A new derivative of ionone from aerial parts of Viola odorata Linn. and its antibacterial role against respiratory pathogens

Abstract

Background

Viola odorata, widely distributed in Eastern and Western Himalaya region is extensively used in treatment of various respiratory ailment, calculous affections and nervous disorders. It is a rich source of alkaloids, terpenes, flavonoids, glycosides, tannins as well as viola-quercitin and salicyclic acid known as natural aspirin. The present study was aimed to isolate bioactive compounds from the aerial parts of V. odorata.

Methods

The isolated compound from aerial parts of V. odorata was identified grounded on observed spectral data including ultraviolet–visible (UV–vis), Fourier-transform infrared, gas chromatography-mass spectroscopy, ¹H and ¹³C-nuclear magnetic resonance. The antibacterial efficacy of isolated compound was determined by disc diffusion method against five bacterial strains namely, Haemophilus influenzae (MTCC 3826), Pseudomonas aeruginosa (MTCC 2474), Staphylococcus aureus (MTCC 1144), Streptococcus pyogenes (MTCC 422) and Streptococcus pneumoniae (MTCC 655) and Minimum inhibitory concentrations (MICs) by a serial dilution method by using 96 well microtiter plates.

Results

Based on spectral data analysis, one new structurally related ionone-like compound known as 3-(2′,4′,6′,6′-tetramethylcyclohexa-1′,4′-dienyl)acrylic acid (1) has been isolated from aerial parts of V. odorata, with significant bioactivity. The antibacterial efficacy of compound 1 ranged between 7.3 ± 0.28 to 9.3 ± 0.28 mm against selected respiratory bacteria at 6 mg/disc concentration. The MICs were recorded at 32–128 μg/ml. Streptococcus pyogenes was fairly resistant and Haemophilus influenzae more sensitive than others.

Conclusions

By this study, it can be concluded that compound 1 has significant bioactive properties against tested microorganisms. It validates its use as a new potential source of natural drug for curing the respiratory diseases caused by selected microorganisms.

Background

Natural products from medicinal plants, either as pure compounds or as standardized extracts, provide a limitless opportunities for novel drugs discovery due to outstanding availability of chemical diversity. Viola odorata Linn. (Violaceae), commonly known as Sweet Violet, English Violet or Common Violet and Gulbanafsa in Hindi, is a medicinal herb natively from Mediterranean countries and Asia Minor. The genus Viola contains about 500 species worldwide. Currently, numerous species and varieties of violet is known and used for the extraction of their natural occurring phytoconstituents. V. odorata has excessive medicinal values. Its aerial parts were reported as antibacterial, antioxidant, anti-inflammatory [1–3], and in treatment of bronchitis, tonsillitis, respiratory catarrh, cough, asthma, calculous affections and nervous disorders [4, 5]. Recent studies have reported the presence of antibacterial cyclic proteins known as cyclotides, the positively charged bracelet cyclotide cycloviolacin O₂ (cyO₂) in V. odorata. It has been found efficient with killing properties against gram negative bacteria including Pseudomonas aeruginosa, Salmonella enterica serovar Typhimurium LT2 [6, 7].

The ionones, dihydroionones, ionols and epoxy ionones are some few representative group of compounds explored in violet flowers [8–10]. They are responsible for well natural fragrance with pleasant woody-floral, violet-ionone like scent characteristics [11]. Ionones and their derivatives are a member of terpene, the naturally occurring unsaturated hydrocarbons whose carbon skeletons are composed of isoprene C5 units. The isomers and its derivatives differ due to presence of double bond at 1 or 2 position, and shifting of methyl group in cyclohexane ring. They occur mainly in plant containing beta-carotene. Based on the close structurally related derivatives of ionone, they could be expected to exhibit similar biological activities.

In present study, qualitative chemical screening of aerial parts of V. odorata has led to isolation of compound 1 named as 3-(2′,4′,6′,6′-tetramethylcyclohexa-1′,4′-dienyl)acrylic acid, a new derivative of ionone, along with its antibacterial efficacy.

Methods

Chemicals and reagents

All the chemicals and solvents, silica gel for column chromatography of mesh 60–120, silica gel-G for TLC, and silica gel 60 F₂₅₄ (0.25 mm layer thickness pre-coated on aluminium back), and iodine resublimed were commercial and purchased from Merck, Aldrich and Fluka and were used as received. All the other solvents and chemical used were of HPLC or analytical grade.

Plant material and isolation procedure

The aerial parts of V. odorata were collected in April, 2012 from Haridwar and authenticated at Botanical Survey of India, Dehradun (BSD No. 113405). Powdered material (1.2 Kg) was extracted successively with petroleum ether (PET), acetone (ACE) and methanol (MeOH) by using Soxhlet apparatus. The yield of MeOH extract was 1.22 % w/w (14.6 g). The MeOH extract was fractionated by column chromatography (CC) on a silica gel (600 × 30 mm, 60–120 mesh size), and sequentially eluted with chloroform (CHCl₃)/ACE with a gradual increment of the CHCl₃ content (v/v 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, each 1000 ml), resulting in 11 lots (A-K) with each 20 fractions (A1-A20). Fraction G6-G20 and H1-H15 showed similar TLC pattern and purified by preparative TLC (prep-TLC) and finally at CHCl₃/MeOH/H₂O mixture (v/v, 75:17:8). A yellowish powdered compound 1 (21 mg) was obtained.

Instruments

UV–vis absorption spectra was recorded in the wavelength range 190–800 nm with Shimadzu UV-1800 series Pharmac-spec UV–vis spectrophotometer (Japan). The signals were acquired four times and the mean signals were taken as the best value of the UV spectra. Before every measurement the blank spectrum was also recorded, and automatically subtracted from the sample spectrum by the instrument software. The IR spectra were recorded in the wave number range 400–4000 cm⁻¹ on Varian 3100 FT-IR spectrophotometer (Perkin Elmer Spectrum Version 10.03.05). The GC-MS spectra were recorded on Varian Saturn (mass spec model). ¹H and ¹³C NMR spectra were recorded on JEOL AL 300 FT-NMR spectrometer. Chemical shifts are given as δ value with reference to tetramethylsilane (TMS) as the internal standard.

Spectroscopic data of compound 1

Yellow powdered compound; UV λ_max (MeOH) nm: 246.00, 202.00, 222.50; IR (KBr) ν_max: 3429.50, 2919.91, 2851.39, 1563.04, 1436.62, 1384.56, 1167.41, 1093.92, 880.64 cm⁻¹; GC-MS: m/z 207 [M + H]⁺ calcd: for C₁₃H₁₉O₂ (207.281); ¹H NMR (300 MHz, DMSO-d ₆ ): δ 7.613 (1H, d, J = 8.14 Hz, H-3), 6.532 (1H, d, J = 8.17 Hz, H-2), 5.406 (1H, s, H-5′), 2.436 (2H, s, H_a-3′, H_b-3′), 1.558 (3H, s, H_a-9′, H_b-9′, H_c-9′), 1.164 (3H, s, H_a-10′, H_b-10′, H_c-10′), 0.757 (6H, s, H_a-7′, H_b-7′, H_c-7′, H_a-8′, H_b-8′, H_c-8′), 3.330 (m, DMSO-d ₆ ); ¹³C NMR (75.45 MHz, DMSO-d ₆ ): δ 171.645 (C-1), 158.716 (C-3), 154.310 (C-5′), 130.834 (C-1´), 122.652 (C-2), 113.833 (C-4´), 107.423 (C-2´), 34.477 (C-3′), 34.405 (C-6′), 31.269 (C-7′), 28.969 (C-8′), 21.722 (C-9′), 21.431 (C-10′), 39.529 (DMSO-d ₆ ).

Antibacterial assay

Microorganisms used

The bacterial strains causing respiratory tract infections namely, Haemophilus influenzae (MTCC 3826), Pseudomonas aeruginosa (MTCC 2474), Staphylococcus aureus (MTCC 1144), Streptococcus pyogenes (MTCC 422) and Streptococcus pneumoniae (MTCC 655) were procured from Institute of Microbial Technology (IMTECH), Chandigarh. Bacterial strains were maintained at 4 °C on nutrient agar medium.

Antibacterial testing

The in vitro antibacterial activity was tested by disc diffusion method [12]. This method depends upon the diffusion of the tested material to such an extent that growth of the added microorganism is prevented entirely in a zone around the disc containing a solution of tested material. Muller Hinton Agar (MHA) was used for antibacterial screening. 6 mg/disc concentration of compound was impregnated on the discs. These discs were placed on the surface of the agar plates already inoculated with selected bacterial organisms. An additional control disc without any sample but impregnated with an equivalent amount of solvent (DMSO) was used in the assay. Efficacy of compound 1 against bacteria was compared with a broad-spectrum antibiotic erythromycin as positive control. Plates were incubated at 37 °C for 24 h in BOD incubator. The antibacterial activity was interpreted from size of diameter of zone of inhibition measured in millimetre (mm).

Determination of Minimum Inhibitory Concentrations (MICs)

The broth microdilution method employed for the determination of minimum inhibitory concentrations (MICs) of compound 1 as according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS) by a serial dilution method by using 96 well microtiter plates. Bacterial strains were cultured at 37 °C in MHA medium. After 18 h incubation, bacterial suspensions were prepared in Mueller Hinton Broth (MHB) and their turbidity was standardized to 0.5 McFarland. Optical density of test strains were confirmed on spectrophotometer (Shimadzu UV-1800 series Pharmac-spec UV–vis spectrophotometer, Japan) at A₆₀₀. The bacterial density was 5 × 10⁵. By using multipipettor, 100 μl of broth medium dispensed into all wells of microtitre plate. Total 11 dilutions (1024–1.0 μg/ml) of compound 1 were prepared by two-fold serial dilution method and 100 μl of each concentration was placed into each well and mixed properly. Column 12 was used as negative control. 5 μl of bacterial cultures were dispensed into wells in column 11 to 1. The plates were incubated at 37 °C. After 18–22 h, the plates were observed and bacterial cultures streaked on culture plates to check their purity.

Results and discussion

Chemistry

Analysis of spectroscopic data revealed that compound 1 is 3-(2′,4′,6′,6′-tetramethylcyclohexa-1′,4′-dienyl)acrylic acid (Fig. 1). Its molecular formula was assigned to be C₁₃H₁₈O₂ [Elemental composition: (in %) C, 75.69; H, 8.80; O, 15.51] according to ¹H NMR, ¹³C NMR and a quasi-molecular ion molecular ion peak in GC-MS in at m/z 207 ([M + H]⁺ calculated for C₁₃H₁₉O₂, 207.281)_.

Fig. 1

Chemical structure of compound 1 i.e. 3-(2′,4′,6′,6′-tetramethylcyclohexa-1′,4′-dienyl)acrylic acid

The UV–vis spectrum of compound 1 [see Additional file 1] indicated the absorption maxima characteristic for π → π^* transitions for CH = CH of aromatic ring (246.00 nm). FT-IR spectrum of compound 1 [see Additional file 2] revealed the absorption band of O–H stretching (3429.50 cm⁻¹), C-H stretching (2919.91, 2851.39 cm⁻¹), C = O stretching (1563.04 cm⁻¹), C-H deformation of alkenes (1436.62, 1384.56 cm⁻¹), C-O stretching of alcohol (1167.41 cm⁻¹), O-H deformation of alcohol (1093.92 cm⁻¹), and out plane deformation of CH in alkene (880.64 cm⁻¹).

The mass spectrum for compound 1 obtained by GC-MS [see Additional files 3 & 4] revealed its molecular formula could be assigned as [M] = [C₁₃H₁₈O₂] with a quasi-molecular ion molecular ion peak at m/z 207 [M + H]⁺ = [C₁₃H₁₉O₂]. Loss of a neutral ethane molecule from quasi-molecular ion generates first daughter ion with m/z 177 which corresponds to [M + H – C₂H₆] = [C₁₁H₁₃O₂]; further fragmentation of first daughter ion and elimination of a neutral C = O molecule generates a stable ion which corresponds to base peak at m/z 149 [M + H – C₂H₆ - CO] = [C₁₀H₁₃O]. Successive fragmentation and loss of CO₂, C₂H₂ and two CH₄ neutral molecules from quasi-molecular ion leads to generate a molecule with m/z 105 corresponding to [M + H – CO₂ – C₂H₂ – 2CH₄] = [C₈H₁₀].

The ¹H NMR spectrum of compound 1 [see Additional file 5] exhibited that it contain two ethylenic protons indicated by two doublets each for 1H at δ_H 7.613 (1H, d, J = 8.14 Hz) and 6.532 (1H, d, J = 8.17 Hz), their spin coupling pattern indicates presence of one cis-di-substituted ethylene moiety in the molecule. It also represented two singlet one for a methine proton which appear at δ_H 5.406 (1H, s) and one for two methylene protons at δ_H 2.436 (2H, s). The compound also illustrated three characteristic singlet for twelve methyl protons at δ_H 1.558 (3H, s), 1.164 (3H, s) and 0.757 (6H, s).

The ¹³C NMR spectrum of compound 1 [see Additional file 6] marked the presence of thirteen signals indicating the presence of thirteen carbon atoms in the molecule. The highest downfield shift at δ_H 171.645 indicates the presence of one carbonyl group (in form of carboxylic acid). It represented six signals between δ_H 158.716 and 107.423 characteristic for six olefinic carbon atoms. The spectrum also showed two signals each for a secondary carbon at δ_H 34.477 and a quaternary carbon at δ_H 34.405. The compound also demonstrated the presence of four methyl carbon which signals at δ_H 31.269, 28.969, 21.722 and 21.431.

Antibacterial activity

The purified compound 1 was tested for antibacterial activity against selected respiratory tract pathogens at 6 mg/ml concentration and showed better inhibitory activities (Table 1). Compound 1 was found most efficient against H. influenzae (9.3 ± 0.28 mm) and S. pneumoniae (8.0 ± 0.50 mm) respectively. While erythromycin showed inhibition ranged between 8.3 ± 0.23 to 13.3 ± 0.47 mm which slightly higher values at similar concentration.

Table 1 Zone of inhibition of compound 1 and reference drug (erythromycin) against respiratory tract pathogens

The MICs for compound 1 were ranged from 32 to 128 μg/ml (Fig. 2). The inhibition was observed similar at 64 μg/ml against H. influenzae and S. aureus, 128 μg/ml against P. aeruginosa and S. pneumoniae, and 32 μg/ml against S. pyogenes.

Fig. 2

Minimum inhibitory concentrations (MICs) of compound 1. The inhibition is noted at a) 64 μg/ml against H. influenzae and S. aureus, b) 128 μg/ml against P. aeruginosa and S. pneumoniae, and c) 32 μg/ml against S. pyogenes

Conclusions

In conclusion, present paper has shown the isolation and structure elucidation of one new derivative of ionone like compound named as 3-(2′,4′,6′,6′-tetramethylcyclohexa-1′,4′-dienyl)acrylic acid (compound 1) from methanol fraction of V. odorata with antibacterial activity. In regards to antibacterial activity, compound 1 was significantly active against tested respiratory tract pathogens including H. influenzae, P. aeruginosa, S. aureus, S. pneumoniae and S. pyogenes respectively.