5-year-old plants of Cinnamomum camphora (L.) Presl were selected, and the fresh CCPL was obtained from upper branches collected during October to November in 2018 from Jinxi (Jiangxi Province, China), Nanchang (Jiangxi Province, China), Chengdu (Sichuan Province, China), Mianyang (Sichuan Province, China), and Kunming (Yunnan Province, China). The plants were identified by Prof. Ming Yang from the Jiangxi University of Traditional Chinese Medicine.

The nutrient broth and nutrient agar were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All solvents and other chemicals of analytical grade were purchased from Sigma Chemicals Co., Ltd.

Each essential oil was obtained from the fresh CCPL (150 g) by using a Clevenger-type apparatus as described by Ait Babahmad et al. (2018). In the extractions, the relationship between leaf mass and water volume was 3/50, at a temperature of 100 °C, for a period of 5 h. The obtained oils were dried over anhydrous sodium sulfate, weighed, and stored at 4 °C until use.

The determination of the chemical composition of the essential oils was made by means of an Agilent 7890A/5975C Gas Chromatography-Mass Spectrometer, equipped with a FID detector and a HP-5 ™ fused silica capillary column (30 m × 0.25 µm× 0.25 µm film thickness). The injector and detector temperature were set at 250 and 230 °C, respectively; while the detector operated in the electron-impact ionization (EI) mode with a mass scan range from m/z 40 to 400 at 70 eV. The ion source temperature was 230 °C. Helium was used as the carrier gas at a flow of 1 mL/min, and split ratio of 1:50. The oven temperature was programmed to start at 50 °C for 1 min, followed by an increase of 5 °C/min to 200 °C, then linearly increased by 10 °C/min to a final temperature of 250 °C, where it remained for 5 min.

The identification of components was mainly based on the comparison of their GC Kovat retention indices (RI), determined by reference to a homologous series of C7-C32 n-alkanes. GC retention times were also analyzed. Computer matching with NIST 11 library and comparison of the fragmentation patterns with those reported in the literature were also performed to ensure accuracy.

All analyses were conducted in triplicate. Data were analyzed using one-way analysis of variance ANOVA and Duncan’s mean comparison test. The software (IBM SPSS Statistics 21) was used to calculate the eigenvalues and cumulative contribution rate of samples from different habitats by principal component analysis. Using Euclidean square distance as the measurement, a cluster analysis of samples from different habitats was carried out. Tukey test was used to detect significant differences (p ≤ 0.05) among the mean values obtained from the antimicrobial activity assay.

The essential oils of CCPL from 5 different habitats in China gave nearly colorless oil with a yield of 2.07% (Nanchang), light yellow oil with a yield of 1.52% (Jinxi), nearly colorless oil with a yield of 1.39% (Chengdu), light yellow oil with a yield of 1.11% (Kunming) and golden oil with a yield of 0.80% (Mianyang). The essential oil from Nanchang had the highest oil yield, while Mianyang had the lowest oil yield. Similar yields (1.83% and 1.3%) of essential oils extracted from CCPL were reported by Chen HP et al. (2014) and Satyal et al. (2013), respectively. The different yield obtained may be explained by different developmental stages and also by the environmental conditions as well as ontogenetic developments which influence the biosynthetic pathway of oil compounds (Jamali et al., 2013; Kizil et al., 2008).

The composition of the essential oils of CCPL were identified using GC-MS. The chemical compositions of the essential oils are reported in Table 1 with their retention index (RIs), the molecular formula and the relative areas of compounds. A total of 30 compounds were identified in the essential oils of CCPL from 5 different habitats. Among these compounds, only 5 compounds were identified in the essential oil of CCPL from Jinxi; while 17 compounds were identified in the essential oil of CCPL from Mianyang, Kunming, Chengdu and Nanchang.

According to the GC-MS quantitative analysis, the collected results highlighted the domination of the essential oil of CCPL from Jinxi by oxygenated monoterpenes, where linalool (96.47%) was distinguished as the chief component. Its proportion was more than twice that of Mianyang’s. Among the 17 compounds identified in the leaves’ essential oil from Mianyang, the main constituents in descending order of content were linalool (42.65%), camphor (26.08%), borneol (5.62%), nerolidol (3.59%), caryophyllene oxide (3.34%), bornyl acetate (3.12%) and eucalyptol (2.33%). The above main components accounted for 86.73% of the total amount of essential oil. A total of 17 components were detected in the essential oil of CCPL from Kunming, which accounted for 95.82%. They were mainly composed of the oxygenated monoterpenes eucalyptol (39.07%) followed by nerolidol (16.9%), 3,3,6-trimethyl-1,5-heptadine-4-one (13.42%) and α-terpineol (8.19%). Moreover, no significant difference was found between the quantitative and qualitative composition of the oils obtained from the CCPL of Chengdu or Nanchang. Comparing the composition of the essential oils of Chengdu and Nanchang, oxygenated monoterpenes (71.55 and 72.67%, respectively) and monoterpene hydrocarbons (26.41 and 24.89%, respectively) were found to be the important constituents in the study. For instance, linalool (42.65-96.47%) was identified as the major compound of the leaves’ essential oil from Jinxi and Mianyang; while 3 others were dominated by eucalyptol (39.07-55.35%). Therefore, combined with the previous studies reported by Chen et al. (2018), the CCPL from Jinxi and Mianyang belonged to the linalool type and 3 others belonged to the cineole type because of rich linalool and eucalyptol, respectively. As natural linalool is an important source for domestic products, cosmetics and fragrance applications, this significant type of Cinnamomum camphora from Jinxi is economically and practically ideal (Amiri et al., 2016; Herman et al., 2016)

In fact, these differences in chemical compounds of essential oils in quantitative and qualitative terms could be caused by several factors such as environment (Mutlu-Ingok et al., 2020), harvest time, local climate, extraction technique and variety (Harkat-Madouri et al., 2015). As essential oil of the Cinnamomum genus (Lauraceae) is an important source for chemical and pharmaceutical use (Dai et al., 2020), various profiles of famous medicinal herbs such as cinnamon (C. cassia Presl), Ceylon cinnamon (C. zeylanicum Bl.), Chai Gui (C. tamala), sassafras (C. porrectum.) and Chuan Gui (C. wilsonii Gamble) were previously described based on their major compounds of essential oils. Also, recent research works were focused on the Cinnamomum genus essential oils containing different major compounds. As shown in Table 2, aromatic compounds and oxygenated monoterpenes were reported as the main constituents in the essential oils of Cinnamomum genus. The essential oil also contained elements which characterize certain genera belonging to the Cinnamomum genus, such as linalool and camphor, which were the basis of the Sect. Camphora (Trew) Meissn. Compounds; whereas eugenol and borneol were the main compounds in Sect. Cinnamomum.

The antimicrobial properties of CCPL essential oils from 5 different habitats in China were assessed against one gram-positive bacteria (S. aureus) and 2 Gram-negative bacteria (E. coli and P. aeruginosa) by the paper disc diffusion method which was used to measure the diameter of the inhibition zone. As shown in Table 5, the obtained data suggested that all essential oils possessed different degrees of inhibition against all tested strains, apart from CCPL EO from Kunming, which had no inhibitory effects on P. aeruginosa. Among the 5 essential oils, Jinxi’s showed the strongest antibacterial properties (with 14.29±0.02mm as IZ) especially when tested against S. aureus; while the other 4 essential oils showed low bacteriostasis (6.30±0.02-8.79±0.09). Furthermore, the EO of Jinxi’s on E. coli and P. aeruginosa had larger inhibition zones (11.33±0.18mm and 7.89±0.09mm, respectively), compared to the other 4 essential oils (from 6.16±0.03 mm to 9.14±0.02 mm and from 6.47±0.15 mm to 7.37±0.02 mm, against E. coli and P. aeruginosa, respectively). Moreover, considering the antibacterial properties, the essential oils from Mianyang, Chengdu and Nanchang showed no statistical differences (p > 0.05) against S. aureus and E. coli, respectively.

Previous studies have shown that the most diverse group of isoprenoids, sesquiterpenes and monoterpenes accounted for the largest proportion of essential oils in plants (Sarikurkcu et al., 2018). In addition, Chen et al. (2018) reported that different major monoterpenoids primarily led to differences in different chemical types of volatile oils. Therefore, the similar antibacterial effects of CCPL essential oils from Chengdu and Nanchang can be attributed to the similar proportion of the major compound eucalyptol (52.2 and 55.35%, respectively). As for the EO from Mianyang, the high concentration of other constituents, such as linalool (42.65%) and camphor (26.08%) also contributed to its synergistic effects on antimicrobial activity. This antibacterial potency of the EO from Jinxi can probably be explained by the known antimicrobial effects of linalool as it was identified as the major compound (96.47%) in the essential oil of CCPL from Jinxi. Indeed, Herman et al. (2016) confirmed that the combination of tested essential oil with linalool showed a greater efficacy than the essential oil and linalool separately against Gram-negative bacteria including P. aeruginosa and E. coli, also against C. albicans (fungus). This conclusion is strongly supported by a study by Schmidt et al. (2012), who proved the antibacterial activity of linalool chemotype essential oil, which can be related to the interaction of the main constituent linalool. The study by Chen et al. (2020) also showed that the essential oil from C. camphora, which is rich in linalool had good activity against MRSA, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Salmonella gallinarum and Escherichia coli.

Generally, comparing the gram-negative bacteria, gram-positive bacteria proved to be more sensitive to the essential oils. As a matter of fact, the essential oils from different habitats had the strongest inhibitory effects on S. aureus and the weakest inhibition on P. aeruginosa. Moreover, Kunming’s oil had the minimal inhibition zones (6.30±0.02mm and 6.16±0.03mm against S. aureus and E. coli, respectively) on both tested strains, and even no effect on P. aeruginosa. This variation in antibacterial behavior could be explained by the fact that the cell wall structure of bacteria was different. Gram-negative has a special component of the outer membrane layer embedded with lipopolysaccharide (Nisar et al., 2018; Otoni et al., 2014). The addition of an outer lipophilic membrane increased the penetration of hydrophobic compounds (linalool, eucalyptol and camphor) through the membrane (Fisher et al., 2006).