Antimicrobial alkaloids from the leaves of Pandanus amaryllifolius

Article history: Received on: 12/08/2015 Revised on: 03/09/2015 Accepted on: 23/09/2015 Available online: 28/10/2015 Chemical investigation on the crude base of the Pandanus amaryllifolius leaves led to the isolation and identification of pandamarilactone-1 (1), pandamarilactone-32 (2), pandamarilactonine-A (3), and pandamarilactonine-B (4). Their structures were elucidated based on 1H and 13C NMR and in comparison with the literature data. Compound 3 was found to be the most active among the four isolates with an MIC of 15.6 g/mL and MBC of 31.25 g/mL against Pseudomonas aeruginosa. This is the first report on the antimicrobial activity of the isolated alkaloids from the genus Pandanus.


INTRODUCTION
The family Pandanaceae or the screw pine family is composed of four monocotyledon genus: the Pandanus, Freycinetia, Sararanga, and Martellindendron (Callmander, 2001;Callmander et al., 2003).The largest among the four, the genus Pandanus of about 700 species, is prevalent in tropical and sub-tropical areas, especially on the Pacific islands, Malaysian islands and Australia.Several of the Pandanus species are recognized as medicinal plants and used in traditional medicines.The P. amaryllifolius commonly known as the fragrant screw pine because of its scented leaf is used to refresh the body, reduce fever, and relieve indigestion.The oil of the leaves is used as purgative, as a treatment for leprosy, stimulant and cures headaches and rheumatism (Cheeptham, 2002;Quisumbing, 1978).The root decoction showed hypoglycemic activity and 4hydroxybenzoic acid was identified as the active compound (Peungvicha et al., 1998).Phytochemical analysis on the leaves and roots of P. amaryllifolius had elaborated mostly the presence of alkaloids (Tan et al., 2010a;Tan et al., 2010b).However, there is a dearth on the pharmacological activity on the identified alkaloids.Based on the ethnopharmacological activities, the isolation and identification of alkaloids and their antimicrobial activities, including the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), will be reported in this paper.

The Plant Material
Fresh leaves of P. amaryllifolius was collected in March 2014 and identified by Miss Ophelia Laurente, botanist of the UST Herbarium.A voucher specimen with accession number USTH-3728 was deposited at the UST Herbarium.

Extraction and isolation
The air-dried and ground leaves (1.1 kg) were percolated with distilled MeOH for a total of 7 L for 3 consecutive days.The combined filtrate was concentrated under reduced pressure to obtain the crude extract (195 g).The crude extract was dissolved in 1M H 2 SO 4 and partitioned with DCM thrice.
The acid layer was basified to pH 8 with the addition of Na 2 CO 3 .The basified solution was extracted with DCM and the collected organic layers were concentrated under reduced pressure to obtain the crude alkaloidal extract.The crude alkaloidal extract was initially subjected to column chromatography using increasing increments of MeOH in CHCl 3 .TLC of the collected fractions resulted to five pooled fractions, PA-1 to PA-5.

Antimicrobial Assay
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the different extracts were determined using microwell assay.Extracts were diluted with DMSO to a concentration of 1 mg/ml, placed in microwells, then serially diluted (1:2) into 8 wells to a final volume of 100 L for each test organism.Three bacteria were used for the assay: S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853.A 100 L of bacterial suspension (1.5 x 10 8 CFU/mL) was added to each well and incubated at 37 o C for 24 hours.The concentration in the last well with no growth after 24 hours was reported as the minimum inhibitory concentration (MIC).
All wells with no growth were then subcultured into nutrient agar (NA) plates to determine the minimum bactericidal concentration.The lowest concentration of extract which did not show bacterial growth in the NA plates after 24 hours was reported as the minimum bactericidal concentration (MBC).All setups were done in triplicate for each extract.
The presence of piperidine- (Nonato et al., 1993), pyrrolidinone-(Sjaifullah andGarson, 1996), pyrrolidine- (Takayama et al., 2002), and indolizidine-type (Cheng et al., 2015) alkaloids have been previously identified from the genus Pandanus.However, no biological activity has been associated yet with those alkaloids.To address this fact and utilizing the ethnopharmacological activities associated with P. amaryllifolius, the crude base and the isolates have been subjected to antimicrobial assay using the microtiter plate diffusion method.Three organisms namely Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were used to determine the minimum inhibitory concentration (MIC) (Table 1) and minimum bactericidal concentration (MBC) (Table 2).Results indicated that pandamarilactonine-A (3) was found to be the most active among the four isolates.The crude base was also found to exhibit an activity against P. aeruginosa.

CONCLUSION
Re-investigation on the leaves of P. amaryllifolius had isolated the piperidine (1 and 2) and pyrrolidine (3 and 4) type alkaloids.This is the first report that their MIC and MBC antimicrobial activities have also been determined against three organisms Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853.

Table 1 :
Minimum Inhibitory Concentration (MIC) of the different isolates.

Table 2 :
Minimum Bactericidal Concentration (MBC) of the different isolates.