Constituents of Essential Oil from Vinca major var. variegata and its Antibacterial Activity

Published in Khimiya Prirodnykh Soedinenii, No. 5, September–October, 2021, pp. 823–825.

The genus Vinca is mainly native to Europe, southwest Asia, and northwest Africa [[1]]. Vinca major var. variegata Loudon, belonging to Apocynaceae, is a Mediterranean species hemicryptophyte that is widely distributed in many European countries such as Romania, Austria, and Bulgaria and that grows on walls or cliffs [[2]]. V. major has been planted widely in China as a leaf-colored ornamental and functional plant [[3]]. The genus Vinca usually can be used to extract and isolate indole alkaloids with diverse structures for medicinal research and development [[1]]. There are some reports on the isolation of alkaloids of the leaves of V. major var. variegata, such as reserpinine, majdine, akuammicine, strictosidine lactam, seudoakuammigine, akuammine, 11-hydroxypolyneuridine, vallesiachotamine, isovallesiachotamine, reserpinine, vincamajine, and 10-hydroxycathofoline [[4]]. Majdine has antioxidant, apoptotic, and antiradical effects [[6]], and vallesiachotamine has antiproliferative effects on human melanoma cells [[7]]. The alkaloid extract has high lipid peroxidation inhibitory, DPPH radical scavenging, and anticholinesterase activities [[1]].

In this study, the chemical compositions of the essential oil from V. major var. variegata leaves and stems were investigated for the first time and used to characterize and compare the constituents of both oils.

Hydrodistillation of V. major var. variegata leaves and stems yielded 0.10% and 0.05% (w/w) volatile oil based on dry weight, respectively. A total of 50 compounds of both essential oils was identified according to their retention indices (RI) as shown in the Table 1. Forty-four volatiles, representing 92.9% of the oil content, were observed in the leaves with five main chemical compounds, (Z)-3-hexen-1-ol (19.7%), cyclohexanone (17.4%), acetic acid n-octadecyl ester (7.4%), benzeneacetaldehyde (6.6%), and o-xylene (5.9%). Meanwhile, 25 compounds were detected in the stems oil, which represented 91.3% of the total composition, with the principal oil constituents being cyclohexanone (17.2%), 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester (14.6%), (Z)-3-hexen-1-ol (9.5%), acetic acid n-octadecyl ester (6.8%), benzeneacetaldehyde (6.0%), and palmitic acid (5.5%). Additionally, GC-MS analysis for both volatile oils revealed that the major type of volatile oil compounds included alcohols (28.0%, 12.8%), esters (14.6%, 28.0%), ketones (17.5%, 17.2%), benzenes (14.3%, 10.4%), aldehydes (7.2%, 6.0%), cycloalkanes (3.9%, 3.6%), straight-chain alkanes (5.3%, 4.6%), phenols (1.2%, 0.1%), terpenoids (0.7, 0.2%), acids (0, 8.4%), and others (0.2%, 0%) for the leaves and stems. A comparison of the constituents of both oils showed that 18 compounds coexisted in the leaf and stem oils and another 32 different compounds. From the MIC of leaves and stems against various pathogenic and drug-resistant microbes, it was obvious that the leaf oil was more active than the stem oil (Table 2). The leaf oil showed strongest activity against Pseudomonas aeruginosa and Proteus. spp. (MIC 0.16 mg/mL) and weakest activity against methicillin-sensitive Staphylococcus aureus and Klebsiella pneumoniae. This may contribute to the reason why the presence of numerous antibacterial ingredients cause leakage of various entities such as ions, amino acids, nucleic acids, ATP, etc., leading to a disturbance and increase in membrane permeability [[8]].

TABLE 1. Constituents of V. major var. variegata Leaf and Stem Essential Oils, %

LRI: Linear retention index; –: not detected.

TABLE 2. MIC (mg/mL) values of leaf and stem oils of V. major var. variegata

Fresh leaves and stems of V. major var. variegata were harvested in May 2020 from the schoolyard of Anhui Xinhua University (31°83′N, 117°19′E, 55 m above sea level), Hefei, China. The botanical identification of the plants was performed by Prof. Q. Z. Li. A voucher specimen (No. AHXH 122) has been deposited in the laboratory of Anhui Xinhua University. The materials were air-dried at room temperature for 24 h and ground into a powder. Dry leaves and stems (500 mg) were immersed in 3 L of distilled water in a 8 L three-necked flask. The leaf and stem oils were extracted by hydrodistillation for 4 h. Then both essential oils were obtained and kept in a refrigerator for further analysis.

The gas chromatography-mass spectrometry (GC-MS) analysis were performed using an Agilent GC 7890A coupled with an Agilent 5975C mass selective detector. The gas chromatograph was equipped with a flame ionization detector (FID) and an HP-5MS capillary column (30 m × 0.25 mm, film thickness 0.25 μm). Helium was used as a carrier gas at a flux of 1 mL/min. Samples of 1 μL were injected using split ratio 1:30. The injector temperature was 250°C. The oven temperature was set as follows: initial temperature 40°C for 1 min, ramp of 5°C/min up to 300°C. MS conditions: electron bombardment ionization energy 60 eV. Mass range was from 25 to 550 atomic mass units. Ion source and interface temperature was 220°C. Solvent delay time was 5 min. The sample was dissolved in 10% cyclohexane.

Identification of the volatile oil compounds was based on matching their mass spectra with standard data by comparison of their mass spectra and retention indices (RI) recorded in the NIST-11 library or the National Institute of Standards and Technology NIST Chemistry Webbook, SRD69, USA. The MS fragmentation patterns were checked with those of other essential oils of known composition (NIST). The relative contents of the identified compounds in the essential oil were qualified by the method of peak area normalization without taking into account the response factors of individual compounds.

Antimicrobial activity of the essential oil of V. major var. variegata was determined using disc diffusion method [[10]]. Strains were obtained from the Second Hospital of Anhui Medical University in Anhui Province of China. The bacterial inocula were made by suspending overnight colonies from Muller–Hinton (MH) agar media in 0.9% saline. Dimethylsulfoxide (DMSO)-soluble essential oils (10% v/v) were dissolved at a concentration of 10 μg/mL and further diluted to 5.00, 2.50, 1.25, 0.62, 0.31, and 0.16 mg/mL. Blank discs containing 20 μL of 10% DMSO were used as controls. Microtiter plates were incubated at 30–35°C for 24 h. The minimum inhibitory concentration (MIC) was determined as the lowest concentration at which growth was inhibited.

This work was supported financially by Natural Science Foundation Project of Anhui Educational Committee (KJ2020A0791).