Nanoemulsified Essential Oil of Melaleuca leucadendron Leaves for Topical Application: In Vitro Photoprotective, Antioxidant and Anti-Melanoma Activities.

Melanoma, primarily caused by solar ultraviolet (UV) radiation, can be prevented by the use of sunscreens. However, the use of synthetic sunscreens raises environmental concerns. Natural compounds with antioxidant photoprotective properties and cytotoxic effects against cancer cells can be promising for the prevention and treatment of melanoma with less environmental effect. This study focuses on Melaleuca leucadendron essential oil (EO) for photoprotection and antitumor applications. EO was hydrodistilled from M. leucadendron leaves with a 0.59% yield. Gas chromatography-mass spectrometry detected monoterpenes and sesquiterpenes. Nanoemulsions were prepared with (NE-EO) and without EO (NE-B) using the phase inversion method, showing good stability, spherical or oval morphology, and a pseudoplastic profile. Photoprotective activity assessed spectrophotometrically showed that the NE-EO was more effective than NE-B and free EO. Antioxidant activity evaluated by DPPH and ABTS methods indicated that pure and nanoemulsified EO mainly inhibited the ABTS radical, showing IC50 40.72 and 5.30 µg/mL, respectively. Cytotoxicity tests on L-929 mouse fibroblasts, NGM human melanocyte, B16-F10 melanoma, and MeWo human melanoma revealed that EO and NE-EO were more cytotoxic to melanoma cells than to non-tumor cells. The stable NE-EO demonstrates potential for melanoma prevention and treatment. Further research is required to gain a better understanding of these activities.

High rates of evolution preceded shifts to sex-biased gene expression in Leucadendron, the most sexually dimorphic angiosperms.

Differences between males and females are usually more subtle in dioecious plants than animals, but strong sexual dimorphism has evolved convergently in the South African Cape plant genus Leucadendron. Such sexual dimorphism in leaf size is expected largely to be due to differential gene expression between the sexes. We compared patterns of gene expression in leaves among 10 Leucadendron species across the genus. Surprisingly, we found no positive association between sexual dimorphism in morphology and the number or the percentage of sex-biased genes (SBGs). Sex bias in most SBGs evolved recently and was species specific. We compared rates of evolutionary change in expression for genes that were sex biased in one species but unbiased in others and found that SBGs evolved faster in expression than unbiased genes. This greater rate of expression evolution of SBGs, also documented in animals, might suggest the possible role of sexual selection in the evolution of gene expression. However, our comparative analysis clearly indicates that the more rapid rate of expression evolution of SBGs predated the origin of bias, and shifts towards bias were depleted in signatures of adaptation. Our results are thus more consistent with the view that sex bias is simply freer to evolve in genes less subject to constraints in expression level.

Melaleuca leucadendron (L.) L. flower extract exhibits antioxidant and photoprotective activities in human keratinocytes exposed to ultraviolet B radiation.

Recently, there has been a demand for the replacement of chemical sunscreens with natural compounds that could prevent or restore UV-induced skin damage. Here, we investigated the photoprotective influence of the Melaleuca leucadendron ethanolic flower extract (EEMec) on factors involved in cellular and molecular UVB-induced oxidative stress in human skin keratinocytes (HaCaT). The phytochemical constituents, antioxidant potential by DPPH assay, content of total phenolic and flavonoid compounds in EEMec were evaluated. HaCaT cells were treated with EEMec followed by irradiation with UVB. CAT activity; GSH and ROS levels; and SOD1, GPx, CAT and COX-2 expression assays were employed to verify the oxidative stress, as well as EEMec effect on transmembrane transport, and pro-inflammatory and pro-apoptotic protein expression. EEMec reverted the viability loss of HaCaT cells after irradiation with UVB, exhibited significant antioxidant capacity and free radical scavenging activity in vitro, inhibited COX-2 expression and ensure protection of DNA-damage. EEMec shown a great photoprotective property to prevent keratinocytes damage induced by UV radiation and, thus a candidate potential to application as an adjuvant in sunscreen formulations as a strategy to reduce risk of sunburn and prevent skin diseases associated with UV-induced inflammation and cancer.

Diversity and functional characterization of antifungal-producing Streptomyces-like microbes isolated from the rhizosphere of cajuput plants (Melaleuca leucodendron L.)

Aims@#The study was undertaken to evaluate the diversity of actinomycetes from the rhizosphere of the cajuput plant (Melaleuca leucodendron L.) using ARDRA, and to examine their in vitro antifungal potency against selected fungi. @*Methodology and results@#A total of 78 Streptomyces-like microbes were isolated from the limestone rhizosphere of cajuput plants and cultured in SN agar medium. The ability to inhibit fungal growth was observed using a dual culture assay. The diversity of the isolates was examined by morphological and genotype profiling using ARDRA (Amplified 16S ribosomal DNA restriction analysis), following which they were assigned to eight groups. Seventeen (21.8%) strains showed the ability to produce the antifungal compound as evidenced by their antagonism in vitro against the tested fungi, namely Saccharomyces cerevisiae BY 47420, Candida albicans CGMCC 2538, Aspergillus flavus NRLL 3357, and Fusarium oxysporum KFCC 11363P in the dual culture assay. One isolate, GMR22, which showed potent antagonism against unicellular and filamentous fungi, displayed 97% 16S rRNA sequence similarity to Streptomyces vayuensis. @*Conclusion, significance and impact of study@#This study demonstrated that a diversity of Streptomyces-like antifungal producing microbes were present in the rhizosphere of cajuput plants in the Wanagama Forests of Indonesia. This rhizosphere represents a potential new source of actinomycetes that produce biologically active compounds. One selected strain (GMR22) has the potential to be developed as a commercial biofungicide.

Vegetation responses to season of fire in an aseasonal, fire-prone fynbos shrubland.

Season of fire has marked effects on floristic composition in fire-prone Mediterranean-climate shrublands. In these winter-rainfall systems, summer-autumn fires lead to optimal recruitment of overstorey proteoid shrubs (non-sprouting, slow-maturing, serotinous Proteaceae) which are important to the conservation of floral diversity. We explored whether fire season has similar effects on early establishment of five proteoid species in the eastern coastal part of the Cape Floral Kingdom (South Africa) where rainfall occurs year-round and where weather conducive to fire and the actual incidence of fire are largely aseasonal. We surveyed recruitment success (ratio of post-fire recruits to pre-fire parents) of proteoids after fires in different seasons. We also planted proteoid seeds into exclosures, designed to prevent predation by small mammals and birds, in cleared (intended to simulate fire) fynbos shrublands at different sites in each of four seasons and monitored their germination and survival to one year post-planting (hereafter termed 'recruitment'). Factors (in decreasing order of importance) affecting recruitment success in the post-fire surveys were species, pre-fire parent density, post-fire age of the vegetation at the time of assessment, and fire season, whereas rainfall (for six months post-fire) and fire return interval (>7 years) had little effect. In the seed-planting experiment, germination occurred during the cooler months and mostly within two months of planting, except for summer-plantings, which took 2-3 months longer to germinate. Although recruitment success differed significantly among planting seasons, sites and species, significant interactions occurred among the experimental factors. In both the post-fire surveys and seed planting experiment, recruitment success in relation to fire- or planting season varied greatly within and among species and sites. Results of these two datasets were furthermore inconsistent, suggesting that proteoid recruitment responses are not related to the season of fire. Germination appeared less rainfall-dependent than in winter-rainfall shrublands, suggesting that summer drought-avoiding dormancy is limited and has less influence on variation in recruitment success among fire seasons. The varied response of proteoid recruitment to fire season (or its simulation) implies that burning does not have to be restricted to particular seasons in eastern coastal fynbos, affording more flexibility for fire management than in shrublands associated with winter rainfall.

Chemical characterization of essential oil from the leaves of Callistemon viminalis (D.R.) and Melaleuca leucadendron (Linn.)

Objective:To isolate and identify the compounds in the essential oils from the leaves of Callistemon viminalis (D.R.) and Melaleuca leucadendron (Linn.) collected in Dakar,Senegal.Methods:The essential oils from the leaves of these two myrtaceaes were extracted by steam distillation and analyzed by gas chromatograph and gas chromatography-mass spectrometer.Results:A total of 34 constituents were identified in the oil of Callistemon viminalis and the major compounds were 1.8-cineole (58.12％),limonene (9.72％),α-terpineol (9.56％),geranial (6.02％),δ-elemene (3.53％),myrcene (2.96％) and α-pinene (2.49％).For the essential oil of Melaleuca leucadendron,43 constituents were identified,and 1.8-cineole (28.87％),epiglobulol (23.06％),α-pinene (12.22％),limonene (11.65％) and α-terpineol (7.06％) were major compounds.Conclusions:Considering properties of the identified major compounds,essential oils of both studied myrtaceae could be used in the medicine field including the food,pharmaceutical and cosmetic industry.

Chemical characterization of essential oil from the leaves of Callistemon viminalis (D.R.) and Melaleuca leucadendron (Linn.)

Objective To isolate and identify the compounds in the essential oils from the leaves of Callistemon viminalis (D.R.) and Melaleuca leucadendron (Linn.) collected in Dakar, Senegal. Methods The essential oils from the leaves of these two myrtaceaes were extracted by steam distillation and analyzed by gas chromatograph and gas chromatography–mass spectrometer. Results A total of 34 constituents were identified in the oil of Callistemon viminalis and the major compounds were 1.8-cineole (58.12%), limonene (9.72%), α-terpineol (9.56%), geranial (6.02%), δ-elemene (3.53%), myrcene (2.96%) and α-pinene (2.49%). For the essential oil of Melaleuca leucadendron, 43 constituents were identified, and 1.8-cineole (28.87%), epiglobulol (23.06%), α-pinene (12.22%), limonene (11.65%) and α-terpineol (7.06%) were major compounds. Conclusions Considering properties of the identified major compounds, essential oils of both studied myrtaceae could be used in the medicine field including the food, pharmaceutical and cosmetic industry.

Total growth and root-cluster production by legumes and proteas depends on rhizobacterial strain, host species and nitrogen level.

Background Root clusters are bunches of hairy rootlets produced by >1800 species in nine families. The possible involvement of micro-organisms in root-cluster formation has produced conflicting results over the last 40 years. In addition, any effect of rhizobacteria on overall plant growth of root-cluster-bearing species remains unknown. Aims To evaluate the effect of seven rhizobacteria on total plant size, and relative cluster production, by three species, and relate outcomes to their indole-3-acetic acid (IAA)-producing ability as part explanation of past disparate results. Methods We grew Leucadendron salicifolium (from South Africa), Viminaria juncea (Australia) and Lupinus albus (Europe) in gnotobiotic, hydroponic culture at two nitrogen (N) levels and inoculated them with seven bacterial strains and harvested the plants after 13 weeks. Key Results Following inoculation with all seven bacteria individually, plant growth sometimes greatly exceeded that of the aseptic controls, but, under other conditions, growth was less than the controls. Leucadendron and Lupinus failed to produce root clusters in the -N aseptic controls and Viminaria in the +N controls that was overcome by inoculating them with selected bacteria. Six bacteria were able to induce far more root clusters than those of the aseptic controls, while all bacteria sometimes suppressed cluster production in other treatments. All nine possible combinations of resource (plant size, indirect) and morphogenetic (relative cluster production, direct) effects were represented among the results, especially positive synergism (larger plants with a greater density of clusters). There was no clear relationship with IAA-producing ability of the seven bacteria, but low IAA strains of Pseudomonas putida and Bacillus magetarium were associated with greatest cluster production. Conclusions While root-cluster formation can sometimes be induced by introducing rhizobacteria to aseptic culture, the growth-promoting properties of apparently beneficial bacteria on general growth and root-cluster production are best described as facultative, as their promotory effects depend on host species, growing conditions and index of plant response used.

The Potent Inhibitors of Protein Tyrosine Phosphatase 1B from the Fruits of Melaleuca leucadendron.

BACKGROUND: Melaleuca leucadendron (Myrtaceae) is a kind of fruit used as Indonesian medicinal component and recorded in Jamu (tonic made of medical herbs) prescription records for the diabetes treatment. Its methanol extract exhibited a strong inhibitory activity with the half maximal inhibitory concentration (IC50) value of 2.05 µg/mL, while it is the same value with positive control RK-682. OBJECTIVE: To isolate the chemical constituents of M. leucadendron and to evaluate their activity against protein tyrosine phosphatase 1B (PTP1B). Further, determine their toxicity potential against T-cell protein tyrosine phosphatase (TCPTP). MATERIALS AND METHODS: Methanol extract was fractionated using silica column chromatography, and the obtained fraction was purified using Sephadex 20-LH. The structure of isolated compounds was identified based on (1)H and (13)Nuclear Magnetic Resonance Spectrometry. Furthermore, the compounds were examined against PTP1B and TCPTP. RESULTS: Methanol extract of M. leucadendron (Myrtaceae) afforded two triterpenes: Betulinic acid and ursolic acid in high quantities. Both compounds exhibited a strong inhibitory activity against PTP1B inhibition with IC50 value of 1.5 and 2.3 µg/mL, respectively (positive control RK-682, IC50 = 2.05 µg/mL). Their activity toward TCPTP, on the other hand, were at 2.4 and 3.1 µg/mL, respectively. Based on this purification work, betulinic acid and ursolic acid presented 7.6% and 2.4%, respectively, as markedly M. leucadendron most potential for betulinic acid source among Indonesian plants. The result should have demonstrated that the antidiabetes of M. dendron could be through the inhibition of PTP1B. SUMMARY: Melaleuca leucadendron is a good source for ursolic acid.Confirming traditional use for type II diabetes via PTP1B inhibition.

Synergistic repellent and irritant effect of combined essential oils on Aedes aegypti (L.) mosquitoes.

This study was designed to compare the behavioral responses of Aedes aegypti to a single essential oil and to a mixture of two or three essential oils using an excito-repellency test chamber. Mixtures were prepared from essential oils extracted from Litsea cubeba (LC), Litsea salicifolia (LS), and Melaleuca leucadendron (ML). In general, the mixture of essential oils produced a much stronger escape response by Ae. aegypti, regardless of the test conditions. No significant difference in escape responses was seen when the mixture of oils was compared with a standard commercial product containing DEET. Greater contact irritancy was seen from mixed oils of LC and LS than with other mixed oils. Mixtures of LC and LS at 0.075% showed the highest synergistic action (65.5% escaped) compared to that with unmixed oil alone at the same concentration (LC/20% and LS=32.2%). In addition, mixtures of LC and LS at 0.075% demonstrated the highest non-contact repellency (62.7%) and showed a greater effect than the use of LC (20%) or LS (20.3%) alone. We conclude that mixtures of two essential oils show potential as active ingredients for mosquito repellents.

Antioxidant and Anti-inflammatory Activities of Butanol Extract of Melaleuca leucadendron L.

Melaleuca leucadendron L. has been used as a tranquilizing, sedating, evil-dispelling and pain-relieving agent. We examined the effects of M. leucadendron L. extracts on oxidative stress and inflammation. M. leucadendron L. was extracted with methanol (MeOH) and then fractionated with chloroform (CHCl3) and butanol (BuOH). Antioxidant activity of the MeOH extract and BuOH fraction were higher than that of both α-tocopherol and butyrated hydroxytoluene (BHT). Total phenol content in the extracts of M. leucadendron L., especially the BuOH fraction, well correlated with the antioxidant activity. The anti-inflammatory activity of BuOH extracts were investigated by lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. The BuOH fraction significantly inhibited LPS-induced NO and PGE2 production. Furthermore, BuOH extract of M. leucadendron L. inhibited the expression of COX-2 and iNOS protein without an appreciable cytotoxic effect on RAW264.7 cells. The extract of M. leucadendron L. also suppressed the phosphorylation of inhibitor κBα (IκBα) and its degradation associated with nuclear factor-κB (NF-κB) activation. Furthermore, BuOH fraction inhibited LPS-induced NF-κB transcriptional activity in a dose-dependent manner. These results suggested that M. leucadendron L. could be useful as a natural anti-oxidant and anti-inflammatory resource.

Host specificity and speciation of Mycosphaerella and Teratosphaeria species associated with leaf spots of Proteaceae.

Species of Mycosphaerella and Teratosphaeria represent important foliicolous pathogens of Proteaceae. Presently approximately 40 members of these genera (incl. anamorphs) have been recorded from Proteaceae, though the majority are not known from culture, and have never been subjected to DNA sequence analysis. During the course of this study, epitypes were designated for several important species, namely Batcheloromyces leucadendri, B. proteae, Catenulostroma macowanii, Mycosphaerella marksii, Teratosphaeria bellula, T. jonkershoekensis, T. parva, and T. proteae-arboreae. Several species were also newly described, namely Batcheloromyces sedgefieldii, Catenulostroma wingfieldii, Dissoconium proteae, Teratosphaeria persoonii, T. knoxdavesii, and T. marasasii. Although accepted as being highly host specific, some species were shown to have wider host ranges, such as M. communis (Eucalyptus, Protea), M. konae (Leucospermum, Eucalyptus), M. marksii (Eucalyptus, Leucadendron), T. associata (Eucalyptus,Protea), and T. parva (Eucalyptus, Protea), which in most cases were found to co-occur with other species of Mycosphaerella or Teratosphaeria on Proteaceae. Furthermore, earlier records of T. jonkershoekensis on Proteaceae in Australia were shown to be representative of two recently described species, T. associata and T. maxii. A phenomenon of underdeveloped, or micro-ascospores was also newly observed in asci of T. maculiformis and T. proteae-arboreae. The exact purpose of asci with two distinct types of ascospores remains to be clarified, as both types were observed to germinate on agar.

Cytokinins in the xylem sap of the dioecious fynbos shrub, Leucadendron rubrum Burm. f.: seasonal fluctuations and their possible interaction with morphological characteristics as expressed in the two sexes.

Brandies of male and female plants of the dioecious Leucadendron rubrum Burm. f. were collected on the site of the Weikamp weather-station in the Jonkershoek State Forest, Stellenbosch, from July 1990 to September 1991. Xylem sap was extracted from the branches by vacuum extraction. HPLC was employed to purify the xylem sap and to separate the cytokinins prior to quantitation by ELISA. The cytokinin levels in the purified plant samples were determined using rabbit antiserum-based ELISAs for the determination of the levels of the cytokinins DHZR and IPA, and a trans-ZR specific monoclonal antibody-based ELISA for the determination of the levels of the cytokinin ZR. It was observed that the levels of the cytokinins DHZR and ZR did not differ significantly between male and female plants but did correlate with the seasonal growth patterns. The levels of the cytokinin IPA, in male and female plants of L. rubrum, not only correlated specifically with the different seasonal growth patterns observed but differed significantly between male and female plants. Peaks in the levels of all three cytokinins. i.e. ZR, DHZR and IPA, occurred during the study period. These peaks corresponded with the phenological development of L. rubrum, specifically with vegetative bud break and inflorescence differentiation and appeared to be synchronized with the growth and development. An attempt was also made to correlate cytokinin levels with the extreme differences in morphological characters exhibited by male and female plants, and possibly to explain to what extent these phytohormones influence sex expression in L. rubrum.