Caryota mitis Lour. palm belongs to Family Arecaceae that contains about 230 genera and 3000 species [1]. The plants of this Family are distributed in tropical and subtropical areas [1]. In folk medicine, the leaves and fruits of C. mitis Lour. are used for treatment of constipation, hemorrhoids, loss of virility, rheumatoid arthritis, vomiting and stomachache [2,3]. The chemical screening Tests on the leaves of C. mitis Lour. showed the presence of different chemical classes; steroids, triterpenoids, flavonoids, saponins and alkaloids [4]. Both piperidine and pyridine alkaloids such as; arecoline, guvacoline, nicotine and ethyl nicotinate were reported in the Family Arecaceae [5]. These alkaloids possess wide range of biological activities include antidepressant [6], anticonvulsant [7], hypoglycemic [8], salivary stimulant [9] and anthelmintic to eradicate worms [10]. In this study; eight of piperidine and pyridine alkaloids (Table 1) are first detected in the genus Caryota mitis Lour. by LC-MS/MS. The fraction of alkaloids showed antibacterial activity (MIC) as shown in Table 2.

Table 1: Retention times and mass spectral data of the chromatographic peaks depicted in Figure 1 obtained from (+)-ESI LC-MS spectral analysis of the constituents of the alkaloid fraction extract.

Table 2: Inhibitory activity (MIC) of the leave extract fractions of C. mitis Lour. at (20 μg/ml).

Materials and chemicals

The leaves of C. mitis Lour. were collected in June 2014 from El- Orman Botanical Garden, Giza, Egypt. The extraction of the alkaloids from the dried powdered leaves of C. mitis Lour. was performed in the Natural Product Chemistry Lab, Faculty of Pharmacy, Assiut University, Egypt. LC-MS and LC-MS/MS analyses of the alkaloid fraction were achieved in the Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, USA. All organic solvents were of HPLC grade and were purchased from Thermo Fisher (Hanover Park, IL, USA).

Extraction of alkaloids

Extraction of alkaloid rich fraction from C. mitis Lour. leaves were performed by acid base extraction method [11]. Ten grams of the dried powdered leaves were defatted by maceration in n-hexane followed by filtration. The marc was then soaked in 70% ethanol at 25°C for 2 hrs. The methanol extract was filtered using Whatman No.1 filter paper and concentrated by Rota vapor under reduced pressure at 40°C. The dried alkaloid fraction (100 mg) was mixed with 300 ml tartaric acid (2% W/V) then filtered. The aqueous filtrate was rendered alkaline at PH 10 by adding 20% NH₄OH. The alkaline aqueous solution was transferred into 1 L separating funnel containing equal amount of dichloromethane. The dichloromethane layer was collected after shaking, concentrated and confirmed for presence of alkaloids by Dragendorff reagent (repeated three time). This alkaloid rich fraction was subjected to LC-MS and LC-MS/MS analyses.

LC-MS and LC-MS/MS analysis

A ZORBAX Eclipse XDB-C18 column (Agilent Technologies, New Castle, DE, USA) (5 μm, 4.6 × 150 mm) was used for separation of the alkaloids. A gradient mobile phase consisting of (A) 0.1% formic acid in water and (B) methanol was used for chromatographic separation of alkaloids at 25°C with flow rate 0.6 mL/min. Identification of compounds was achieved by using an Agilent 6520 Q-TOF mass spectrometer equipped with a 1220 RRLC system (Agilent Technologies, Little Falls, DE, USA) and electrospray ion (ESI) source was used for detection of plant alkaloids. ESI-MS analysis was performed with a capillary voltage of 3200 and 3400 V, respectively [12]. MS/MS experiments were carried out under similar LC-MS conditions described above with collision energies of 20 eV.

Antibacterial and antifungal assays

Preparation of extracts for antimicrobial study: Fourty mg of each fraction (total extract, n-hexane, chloroform, n-butanol, aqueous and alkaloid fraction) were separately reconstituted in the least amount of mixture of DMSO (Dimethylsulfoxide) and Water (1:1% v/v) and the volume was completed to 2 ml with DMSO: H₂O (20 mg/ml). DMSO: H2O mixture served as a negative control.

Antibacterial assay: The inoculum size of each test strain was standardized according to the national committee for the clinical laboratory standard (NCCLS). Strains were grown in Mueller-Hinton broth or Mueller-Hinton agar medium at 37°C. A single isolated colony of each bacteria was picked from the agar plate culture and suspended in 0.9% (w/v) sterile aqueous saline solution to prepare bacterial suspensions of 0.5 McFarland standard and diluted to give a final concentration of 10⁸ CFU/ml. Antibacterial screening of C. mitis Lour. leaf extracts were investigated using agar well diffusion method [13,14]. Bacterial suspensions of either Staphylococcus aureus or E.coli strains were mixed with Sabouraud agar (20 ml) in sterile petri dishes (9 cm in diameter) and the agar plates were allowed to solidify. After solidification, wells were made in the agar plates using a poremaker of size 10 mm and filled with 100 μL of different extracts (20 mg/ml). Ampicillin (10 μg/ml) and Gentamicin (5 μg/ml) were used as positive controls, while DMSO:H₂O mixture (1:1) served as a negative control. Plates were incubated at 37°C for 24 h and the diameters of inhibition zone were measured using a digital caliber. Inhibition zone diameter was an indicator for the antibacterial activity of the studied solutions.

Antifungal activity: The antifungal activity of the tested extracts was evaluated by the agar dilution method [15]. Using Emmon's Sabouraud Dextrose Agar (ESDA) as a growth medium. Stock solutions of the test extracts were prepared at initial concentration of 20 mg/ml in DMSOWater mixture (1:1), and the reference standard antifungal drug (clotrimazole) was prepared in the same mixture at concentrations (0.1-100 mg/ml) and incorporated into the growth medium and plates were poured. ESDA incorporating only DMF was used as a negative control. Plates were inculated with 0.05 ml of the fungal suspension (approximately 5-10 conidia or hyphal element/ml 0.9% normal saline) and incubated at 30°C until macroscopically visible growth appeared in the control (48-96 hrs post inoculation).

Determination of the minimal inhibitory concentration (MIC): MIC of C. mitis Lour. leaf extracts were determined against Staphylococcus aureus and E. coli strains using microtiter broth dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2012). Sterile 96-well rounded bottom plates were used. Samples were vortexed before the experiment to ensure homogenous distribution of the particles. Mueller Hinton broth (100 μl) was dispensed into all wells of the microtiter plate. Each tested extract was added to the first well and 10 serial two-fold dilutions were made (20-0.039 mg/ml). Bacterial suspensions were added into all wells except for control wells. Plates were incubated in 37°C for 24 h and evaluated for the minimum inhibitory concentration by visual examination of the culture turbidity.

Identification of alkaloids

Eight different peaks appeared in the Extractive Ion Chromatogram (EIC) of C. mitis Lour. (Figure 1), the peaks were prescribed according to their retention times and were identified by LC-MS and LC-MS/MS analyses. Based on accurate mass measurement, molecular formula, double bond equivalent (DBE), MS/MS fragmentation (Table 1) and comparison with previously available MS data, alkaloids of C. mitis Lour. were identified.

Figure 1: Extractive ion chromatogram (EIC) of alkaloids fraction of Caryota mitis acquired in (+)-ESI LC-MS.

Compound 1 appeared at (tR=3.406 min) and displayed a protonated molecule at m/z 163.1231 [M+H]⁺ with molecular formula C10H14N2, DBE=5 supporting one cyclic ring and a pyridine nucleus. MS/MS fragmentation (Figure 2) showed peaks at m/z 134.0987 [M +H- 29.0244]+ empathized loss of N-CH₃ group, m/z 120.0814 [M+HNC₂H₅]⁺, m/z 106.0662 [M+H-C₃H₇N]⁺ and m/z 79.0556 for pyridinium cation [C₅H₄N]⁺ which confirmed the MS fragmentation pattern of nicotine [16] previously isolated from Nicotiana tabaccum L. [17] and this is the first report in the genus Caryota.

Figure 2: Tandem mass spectrometry fragmentation of nicotine (1), methyl N-methylpiperidine-3-carboxylate (2) and propyl N-methyl piperidine-3-carboxylate (3). ethyl N-methyl piperidine-3- carboxylate (4).

Compound 2 eluted at (tR=15.117 min) and displayed a protonated molecule at m/z 158.1168 [M+H]+ with molecular formula C₈H₁₅NO₂, DBE=2 supporting one cyclic ring and a carbonyl group. MS/MS fragmentation (Figure 2) showed peaks at m/z 143.0702 [M+H-CH3]+, m/z 99.1027 [M+H-59.0141]⁺ corresponding to loss of methyl carboxylate group and this confirmed the MS fragmentation pattern of methyl N-methyl piperidine-3-carboxylate previously detected in Areca catechu L. [5] and this is the first report in the genus Caryota.

Compound 3 with (tR=15.585 min) appeared as a protonated molecule at m/z 186.1484 [M+H]⁺ with molecular formula C₁₀H₁₉NO₂, DBE=2 expressed by presence of one cyclic ring and a carbonyl group. MS/MS fragmentation (Figure 2) showed peaks at m/z 171.0732 [M+H-CH₃]⁺, m/z 157.0836 [M+H-C₂H₅]⁺, m/z 143.0725 [M+H-C₃H₇]⁺, m/z 99.1020 [M+H-C₄H₇O₂]⁺ explaining loss of propyl carboxylate group. This MS fragmentation pattern helped us to identify the compound as propyl N-methyl piperidine-3-carboxylate suggesting it to be a new compound that is firstly isolated from the genus Caryota.

Compound 4 appeared at (tR=15.662 min) exhibited a protonated molecule at m/z 172.1326 [M+H]⁺ with molecular formula C₉H₁₇NO₂, DBE=2 confirmed the presence of one cyclic ring and a carbonyl group. MS/MS fragmentation (Figure 3) showed peaks at m/z 157.0828 [M+H-15.0498]⁺ with loss of methyl group, m/z 143.0715 [M+HC₂H₅⁺, m/z 99.1018 [M+H-C₃H₅O₂]⁺ corresponding to loss of ethyl carboxylate group and matched with MS fragmentation pattern of ethyl N-methyl piperidine-3-carboxylate previously detected in Areca catechu L. [5] and this is the first report in the genus Caryota .

Figure 3: Tandem mass spectrometry fragmentation of ethyl Nmethyl piperidine-3-carboxylate (4), guvacoline (5) and ethyl Nmethyl- l,2,5,6-tetrahydro-pyridine-3-carboxylate (6).

Compound 5: (tR=15.749 min) resulted in a protonated molecule at m/z 142.0858 [M+H]⁺ with molecular formula C₇H₁₁NO₂, DBE=3, which mean that one monounsaturated cyclic ring and a carbonyl group are present. MS/MS fragmentation (Figure 3) exhibited peaks at m/z 127.0745 [M+H-CH₃]⁺, m/z 83.0502 [M+H-59.0356]⁺ with loss of methyl carboxylate group which is similar to MS fragmentation of guvacoline previously identified in Areca catechu L. [5] and this is the first report in the genus Caryota.

Compound 6: eluted at (tR=15.850 min) showed a protonated molecule at m/z 170.1163 [M+H]⁺ with molecular formula C9H15NO2, DBE=3 supporting the presence of one monounsaturated cyclic ring and a carbonyl group. MS/MS fragmentation (Figure 3) showed peaks at m/z 141.1009 [M+H-29.0154]⁺ with loss of ethyl group, m/z 97.0679 [M+H-73.0484]⁺ with loss of ethyl carboxylate group, m/z 82.0654 [M +H-C₄H₈O₂]⁺ that confirm MS fragmentation of Ethyl N-methyl-l, 2,5,6-tetrahydro-pyridine-3-carboxylate previously identified in Areca catechu L.[5] and this is the first report in the genus Caryota.

Compound 7: (t_R=17.376 min) displayed a protonated molecule at m/z 156.1016 [M+H]⁺ with molecular formula C₈H₁₃NO₂, DBE=3 supporting the presence of one monounsaturated cyclic ring and a carbonyl group. MS/MS fragmentation (Figure 4) showed peaks at m/z 141.0917 [M+H- CH₃]⁺, m/z 97.0664 [M+H-59.0352]⁺ with loss of methyl carboxylate group, m/z 82.0663 [M+H-C₃H₆O₂]⁺ which is similar to MS fragmentation of arecoline previously detected in Areca catechu L. [5] and this is the first report in the genus Caryota.

Figure 4: Tandem mass spectrometry fragmentation of arecoline (7) and Ethyl nicotinate (8).

Compound 8: (tR=21.353 min) displayed a protonated molecule at m/z 152.0681 [M+H]⁺ with molecular formula C₈H₉NO₂, DBE=5 confirming one cyclic ring and a pyridine nucleus. MS/MS fragmentation (Figure 4) showed peaks at m/z 123.0486 [M+H-29.0195]⁺ with loss of ethyl group, m/z 79.0542 [M+H-73.0139]⁺ with loss of ethyl carboxylate group, which was in accordance with the MS fragmentation pattern of ethyl nicotinate previously identified in Areca catechu L. [5] and this is the first report in the genus Caryota.

Antibacterial and antifungal activities

The leaf different extracts including the alkaloid fraction showed promising antibacterial and antifungal activities as shown in Table 2. Gentamicin (5 μg/ml) and ampicillin (10 μg/ml) were used as reference antibiotics. All the tested bacterial and fungal strains showed different susceptibility to each extract. The n-hexane and chloroform fractions demonstrated weak activity against the tested strains with zone of inhibition range from 3-5 mm. The total and aqueous fractions exhibited moderate bactericidal activity against both S. aureus and E. coli relative to the positive controls and caused inhibition zone of 7-10 mm diameter (MIC; 2.5 and 5 mg/ml). The ethyl acetate fraction displayed the strongest bactericidal activity against S. aureus , relative to the positive control (Ampicillin) and caused inhibition of 20 mm diameter (MIC; 2.5 mg/ml), and also showed moderate activity against E. coli relative to control (gentamycin) and caused zone of inhibition of 11 mm diameter. Both n-butanol and alkaloids fractions demonstrated strong bactericidal activity against both S. aureus and E. coli relative to positive controls and exhibited inhibition zones of 17-19 and 14-18 mm diameters respectively, (MIC; 2.5 and 5 mg/ ml, respectively).

The results of the antifungal study against C. albicans strains revealed that the alkaloids fraction has powerful antifungal activity with zone of inhibition of (16 mm MIC; 2.5 mg/ ml) more than the nbutanol and aqueous fractions with zone of inhibition (12 mm MIC; 5 mg/ ml). The other extracts exhibited no antifungal activity.

Caryota mitis Lour. is a member of Family Arecaceae that biosynthesize alkaloids among their secondary metabolites. Using LCMS and LC-MS/MS analyses, eight alkaloids were identified and firstly reported in C. mitis according to their accurate mass measurement, molecular formula, double bond equivalent (DBE), MS/MS fragmentation (Table 1) and comparison with previously available MS data. Alkaloids fractions demonstrated strong antibacterial and antifungal activities against tested bacteria.

Authors are indebted to the Joint Supervision Program of Egypt (2013-2015) for financial support to A. M. Zaher at the Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, USA. Authors are thankful to Dr. Mohamed A Mokhtar, Department of Microbiology and Immunity, Faculty of Medicine, Assiut University, Assiut, Egypt for his great help in the antibacterial and antifungal assays.