A New Amide From Piper maingayi Hk.F. (Piperaceae)

Abstract

Phytochemical investigation from the leaves and stem extracts of Piper maingayi Hk.F. resulted in the isolation of a new amide, N-isobutyl-15-(3″,4″-methylenedioxyphenyl)-2E,4E,12Z-pentadecatrienamide (1), together with sesamin (2), piperumbellactam A (3), cepharadione A (4), cepharadione B (5), piperolactam A (6), oleic acid (7), methyl linolenate (8), β-sitosterol (9), butyl dodecanoate (10), and isovanillic acid (11). Their structures were established by the analysis of their spectroscopic (1D and 2D NMR) and spectrometric (MS) data, as well as by comparison with those reported in the literature. Compound (1) exhibited strong inhibition against lipoxygenase (15-LOX) at IC₅₀ 42.5 μM and antibacterial activity toward Bacillus subtillis and Pseudomas aeruginosa with minimum inhibitory concentration (MIC) value of 250 μg/mL each.

Piper is the largest genus in the Piperaceae family. This pantropical genus is estimated to contain 2000 species dispersed widely in American and Asian tropic including Indian, Indonesian, and Malaysian tropical rainforest. ¹ Most species of Piper appeared to be restricted to altitudes ranging from 0 to 2500 m, and very few occurred above 3000 m which grow in wet and shaded places. ^1,2 This genus is usually erect or scandent herbs, shrubs, or infrequently trees. ^3,4 The structure is rather uniform morphologically, with simple alternate leaves and joined stems with enlarged nodes and possessed aromatic or pungent smell. Many produce pearl bodies on the leaves or stems, but the most distinctive morphological feature is the production of inflorescences of tiny seeds packed into upright or pendant spikes. ⁵ It has provided many past and present civilizations with a source of medicines and food spices. The well-known species as stated previously, Piper betle, Piper nigrum, and Piper sarmentosum, had been brought up to the highest level of usage in perfumery and herbal products. Chemical studies carried out on Piperaceae species have revealed the presence of several compounds including alkaloids/amides, propenylphenols, lignans, neolignanas, terpenes, steroids, kawapyrones, piperolides, chalcones, dihydrochalcones, flavones, flavanones, and miscellaneous compounds. ³ Amide derivatives are frequently isolated from Piper species, and many of them exhibit diverse biological activities, such as antimicrobial, antitubercular, antitumor, and cytotoxicity. ^{6 -9}

A study on the essential oil of Piper maingayi leaf has been reported by Sirat et al. ¹⁰ Our first study on the fruits and stem essential oils obtained from P. maingayi has resulted in the identification of 34 and 18 components accounting for 83.6% and 78.7% of the total amount, respectively. The main constituents of stems essential oil were β-caryophyllene (26.2%), while the fruits essential oil was dominated by δ-cadinene (22.6%) and β-caryophyllene (18.8%). The stem and fruit oils were found to be effective scavengers of the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical with an IC₅₀ values at 12.6 and 13.9 µg/mL and 2,2-diphenyl-1-picrylhydrazyl assay with IC₅₀ 14.9 and 20.8 µg/mL, respectively. The tyrosinase inhibition activity (1 mg/mL) was found to be significant for stem oil (65.5 ± 0.2%), but lower than the positive control, kojic acid (91.3 ± 0.1%). ¹¹ With regard to the phytochemicals investigation, only one short communication on the phytochemicals of P. maingayi has been published by Ahmad et al. ¹² Thus far, in the aspect of biological activity, none has been reported on P. maingayi. The paucity of the studies on the biological active metabolites of P. maingayi justify our exploration of the phytochemical constituents from this species in an attempt to enhance the acceptability of herbal medicines for the development of pharmaceutical and herbal formulation documentations.

Compound (1) was isolated as a white amorphous solid (7.3 mg) with m.p. 132°C to 137°C from stem n-hexane extract (PMSH) fractions. The thin-layer chromatography (TLC) plate showed a dark violet spot after spraying with vanilin-sulfuric acid reagent. The IR spectrum revealed the characteristic stretching frequencies of dienamide grouping at 3298 cm⁻¹ (N-H stretch), 1654 cm⁻¹ (C=O), and 1628 and 1610 cm⁻¹ (C=C olefinic). Compound (1) exhibited distinctive signals in the ¹H NMR spectra for a long-chain amide with a significant N-isobutyl amide moiety which were observed at δ 0.94 (6H, d, J = 6.8 Hz, H-3′ and H-4′), 1.82 (1H, m, H-2′), 3.16 (2H, t, J = 6.8 Hz, H-1′), and 5.53 (N-H, 1 H, br.s). The presence of 3 sets of olefinic protons attributed for 2 trans-olefinic and 1 for cis-olefinic protons was also revealed in the ¹H NMR spectrum. The trans-olefinic protons of H-2 resonated at δ 5.77 were observed as doublet (15.2 Hz) which coupled with H-3 (7.21, dd, J = 15.2, 10.0 Hz). The alternate trans-olefinic of H-4 (6.12, dd, J = 15.2, 10.0 Hz) and H-5 (6.07, dd, J = 15.2, 6.4 Hz) were also observed. An olefinic bond represented by H-12 and H-13 was verified at δ 5.43 (2H, dt, J = 8.4, 4.8 Hz). The small coupling constant (8.4 Hz) concluded the third double bond as cis-relationship. The reported chemical shifts of H-12 and H-13 of the trans-olefinic of the isomeric compound were observed as the doublet of triplets of 15.0 and 10.0 Hz at δ 5.41. ¹³ The long-chain methylenes attributed for H-8 to H-10 were observed at δ 1.30 integrated as 6 H. The aromatic attached protons of H-15 were resonated at δ 2.60 (t, J = 7.2 Hz) which were coupled with H-14 (m) at δ 2.27. The aromatic system displayed as a tri-substituted pattern represented by proton H-6″ which was resonated at δ 6.63 with doublet of doublets (J = 8.0, 1.6 Hz) and ortho-coupled with H-5″ at δ 6.73 (d, J = 8.0 Hz) and meta-coupled with H-2″ at δ 6.69 (d, J = 1.6 Hz). A total of 25 signals were observed in the ¹³C NMR spectrum and DEPT spectra. Characterization of 2 methyl carbons at δ 20.1 (C-3′ and C-4′), as well as 6 methine signals attributed to 3 olefinic bonds, were observed at δ 121.8 (C-2), 143.1 (C-3), 128.2 (C-4), 141.3 (C-5), 129.3 (C-12), and 131.1 (C-13). The carbonyl carbon was ascribed at δ 166.4. In addition, the presence of methylenedioxy carbon appeared at δ 100.7 with methine aromatic carbons at δ 108.0 (C-2″), 108.9 (C-5″), and 121.1 (C-6″), correspondingly. Three quaternary carbons of the aromatic system were observed at δ 136.1 (C-1″), 147.4 (C-3″), and 145.5 (C-4″).

The assignments of the olefinic protons were further substantiated by the COSY spectrum. Thus, signals for olefinic protons of H-3 (δ 7.21) were coupled with its neighboring proton of H-4 (δ 6.12) which then coupled with H-2 (δ 5.77). Meanwhile, H-1′ was also observed to couple with proton of amide system (N-H, δ 5.53) and H-2′ at δ 1.82. The cis-olefinic protons which were assigned to H-12 and H-13 were observed to couple with H-14 (δ 2.28). Thus, the coupling pattern of this isobutylamide was consistent with the proton and carbon assignation position. The HMQC spectrum showed direct correlation of the assigned H-2 (δ 5.77) with C-2 (δ 121.8), H-3 (δ 7.21) with C-3 (δ 143.1), H-4 (δ 6.12) with C-4 (δ 128.2), and H-5 (δ 6.07) with C-5 (δ 141.3). Meanwhile, the cis-olefinic H-12 and H-13 were also observed to have direct correlation with individual carbon signal at δ 129.3 (C-12) and 131.1 (C-13), respectively. The HMBC spectrum reconfirmed the position of cis-olefinic protons H-12 and H-13 because C-12 was observed to possess a strong correlation with H-14 and H-11. Meanwhile, H-15 was also observed to have a long distance correlation with quaternary C-3″ (δ 147.4) and C-12 (δ 129.3), while H-6 (δ 2.16) was found to correlate with C-4 (δ 128.2) and C-5 (δ 141.3). The methyl group at H-3′ and H-4′ were observed to correlate with C-2′ (δ 28.6) and C-1′ (δ 46.9). Meanwhile, H-1′ (δ 3.16) was correlated with J ³ distance with C=O unsaturated carbonyl carbon (δ 166.4). The mass spectrum showed a peak corresponding to the molecular formula C₂₆H₃₇NO₃, based on the [M]⁺ ion signal at m/z 411.2789 (calc. 411.2785). ¹³

The NMR data (Table 1) of compound (1) were compared with the reported N-isobutyl-15-(18,19-methylenedioxyphenyl)-2E,4E,12E-pentadecatri-enamide from Piper ridleyi which had a double bond possessing trans configuration at H-12. ¹³ Thus, from the published discussed data, compound (1) was assigned as N-isobutyl-15-(3″,4″-methylenedioxyphenyl)-2E,4E,12Z-pentadecatrienamide (Figure 1). This compound was characterized as a new compound and has not been published elsewhere. Compound (1) showed a moderate antibacterial activity with minimum inhibitory concentration (MIC) value of 250 µg/mL against Bacillus subtilis and Pseudomonas aeruginosa. Study on anti-inflammatory activity was carried out using 15-LOX enzymatic assay. Compound (1) exhibited the strongest inhibition against 15-LOX at IC₅₀ 42.52 µM.

Figure 1

Chemical structure of compound (1).

Table 1

NMR Data for Compound (1).

Carbon	δ H a	δ C b
2	5.77 (1H, d, J = 15.2 Hz)	121.8
3	7.21 (1H, dd, J = 15.2, 10.0 Hz)	143.1
4	6.12 (1H, dd, J = 15.2, 10.0 Hz)	128.2
5	6.07 (1H, dt, J = 15.2, 6.4 Hz)	141.3
6	2.16 (2H, m)	32.9
7	1.39-1.48 (2H, m)	28.8-29.4
8-10	1.28-1.36 (6H, m)	28.8-29.4
11	1.98 (2H, m)	32.5
12	5.43 (2H, dt, J = 8.4, 4.8 Hz)	129.3
13	5.43 (2H, dt, J = 8.4, 4.8 Hz)	131.1
14	2.27 (2H, m)	34.7
15	2.60 (2H, t, J = 7.2 Hz)	35.9
1″	-	136.1
2″	6.69 (1H, d, J = 1.6 Hz)	108.0
3″	-	147.4
4″	-	145.5
5″	6.73 (1H, d, J = 8.0 Hz)	108.9
6″	6.63 (1H, dd, J = 8.0, 1.6 Hz)	121.1
1′	3.16 (2H, t, J = 6.5 Hz)	46.9
2′	1.82 (1H, m, J = 6.8 Hz)	28.6
3′	0.94 (3H, d, J = 6.7 Hz)	20.1
4′	0.94 (3H, d, J = 6.7 Hz)	20.1
NH	5.53 (1H, br.s)	-
OCH2O	5.93 (2H, s)	100.7
C = O	-	166.4

^aExperimental data measured at 400 MHz in CDCl₃

^bExperimental data measured at 100 MHz in CDCl₃

Experimental

Plant Materials

Piper maingayi was collected from Hutan Simpan Fraser, Fraser Hill, Perak, in January 2014, and identified by Shamsul Khamis from Institute of Biosience (IBS), Universiti Putra Malaysia (UPM). The voucher specimens (SK2329/14) were deposited at the Herbarium of IBS, UPM.

Extraction and Isolation

The dried leaves (697 g) and stems (539 g) were sequentially subjected to cold extraction with n-hexane (5.0 L), EtOAc (5.0 L), and MeOH (5.0 L) successively. The n-hexane extract (PMLH) (9.34 g), EtOAc extract (PMLE) (17.43 g), and MeOH extract (PMLM) (6.95 g) of leaves were fractionated using vacuum liquid chromatography (VLC) (70.0 g silica, column size 6.0 cm × 4.0 cm). PMLH and PMLE were fractionated and eluted with n-hexane, CHCl₃, EtOAc, and MeOH by increasing polarity to give 28 and 17 fractions. Fractionation of PMLH gave 3 combined fractions, F 2-12, F 8-23, and F 24-28, whereas PMLE were pooled into 5 fractions, F 1-3, F 5-9, F 10-11, F 12-13, and F 14-17. The fractions were combined based on TLC profiles. Fraction F 2-12 was subjected to column chromatography (CC) afforded oleic acid (7) (yellowish oil; 51.3 mg). Fraction F 8-23 was recrystallized to afford methyl linolenate (8) (yellowish oil; 32.4 mg) and fraction F 24-28 was subjected to repetitive CC to afford sesamin (2) (colorless needle; 12.4 mg). The combined fraction F 1-3 from PMLE was crystallized to afford β-sitosterol (9) (white crystalline needle; 11.8 mg); while fraction F 5-9 has afforded butyl dodecanoate (10) (white amorphous; 7.9 mg) and sesamin (2) by repetitive CC, Fraction F 10-11 was subjected to CC to afford oleic acid (7); Fraction F 12-13 has afforded isovanillic acid (11) (white solid; 5.1 mg); and fraction F 14-17 was subjected to repetitive CC to afford piperumbellactam A (3) (yellow needle; 0.9 mg), and cepharadione A (4) (orange needle; 3.1 mg). Application of recycle-HPLC of complex mixture from fraction F 14-17 has led to the isolation of 2 compounds which were cepharadione B (5) (light orange needle; 0.6 mg) and piperolactam A (6). Fractionation of the n-hexane stem extract, PMSH (8.81 g), from the stems of P. maingayi with n-hexane-CHCl₃/CHCl₃-EtOAc using VLC (70 g silica, column size 6.0 cm × 4.0 cm) afforded 12 fractions and fractions with similar TLC profiles were combined to yield 2 fractions F 6-9 and F 10-12. Fraction F 6-9 was subjected to recycle-HPLC with CHCl₃ as eluent to yield methyl linolenate (8). Fraction F 10-12 was subjected to CC to afford sesamin (2), while subfraction F 133-136 which was combined from CC of F 10-12 was subjected to Versa Flash in n-hexane:CHCl₃ as eluents to give (1). VLC fractionation (70 g silica, column size 6.0 cm × 4.0 cm) has afforded 21 fractions. Fraction F 10-12 was subjected to CC to afford subfraction F 80-103 which was recrystallized to afford piperumbellactam A (3). Fraction F 13-21 which was fractionated using Versa Flash in n-hexane:CHCl₃ has yield isovanillic acid (11).

Antibacterial—MIC

Minimum inhibition concentration was verified by using micro broth dilution technique. ^14,15 This test was conducted in a sterile 96-well round bottom microplate. The compound was diluted in dimethylsulfoxide (DMSO) to obtain a sample stock solution with a concentration of 1 mg/mL. The bacteria (50 µL) was added to the sample solution (50 µL) with concentration ranging from 1000 to 7.81 µg/mL obtained from 2-fold serial dilution with nutrient broth (NB). The microplates were then incubated for 24 hours at 37°C for Staphylococcus aureus, Escherichia coli, and P. aeruginosa and 30°C for B. subtilis. Streptomycin sulfate (0.1 mg/mL) was employed as a positive control in this assay and was treated similar to the sample. Inoculum viability wells that contain bacteria (50 µL) and NB (50 µL) supplemented with Tween 80 (0.02% v/v) and sterility control wells that contain only the sample (50 µL) in 2-fold dilution were reserved in each microplate. 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium (INT) staining method was chosen to evaluate the growth of bacteria in the wells. INT solution (0.2 mg/mL) was prepared freshly and should be kept away from light. After 24 hours of incubation, INT solution (25 µL) was added to all wells followed by incubation for at least 30 minutes. Bacteria growth in the well was indicated by the formation of reddish-pink color, while clear well indicated the inhibition of bacterial growth by the sample.

Lipoxygenase (15-LOX) Inhibitory Activity

The reagents were prepared according to the standard protocol (Lipoxygenase inhibitor screening assay kit, item no. 760 700 Cayman Chemicals Co., Ann Arbor, MI). ¹⁶ Stock solutions of sample were prepared to obtain the concentrations of 1000 to 62.5 μg/mL in the respective wells. The prepared solutions were then introduced onto 96-well plates where the cells were distributed as blank 1A-2A-1D (triplicate), positive control 1B-2B (duplicate), and 100% initial activity wells 1C-2C-2D (triplicate). The remaining wells were designated for inhibitor (compound) solutions in duplicate. The addition of the reagents was done according to the standard protocol in which 100 µL assay buffer was added to the blank wells, whereas 90 µL lipoxygenase (15-LOX) enzyme and 10 µL assay buffer were added to the positive control wells. About 90 µL lipoxygenase enzyme and 10 µL solvent (DMSO) were added to wells with 100% initial activity. The inhibitor (compound) wells were charged with 90 µL lipoxygenase enzyme and 10 µL respective stock (compound) solutions. The reaction was initiated by adding 10 µL of the substrate (AA) to all wells. The plate was then shaken for 5 minutes on an orbital shaker. Ultimately, 100 µL of chromogen solution (prepared according to the standard protocol) was added to each well to stop enzyme catalysis. The plate was incubated for 30 minutes and was read at 500 nm. The percentage inhibition (I%) of the compound was calculated using the following equation:

IA_inhibitorA_initial

where A _initial is the absorbance of 100% initial activity wells without sample and A _inhibitor is the absorbance of compound/reference. Analyses were expressed as mean±SD of triplicates.

Statistical Analysis

Data obtained from biological activities are expressed as mean values. Statistical analyses were carried out by employing one-way ANOVA (P > 0.05). A statistical package (SPSS version 11.0) was used for the data analysis.

N-Isobutyl-15-(3″,4″-methylenedioxyphenyl)-2E,4E,12Z-pentadecatrienamide (1)

White amorphous.

Rf: 0.35 (n-Hexane: CHCl₃; 3:2).

IR (KBr): 3298, 1654, 1628, 1610 cm⁻¹.

¹H (400 MHz, CDCl₃) and ¹³C NMR (100 MHz, CDCl₃): Table 1. HR-EI-MS: m/z 411.2789 [M]⁺ ; calcd for C₂₆H₃₇NO₃, 411.2785. Spectroscopic data was available in Supplementary Material.

Supplemental Material

Supplementary data - Supplemental material for A New Amide From Piper maingayi Hk.F. (Piperaceae)

Supplemental material, Supplementary data, for A New Amide From Piper maingayi Hk.F. (Piperaceae) by Nur Athirah Hashim, Farediah Ahmad, Wan Mohd Nuzul Hakimi Wan Salleh, and Shamsul Khamis in Natural Product Communications