Novel Amides Derivative with Antimicrobial Activity of Piper betle var. nigra Leaves from Indonesia

Abstract

Piper betle var. nigra is a tropical plant closely related to the common piper. P. betle has also been dubbed a promising source of natural antioxidants in herbal health products, antibacterial, antifungal, antimalarial, cytotoxic activity against the cancer cell lines K562 and HL-60, and antileishmanial. The aim of this study to observation Antimicrobial activity and isolation of chemical compound. The antimicrobial activity of P. betle extract was performed by well diffusion method against two oral pathogenic bacteria (Streptococcus mutans and Streptococcus sanguinis) and opportunistic pathogenic yeast (Candida albicans). The inoculum (bacterial and yeast suspension) was prepared from a 24-h culture on NB for bacterial suspension and on TSB for yeast suspension. Extraction and isolation using various method of chromatography. Isolated compounds were characterized by spectroscopic means. Our study showed antimicrobial activity from crude ethanol extract of leaves P. betle L. var. nigra against two oral pathogenic bacteria and opportunistic pathogenic yeast with concentration 0.5% and 1%. The first report of two new amides derivatives, piperenamide A (1) and piperenamide B (2) in P. betle L. var. nigra.

1. Introduction

Piper betle a piper species have a simple profile contain very diverse suites of secondary metabolites and responsible for their use in traditional medicines to treat several disease [1]. Piper betle L. var. nigra or black betle (in Indonesia known as Sirih Hitam) is a tropical plant closely related to the common piper and belongs to the Piperaceae family and the genus of piper. This genus consists of five subgenera and approximately 1400 species spread throughout tropical and subtropical regions and widely cultivated in Indonesia, India, Sri Lanka, Malaysia, Thailand, Taiwan, and other Southeast Asian countries and has a long history of over 2000 years. This plant use for decoration and medicinal plants. P. betle is one of the most potent medicinal herbs that has been used over the years. In addition to the large number of beneficial properties, P. betle has also been dubbed a promising source of natural antioxidants in herbal health products, antibacterial, antifungal, antimalarial, cytotoxic activity against the cancer cell lines K562 and HL-60, and antileishmanial [2,3,4,5,6,7,8]. Chemical composition from Piper betle L. var. nigra included caryophyllene, cadinene, γ-lactone, allyl catechol, p-cymene, eugenol methyl ether, 4-allyl resorcinol, stigmast-4-en-3,6-dione, and aristololactam A-II, and essential oils such as chavicol, chavibetol, chavabetyl acetate, eugenol, eugenyl acetate, safrole, (E) Isoeugenol, and B-caryophyllen, pipercerebrosides A and B, and amides alkaloid [8,9,10]. Amides as a class of typical constituents. More than 300 members of amide alkaloids are already found in species of the genus Piper. It seems most of them have possible bioactivity, such as antifungal, antiepileptic, antidepressant, hepatoprotective, and antiplatelet aggregation activities [11]. Isobutyl amides are one of the most frequently known classes o the amides in the plants. These amides are primarily found as long chain conjugates in piper genus. Arboreumine, pellitorine, fagaramide, brachystamides-C, D, E, retrofractamide-D, N-isobutyl-4-hexanoyl-hydroxypyrrolidin-1-one, (±)-threo-N-isobutyl-4,5-dihydroxy-2E-octaenamide, scutifoliamide A, B, hoffmannseggiamide A, B, and cyclopipperettine [12,13].

In this study we reported the isolation and elucidation of the chemical structures two novel amide derivatives compound and evaluated the antimicrobial activity effect of the crude ethanol extract of Piper betle var. nigra leaves from Indonesia. Antimicrobial assay to be carried out for the activity of ethanol extract from P. betel leaves against the activities of antimicrobial against two oral pathogenic bacteria (Streptococcus mutans and Streptococcus sanguinis) and opportunistic pathogenic yeast (Candida albicans).

2. Results and Discussion

2.1. Antimicrobial Activity

Table 1. Growth inhibition diameters of Candida albicans, Streptococcus mutans, and Streptococcus sanguinis by Piper betle var. nigra extract.

Microbial	Inhibition Diameters ± SD (mm)
	Extract Concentration of 0.5%	Extract Concentration of 1%
Candida albicans	16.7 ± 0.8	18.1 ± 0.7
Streptococcus mutans	18.2 ± 1.8	19.6 ± 1.9
Streptococcus sanguinis	9.9 ± 1.8	12.3 ± 1.1

shows the antibacterial activity of P. betle extract against two oral pathogenic bacteria and an oral opportunistic fungal. P. betle extract in all concentration from 0.5% to 1% inhibited growth of S. mutans, S. sanguinis, and C. albicans with increasing diameters of inhibition observed with increasing concentration of P. betle extract (Figure 1). S. mutans was the most sensitive strain compared to S. sanguinis and C. albicans against P. betle extract (0.5%) with inhibition diameters of 18.2 mm, 9.9mm, and 16.7 mm, respectively. The difference in sensitivity of these organisms is probably due to different cell types. Candida (yeast) cell types are different from streptococci (bacteria). This causes differences in sensitivity to P. betle extract. Sensitivity of S. mutans are more susceptible than S. sanguinis. The possible causes are many factors, including differences in virulence. In 2015, Azizi et al. assed the sensitivity of S. mutans and S. sanguinis to Zingiber officinale. They showed that S. mutans more susceptible than S. sanguinis with MIC value of 0.02 and 0.3 mg/mL, respectively [14]. In addition, their sensitivity to chlorhexidine were also reported by Medina-Flores et al. (2016), where S. mutans more susceptible than S. sanguinis with growth inhibition 23.97 mm and 19.80 mm, respectively. Alkaloid, terpenoid, flavonoid, polyphenols, tannin, and saponin compounds have been identified from P. betle leaf extract [15]. Flavonoids, polyphenols, and tannins are known to have antibacterial activity with at least five possible mechanisms: damage the cell membrane permeability, inhibit protein synthesis, damage the bacterial cell wall, inhibit ATP synthesis, and interfere with cell [16]. The antibacterial activity that we obtained is thought to be derived from the activity of the isolate compounds of Piperamide A and B was isolated from Piper betle var. nigra. This is as has been reported from other plants of the Piper genus because of the similarity in structure to the compounds we obtained although further testing is needed to ensure the strength of the antibacterial activity of these compounds [17,18,19].

Table 2. Growth inhibition diameters of Candida albicans, Streptococcus mutans, and Streptococcus sanguinis by Piper betle var. nigra isolate piperamide A and B.

Microbial	Inhibition Diameters ± SD (mm)
	Piperamide A	Piperamide B
	Concentration of 0.02%	Concentration of 0.02%
Candida albicans	-	-
Streptococcus mutans	-	-
Streptococcus sanguinis	-	-

shows none of the tested samples of Piperamide A and B showed antimicrobial activity against S. mutans, S. sanguinis, and C. albicans up to 0.02%. These result from amides derivative showed the same results in tests for cyclopipperetine on the Piper nigrum [13]. In recent years, Candida species has demonstrated resistance to many synthetic medications, indicating the need for new antifungal drugs with less side effects to treat candidiasis effectively. Several experiments used natural substances against multiresistant strains and in particular against azole resistant candidiasis have indicated that certain species of plants have promising antimicrobial compounds from natural substances [20,21,22].

2.2. Isolation of Crude Ethanol Extracf of Piper betle var. nigra

The EtOH extract from the leaf of P. betle L. var. nigra was chromatographed over a vacuum-liquid chromatographed (VLC) column, packed with silica gel 60 by gradient elution. The VLC fractions were repeatedly subjected to normal and reverse phase column chromatography, as well as preparative TLC on silica gel GF₂₅₄ to accommodate compounds 1–2.

Piperenamide A (1) was observed as a colorless amorphous solid, with its molecular composition established as C₁₈H₂₃NO₃, based on HR-TOFMS. This showed a [M + H]⁺ ion peak at m/z 302.1749 (calcd. C₁₈H₂₄NO₃ m/z 302.1756), requiring to eight degrees of unsaturation (Figure 2). The UV spectrum showed maximum absorption at 270 and 241 nm, indicating the presence of a benzene and conjugated alkene. The IR spectrum showed bands which were ascribed to an amine (ν_max 3295 cm⁻¹), amide carbonyl (ν_max 1656 cm⁻¹), isolated double bond conjugated (ν_max 1611 and 1504 cm⁻¹), and ether groups (ν_max 1241 cm⁻¹). Furthermore, the ¹H-NMR spectrum (

Table 3. NMR data compound 1–2 (500 MHz for ¹H dan 125 MHz for ¹³C).

Position Carbon	1		2
	13C-NMR	1H-NMR	13C-NMR	1H-NMR
	δC (mult.)	δC [(ΣH, mult, J(Hz)]	δC (ppm)	δC [(ΣH, mult, J(Hz)]
1	169.1 (s)	-	168.4 (s)	-
2	48.0 (t)	3.06 (2H, d, 6.7)	47.3 (t)	3.10 (2H, d, 6.7)
3	29.7 (d)	1.78 (1H, qt, 6.7)	28.9 (d)	1.82 (1H, qt, 6.7)
4	20.1 (q)	0.90 (3H, d, 6.7)	20.1 (q)	0.94 (3H, d, 6.7)
5	20.1 (q)	0.90 (3H, d, 6.7)	20.1 (q)	0.94 (3H, d, 6.7)
1′	123.3 (d)	5.92 (1H, dd, 4.6; 15.1)	122.6(d)	5.96 (1H, dd, 4.6; 15.1)
2′	141.9 (d)	7.02 (1H, dd, 10.6; 15.1)	141.2 (d)	7.12 (1H, dd, 10.6; 15.1)
3′	142.8 (d)	6.09 (1H, dd, 10.6; 15.2)	142.0 (d)	6.12 (1H, dd, 10.6; 15.2)
4′	130.2 (d)	6.18 (1H, dd, 7.2; 15.2)	129.6 (d)	6.19 (1H, dd, 7.2; 15.2)
5′	36.1 (t)	2.44 (2H, dd, 7.2; 7.5)	35.4 (t)	2.39 (2H, d, 7.2)
6′	35.9 (t)	2.66 (2H, t, 7.5)	-	-
1″	136.5 (s)	-	135.8 (s)	-
2″	109.7 (d)	6.69 (1H, d, 1.5)	109.8 (d)	6.73 (1H, d, 1.5)
3″	149.0 (s)	-	148.3 (s)	-
4″	147.9 (s)	-	146.4 (s)	-
5″	108.8 (t)	6.69 (1H, d, 7.9)	108.2 (t)	6.73 (1H, d, 7.9)
6″	122.3 (d)	6.63 (1H, dd, 1.5, 7.9)	121.6 (d)	6.68 (1H, dd, 1.5, 7.9)
OCH2O	102.0 (t)	5.88 (2H, s)	101.2 (t)	5.91 (2H, s)
NH	-	4.62 (1H, br.s)	-	4.65 (1H, br.s)

) showed two primary methyls at δ_H 0.90 (6H, d, 6.7 Hz, Me-4 and 5), four sp2 methine protons at δ_H 5.92 (1H, dd, J = 4.6; 15.1 Hz, H-1′), 7.02 (1H, dd, J = 10.6; 15.1 Hz, H-2′), 6.09 (1H, dd, 10.6; 15.2 Hz, H-3′), and 6.18 (1H, dd, 7.2; 15.2 Hz, H-4′), suggested the presence of an α,β,γ,δ-unsaturated secondary amide, two trans double bonds (J1′,2′ = 15.1 Hz, J_3′,4′ = 15.2 Hz), one cis double bonds (J_2′,3′ = 10.6 Hz), tri-substituted benzene at δ_H 6.63 (1H, dd, J = 1.5; 7.9 Hz, m; o, H-6″), 6.69 (1H, d, 1.5 Hz, m, H-2″), and 6.69 (1H, d, 7.9 Hz, m, H-5″), a methylene dioxyphenyl δ_H 5.88 (2H, s) and some aliphatic signals in the upfield region (Supplementary Materials). Comparison of the NMR data of 1 with those of guineensine [23] shows the same amide chain but with different N-substituent positions. The N-substituent of 1 position is attached to the olefin proton resonances, this is indicated δ_H 3.06 (2H, d, 6.7 Hz, H-2) no correlation to δ_H 4.62 (NH s), δ_H 5.92 (1H, dd, J = 4.6; 15.1 Hz, H-1′) and guineensine position is attached to the iso-butyl, this is indicated δ_H 3.16 (2H, t, 6.4; 12.9 Hz, H-1′) correlation to δ_H 5.60 (NH brs), δ_H 5.76 (1H, d, 15.0 Hz, H-2). The ¹³C NMR together with the DEPT spectra revealed eighteen carbons consisting of an amide carbonyl at δC 169.1, α,β,γ,δ-unsaturated secondary amide at δ_C 123.3, 141.9, 142.8 and 130.2, tri-substituted benzene at δ_C 136.5, 109.7, 149.0, 147.9, 147.9, 108.8, and 122.3, carbon methylene dioxyphenyl at δ_C 102.0, two methyls at δc 20.1 (Me-4) and 20.1 (Me-5). The ¹H-¹H COSY spectrum of compound 1 showed correlations in H2-H3-H4 and H5, H1′-H2′-H3′-H4′-H5′-H6′ and H5″-H6″, supporting the presence of a secondary amide [10]. The HMBC correlations from H-2 to C-1, C-3, C-4 and C-5, H-1′ to C-1 and C-3′, H-2′ to C-4, H-3′ to C-1 and C-5, H-4′ to C-2′ and C-6′, H-5′ to C-3′ and C-1″, H-6′ to C-4′, C-2″ and C-6″, H-2″ to C-4′, C-4″ and C-6″, H-5″ to C-1″ and C-3″, H-6″ to C-6′, C-2″ and C4″, and OCH₂O to C-3″ and 4″, which was verified by correlations observed in the ¹H-¹H COSY and HMBC spectra (Figure 3) Therefore, the structure of compound 1 was elucidated as the new amide and namely as piperenamide A (Figure 2).

Piperenamide B (2) was observed as a colorless amorphous solid, with its molecular composition established as C₁₇H₂₁NO₃, based on HR-TOFMS. This showed a [M + H]⁺ ion peak at m/z 288.1567 (calcd. C₁₇H₂₂NO₃ m/z 288.1521), requiring to eight degrees of unsaturation (Figure 2). The UV spectrum showed maximum absorption at 270 and 241 nm, indicating the presence of a benzene and conjugated alkene. The IR spectrum showed bands which were ascribed to an amine (ν_max 3235 cm⁻¹), amide carbonyl (νmax 1690 cm⁻¹), isolated double bond conjugated (ν_max 1610 and 1504 cm⁻¹), and ether groups (ν_max 1241 cm⁻¹). The NMR spectra of two was very similar to those of one (Table 3), except with lost of methylene at δ_H 2.66 (2H, t, 7.5 Hz, H-6′); δ_C 35.9, C-6′) and methylene at δ_H 2.44 (2H, dd, 7.2; 7.5 Hz, H-5′), while two, methylene at δ_H 2.39 (2H, d, 7.2 Hz, H-5′), indicating that 2 was a demethylene derivative of one. In the HMBC spectrum, methylene correlations from H-5′ to C-1″, C-2″ and C-6″, suggest methylene δ_H 2.39; δ_C 35.4, directly apply to benzene (Supplementary Materials). Therefore, leading to the structure of 2 had been elucidated as the new amide, namely as piperenamide B (Figure 2).

3. Materials and Methods

3.1. General Experimental Procedures

UV spectra was measured using a TECAN Infinite M200 pro, with MeOH. The IR spectra and mass spectra were recorded on a SHIMADZU IR Prestige-21 in KBr and Waters Xevo QTOF MS, respectively. Using an NMR JEOL ECZ-500 and Variant Unity INOVA-500 Spectrometer (Agilent Technologies, Santa Carla, CA, USA), the NMR data was recorded at 500 MHz for ¹H and 125 MHz for ¹³C, using TMS as internal standard. Column chromatography was conducted on the silica gel 60 (70–230 and 230–400 mesh, Merck, Kenilworth, NJ, USA), after which TLC analysis was carried out on 60 GF₂₅₄ (Merck, 0.25 mm) using various solvent systems, in order to detect spots by irradiating under ultraviolet-visible light (257 and 364 nm) and heating of silica gel plates, sprayed with H₂SO₄ in EtOH (10%).

3.2. Plant Material

The leaf of P. betle L. var. nigra were collected from Samarinda, East Kalimantan, Indonesia in June 2020. Futhermore, the plant was identified By Dr. Atik Retnowati a staff of the Bogoriense Herbarium, Bogor, Indonesia. Finally, a voucher specimen (No. 749/IPH.1.01/If.07/VII/2020) was deposited at the Herbarium.

3.3. Extraction and Isolation

Method for extraction and isolation was referred to supriatno et al. (2018) [24]. The dried ground leaf (473.21 g) of P. betle L. var nigra was extracted with ethanol 70% (14 L), at room temperature for 7 days. After removal of the solvent under vacuum, the viscous concentrated EtOH extract (14.61 g) was obtained. The EtOH extract (14.61 g) was fractionated by column chromatography on silica gel, using a gradient of n-hexane, EtOAc and MeOH (10% stepwise) resulting into eight fractions (A–H). Fraction D (2.32 g) was subjected to column chromatography on silica gel using n-hexane-CHCl₃-EtOAc (5% stepwise), as eluting solvents to afford seven subfractions (D1–D7). Subfraction D3 (882.2 mg) was chromatographed on a column of silica gel, eluted with n-hexane: EtOAc (7:3), to give seven subfractions (D3A–D3G). Similarly, subfraction D3D (120.3 mg) was chromatographed on silica gel eluted with n-hexane: CHCl₃: EtOAc (7:2:1), to give 1 (8.2 mg). Subfraction D3E (90.1 mg) was chromatographed on silica gel eluted with petroleum ether: CHCl₃ (4.5:5.5), to give 2 (2.8 mg) [24].

3.3.1. Piperenamide A (1)

Colorless amorphous solid; mp 185−190 °C; UV (MeOH) λmax (log ε) 270 (4.01) and 241 (3.91) nm; IR (KBr) vmax 3295, 1656, 1611, 1504, 1490, 1443, 1141 and 1030 cm⁻¹; HR-TOFMS m/z 302.1751 [M + H]⁺, (calcd. C₁₈H₂₃NO₃ m/z 301,1749); ¹H-NMR (CDCl₃, 500 MHz), see Table 1; ¹³C-NMR (CDCl₃, 125 MHz), see Table 1.

3.3.2. Piperenamide B (2)

Colorless amorphous solid; mp 199−211 °C; UV (MeOH) λ_max (log ε) 268 (3.91), and 219 (3.67) nm; IR (KBr) v_max 3235, 1690, 1610, 1504, 1480, 1443, 1141 and 1030 cm⁻¹; HR-TOFMS m/z 288.1567 (calcd. C₁₇H₂₂NO₃ m/z 288.1521) ¹H-NMR (CDCl₃, 500 MHz), see Table 1; ¹³C-NMR (CDCl₃, 125 MHz), see Table 1.

3.4. Antimicrobial Activity

3.4.1. Material

Liquid media for this study were nutrient broth (NB; Oxoid, Hampshire, UK) and tryptic soy broth (TSB; Merck). Solid media were Mueller Hinton Agar (MHA; Oxoid) and Tryptic soy agar (TSA; Merck)

3.4.2. Method

The antimicrobial activity of P. betle extract was performed by well diffusion method against two oral pathogenic bacteria (Streptococcus mutans, Streptococcus sanguinis) and opportunistic pathogenic yeast (Candida albicans). The inoculum (bacterial and yeast suspension) was prepared from a 24-h culture on NB for bacterial suspension and on TSB for yeast suspension. Each of inoculum was diluted with sterile physiological solution (0.9%) to 108 CFU/mL (McFarland standard 0.5). 20 mL of each agar media were melted (NA media for bacterial and TSA media for yeast), cooled to 50 °C and then inoculated with 0.2 mL of the microbial suspension. The inoculated agar was poured into sterile petri dish, and then allowed to cool down on a leveled surface. Once the media had compacted, two wells were cut out of the agar, each 6 mm in diameter. Then, 30 µL of the extract sample (concentration of 0.5 and 1%) were added into each well and incubated for 24 h at 36 °C ± 1°C under aerobic condition. Inhibition of microbial growth was measured in mm using SCAN 500^® tools. Tests were performed in duplicate.

4. Conclusions

The presents study showed antimicrobial activity from crude ethanol extract of leaves P. betle L. var. nigra. The first report of two new amides derivatives, piperenamide A (1) and piperenamide B (2) in P. betle L. var. nigra.