Abstract
The chemical profiles of essential oils (EOs) of 7 Cinnamomum species (C. sulphuratum, C. perotettii, C. verum, C. wightii, C. camphora, C. glanduliferum, and C. malabatrum) composed from assorted parts of Western Ghats were analyzed. The antioxidant, antibacterial, and larvicidal potentialities of 7 essential oils were also investigated. Cinnamomum verum was observed to have the maximum yield (1.96%) of EO followed by C. malabatrum (1.71%). GC-MS analysis of EOs reported the highest amount of eugenol (38.04%), shyobunol (13.31%) in C. verum, whereas C. perrottetii identified 14–42 phytocompounds including terpenes, sesquiterpenes, monoterpenes, diterpenes, and phenylpropanoids, esters, aldehydes, and fatty acids. In the antibacterial activity, the higher zone of inhibition of C. malabatrum showed against S. pyogenes (28 ± 2.5 mm) and E. faecalis (27 ± 1.24 mm) followed by C. verum has shown notable inhibition zone against K. pneumoniae (26 ± 1.15 mm) and S. flexneri (24.33 ± 0.88 mm), with MIC value against C. verum, showed MIC value (1.17 ± 0.34 μg/mL) against S. flexneri, S. pyogenes, and K. pneumonia. All the Cinnamomum oil samples exhibited appreciable antioxidant radical scavenging activities with IC50 values ranged 10.83–15.06 μg/mL. All the oil samples showed promising larvicidal activity against the late-third instar larvae of Aedes aegypti. Extending the present study with in vivo and in vitro animal studies will confirm the potentiality of Cinnamomum oils in the field of biomedicine to detect novel medicines.
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1 Introduction
Cinnamomum is reported to have higher medicinal value and larvicidal properties which can be considered a commercial cultivable species for the rural economy [1]. EOs are volatile lipophilic compounds secreted through aromatic plant cells with diverse morphology, synthesized through numerous plant organs such as buds, branches, stems, roots, berries, seeds, flowers, bark, and wood, and stored in secretory cells, channels, cavities, trichomes, or epidermal cells [2]. The secretory cells range from highly specialized to non-specialized cells, osmophores, ducts, and cavities reported in dissimilar plant parts are the repositories of Eos [3]. EOs is short-molecular-weight compound mixtures with a strong odor obtained from aromatic plants [4, 5]. They are the mixtures of a large number of compounds having differentiated structures mainly of terpenes or phenylpropanoid derivatives. EOs is significant derived metabolites reported in aromatic and medicinal plants used in fields and industries such as pharmaceuticals, cosmetics, food, and aromatherapy [6]. EOs obtained from plant sources are the best alternative to synthetic oils used in human health care. They are also reported as the best food additives to retain the quality and value of nutrition [7]. Moreover, spice and aromatic plants are reported to be prosperous sources of volatile compounds. Many of these secondary metabolites are approved for human consumption through the United States Food and Drug Administration, widely used in flavors and fragrances, food preservation, and pharmaceutical industries [8].
The different compounds in EO samples are recognized to have acquired an assortment of biological significance, which capacity has been accountable for therapeutic consequences such as insecticidal, anti-allergic, anti-inflammatory, antioxidant, and anticancer properties, along with their antimicrobial characteristics [9,10,11]. Multidrug resistance and side effects of antibiotics increased the interest and need for developing new antimicrobial agents from plants [12].
The antimicrobial activity of EO is also proposed to the morphological changes produced in bacteria by the volatile compounds, which modulate bacterial growth and are capable of trapping the free radicals and preventing oxidation [8]. The essential oils from cinnamon improve the activity of antioxidant enzymes by reducing the reactive oxygen-free radical [4]. Many studies on EO reported that the antioxidant activity is unpaid to the existence of several monoterpenes and sesquiterpene compounds [13, 14]. By the action of an antibacterial representative on bacteria, their ROS content increases. When it exceeds the antioxidant defense ability, the protein and further intracellular substances of the bacteria get injured and hence affect the metabolic process of the bacteria [15]. Also, the EOs of several plant species have been documented to exhibit toxicity effects against a few species of mosquitoes. Only substantial numbers of studies have been attempted on the usage of EOs of cinnamon as food additives and medicines. The comparative analysis of volatile composition among the species helps to understand the medicinal values of the EO-yielding species of a group [16].
The genus Cinnamomum contains shrubs and evergreen trees belonging to the Lauraceae family consisting of about species are 250 in subtropical and tropical regions, mainly in Asia, and a few in central and South America and Australia [17]. The bark of Cinnamomum is widely employed as a spice and denoted as a highly valued oldest spice which ranks second next to pepper and is commonly used for flavoring, preservation, cosmetics, and perfumery industries and in preparation of various ayurvedic and allopathic medicines [18]. The EOs from the species of Cinnamomum are reported as an important source of chemicals with pharmaceutical uses and hence, current investigate works were alerted on Cinnamomum ([19, 20]. The plants of this genus are reported with numerous pharmacological properties; antioxidant, antibacterial, and anti-inflammatory properties [21, 22]. The occurrence of bulky amounts of phytoconstituents such as camphor, α-terpineol, linalool, eucalyptol, and borneol in the EO is accountable for their biological activities [20]. The EOs of Cinnamomum are reported to be carminative and expectorant; the bark oil is an anthelmintic, aphrodisiac, and tonic and also useful in treating bronchitis, parched mouth, itching, diarrhoea, piles, flatulence, headache, heart, urinary diseases, etc. [23].
The current study of seven Cinnamomum species C. verum, C. sulphuratum, C. perrottetii, C. camphora, C. wightii, C. glanduliferum, and C. malabatrum selected and focused for the identification of secondary metabolites in essential oils by gas chromatography mass spectroscopy (GC-MS), extracted essential oils targeted pharmacological applications like free radical scavenging assay by DPPH method, antibacterial activity against two gram-positive bacteria, such as Streptococcus pyogenes and Enterococcus faecalis and four gram-negative bacteria, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella pneumoniae, and Shigella flexneri human pathogenic bacteria and larvicidal activity against Aedes aegypti.
2 Materials and methods
2.1 Plant materials
Fresh leaves of seven species of Cinnamomum, Cinnamomum verum J. Presl., Cinnamomum sulphuratum Nees., Cinnamomum perrottetii Meisn., Cinnamomum camphora (L.) J. Presl., Cinnamomum wightii Meisn., Cinnamomum glanduliferum (Wall.) Meisn., and Cinnamomum malabatrum (Burm.f.) Presl. were composed of dissimilar forest areas of the Western Ghats. The collected leaf specimens were properly identified with standard journalism and authenticated with official voucher specimens (Fig. 1). The receipt specimens of all the studied species (Cinnamomum verum J. Presl.: GUD 942, C. sulphuratum Nees.: GUD 943, C. perrottetii, Meisn.: GUD 944, C. camphora (L.) J. Presl.: GUD 945, C.wightii Meisn.: GUD 946, C. glanduliferum (Wall.) Meisn.: GUD 947, and C. malabatrum (Burm.f.) Presl.): GUD 948) were placed in the herbarium (GUD) of the Gandhigram Rural Institute (Deemed to be University), Department of Biology, Gandhigram, Dindigul (DT), Tamil Nadu.
2.2 Isolation of essential oils
The fresh leaves (200–500 g) of all the species of Cinnamomum were cut into small pieces (2–3 cm) individually and subjected to hydro-distillation with the Clevenger-type equipment (Borosilicate Glass) for about 4–6 h at ambient temperature (60–80 °C), the yield of EOs was calculated. The EOs collected from each species were dried over anhydrous Na2SO4 and then stored at 4 °C in a dark place for further analysis and further work [12, 13].
2.3 Analysis using gas chromatography and mass spectrometry (GC-MS)
The essential oil samples were diluted in n-hexane (10 μL/300 μL), and 10 μL of each solution was added to a split-splitless injector attached to an HP-5 column (30 m × 0.32 mm, film thickness 0.25 μm), which was used in a spit-mode gas chromatography experiment on an Agilent Technologies 7890 apparatus. As a carrier gas, helium (1 mL/min/210 °C) was employed. The column temperature was linearly programmed from 40 to 260 °C at 4 °C/min, while the injector and detector temperatures were set at 250 and 280 °C, respectively. Without using any correction variables, the percentage composition was calculated from the peak areas. The HPG 1800 C Series II GCD analytical instrument with an HP-5MS column was used to perform the GC-MS. Re-recorded mass spectra in the m/z 40–450 range were done so in EI mode (70 eV). By calculating the oils’ Kovats retention index (RI) and comparing their mass spectra to reference compounds from the Nirst and Willey libraries, the volatile components of the oils were located [12].
2.4 Antibacterial activities of essential oils
Two gram-positive bacteria, such as Streptococcus pyogenes (MTCC 442) and Enterococcus faecalis (MTCC 439), and four gram-negative bacteria, Pseudomonas aeruginosa (ATCC 27853), Enterobacter aerogenes (MTCC 111), Klebsiella pneumoniae (MTCC 432), and Shigella flexneri (MTCC 1457) were used. The microbial cultures were obtained from the Laboratory of the Department of Biology, The Gandhigram Rural Institute, Dindigul, India. The diameter of the zone of inhibition of EOs was evaluated based on the qualitative determination of the antibacterial activity of essential oils [14]. Different concentrations (50–150 μg/mL) of sample solutions were added (50–90 μL) to each well, and the plates were incubated at respective temperatures of 37 °C for 24 h. Gentamicin (Laborate Pharmaceuticals India Ltd.) (10 μg/mL) and 1% DMSO (SRL chemical) were used as the experimental +ve and −ve controls. The triplicates were maintained, and the inhibition zone diameter (IZD) was observed and calculated for every organism and expressed the value as mean ± S.E. [14].
2.5 MIC (minimal inhibitory concentration) of EOs determination
The testing of MIC, seven essential oil samples selected, and the bacterial strains was determined through the method of microdilution as prearranged through the CLSI method by using 96-well microtiter plates (Tarsons trust delivered, Cat. No. 980040) with some modifications [24,25,26], minimal concentrations of essential oil samples that inhibit or kill the microorganisms were resoluted through the dilution of broth method. Each EO was subjected to dilution to 1 mg/mL for testing, followed by a series of two-fold dilutions from 150 to 0.29 μg/mL. Gentamicin (Laborate Pharmaceuticals India Ltd.) (1 mg/mL) and DMSO (SRL chemical) (100%) were taken as positive and negative controls respectively. The early bacterial inoculums were attuned to 5 × 106 CFU/mL and in a sterile 96-well microplate, nutrient broth (95 μL) as well as respective pathogenic culture (1 × 105 cells/ mL), were added into each well. The oil samples 100 μL were added to consecutive wells and incubated at 37 °C for 18 h. For MIC determination, resazurin (10 μL, 0.02% (w/v)) was transferred to each well followed by 2–4 h of further incubation. Incubated then wells were subjected to illustration evaluation of color changes that imply bacterial growth. The color changes indicated resazurin (HiMedia) reduction (blue/purple) to resorufin (pink). The 570 nm absorbance was read by Robonik Readwell Touch Automatic ELISA plate analyzer. The low concentration at which absolute inhibition of MIC values was recorded; all the experimental trials were performed in triplicates [16].
2.6 Antioxidant activities of Eos
The capability of the DPPH radical scavenging assay of EO was also analyzed spectrophotometrically. DPPH (0.1 mM; 1 mL) solution was added to different concentrations (10–50 μg/mL) of sample solution and then incubated for about 30 min. The reaction mixtures were read by a spectrophotometer (GENESYS 180 UV – Visible spectrophotometer) at 517 nm. The standard ascorbic acid (Sigma-Aldrich) in various concentrations (10–50 μg/mL), the DPPH radical scavenging activity was calculated against sample concentrations, and IC50 values were obtained from the graph [24, 27,28,29].
2.7 Larvicidal bioassay
The larvicidal assay was experimented on the 3rd instar larvae of Aedes aegypti as per the methodology of WHO with minor modifications [30]. The larval colonies of A. aegypti were collected from ICMR, Madurai, CRME (Centre for Research in Medical Entomology). Dissimilar test concentrations (50–250 μg/ml) from the stock essential oils were prepared with methanol (0.1 mM) (HiMedia). The appropriate volume of methanol dissolved in distilled water has served as a negative control. Ten healthy 3rd instar larvae were released into every glass beaker containing the respective examination solution. The controls tested were also done in a similar with each duplicate. The death rate of larvae was intended after one hour of the revelation period with an interval of 1 h up to 10 h. The motionless and unreactive larvae were considered dead. The departed larvae were noted and counted, and the regular % of transience was considered using Abbott’s procedure.
where X is the survival % in the larvae population control, and Y is the % of survival in the larvae population treated.
2.8 Statistical analysis
The mortality of the larvae at various concentrations was analyzed by the probit analysis method described by Raymond in 1985. The attentiveness that caused 90% and 50% death (LC50) have resulted in assurance intervals for mathematically important comparisons. The significance of the differences between the treatments versus concentrations was established by Oneway ANOVA and p values < 0.05, were measured as noteworthy. The data were analyzed statistically and completed using IBM SPSS (Statistics version 21), and the graphs were plotted using ggplot 2 version 3.3.6. in R version 4.1.1 [24].
3 Results and discussion
3.1 Chemical composition of essential oils
The present study confirmed that species of Cinnamomum were rich repositories of terpene compounds and oxygenated derivatives. A comparison between the chemical compositions of oils with the previous reports has indicated the compositional differences of essential oils according to the circulation and components accumulation in the same plant parts. The EOs isolated from Cinnamomum leaves were characterized by a diverse group of sesquiterpenes with varying areas of distribution. Few previous studies attempted on essential oils have shown variation in the chemical constituents from the same plant part, this variation was due to the influence of ecological factors such as climate and soil [31,32,33] genetic and developmental factors [33, 34] and also depends on the geographical region where the plant grow [35,36,37].
The yield of EOs from Cinnamomum species mottled from 0.142 to 1.96% v/w. The greatest surrender was obtained from C. verum (1.96% v/w) followed by C. malabatrum (1.71%), C. sulphuratum (1.17% v/w), C. wightii (1.014% v/w), C. camphora (0.32% v/w), and C. glanduliferum (0.171% v/w) whereas it was minimum yield from C. perrottetii (0.142% v/w). The higher quantity was obtained from C. verum (1.96% v/w) and C.sulphuratum (1.18% v/w) making these species a new natural potential source of volatile aromatic compounds. The GC-MS results demonstrated that sesquiterpenes were the principal class of compound in C. sulphuratum, C. perrottetii, C. wightii, C. camphora, and C. glanduliferum. In contrast to that, monoterpenes were predominantly reported in C. verum and esters in C. malabatrum. The predominant volatile components detected from the species of Cinnamomum were eugenol (38.04%), shyobunol (13.31%), isocaryophyllene (10.36%) along with germacrene D (7.89%), β-caryophyllene (11.33%), α-cadinol (10.7%), and Isoeugenol acetate (27.95%) in C. verum, C. perrottetii, C. camphora, C. sulphuratum, C. wightii, C. glanduliferum, and C. malabatrum, respectively (Table 1).
A total of 42 phytoconstituents were detected from C. sulphuratum including 31 sesquiterpenes, 7 sesquiterpenoids, and 4 monoterpenes. Germacrene D was established as the chief compound eluted at a retention time of 17.95 with a 7.89 % distribution. Furthermore, β-selinene (5.66%) and α-selinene (5.58%) were detected at the elution time of 18.128 and 18.266, respectively. Among the identified phytocompounds, the minor bioactive compounds of the essential oil were allo-ocimene (0.34%) and α-cubebene (0.32%) which were detected at the elution times 12.33 and 23.12 (Table 2 and Fig. 2a). The C. sulphuratum EO chemical composition from Kerala were rich with benzyl benzoate and another accession from Kerala has reported with (E)-cinnamyl acetate, linalool and benzyl benzoate as main constituents. C. sulphuratum from Kodagu district and Gudalur, Nilgiri region of Tamil Nadu showed dissimilarities of chemical constituents, in which α-phellandrene, (E)-caryophyllene, linalool, and benzyl benzoate were the major compounds from Kodaku [102] and (E)-cinnamyl acetate, linalool, α-cadinol, benzyl benzoate, α-cadinene, and α-muurolol from Gudulur. The Northeast India leaf has also shown disparity with a significant presence of geranial, neral, and geraniol [103].
Out of 35 compounds reported from the E of C. perrottetii, 17 sesquiterpenes contributed to the main division of the EOs, along with 8 sesquiterpenoids, 7 monoterpenes, 2 monoterpenoids, and 1 diterpene. Shyobunol (13.31%) was detected as the dominant compound recorded at the elution time of 22.27 followed by caryophyllene (10.91%), α-Cadinol (10.86%), germacrene-D (8.9%), viridiflorol (5.96%), and germacrene D-4-Ol (4.43%) at the retention time of 16.7, 21.49, 17.98, 20.30, and 19.91, respectively (Table 2 and Fig. 2b). Schmidt et al. [104] and Boniface et al. [105] reported eugenol (74.9%) as the basic constituent of the C. perrottetii oil from Srilanka. Although, (E)-cinnamaldehyde (37.6%), cinnamyl benzoate (16.4%), and cinnamyl acetate (23.7%), as major constituents of the fresh leaves of C. perrottetii. The studies carried out by Unlu et al. [106] found that the major compounds C. perrottetii oil were (E) (E)-cinnamyl acetate (7.44%), cinnamaldehyde (68.95%), and benzaldehyde (9.94%). When compared to data observed from the current study with prior reports, the major components of C. perrottetii, have indicated that there was no similarity recorded in the principle component, shyobunol (13.31%) from the EOs.
Volatile phytocompounds 29 were identified from the EO of C. verum including 9 monoterpenes, 7 sesquiterpenes, 5 esters, 4 phenylpropanoids, 3 sesquiterpenoids, and 1 monoterpenoid. Eugenol (38.04%), one of the important phenylpropanoids was prominently detected at the elution time of 15.593. Cinnamyl acetate (22.36%), benzyl alcohol (8.13%), and cis-cinnamaldehyde (4.81%) were the other compounds subsequently eluted at 17.42, 11.344, and 13.603 retention times (Table 2 and Fig. 2c).
The EO chemical combination of C. verum collected from various geographical regions confirmed the dissimilarities of phytocompounds, in which (E)-linalool (2.30%), eugenol (86.02%), and caryophyllene (5.70%) recorded from the leaf samples of Fiji islands [107]. By the present study, C.verum essential oil from Palni hills, Tamil Nadu has also detected eugenol as a major compound (3.9%) [108]. C. verum essential oil from Brazil recorded linalool (5.4%), E-cinnamaldehyde (4.0%), β-phellandrene (3.4%), eugenol (3.4%), α-pinene (3.9%), and benzaldehyde (2.7%) as major constituents [109] and the Vietnam samples reported linalool (22%), caryophyllene oxide (5.6%), methyl eugenol (0.7%), β-bisabolene (7.7%), eugenol (0.1%), bicyclogermacrene (11.2%), (E)-nerolidol (1.3%), and γ-cadinene (4.0%) as significant compounds [110]. The present study has reported eugenol and benzyl benzoate compared to the previous studies [23, 109, 111,112,113].
GC-MS chromatogram of essential oil of C. wightii has detected 45 compounds, of which, 25 were sesquiterpenes and the rest of them were categorized into 6 sesquiterpenoids, 5 monoterpenes, 2 monoterpenoids, together with 2 esters, 2 fatty acids, 2 aldehydes, and 1 diterpene(Table 2 and Fig. 2d). β-caryophyllene was reported with an area of 11.33% which has eluted at 16.73 along with α cadinol. 𝛿-cadinene (5.14%), γ-muurolene (4.49%), globulol (4.46%), and allo-ocimene (4.11%) were further recognized based on the elution time. The oil leaf EO from seven different accessions of the Western Ghats showed a disparity in the chemical composition compared to the present study with the presence of monoterpenes such as β-phellandrene, α-pinene, linalool, limonene, p-cymene, methyl eugenol, (E)-caryophyllene, elemicin, α-phellandrene, and spathulenol [114].
Among 41 phytocompounds reported from the essential oil of C. camphora, 24 sesquiterpenes 5 sesquiterpenoids, 4 monoterpenes, 3 monoterpenoids, 2 diterpenes, 2 aldehydes, and 1 fatty acid were reported. Isocaryophyllene showed a major peak at a maintenance time of 16.65 with a relation profusion of 10.36%. The caryophyllene oxide (8.74%) and α-cardinal (8.71%) were reported as leading compounds eluted at 20.03 and 21.43 (Table 2 and Fig. 2e). By analyzing the result of GC-MS data of C. camphora, phytone (0.19%), piperitone (0.16%), and benzaldehyde (0.14%) were the meagre compounds eluted at 24.62, 12.98, and 12.72 retention times respectively. The C. camphora compound composition of oil has differed considerably from the previous reports by having isocaryophyllene (10.36%), β-caryophyllene oxide (8.74%), and α-cadinol (8.71) as the major constituents. However, all the phytocomponents reported by this study were similar to the previous study reports at different geographical regions [43, 115,116,117,118,119]. Comparatively, a different compound composition of C. camphora essential oil from China was reported with borneol as the major constituent, and another study reported a dissimilar composition with eucalyptol (16.8%), isoborneol (8.1%), camphor (5.0%), linalool (26.6%), and α-terpineol (8.7%), and β-phellandrene (5.1%) as major compounds [120].
Furthermore, 32 compounds were reported from C. glanduliferum, with 18 sesquiterpenes and 7 sesquiterpenoids, 2 monoterpenoids, 4 monoterpenes, 1 diterpene, and 1 fatty acid, α-cadinol has been observed from the large area of distribution with an area percentage of 10.7% followed by β-caryophyllene (9.35%), β-elemene (6.31%), 𝛿-muurolene (5.11%), and α-cadinene (5.9%) at the retention time of 16.65, 15.95, 21.14, respectively (Table 2 and Fig. 2f). The C. glanduliferum EO composed in various geographical regions showed different composition in which, (E)-nerolidol (52.2 %) and caryophyllene oxide (6.0%) was recorded in samples from the Chessa area of Arunachal Pradesh [121], α-terpineol (9.40 %), α-pinene (20.28 %), 1, 8-cineole (41.42 %), and germacrene D-4-ol (6.10 %) from Uttarakhand [122], eucalyptol (59.44%), sabinene (14.99%), a-pinene (5.27%), a-terpineol (6.44%), β-elemene, and β-pinene (3.75%) from Al-Zohria Garden, Cairo, Egypt [123], and a new benzyl benzoate affluent chemotype was reported from Southern Western Ghats, India [36].
C. malabatrum leaf essential oil was reported with a total of fourteen compounds consisting of esters, sesquiterpenes, phenyl propanoids, monoterpene, and sulfone compounds. Isoeugenol acetate (27.95% at retention time 15.14), (E)-cinnamyl acetate (4.5% at retention time 17.075), cinnamaldehyde (4.28% at retention time 13.304), and benzyl acetate (1.02% retention time 10.68) were the major compounds (Table 2. and Fig. 2g). A previous study in C. malabatrum essential oil reported cinnamyl acetate and benzyl benzoate which were also detected in the present study [41].
By comparing the present attempt with the previous studies, the principle components of EO of species of Cinnamomum have varied due to geographical distribution and season of procurement. Nevertheless, the dissimilarity in the yield of EO and its chemical composition among the plants comes to analogous taxonomic groups anticipated to vary across agro-climatic and geographical conditions. It is as well as influenced by numerous external and internal environmental factors [2, 124, 125].
3.2 Antibacterial activity of EOs
The antibacterial potentialities of EOs from seven species of Cinnamomum were evaluated against 6 selected bacterial strains through the agar well method (diffusion). The results indicated that there has been a significant increase in the inhibition of bacterial growth with an increase in oil concentrations. The growth of microorganisms was inhibited and a maximum zone of inhibition (32 ± 0.88 mm) in Gentamicin. The leaf oils of Cinnamomum spp. were shown a synergistic effect with an inhibition zone alongside all experienced microorganisms (Fig. 3).
C. verum has shown notable inhibition zone against E. faecalis (19 ± 0.57 mm), S. flexneri (24.33 ± 0.88 mm), S. pyogenes (23.33 ± 0.66 mm), K. pneumoniae (26 ± 1.15 mm), P. aeruginosa (21.33 ± 1.33 mm), and E. aerogenes (21.66 ± 1.0 mm) at higher concentration (150 μg/ml). Among the screened bacteria, K. pneumoniae exhibited higher antibacterial activity with the highest zone of inhibition (26 ± 1.15 mm) (Table 3). S. pyogenes showed higher susceptibility to the essential oils isolated from C. sulphuratum with a higher zone of inhibition (23.33 ± 1 mm). E. aerogenes exhibited a moderate inhibition zone (21.66 ± 0.88 mm) followed by S. flexneri (18.33 ± 0.33 mm) and P. aeruginosa (18.33 ± 1.2 mm) (Figs. 4 and 5).
Cinnamomum perrottetii exhibited very similar trends of antibacterial activities and S. pyogenes (19.66 ± 1.33 mm) showed the prevailing zone of inhibition. E. faecalis and E. aerogenes were recorded with an almost similar zone of inhibition (18 ± 0.57 mm and 18.33 ± 0.88 mm) followed by P. aeruginosa and S. flexneri (16.33 ± 1.2 mm and 16 ± 1 mm). Among the tested oil samples, C. wightii and C. camphora exhibited less susceptibility to selected pathogens. C. wightii displayed a moderate zone of inhibition for all screened bacteria and the highest resistance was recorded in E. aerogenes (24.33 ± 0.33 mm). The essential oil of C. camphora showed higher inhibitory activity against S. flexneri (19 ± 1 mm) and P. aeruginosa (16.66 ± 0.66 mm), S. pyogenes and K. pneumoniae with an equal value of inhibition at higher concentrations. Among the screened strains, E. faecalis and E. aerogenes exhibited 100% resistance against essential oil isolated from C. camphora (Figs. 6, 7, and 8).
C. malabatrum EO has shown a minimum inhibition zone of about 11 ± 0.58 mm, 11 ± 0.47 mm, and 20 ± 1.15 mm against S. flexneri, K. pneumoniae, and S. pyogenes at initial concentration (50 μg/mL). By increasing the oil concentration to 100 μg/mL, there was an increased antibacterial activity with an inhibition zone. Maximum zone of inhibition was noticed at higher concentration (150 μg/mL) against all microbial species such as K. pneumoniae (18 ± 2.08 mm), S. flexneri (20 ± 1.18 mm), S. pyogenes (28 ± 2.5 mm), E. faecalis (27 ± 1.24 mm) P. aeruginosa, (16 ± 1.56 mm), and E. aerogenes (16 ± 2.1 mm) (Figs. 9 and 10).
The data observed by the agar well dispersion assay of the essential oil suggested that the samples have synergistic antibacterial potentialities alongside gram +ve and −ve bacteria. The results suggested that S. flexneri, K. pneumoniae, and E. aerogenes were found to be more susceptible when compared with other selected strains [107]. E. faecalis and E. aerogenes showed higher resistance against all essential oils of Cinnamomum [108]. Nonetheless, all the oils exhibited antibacterial activity against screened bacteria and the inhibition zones were more eminent in C. verum followed by C. malabatrum, C. sulphuratum, C. glanduliferum, C. perrottetii, C. wightii, and C. camphora. Essential oils are a rich repository of volatile constituents such as phenol-derived aromatic and aliphatic compounds, terpenes, and terpenoids responsible for bactericidal activities [109]. Several studies have revealed that cinnamon has a strapping and reliable inhibitory effect against human pathogens [67, 126,127,128,129,130,131,132]. Previous studies have also confirmed that the phytocomponents, sesquiterpenes, cinnamaldehyde, and eugenol are accountable for the antibacterial activity of cinnamon oil [132,133,134,135]. Also, the bactericidal action mechanisms of cinnamaldehyde and eugenol of essential oils were recorded [136].
Gram -ve organisms are supposed to be less responsive to EOs than gram-positive bacteria [10, 14]. However, the present study has revealed equal inhibitory action of all oil samples with clear zones of inhibition that portray higher antibacterial efficacy. The essential oils are considered antimicrobials due to the hydrophobic character compounds are cause the destruction of cell structures, leading to the escape of the cell membrane and increasing the membrane permeability [132, 137] and thereby in a cascade type of action that affects other cellular structures. The essential oils damage lipids and proteins by getting coagulated in the cytoplasm of the bacterial cell [138, 139].
3.3 Minimal inhibitory concentration (MIC) determination of EOs
C. verum showed a similar MIC value (1.17 ± 0.34 μg/mL) against S. flexneri, S. pyogenes, and K. pneumonia and it was highest against E. faecalis (9.375 ± 0.95 μg/ml). Klebsiella sp. membrane was destroyed by inducing oxidative damage by C. verum bark essential oil [140]. Apart from bark, EOs obtained from flowers and leaves also act as strong antibacterial agents against both gram +ve and −ve bacteria [141]. C. glanduliferum exhibited the least MIC value of 2.34 ± 0.67 μg/mL against E. aerogenes. E. faecalis and S. pyogenes exhibited the MIC value of 18.75 ± 0.63 μg/mL and 18.75 ± 0.46 μg/mL. The MIC value (3.43 μg/mL) was observed against P. aeruginosa and C. glanduliferum. C. sulphuratum showed minimum inhibition at concentrations ranging from 1.17 to 18.75 μg/mL against S. pyogenes and E. aerogenes, S. flexneri, K. pneumonia, and P. aeruginosa. The essential oil of C. wightii exhibited MIC values of 9.375 ± 0.87, 1.17 ± 0.97, 9.375 ± 1.23, etc. μg/mL against E. faecalis, S. flexneri, S. pyogenes, etc. Marasini et al. [142] reported MIC value of 49 μg/mL against S. pyogenes by C. camphora extracts however, the present study showed more inhibitory effect compared to previous studies. C. camphora essential oil showed less inhibitory activity against E. faecalis and Enterobacter aerogenes compared to the other seven essential oils at higher concentrations (150 μg/mL). C. perrottetii exhibited varying minimum inhibitory concentrations against bacterial species with higher efficiency against S. pyogenes (2.34 μg/mL) and low efficiency against K. pneumonia with MIC value of 18.75 μg/mL. The most effective microbial inhibitory activity was observed in C. malabatrum leaf essential oil against E. faecalis and S. pyogenes with MIC value of about 1.17 μg/mL.
3.4 Free radical scavenging (antioxidant) activity of EOs
The antioxidant properties of the EOs were found promising, highly comparable with Standard (Ascorbic acid), and found to be significantly augmented with the augment in attentiveness. The antioxidant activity of EOs was recorded in terms of IC50 value and the minimum IC50 value corresponds to a greater potency [143]. All the EOs exhibited appreciable inhibition with IC50 values ranging 10.83–15.06 μg/mL which was secure to IC50 value (18.33 μg/mL) of the standard. C. verum was observed as a prospective foundation of antioxidants and recorded with the lowest IC50 value (10.83 μg/mL) followed by C. perrottetii (11.63 μg/mL), C. glanduliferum (13.02 μg/mL), C. camphora (13.04 μg/mL), C. sulphuratum(13.58 μg/ml), and C. wightii (15.06 μg/mL) (Fig. 11). However, C. malabatrum has recorded a higher IC50 value of about 15.86 μg/mL, since it was lower than standard (ascorbic acid).
The magnitude of antioxidant activity of volatile oils is generally attributed to the effects caused through the interaction of all phytoconstituents of the oil and also by the number of compounds that have been involved. Among all the oil samples studied, C. verum exhibited maximum antioxidant activity. Terpenes are considered to be a potential natural antioxidant and the main components of the aromatic plants Eos [144]. Thus, this movement could be accredited to the terpene oil content which is in agreement with the reports of Palozza and Krinsky [145]. GC-MS results of oils have also confirmed that sesquiterpenes were the principal compound of all the species studied, whereas, monoterpenes and phenylpropanoids were predominantly reported in C. verum.
DPPH scavenging activity was observed to be increased with an augment in the phenolic components such as flavonoids, phenolic diterpenes, and phenolic acids. Essential oils contain relatively higher amounts of phenolic compounds, which play a significant responsibility in antioxidant activity by acting as H donors [146, 147] In vitro studies have confirmed the antioxidant effects of EOs. However, previous reports suggested that synergistic interaction between major and minor compounds attributed to the hydrogen-donating capacity which results in the antioxidant activity of monoterpenes [148, 149] sesquiterpenes [150], phenolic compound, Eugenol [74, 151,152,153,154] reported with significant concentrations. Several studies have proven the potency of the phenolic derivative in which eugenol has the most powerful antioxidant activity and radical-scavenging activity [155, 156].
3.5 Larvicidal activity of EOs of Cinnamomum spp.
The toxicity effects of EOs of seven Cinnamomum species were analyzed beside fourth instar larvae of Aedes aegypti. All the oil samples exhibited promising larvicidal activity and the results were recorded with statistical data regarding LC50, LC90, and confidence limits by probit analysis using PROBIT software SPSS. The mortality percentages of third instar larvae of A. aegypti tested against essential oils were recorded as dose-dependent by increased with increasing concentrations. The lethal efficiency of oils in the concentration with 90% mortality (LC90), 50% death (LC50), and 95% confidence period values were calculated. The positive control temephos showed LC50 and LC90 values at 0.021 μg/mL (0.020–0.023) and 0.042 μg/mL (0.035–0.05). Negative control (DMSO) has not shown mortality and is active at ≤ 10 h exposure. The strong larvicidal activity was observed in C. verum with the most effective LC50 and LC90 values of 101.87 μg/mL (56.21–147.54 μg/mL) and 266.34 μg/mL (181–351.78 μg/mL) respectively. C. sulphuratum exhibited 104.579 μg/mL (66.39–141.05 μg/mL) and 242.61 μg/mL (159.544–325.687 μg/mL) LC90 and LC50 values respectively (Table 4).
The oils of C. perrottetii and C. camphora showed an almost similar susceptibility rate against the larvae of A. aegypti. C. perrottetii has shown moderate activity whereas C. wightii showed mild larvicidal activity with LC50 and LC90 values. Among the evaluated samples, A. aegypti exhibited more susceptibility to essential oils of C. glanduliferum with higher LC50 and LC90 values. The oil of C. verum has shown more sensitive to larvicidal activity with a high percentage of mortality. The consequence of the differentiations between the control and test studies was evaluated through one-way ANOVA, and p-value < 0.05 which were considered highly important. C. malabatrum oil had an LC50 value of 105.54 μg/mL with a lower assurance boundary (LCC) of 81.945 μg/mL and an higher assurance limit (UPL) of 128.545 μg/mL concentrations. LC90 value of 253.977 μg/mL (180.196–631.575) were obtained (Table 4).
There were only a few studies conducted on the larvicidal efficiency of EO in selected Cinnamomum species [141, 157, 158]. The effectiveness of these oils with a larvicidal property must be emphasized by oils the chemical compositions. In most of the oils of Cinnamomum, the dominant constituents were found as terpene derivatives and phenylpropanoids, which are responsible for larvicidal potentiality. Similar results were reported in essential oils with a higher content of phenylpropanoids [159], eugenol [160,161,162,163] and sesquiterpenes [164]. The larvicidal activity of a few species of Cinnamomum was corroborated with the present findings [158, 165,166,167,168,169,170,171,172,173].
Efficient larvicidal activity of larval and adult individuals of A. aegypti essential oil-based terpene compounds. Moreover, the structural characteristics of essential oils also contributed considerably to the consideration of larvicidal activity. The structural and activity associations of monoterpenes [174] and eugenol [162] derivatives alongside A. aegypti were supported by present findings. The results of our current study have demonstrated the potential alternative sources of mosquito larvicides, therefore, the plant-based essential oil compounds are competent when compared to existing synthetic chemicals which can be used to control the population of Aedes aegypti. Primarily essential oils affect the midgut epithelium and secondarily the malpighian tubules and gastric caeca in the mosquito larvae [175, 176] and also through physiology paralyzing and osmoregulation systems of the organisms [177].
4 Conclusion
The current study recorded the list of phytocompounds from the leaf EOs of seven species of Cinnamomum, C. sulphuratum, C. perotettii, C. verum, C. wightii, C. camphora, C. glanduliferum, and C. malabatrum. The GC-MS analysis of Eos indicated that the highest amount of eugenol and shyobunol was recorded in C. verum and C. perrottetii. In antibacterial activity, the higher zone of inhibition of Eos of C. malabatrum showed against S. pyogenes and E. faecalis followed by C. verum has shown a notable inhibition zone against K. pneumoniae . The total antioxidant assessed by IC50 values and larvicidal activities against Aedes aegypti indicated the biomedical efficiency of all seven species studied. Due to its higher efficiency, the multipurpose tree C. verum can be cultivated as a commercial crop instead of utilizing natural resources for the pharmaceutical industry for the development of drugs. Further, this research can be extended with in vivo and in vitro animal studies to confirm the potentiality of seven species of Cinnamomum in the field of biomedicine to detect novel medicines.
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We thank the National Medicinal Plant Board, New Delhi for providing financial support through a major research project (F.No. Z. 18017/187/CSS/ R&D/TN-01/2023-24- NMPB-IV A).
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Saranya Surendran; execution of laboratory studies, preparation of the manuscript; Smija, K.P: execution and assistance of laboratory studies; Raju Ramasubbu: planning of research problem, work designed, lead, review and editing of the manuscript, Azhagu Madhavan Sivalingam and Arjun Pandian reviewed, edited revised article.
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Surendran, S., KP, S., Pandian, A. et al. Analysis of chemical composition and biological efficiency of leaf essential oils isolated from seven species of Cinnamomum of the Western Ghats, India. Biomass Conv. Bioref. (2024). https://doi.org/10.1007/s13399-024-05653-8
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DOI: https://doi.org/10.1007/s13399-024-05653-8