[Essential oil of Cinnamomum camphora mitigates depression-like behavior in mice by regulating Nrf2/HO-1 pathway and inhibiting inflammatory cytokines].

This study aims to investigate the essential oil(EOL) of Cinnamomum camphora regarding its anti-depression effect and mechanism in regulating inflammatory cytokines and the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1) pathway. A mouse model of depression was established by intraperitoneal injection of lipopolysaccharide(LPS). Open field, elevated plus maze, and forced swimming tests were carried out to examine mouse behaviors. Western blot and qRT-PCR were employed to determine the expression of proteins and genes in the Nrf2/HO-1 pathway in the hippocampus. The levels of tumor necrosis factor(TNF)-α, interleukin(IL)-6, and IL-1ß in the serum were measured by enzyme-linked immunosorbent assay(ELISA). The changes of apoptosis in mouse brain were detected by Tunel staining. Compared with the blank control group, the model group showed shortened distance travelled and time spent in the central zone and reduced number of entries in the central zone in the open field test. In the elevated plus maze test, the model group showed reduced open arm time(OT%) and open arm entries(OE%). In the force swimming test, the model group showed extended duration of immobility compared with the blank control group. Compared with the model group, the treatment with EOL significantly increased the distance travelled and time spent in the central zone and increased the number of entries in the central zone in the open field test. In addition, EOL significantly increased the OT% and OE% in the elevated plus maze and shor-tened the immobility duration in the forced swimming test. The model group showed lower expression levels of Nrf2 and HO-1 and hig-her levels of TNF-α, IL-6, and IL-1ß than the blank control group. Compared with the model group, the treatment with EOL up-regulated the expression levels of Nrf2 and HO-1 and lowered the levels of TNF-α, IL-6, and IL-1ß. The Tunel staining results showed that the apoptosis rate in the brain tissue of mice decreased significantly after the treatment with EOL. To sum up, EOL can mitigate the depression-like behaviors of mice by up-regulating the expression of Nrf2 and HO-1 and preventing hippocampal inflammatory damage. The findings provide empirical support for the application of EOL and aromatherapy in the treatment of depression.

Efficacy of a vaginal suppository formulation prepared with Acacia arabica (Lam.) Willd. gum and Cinnamomum camphora (L.) J. Presl. in heavy menstrual bleeding analyzed using a machine learning technique.

Objective: This study aims to determine the efficacy of the Acacia arabica (Lam.) Willd. and Cinnamomum camphora (L.) J. Presl. vaginal suppository in addressing heavy menstrual bleeding (HMB) and their impact on participants' health-related quality of life (HRQoL) analyzed using machine learning algorithms. Method: A total of 62 participants were enrolled in a double-dummy, single-center study. They were randomly assigned to either the suppository group (SG), receiving a formulation prepared with Acacia arabica gum (Gond Babul) and camphor from Cinnamomum camphora (Kafoor) through two vaginal suppositories (each weighing 3,500 mg) for 7 days at bedtime along with oral placebo capsules, or the tranexamic group (TG), receiving oral tranexamic acid (500 mg) twice a day for 5 days and two placebo vaginal suppositories during menstruation at bedtime for three consecutive menstrual cycles. The primary outcome was the pictorial blood loss assessment chart (PBLAC) for HMB, and secondary outcomes included hemoglobin level and SF-36 HRQoL questionnaire scores. Additionally, machine learning algorithms such as k-nearest neighbor (KNN), AdaBoost (AB), naive Bayes (NB), and random forest (RF) classifiers were employed for analysis. Results: In the SG and TG, the mean PBLAC score decreased from 635.322 ± 504.23 to 67.70 ± 22.37 and 512.93 ± 283.57 to 97.96 ± 39.25, respectively, at post-intervention (TF3), demonstrating a statistically significant difference (p < 0.001). A higher percentage of participants in the SG achieved normal menstrual blood loss compared to the TG (93.5% vs 74.2%). The SG showed a considerable improvement in total SF-36 scores (73.56%) compared to the TG (65.65%), with a statistically significant difference (p < 0.001). Additionally, no serious adverse events were reported in either group. Notably, machine learning algorithms, particularly AB and KNN, demonstrated the highest accuracy within cross-validation models for both primary and secondary outcomes. Conclusion: The A. arabica and C. camphora vaginal suppository is effective, cost-effective, and safe in controlling HMB. This botanical vaginal suppository provides a novel and innovative alternative to traditional interventions, demonstrating promise as an effective management approach for HMB.

Adjusting function of camphor on primary metabolism in Cinnamomum camphora stressed by high temperature.

Cinnamomum camphora has great economic value for its wide utilization in traditional medicine and furniture material, and releases lots of monoterpenes to tolerate high temperature. To uncover the adjusting function of monoterpenes on primary metabolism and promoting their utilization as anti-high temperature agents, the photosynthetic capacities, primary metabolite levels, cell ultrastructure and associated gene expression were surveyed in C. camphora when it was blocked monoterpene biosynthesis with fosmidomycin (Fos) and fumigated with camphor (a typical monoterpene in the plant) under high temperature (Fos+38 °C+camphor). Compared with the control (28 °C), high temperature at 38 °C decreased the starch content and starch grain size, and increased the fructose, glucose, sucrose and soluble sugar content. Meanwhile, high temperature also raised the lipid content, with the increase of lipid droplet size and numbers. These variations were further intensified in Fos+ 38 °C treatment. Compared with Fos+ 38 °C treatment, Fos+ 38 °C+camphor treatment improved the starch accumulation by promoting 4 gene expression in starch biosynthesis, and lowered the sugar content by suppressing 3 gene expression in pentose phosphate pathway and promoting 15 gene expression in glycolysis and tricarboxylic acid cycle. Meanwhile, Fos+ 38 °C+camphor treatment also lowered the lipid content, which may be caused by the down-regulation of 2 genes in fatty acid formation and up-regulation of 4 genes in fatty acid decomposition. Although Fos+ 38 °C+camphor treatment improved the photosynthetic capacities in contrast to Fos+ 38 °C treatment, it cannot explain the variations of these primary metabolite levels. Therefore, camphor should adjust related gene expression to maintain the primary metabolism in C. camphora tolerating high temperature.

Allergic contact dermatitis from essential oil in consumer products: Mode of uses and value of patch tests with an essential oil series. Results of a French study of the DAG (Dermato-Allergology group of the French Society of Dermatology).

OBJECTIVE: To analyse the clinical characteristics and sensitivity of an essential oil patch test series (EOS) in patients sensitized to their own essential oils (EOs). METHOD: We analysed the clinical data and patch test results obtained with the European baseline series (BSE) and an EOS, as well as the mode of use of EOs, through a questionnaire included in the patient file. RESULTS: The study included 42 patients (79% women, average age 50 years) with allergic contact dermatitis (ACD), 8 patients required hospitalization. All patients were sensitized to the EO they used, primarily lavender (Lavandula augustifolia, 8000-28-0), tea tree (Melaleuca alternifolia leaf oil, 68647-73-4), ravintsara (Cinnamomum camphora oil, 92201-50-8), and 2 cases were attributed to helichrysum (helichrysum italicum flower absolute, 90045-56-0). 71% had positive patch tests to fragrance mix I or II, 9 only to the EOS and 4 only with their personal EO. Interestingly, 40% of patients did not spontaneously mention the use of EOs, and only 33% received advice on their use at the time of purchase. CONCLUSION: Patch tests with the BSE, limonene and linalool HP, and oxidized tea tree oil is sufficient to detect most EO-sensitized patients. The most important is to test the patient's own used EOs.

[Systematic identification of chemical forms of key terpene synthase in Cinnamomum camphora].

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.

The Mosquito Larvicidal Activity of Lignans from Branches of Cinnamomum camphora chvar. Borneol.

The chemical investigation of branches of Cinnamomum camphora chvar. Borneol guided by mosquito larvicidal activity led to the isolation of fourteen known lignans (1-14). Their structures were elucidated unambiguously based on comprehensive spectroscopic analysis and comparison with the literature data. This is the first report of these compounds being isolated from branches of Cinnamomum camphora chvar. Borneol. Compounds 3-5 and 8-14 were isolated from this plant for the first time. All compounds isolated were subjected to anti-inflammatory, mosquito larvicidal activity and cytotoxic activity evaluation. Compounds (1-14) showed significant mosquito larvicidal activity against Culex pipiens quinquefasciatus with lethal mortality in 50% (LC50), with values ranging from 0.009 to 0.24 µg/mL. Among them, furofuran lignans(1-8) exhibited potent mosquito larvicidal activity against Cx. p. quinquefasciatus, with LC50 values of 0.009-0.021 µg/mL. From the perspective of a structure-activity relationship, compounds with a dioxolane group showed high mosquito larvicidal activity and have potential to be developed into a mosquitocide.

Acute Oral Toxicity and Genotoxicity Test and Evaluation of Cinnamomum camphora Seed Kernel Oil.

Cinnamomum camphora seed kernel oil (CCSKO) is one of the important natural medium chain triglycerides (MCT) resources, with more than 95.00% of medium chain fatty acids found in the world, and has various physiological effects. However, CCSKO has not been generally recognized as a safe oil or new food resource yet. The acute oral toxicity test and a standard battery of genotoxicity tests (mammalian erythrocyte micronucleus test, Ames test, and in vitro mammalian cell TK gene mutation test) of CCSKO as a new edible plant oil were used in the study. The results of the acute oral toxicity test showed that CCSKO was preliminary non-toxic, with an LD50 value higher than 21.5 g/kg body weight. In the mammalian erythrocyte micronucleus test, there was no concentration-response relationship between the dose of CCSKO and micronucleus value in polychromatic erythrocytes compared to the negative control group. No genotoxicity was observed in the Ames test in the presence or absence of S9 at 5000 µg/mL. In vitro mammalian cell TK gene mutation test showed that CCSKO did not induce in vitro mammalian cell TK gene mutation in the presence or absence of S9 at 5000 µg/mL. These results indicated that CCSKO is a non-toxic natural medium-chain oil.

Phytotoxicity of Chemical Compounds from Cinnamomum camphora Pruning Waste in Germination and Plant Cultivation.

Much previous research has indicated most composts of pruning waste are characterized by potential phytotoxicity, it is highly correlated with the chemical compounds of raw materials. Cinnamomum camphora, a common kind of pruning waste in Southeast Asia and East Asia, is characterized by intense bioactivities due to complex chemical components. This study investigated the potential phytotoxicity of C. camphora pruning waste in light of germination and higher plant growth. C. camphora extracted from leaves completely inhibited seed germination and still showed suppression of root elongation at an extremely low dosage. C. camphora extract also displayed significant inhibition of nutrient absorption in tomato seedlings, including moisture, available nutrients (N, P and K) and key microelements (Fe, Mn, Zn and S). The gene expression of aquaporins and transporters of nitrate and phosphate was significantly up-regulated in roots. This could be regarded as a positive response to C. camphora extract for enhancing nutrient absorption. Moreover, the severe damage to the plasma membrane in roots caused by C. camphora extract might seriously affect nutrient absorption. Camphor is the main component of the C. camphora extract that may induce the phytotoxicity of plasma membrane damage, resulting in the inhibition of nutrient absorption and low biomass accumulation. This study provided a new understanding of the ecotoxicological effects of C. camphora pruning waste, indicating that the harmless disposal of pruning waste requires much attention and exploration in the future.

Physiological and Transcriptome Analyses Revealed the Mechanism by Which Deferoxamine Promotes Iron Absorption in Cinnamomum camphora.

Iron deficiency causes chlorosis and growth inhibition in Cinnamomum camphora, an important landscaping tree species. Siderophores produced by plant growth-promoting rhizobacteria have been widely reported to play an indispensable role in plant iron nutrition. However, little to date has been determined about how microbial siderophores promote plant iron absorption. In this study, multidisciplinary approaches, including physiological, biochemical and transcriptome methods, were used to investigate the role of deferoxamine (DFO) in regulating Fe availability in C. camphora seedlings. Our results showed that DFO supplementation significantly increased the Fe2+ content, SPAD value and ferric-chelate reductase (FCR) activity in plants, suggesting its beneficial effect under Fe deficiency. This DFO-driven amelioration of Fe deficiency was further supported by the improvement of photosynthesis. Intriguingly, DFO treatment activated the metabolic pathway of glutathione (GSH) synthesis, and exogenous spraying reduced glutathione and also alleviated chlorosis in C. camphora. In addition, the expression of some Fe acquisition and transport-related genes, including CcbHLH, CcFRO6, CcIRT2, CcNramp5, CcOPT3 and CcVIT4, was significantly upregulated by DFO treatment. Collectively, our data demonstrated an effective, economical and feasible organic iron-complexing agent for iron-deficient camphor trees and provided new insights into the mechanism by which siderophores promote iron absorption in plants.

Effects of Cinnamomum camphora Leaves Extracts-Flocculants Composite Algaecide on Microcystis aeruginosa Growth and Microcystins Release.

In this study, a composite algaecide containing flocculants and Cinnamomum. camphora leaves extracts (CCCLE) were synthesized. The inhibition and flocculation effects on Microcystis aeruginosa (M. aeruginosa) were investigated, and the release of microcystin-LR (MC-LR) was determined. Results showed that the CCLEC composite algaecide was effective for the inhibition and flocculation of M. aeruginosa, and the optimal dose of CCLEC composite algaecide was 1.8%, which resulted in an algae inhibition ratio of 98.00% and a flocculation efficiency of 99.44% within 5 days of M. aeruginosa culturing. Besides, the total amount of MC-LR decreased by 80.04% on day 20 compared with the control group, while the concentration of intracellular MC-LR on day 5 was 36.69 µg L-1, which was related to a portion of cells underwent apoptosis-like cell death under CCLEC composite algaecide stress. The results of this study may improve our understanding of the M. aeruginosa control by CCCLE composite algaecide.

Effects of preheat treatment and polyphenol grafting on the structural, emulsifying and rheological properties of protein isolate from Cinnamomum camphora seed kernel.

In this study, protein isolate (PI) and purified polyphenol extract (PPE) were prepared from Cinnamomum camphora seed kernel (CCSK). The effects of preheat treatment (50-90 °C) combined with polyphenol grafting (5 % PPE, w/w) on the structural, emulsifying and rheological properties of PI were investigated. Results demonstrated the preheat treatments at 80 and 90 °C significantly increased the extent of protein aggregation of PI. Fluorescence spectra and thermal behavior analysis revealed that preheat-treated PI exhibited more compact structure and higher thermal stability. Moreover, the emulsifying stability and apparent viscosity of PI were enhanced after preheat treatments at 50, 60 and 70 °C. After modification by PPE, the secondary structural changes of preheat-treated PI were confirmed by FTIR. PPE modification improved the thermal stability and antioxidant activities of preheat-treated PI. These results provide a novel way to combine the advantages of preheat treatment and polyphenol grafting in developing a novel protein ingredient.

[Chemical constituents from the leaves of Cinnamomum camphora var. linaloolifera and their anti-inflammatory activities].

Thirteen compounds were isolated and purified from the leaves of Cinnamomum camphora by the macroporous resin,silica gel,and Sephadex LH-20 column chromatographies. Those compounds were further identified by IR,UV,MS,and NMR techniques:( 2 S)-1-( 3″,4″-methylenedioxy phenyl)-3-( 2',6'-dimethoxy-4'-hydroxyphenyl)-propan-2-ol( 1),( 2 R,3 R)-5,7-dimethoxy-3',4'-methylenedioxy flavanol( 2),9-hydroxysesamin( 3),sesamin( 4),piperitol( 5),kobusin( 6),(-)-aptosimon( 7),acuminatolide( 8),1ß,11-dihydroxy-5-eudesmene( 9),lasiodiplodin( 10),vanillin( 11),p-hydroxybenzaldehyde( 12),and p-hydroxybenzoic acid ethyl ester( 13). Compound 1 was a novel compound,and compounds 2,6,7,9 and 10 were isolated from Cinnamomum plants for the first time. Compounds 4,7 and 10 were found to possess good inhibitory effect on IL-6 production in LPS-induced BV2 cells at a concentration of 20 µmol·L-1 in the in vitro bioassay,with inhibition rates of 51. 26% ± 4. 13%,67. 82% ± 3. 77% and85. 81%±1. 19%,respectively.

Study on the Composition and Physiological Activity of the Essential Oils and Extracts of Cinnamomum camphora Fruit.

Supercritical carbon dioxide (SC-CO2 ), hydrodistillation (HDO), ethanol extraction (EE), and petroleum ether extraction (PE) were used to extract the essential oil and extracts of Cinnamomum camphora fruit in this study. Gas chromatography-mass spectrometry was used to identify the volatile components of essential oils and extracts, and 63 compounds were identified. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assay and Ferric reducing ability of plasma (FRAP) assays and the inhibition experiment of bacteria and fungi (Staphylococcus aureus (S. aureus), Hay bacillus (H. bacillus), Escherichia coli (E. coli), Aspergillus niger (A. niger), Candida albicans (C. albicans)) showed these essential oils and extracts indicated antioxidant and antibacterial activities. S. aureus was the most sensitive to the essential oil (MIC=0.08 mg/ml). Combined with the Brine Shrimp Lethality Test (BSLT) experiment, HDO (LD50 =68.21 µg/ml) was considered to have the most potential natural preservative. Subsequently, the inhibitory mechanism of HDO on bacteria and fungi was explored through extracellular conductivity and SEM, and the possibility of HDO to preserve the freshness of bananas was verified through banana shelf-life experiments. The results suggested these essential oils and extracts of Cinnamomum camphora fruit indicated effectively inhibit the growth of microorganisms on the surface of bananas, extend the shelf-life, and have the potential to become a natural antiseptic ingredient.

The anti-inflammatory potential of Cinnamomum camphora (L.) J.Presl essential oil in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Borneol was widely used in traditional Chinese medicine formulas due to its pharmacological activities, e.g. sedative, anti-inflammatory, and anti-ischemic properties. Cinnamomum camphora (L.) J.Presl essential oil (BEO) is a by-product of natural crystalline borneol (NCB) production obtained by steam distillation of Cinnamomum camphora (L.) J.Presl leaves, and borneol was the main component of BEO. This study aims to investigate the anti-inflammatory effect of BEO and its corresponding mechanisms through in vitro and in vivo studies. MATERIALS AND METHODS: Human erythrocyte membrane stability assay and the acute inflammation murine model (xylene-induced ear edema) were chosen to evaluate the anti-inflammatory effect of BEO. Expression of inflammatory mediators, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor α (TNF-α) was determined by real-time quantitative polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assays (ELISA). The functional compounds in the BEO were identified by using gas chromatography-mass spectrometry (GC-MS). The steady-state transdermal diffusion rates of BEO and BEO nano-emulsion with were also determined in this study. Cytotoxicity of BEO was analyzed by cell counting kit-8 (CCK-8) assay. RESULTS: The BEO showed a high human erythrocyte membrane stabilization by inhibiting heat-induced hemolysis (IC50 = 5.29 mg/mL) and hypotonic solution-induced hemolysis (IC50 = 0.26 mg/mL) in vitro. The BEO was topically applied to mice auricles, both single and repeated administration significantly reduced xylene-induced auricle swelling (p < 0.0001). Expression of inflammatory mediators, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor α (TNF-α) in serum and tissue was significantly downregulated (p < 0.05), so as to the mRNA expression of IL-1ß (p＜0.05) and TNF-α (p < 0.001). A total of 43 components were identified and quantified by GC-MS. The most abundant was borneol [178.3 mg/mL, 20.9% (m/v)], followed by ß-caryophyllene (116.3 mg/mL), camphor (115.2 mg/mL), and limonene (89.4 mg/mL). For determining the skin permeability of BEO, the steady-state transdermal diffusion rates of BEO and BEO nano-emulsion were determined to be 6.7 and 8.9 mg/cm2·h, respectively. CONCLUSION: It is suspected that the anti-inflammatory effects in vivo and in vitro were derived from the above-mentioned components in the BEO. These findings will facilitate the development of BEO as a new and natural therapeutic agent for inflammatory skin conditions.

Anti-Inflammatory Property of the Essential Oil from Cinnamomum camphora (Linn.) Presl Leaves and the Evaluation of Its Underlying Mechanism by Using Metabolomics Analysis.

Cinnamomum camphora (Linn.) Presl has been widely used in traditional Chinese medicine for a variety of purposes. Our previous study indicated the antibacterial mechanism of the essential oil (EO) from C. camphora leaves; however, its anti-inflammatory activity and the underlying mechanism have not been clearly demonstrated. Thus, the present study investigated its anti-inflammatory property. Our data revealed that EO significantly decreased the release of nitric oxide (NO) and the mRNA expression of inducible NO synthase (iNOS) in lipopolysaccharide (LPS)-induced BV2 microglial cells. EO also attenuated LPS-induced increase in the mRNA expression and secretion of inflammatory cytokines including interleukin-6 (IL-6), IL-18, IL-1ß and tumor necrosis factor-α (TNF-α). Furthermore, the metabolic profiles of LPS-induced BV2 microglial cells treated with or without EO were explored. Thirty-nine metabolites were identified with significantly different contents, including 21 upregulated and 18 downregulated ones. Five pathways were enriched by shared differential metabolites. Compared with the control cells, the glucose level was decreased, while the lactate level was increased, in the culture supernatant from LPS-stimulated cells, which were reversed by EO treatment. Moreover, compared to the LPS-treated group, the activities of phosphofructokinase (PFK) and pyruvate kinase (PK) in EO group were decreased. In summary, the current study demonstrated that EO from C. camphora leaves acts as an anti-inflammatory agent, which might be mediated through attenuating the glycolysis capacity of microglial cells.

Optimization and Characterization of Microwave-Assisted Hydro-Distillation Extraction of Essential Oils from Cinnamomum camphora Leaf and Recovery of Polyphenols from Extract Fluid.

In this study, the efficiency of microwave-assisted hydro-distillation (MAHD) to extract essential oil from Cinnamomum camphora leaf, and the recovery of polyphenols from extract fluid were investigated. The effects of microwave power, liquid-to-material ratio, and extraction time on the extraction efficiency were studied by a single factor test as well as the response surface methodology (RSM) based on the central composite design method. The optimal extraction conditions were a microwave power of 786.27 W, liquid-to-material ratio of 7.47:1 mL/g, and extraction time of 35.57 min. The yield of essential oil was 3.26 ± 0.05% (w/w), and the recovery of polyphenols was 4.97 ± 0.02 mg gallic acid equivalent/g dry weight under the optimal conditions. Furthermore, the comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) was used to characterize the essential oils of fresh and fallen leaves, and 159 individual compounds were tentatively identified, accounting for more than 89.68 and 87.88% of the total contents, respectively. The main ingredients include sabinene, l-ß-pinene, ß-myrcene, α-terpineol, 3-heptanone, and ß-thujene, as well as δ-terpineol and 3-heptanone, which were first identified in C. camphora essential oil. In conclusion, the MAHD method could extract essential oil from C. camphora with high efficiency, and the polyphenols could be obtained from the extract fluid at the same time, improving the utilization of C. camphora leaf.

[Cloning and expression analysis of 5-phosphomevalonate kinase gene (CcPMK) in Cinnamomum camphora].

The 5-phosphomevalonate kinase(PMK) is a key enzyme in mevalonate(MVA) pathway which reversibly catalyzes the phosphorylation of mevalonate 5-phosphate(MVAP) to form mevalonate-5-diphosphate(MVAPP) in the presence of ATP and divalent metal ion such as Mg~(2+). In this research, on the basis of the transciptome database of Cinnamomum camphora, the PMK was cloned by cDNA from C. camphora, and was named CcPMK(GenBank number KU886266). The ORF of CcPMK was composed of 1 545 bp, encoding 514 amino acids. The bioinformatics analysis of CcPMK indicated that the molecular weight of the encoded protein was 56.14 kDa, with a theoretically isoelectric point of 7.64, and there was no signal peptide and transmembrane structure in putative protein. By multiple sequence alignment and phylogenetic tree analysis, we found that similarity between CcPMK and PMK amino acid sequence of other plants was as high as 75%. Among the similar sequences, 45% of them belonged to the alpha helix, while 16% belonged to the beta strand. CcPMK obtained 3 PMK protein family motifs and 1 ATP binding site Gly-Leu-Gly-Ser-Ser-Ala-Ala, and its 3 D structure contained a catalytic pocket structure, proving CcPMK as a member of PMK gene family. The result of phylogenetic tree showed that CcPMK was closely related to monocotyledon plants such as Phonenix dactylifera. The results of the Real-time PCR indicated that the expression level of CcPMK in borneol type was higher than that in linalool type, cineol type, iso-nerolidol type and camphor type. CcPMK expressed highest in roots and lowest in branches. Our results revealed that the expression level of CcPMK was different among five chemical types and different plant tissues, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.

Metabolomics analysis to evaluate the antibacterial activity of the essential oil from the leaves of Cinnamomum camphora (Linn.) Presl.

ETHNOPHARMACOLOGY RELEVANCE: Cinnamomum camphora (Linn.) Presl (C. camphora) is one of the oldest herbal medicines used as a traditional medicine, owning a wide range of biological functions including anti-bacterial, anti-oxidative, anti-fungal, anti-inflammatory, insecticidal and repellent activities. OBJECTIVE: The aim of this study was to investigate the antibacterial activity and mechanism of action of the essential oil (EO) from C. camphora. MATERIALS AND METHODS: The EO was isolated from the leaves of C. camphora by hydrodistillation, and the chemical compositions of the EO were analyzed by gas chromatography-mass spectrometry (GC-MS). The minimum inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) values of the EO were estimated by the microbroth dilution method. Growth curve was investigated by turbidimetry. Apoptosis was measured by flow cytometry. Morphological change of bacteria was observed by field emission scanning electron microscopy and transmission electron microscopy. The integrity of cell membrane was evaluated by NanoDrop and BCA Protein Assay Kit. The methicillin-resistant Staphylococcus aureus (MRSA) metabolic profile in the presence of the EO was explored by GC-MS-based metabolomics. Isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KGDH), succinic dehydrogenase (SDH) and malic dehydrogenase (MDH) activities were detected by commercial kits. RESULTS: The main components of the EO from the leaves of C. camphora were identified to be linalool (26.6%), eucalyptol (16.8%), α-terpineol (8.7%), isoborneol (8.1%), ß-phellandrene (5.1%), and camphor (5.0%). The EO had good activity against MRSA, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Salmonella gallinarum and Escherichia coli. MRSA was selected as the model bacterium to illustrate antibacterial mechanism of action of the EO, and the MIC and MBC values was 0.8 and 1.6 mg/mL, respectively. Apoptosis rate of MRSA increased in a concentration-dependent manner after the addition of EO. The cell morphology was damaged by the EO. There were 74 significantly different metabolites, including 29 upregulated and 45 downregulated metabolites in the result of metabolomics evaluation. Seven pathways were enriched by shared differential metabolites. The EO enhanced the activity of ICDH by 47.35%, while weaken MDH, SDH and α-KGDH by 72.63%, 31.52% and 63.29%, respectively. CONCLUSIONS: The EO from C. camphora showed anti-MRSA activity via damaging cell membranes and disturbing the amino metabolism.

Two spiroketal derivatives with an unprecedented amino group and their cytotoxicity evaluation from the endophytic fungus Pestalotiopsis flavidula.

Two new spiroketal derivatives with an unprecedented amino group, 2'-aminodechloromaldoxin (1) and 2'-aminodechlorogeodoxin (2), along with one known analogue dechloromaldoxin (3), were isolated from the plant endophytic fungus Pestalotiopsis flavidula. Their structures were elucidated on the basis of extensive spectroscopic analysis. The purification was cytotoxicity-guided which indicated the extract, fractions and compounds were evaluated in vitro for anti-proliferative activity against a panel of human cancer cell lines. The results showed compounds 1 and 2 with moderate cytotoxicity while 3 was inactive, which suggested -NH2 group might play a very important role for their cytotoxicity. This is the first study for P. flavidula and the first time to report the spiroketal derivatives as alkaloids from the Pestalotiopsis genus.

Algicidal properties of extracts from Cinnamomum camphora fresh leaves and their main compounds.

Plant allelochemicals are considered as the source of effective, economic and friendly-environmental algaecides. To uncover the anti-algal activities of Cinnamomum camphora fresh leaves and their main algicidal agents, we investigated the inhibitory effects of water and methanol extracts from C. camphora fresh leaves on Microcystis aeruginosa and Chlamydomonas reinhardtii cell growth, analyzed the composition of the water and methanol extracts, and determined the main compounds in extracts on the growth of the two algae and their anti-algal mechanism from photosynthetic abilities. Water and methanol extracts from C. camphora fresh leaves can inhibit M. aeruginosa and C. reinhardtii cell growth, and methanol extracts showed stronger inhibitory effects, due to their more compounds and higher molar concentration. There were 23 compounds in the water extracts, mainly including terpenoids, esters, alcohols, and ketones. Compared to the water extracts, 9 new compounds were detected in the methanol extracts, and the molar concentration of total compounds in methanol extracts increased by 1.3 folds. Camphor, α-terpineol and linalool were 3 main compounds in the water and methanol extracts. Their mixture (1: 3: 6) and individual compound showed remarkable inhibition on M. aeruginosa and C. reinhardtii cell growth. The degradation of photosynthetic pigments and the reduction of maximum quantum yield of photosystem II (PSII) photochemistry, coefficient of photochemical quenching as well as apparent electron transport rate in C. reinhardtii cells aggravated gradually with increasing the concentration of the mixture and individual compound, while the non-photochemical dissipation of absorbed light energy increased gradually, which led to the decline of photosynthetic abilities. This indicated that camphor, α-terpineol and linalool were 3 main algicidal agents in C. camphora fresh leaf extracts, and they inhibited algal growth by inducing photosynthetic pigment degradation and declining PSII efficiency. Therefore, C. camphora fresh leaf extracts and their main components have potential utilization values as algaecides.