Isolation, identification, structural elucidation and bioactivity of Heneicos-1-ene from Coriandrum sativum L. foliage

Coriander foliage is a distinctive spice employed on a daily basis in curry and other Indian traditional food preparations mainly for the unique flavour attributes and health benefits. Radical scavenging activity has been demonstrated previously for coriander foliage. However, specific molecules responsible were not identified. A new molecule was isolated via chromatographic technique, and its structure was established by employing multinuclei and multidimensional NMR and HRMS techniques. The identified molecule Heneicos-1-ene was also screened for radical scavenging activity and antimicrobial activity, wherein it displayed radical scavenging activity of 89.6 ± 0.62% at 200 ppm, and also exhibited substantial antimicrobial activity against E. coli and Salmonella typhi.

Isolation, identification and structural elucidation. Separation using thin layer chromatography. Isolation of the molecule was carried out by thin layer chromatography (TLC) from the extract prepared from dried coriander foliage using 70% ethanol as solvent. TLC aluminium plates (20 × 20 cm) pre-coated with silica gel 60 F 254 were obtained from Merck (Darmstadt, Germany). Developing systems of different composition were tried and optimized to get good separation. Sample solutions were applied to the plates using a capillary tube. All TLC plates were developed using an initial mobile phase of methanol: chloroform (30:70 v/v) followed by twice elution with methanol: chloroform (10:90 v/v). Linear ascending plate development was performed in a suitable chromatographic tank previously saturated for one hour with the developing mobile phase, and the scanning was carried out at 270 nm. The bands obtained were carefully excised from the plate and extracted using 2 mL chloroform. All the chloroform extracts were then pooled; dried in a rotary evaporator (Heidolph, Germany) at 30-35 °C under reduced pressure (40 millibars) and redissolved in a known volume of chloroform.
Spectroscopic measurement. The extracts were subjected to high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy for structural elucidation and identification of the molecule. Solution NMR spectral data recorded on a Bruker Avance instrument having the 1 H frequency of 500 MHz and 13 C frequency of 125 MHz. NMR spectral analysis was accomplished by employing the combination of experiments consisting of 1D NMR techniques 1 H, 13 C{ 1 H}, and 2D NMR techniques involving HSQC (heteronuclear single quantum coherence), HMBC (heteronuclear multiple bond correlation), and TOCSY (total correlation spectroscopy). Mass spectra were recorded in SCIEX 5600 Q-TOF instrument.
Determination of free radical scavenging activity by DPPH. The free radical scavenging activity of isolated Heneicos-1-ene from coriander foliage was measured by the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method 4 . The various concentrations (50, 100, and 200 ppm) of extracts were taken in different test tubes. The volume was made up to 1 mL by using respective solvent. Then 4 mL of 0.1 mM methanol solution of DPPH was added to each test tube and vortexed. The tubes were stored for 20 minutes in the dark at 21 ± 2 °C temperature. Control was prepared as above without Heneicos-1-ene, and the respective solvent was used for the baseline correction. The variation in the absorbance of the samples was measured at 517 nm. BHA was used as a standard antioxidant for comparison. The average values of the three analyses were taken. The free radical scavenging activity was expressed as the inhibition percentage and was calculated using the following formula- A 100 control s ample control where, A sample and A control are the equilibrium absorbance of the test sample and the control, respectively.
Determination of antimicrobial activity. Disc diffusion assay. The antibacterial activity assays were carried out using disk diffusion method 5 and spot on lawn method using Staphylococcus aureus, Escherichia coli, Micrococcus luteus, Salmonella typhi and Listeria monocytogens as test organisms. The Muller-Hinton agar plates inoculated with equal to 0.5 McFarland turbidity of pathogen bacteria by sterile swab. Standard blank disk (6.0 mm diameter) was put on a plate and inoculated with 50 µL of the isolated component (10 mg). All Petri plates were incubated for 24 hours at 37 °C. Zones of inhibition measured after 24 hours. The control discs contained DMSO (−ve) and Ampicillin (+ve).
Spot on lawn assay. The assay was carried out on Muller Hinton agar. Muller Hinton agar plates were inoculated with different pathogen bacteria matched to 0.5 McFarland turbidity of by sterile swab. 10 µL of the isolated component was spotted on the media and incubated at 37 °C.

Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC).
The antimicrobial activity of the isolated compound was determined by adjusting the turbidity of the bacterial pathogen to match a McFarland No. 0.5 standard (~10 7 CFU/mL) and used for the assay 6 . A stock solution of purified compound (2 mg/mL) was prepared and serially diluted in a Muller Hinton broth (100 µL) medium dispensed in a 96-well cell culture plate to obtain a concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.781 µg/mL. Each well was further added with 100 µL of inoculum. The control wells were maintained with and without samples. Ampicillin (10 µg/mL) was used as reference antibiotic. The plates were sealed using parafilm and incubated for 24 hours at 37 °C. The turbidity of the broth was measured at 600 nm using titer plate reader (Thermo Scientific, USA). The minimum concentration of compound at which it inhibited the visible growth of pathogenic bacteria was taken as minimum inhibitory concentration (MIC). MBC is the lowest concentration of compound preventing the visual growth of the bacterial cultures and reduced the pathogen by ≥99.9%, irrespective of counts of survivors at higher antibiotic concentrations.

Results and Discussion
Structural elucidation. Structural elucidation was carried out by employing high-resolution mass spectrometry (HRMS) and 1 H, 13 C, HMBC, HSQC and TOCSY NMR experiments. HRMS ESI-positive mode spectrum did not reveal the occurrence of molecular ion peak. However, it exhibited a peak pattern corresponding to 302.3049 assigned to (M-CH 3 + Na) + , whereas the dominant peak pattern{please refer Figure (Fig. 4). Heneicos-1-ene is synthetically made by decarbonylation reaction of docosanoic acid (CH 3 (CH 2 ) 20 COOH) with Vaska's complex utilizing potassium iodide and acetic anhydride as additive 7 . It has also been made using enzymatic oxidative decarboxylation 8 . This molecule forms the part of essential oil of blue-coloured hybrid tea rose flowers 9 . NMR Data. 1    In-vitro antibacterial activity. Antibacterial activity of both aqueous crude extract and pure compound (Heneicos-1-ene) dissolved in chloroform were tested in-vitro by using disc diffusion method, spot on the lawn and minimal inhibition concentration method. Heneicos-1-ene has inhibited the growth of E. coli and Salmonella typhi at the concentration of 10 mg with a zone of inhibition 8.9 ± 0.079 mm and 11 ± 0.025 mm, respectively ( Table 1). The zone of inhibition of the Heneicos-1-ene was more prominent against Salmonella typhi than E. coli (Fig. 6). There was no significant antibacterial activity observed against the other three tested bacterial pathogens    With regards to the antibacterial activity, the Minimal Inhibition Concentration (MIC) values of Heneicos-1-ene against E. coli and Salmonella typhi of the compound were confirmed using broth dilution microtiter system and expressed data as the mean ± standard deviation (SD, n = 3). The antimicrobial activity of Heneicos-1-ene was found in the range of 25 to 50 µg/mL and the minimal bacterial concentration (MBC) was in the range of 50-100 µg/mL (Table 2) for E. coli and Salmonella typhi. MBC were generally within a two-fold dilution of the MICs. Since the Heneicos-1-ene did not show any inhibition zone against other pathogens, the MIC and MBC were not determined.
Several bioactive peptides, volatile compounds 10 and essential oils 11,12 isolated from coriander have been accounted for a broad range of antimicrobial activity. However, our study for the first time characterizes and confirms the antimicrobial activity of Heneicos-1-ene against E. coli and Salmonella typhi and possesses a promising role in maintaining the shelf life of the food products by preventing spoilage.

Conclusion
We isolated Heniocose-1-ene from a natural food source coriander foliage for the first time. Heniocose-1-ene exhibited excellent radical scavenging and antimicrobial activity.

Data Availability Statement
Complete data of the manuscript is available with Corresponding author.