Punigratane, a novel pyrrolidine alkaloid from Punica granatum rind with putative efflux inhibition activity

Abstract A new pyrrolidine alkaloid named Punigratane was isolated from the rind of Punica granatum. This is the first report of a pyrrolidine-like structure from the rind. The activity of this compound was tested in a representative MDR Klebsiella pneumoniae strain which exhibited high efflux pump activity. At a concentration of 6 mg, this compound Punigratane was found to have efflux inhibition activity.


Introduction
Bacterial multidrug resistance (MDR) is a critical issue in health management leading to escalated costs and high morbidity (WHO report 2014). One of the major contributing factors to MDR is increased efflux activity of the bacterial membrane.
Use of efflux pump inhibitors (EPI) is an attractive strategy to combat this problem as it can be applied to a broad spectrum of antibiotics.
CCCP (carbonyl cyanide m-chlorophenylhydrazone) and PAβN (phenyl-arginine β-naphthylamide) are well-characterized EPI's of the RND efflux pump (specific to Gram negative bacteria) but cannot be commercially used due to their toxicity (Aparna et al. 2014).
Efflux pump inhibition activity has been reported from various plant extracts. Putative NorA EPI of Staphylococcus aureus have been identified from natural sources -reserpine and berberine alkaloids, flavones from Artemisia annua, phenolic metabolites from Dalea versicolor (Stavri et al. 2007). Pyrrolidine ring containing compounds like 2,8-dimethyl-4-(2′-pyrrolidinoethyl)-oxyquinoline have been found to act as potential RND efflux inhibitors (Chevalier et al. 2004). However, no inhibitor has been released for commercial use.
Punica granatum, belonging to the family lythraceae, is a fruit that is rich in compounds like tannins, alkaloids and phenolics that have a number of biological activities (Salah El Dine et al. 2014;Maione et al. 2015). P. granatum extracts are known to have antibacterial activity (Dey et al. 2012). While studies have shown that P. granatum extracts have the ability to inhibit NorA efflux pumps in S. aureus (Braga et al. 2005), single compounds that can act as EPIs are yet to be discovered.
This study describes the bioactivity-guided separation of the P. granatum rind extract resulting in the identification of Punigratane, a new pyrrolidine derivative with potential efflux pump inhibition activity. This is the first report of a pyrrolidine derivative from the rind of P. granatum with potential RND pump inhibition activity.

Results and discussion
The rind of P. granatum Bhagwa variety (1.5 kg) was shade dried, ground and macerated with ethanol to yield a crude ethanol extract. This extract was sequentially extracted with hexane, chloroform and butanol. The butanol fraction (9.0 g) which had the highest efflux inhibition activity was subjected to repeated column chromatography over silica gel and was further purified by preparative TlC to obtain a pure compound Punigratane (60 mg) ( Figure  1). The structure was identified and confirmed to be 2,5-diheptyl-N-methylpyrrolidine by IR, MS and NMR spectroscopic techniques.
Punigratane had a molecular formula of C 19 H 39 N, determined by HR-EI-MS: m/z 281.1098 [M+] (calculated 281.30844), indicating one degree of unsaturation or one ring. The odd molecular mass indicated presence of odd number of nitrogen. The IR spectrum showed absence of hydroxyl, carbonyl and aromatic system. 13 C NMR in CDCl 3 displayed only nine signals which led to presume a symmetry in the structure in order to account for the molecular weight. The absence of peaks above 60 ppm indicated the absence of aromatic rings or any unsaturation. δ at 14.12 indicated the presence of Methyl groups (CH 3 ) and δ at 59.54 indicated the presence of Methine groups (CH). The δ values from 22 to 31 indicate the presence of multiple methylene groups. The methyl group attached to the electronegative nitrogen atom showed a peak at δ 38.16. The 1 H NMR spectroscopic data displayed signals for terminal methyl groups at δ 0.87 (t, 6H, J = 7.0 Hz, H-11, 18) and the methyl group attached to nitrogen was shifted to δ 2.37 (s, 3H). The CH peak near the nitrogen of the pyrrolidine ring was found at δ 2.66 (m, 2H). The structure was further confirmed by simulation of 1 H and 13 C NMR using Chemdraw and an open source software nmrdb (Steinbeck et al. 2003;Banfi & Patiny 2008).
Pyrrolidines like hygrine and norhygrine have been found in the bark of the root of P. granatum (Wang et al. 2010). This is the first report of pyrrolidine containing compound from the rind of P. granatum. Structurally similar compounds like 2,5-dialkyl pyrrolidines have been identified in venomous ants of Solenopsis and Monomorium spp (Blum 1984). These compounds have been found to have insecticidal activity. 2-Butyl-5-heptylpyrrolidine produced by Streptomyces longispororuber was shown to act as a potent sigma receptor ligand (Kumagai et al. 2000). This is the first report of bacterial efflux inhibition activity by 2,5-diheptyl-N-methylpyrrolidine (Punigratane). The efflux pump inhibition assay was carried out in a representative MDR Klebsiella pneumoniae strain U25 that exhibited high efflux pump activity. In this assay, the accumulation of norfloxacin within the cell is estimated; an increased norfloxacin accumulation within the bacterial cell on addition of a compound, indicates EPI activity. On addition of the compound Punigratane (6 mg), an increase in norfloxacin accumulation was noted in the cell (Figure 2). Therefore, we hypothesise that the compound Punigratane is a putative efflux pump inhibitor.

Extraction and isolation
The rind of P. granatum variety Bhagwa was obtained from M/S 'Sam Agritech, Hyderabad' . The product was aseptically separated and shade dried. The dried material (1.5 kg) was ground, sieved and macerated with ethanol in the ratio 1:3 and kept for 5 days with intermittent shaking. The extract was filtered with Whatmann number 1 filter paper (125 mm) and collected. It was then placed in a rotary vacuum evaporator and concentrated under pressure to give 100 g of a viscous material. 90 g of this crude extract was then dissolved in water and sequential extraction was carried out resulting in hexane, butanol and water fractions which were concentrated in rotary vacuum evaporator (Shukla et al. 2010). 9 g of the butanol fraction was loaded onto a silica gel column (Hi-media Silica gel 60-200 mesh,  3 × 100 cm) and eluted with solvent system (Ethyl acetate:Dicholoromethane:Methanol -2.5:2:0.4). Fraction 130-140 which had the best activity was further separated to obtain pure compound Punigratane. It was further ascertained to be a single spot through GCMS and TlC. Structure of the compound was ascertained by IR (Perkin-Elmer Spectrum RX-I FT-IR spectrometer in the range of 4000-400 cm −1 ), Mass (JEOl GC Mate spectrometer) and NMR spectroscopy (500 MHz FT NMR Spectrometer-BRUKER AV III) as 2,5-diheptyl-N-methylpyrrolidine.

Norfloxacin accumulation assay
Efflux inhibition activity was determined by the norfloxacin accumulation assay in a high effluxing K. pneumoniae strain U25 (li et al. 2002). This bacterial strain was isolated from a urine sample from a tertiary care hospital in Chennai, India. The strain was confirmed to be multidrug-resistant strain as per the Clinical and laboratory Standards Institute guidelines. The bacterial strain was grown in the lB broth supplemented with 40 mM potassium lactate for 12 h at 37 °C. Bacterial pellets were washed with 20 ml of 0.2 M MOPS Tris buffer (pH 7.0) containing 10 mM MgSO4. After centrifugation at 10,000 rpm for 3 min at 4 °C, the washed bacterial pellets were suspended in the same buffer to 50 mg (wet weight)/ml. The assay mixture contained cells (10 mg (wet weight)/ml) in the same buffer and 10 mM potassium lactate. After incubation in shaker incubator at 37 °C for 5 min, 3 ml of the sample was collected and kept in ice. To the remaining 27 ml of the assay mixture, 270 μl of 10 mM norfloxacin (100 mM, final concentration) was added to initiate the assay. Samples (3 ml each) were taken at 5 min intervals up to 45 min, centrifuged at 10,000 rpm for 3 min at 4 °C, and washed once with the same buffer. After 15 min of initiating, 1 ml of 6 mg Punigratane was added to the assay mixture. The pellet was suspended in 3 ml of 100 mM glycine-HCl (pH 3.0). The suspension was shaken vigorously for 1 h at room temperature to release their fluorescent contents and then centrifuged at 15,000 rpm for 10 min at room temperature. The fluorescence of supernatants was measured (EX-excitation at 281 nm and EM-emission at 440 nm) with a Shimadzu RF-5301pc fluorescence spectrophotometer.

Conclusion
Identification of EPI from natural sources is a prime research area. This is the first report of a novel pyrrolidine alkaloid from the rind of P. granatum that has a putative efflux inhibition activity. This compound can be used as an adjuvant with antibiotics and can contribute to the battle against MDR.

Supplementary Material
Experimental details related to this article along with spectroscopic analysis and structure elucidation are available online (see supplementary data online only).