and BuTChI as substrates, respectively. Briefly, 8.3 μL of either blood cell lysate or plasma dilutions (at least 6 different concentrations), 283 μL of 5 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) and 8.3 μL of either the sample dilution in dimethyl sulfoxide (DMSO) (40 mM, 10 mM, 4 mM, 1 mM, 0.4 mM and 0 mM) were added to the semi-micro cuvette. The reaction was initiated by addition of 33.3 μL 10 mM substrate (ATChI or BuTChI). The final proportion of DTNB and substrate was 1 : 1. The increase of absorbance (ΔA) at 436 nm for AChE and 412 nm for BuChE was measured for 1 min at 37 °C using a spectrophotometer (Synergy™ HT Multi-Detection Microplate Reader). Each measurement was repeated 6 times for every concentration of enzyme preparation. The % inhibition was calculated according to the formula: , where ΔA_Bl is the increase of absorbance of the blank sample and ΔA_Sa is the increase of absorbance of the measured sample. Inhibition potency of the tested compounds was expressed as IC₅₀ value (concentration of inhibitor, which causes 50% cholinesterase inhibition).

Inhibition mechanism of AChE

The procedure for determination of the inhibition mechanism was similar to that for determination of IC₅₀, with a difference in that uninhibited and inhibited reactions were observed for three different concentrations of acetylthiocholine (20 μM, 40 μM, 60 μM). The dependence of absorbance (412 nm) vs. time was measured and the reaction rate was calculated for all reactions (uninhibited and inhibited). Then, a Lineweaver–Burk plot was constructed and K_m and V_m values were calculated. Each measurement was performed in duplicate.

Prolyloligopeptidase assay

Prolyl oligopeptidase (POP; EC 3.4.21.26) was dissolved in phosphate buffered saline (PBS; 0.01 M Na/K phosphate buffer, pH 7.4, containing 137 mM NaCl and 2.7 mM KCl); the specific activity of the enzyme was 0.2 U mL⁻¹. The assay was performed in standard polystyrene 96-well microplates with a flat and clear bottom. Stock solutions of tested compounds were prepared in dimethyl sulfoxide (DMSO; 10 mM). Dilutions (10⁻³ to 10⁻⁷ M) were prepared from the stock solution with deionized H₂O; the control was performed with the same DMSO concentration. POP substrate, (Z)-Gly-Pro-p-nitroanilide, was dissolved in 50% 1,4-dioxane (5 mM). For each reaction, PBS (170 μL), tested compound (5 μL), and POP (5 μL) were incubated for 5 min at 37 °C. Then, substrate (20 μL) was added and the microplate was incubated for 30 min at 37 °C. The formation of p-nitroanilide, directly proportional to the POP activity, was measured spectrophotometrically at 405 nm using a microplate ELISA reader (Multi-mode microplate reader Synergy 2, BioTek Instruments Inc., Vermont, USA). Inhibition potency of tested compounds was expressed as IC₅₀ value (concentration of inhibitor which causes 50% POP inhibition).

MTT cytotoxicity assay

Cell viability was measured using the MTT cytotoxicity assay originally developed by Mosmann.³⁸ The assay for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) involves the quantification of viability of cells by the ability of living cells' mitochondrial succinic dehydrogenase to reduce the yellow dye MTT to a blue insoluble product formazan. Briefly Caco-2, HT-29 and FHs-74 Int cells were seeded in 96-well plates at a density of 2.5 × 10³ cells per well. After 24 h, the cells were treated with two-fold serial diluted alkaloids (0.01–100 μM mL⁻¹ Caco-2, HT-29 and 3.12–100 μM mL⁻¹ FHs-74 Int) for 72 h. At the end of incubation, the spent medium in each well was replaced by fresh DMEM medium containing MTT (Sigma-Aldrich, St. Louis, MO, USA) reagent (1 mg mL⁻¹) and plates were incubated for an additional 2 h at 37 °C. Two hours later, the culture supernatants were aspirated and the formazan product was dissolved in 100 μL of DMSO (Sigma-Aldrich, St. Louis, MO, USA). The absorbance was then measured at 555 nm using a Tecan Infinite M200 spectrometer (Tecan Group, Männedorf, Switzerland) and the % mortality for concentrations of each alkaloid were plotted and used to determine the 50% inhibitory concentration (IC₅₀ value). Statistical analysis was performed using Magellan™ software (Tecan Group, Männedorf, Switzerland) and Microsoft Office Excel 2003 (Microsoft, Redmond, WA, USA), from the data of three different experiments. Vinorelbine ditartrate salt hydrate (Sigma-Aldrich, Prague, Czech-Republic) was used as positive control in experiments.

Acknowledgements

This project was supported by grants SVV UK 260 292, Charles University grant No. 17/2012/UNCE, Ministry of Defense of the Czech Republic – “Long-term organization development plan 1011”, and Grant Agency of the Czech University of Life Sciences Prague CIGA 20132035.

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