Chemical and biological characterization of Melaleuca subulata (Cheel) Craven leaves' volatile constituents supported by chemometric analysis and molecular docking.

BACKGROUND: The genus Melaleuca (Myrtaceae) comprises dozens of essential oil (EO)-rich species that are appreciated worldwide for their various medicinal values. Additionally, they are renowned in traditional medicine for their antimicrobial, antifungal, and other skin-related activities. The current study investigated the chemical profile and skin-related activities of volatile constituents derived from M. subulata (Cheel) Craven (Synonym Callistemon subulatus) leaves cultivated in Egypt for the first time. METHODS: The volatile components were extracted using hydrodistillation (HD), headspace (HS), and supercritical fluid (SF). GC/MS and Kovat's retention indices were implemented to identify the volatile compounds, while the variations among the components were assessed using Principal Component Analysis and Hierarchical Cluster Analysis. The radical scavenging activity was assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC) and ß-carotene assays. Moreover, the anti-aging effect was evaluated using anti-elastase, and anti-collagenase, while the antimicrobial potential was deduced from the agar diffusion and broth microdilution assays. Lastly, the molecular docking study was executed using C-docker protocol in Discovery Studio 4.5 to rationalize the binding affinity with targeted enzymes. RESULTS: The SF extraction approach offered the highest EO yield, being 0.75%. According to the GC/MS analysis, monoterpene hydrocarbons were the most abundant volatile class in the HD oil sample (54.95%), with α-pinene being the most copious component (35.17%). On the contrary, the HS and SF volatile constituents were pioneered with oxygenated monoterpenes (72.01 and 36.41%) with eucalyptol and isopulegone being the most recognized components, representing 67.75 and 23.46%, respectively. The chemometric analysis showed segregate clustering of the three extraction methods with α-pinene, eucalyptol, and isopulegone serving as the main discriminating phytomarkers. Concerning the bioactivity context, both SF and HD-EOs exhibited antioxidant effects in terms of ORAC and ß-carotene bleaching. The HD-EO displayed potent anti-tyrosinase activity, whereas the SF-EO exhibited significant anti-elastase properties. Moreover, SF-EO shows selective activity against gram-positive skin pathogens, especially S. aureus. Ultimately, molecular docking revealed binding scores for the volatile constituents; analogous to those of the docked reference drugs. CONCLUSIONS: M. subulata leaves constitute bioactive volatile components that may be indorsed as bioactive hits for managing skin aging and infection, though further in vivo studies are recommended.

Melaleuca rugulosa (Link) Craven Tannins: Appraisal of anti-inflammatory, radical scavenging activities, and molecular modeling studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Genus Melaleuca or tea tree species are well known to be an important source of biological active oils and extracts. The biological significance appears in their usage for treatment of several clinical disorder owing to their traditional uses as anti-inflammatory, antibacterial, antifungal, and cytotoxic activities. AIM OF THE STUDY: Our study aimed to investigate the metabolic profile of the M. rugulosa polyphenol-rich fraction along with determination of its anti-inflammatory potential, free radical scavenging and antiaging activities supported with virtual understanding of the mode of action using molecular modeling strategy. MATERIALS AND METHODS: The anti-inflammatory activity of the phenolic rich fraction was investigated through measuring its inhibitory activity against inflammatory mediators viz tumor necrosing factor receptor-2 (TNF-α) and cyclooxygenases 1/2 (COX-1/2) in a cell free and cell-based assays. Moreover, the radical scavenging activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC) and ß-carotene assays, while the antiaging activity in anti-elastase, anti-collagenase, and anti-tyrosinase inhibitory assays. Finally, the biological findings were supported with molecular docking study using MOE software. RESULTS: The chromatographic purification of the polyphenol-rich fraction of Melaleuca rugulosa (Link) Craven afforded fourteen phytoconstituents (1-14). The anti-inflammatory gauging experiments demonstrated inhibition of inflammatory-linked enzymes COX-1/2 and the TNF-α at low µg/mL levels in the enzyme-based assays. Further investigation of the underlying mechanism was inferred from the quantification of protein levels and gene expression in the lipopolysaccharide (LPS)-activated murine macrophages (RAW264.7) in vitro model. The results revealed the reduction of protein synthesis of COX-1/2 and TNF-α with the down regulation of gene expression. The cell free in vitro radical scavenging assessment of the polyphenol-rich fraction revealed a significant DPPH reduction, peroxyl radicals scavenging, and ß-carotene peroxidation inhibition. Besides, the polyphenol-rich fraction showed a considerable inhibition of the skin aging-related enzymes as elastase, collagenase, and tyrosinase. Ultimately, the computational molecular modelling studies uncovered the potential binding poses and relevant molecular interactions of the identified polyphenols with their targeted enzymes. Particularly, terflavin C (8) which showed a favorable binding pose at the elastase binding pocket, while rosmarinic acid (14) demonstrated the best binding pose at the COX-2 catalytic domain. In short, natural polyphenols are potential candidates for the management of free radicals, inflammation, and skin aging related conditions. CONCLUSION: Natural polyphenols are potential candidates for the management of free radicals, inflammation, and skin aging related conditions.

Ethnopharmacological impact of Melaleuca rugulosa (Link) Craven leaves extract on liver inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Melaleuca species have been used by many ethnic communities for the management and treatment of several ailments as hemorrhoids, cough, skin infections, rheumatism, sore throat, pain, inflammation, and digestive system malfunctions. However, the detailed mechanistic pharmacological effect of Melaleuca rugulosa (Link) Craven leaves in the management of liver inflammation has not been yet addressed. AIM OF THE STUDY: The present study aimed to evaluate the anti-inflammatory, antioxidant, and antiapoptotic capacities of the aqueous methanol extract of M. rugulosa leaves in relevance to their flavonoid content using an appropriate in vivo model. MATERIALS AND METHODS: The aqueous methanol extract of M. rugulosa leaves was administered to the rats at three non-toxic doses (250, 500, and 1000 mg/kg) for seven days prior to the initiation of liver-injury induced by paracetamol (3 g/kg). Liver enzymes including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were evaluated in serum samples. The oxidative stress markers including reduced glutathione (GSH), malondialdehyde (MDA), and nitric oxide (NO) levels as well as the inflammatory markers such as tumour necrosis factor-alpha (TNF-α) and nuclear factor-kappa B (NF-κB), were assessed in liver homogenate. The results were supported by histopathological and immuno-histochemical studies. The phytochemical investigation of the flavonoid-rich fraction of the aqueous methanol extract was accomplished using different chromatographic and spectroscopic techniques. RESULTS: The aqueous methanol extract of M. rugulosa leaves showed a powerful hepatoprotective activity evidenced by the significant reduction of MDA and NO levels, as well as increasing GSH and catalase activity. Moreover, the extract exhibited anti-inflammatory and antiapoptotic activities witnessed by decreasing TNF-α, NF-κB, iNOS, p-JNK, caspase-3, BAX, and increasing Bcl-2 levels. Moreover, the pretreatment of rats with all doses of M. rugulosa leaves extract showed a significant decrease in liver weight/body weight (LW/BW) ratio, and total bilirubin induced by paracetamol. On the other hand, the chromatographic separation of the flavonoid-rich fraction afforded twenty known flavonoids namely; iso-orientin (1), orientin (2), isovitexin (3), vitexin (4), quercetin-3-O-ß-D-glucuronid methyl ether (5), quercetin-3-O-ß-D-mannuronpyranoside (6), isoquercetin (7), quercitrin (8), kaempferol-3-O-ß-D-mannuronopyranoside (9), kaempferol-7-O-methyl ether-3-O-ß-D-glucopyranoside (10), guaijaverin (11), avicularin (12), kaempferide-3-O-ß-D-glucopyranoside (13), astragalin (14), afzelin (15), luteolin (16), apigenin (17), quercetin (18), kaempferol (19), and catechin (20). CONCLUSION: The aqueous methanol extract of M. rugulosa leaves showed potential hepatoprotective, antioxidant, and anti-inflammatory activities against paracetamol-induced liver inflammation which is correlated at least in part to its considerable phenolic content.

Polyphenolic profile and ethno pharmacological activities of Callistemonsubulatus (Cheel) Craven leaves cultivated in Egypt.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Callistemon (syn. Melaleuca) of the myrtle family (Myrtaceae) has been documented as an integral part in the ethnobotanical system of the indigenous people of Australian mainland and many of its islands. Several Callistemons including the species subulatus were used by aboriginal Australians for making rafts, roofs for shelters, bandages, and food recipes, in addition to the management of wounds, infections, pain, cough, bronchitis, and gastrointestinal tract (GIT) disorders. AIM OF THE STUDY: The current study is designed to document the therapeutic effect of the aqueous methanolic extract (AME) of C. sabulatus Chell (syn. M. sabulata) leaves in the management of diarrhea and pain. Also, its influence on additional pharmacological modalities that are related to oxidative stress just as skin aging. Ultimately, the polyphenolic profile of the extract is disclosed and correlated to the aforementioned bioactivities. MATERIALS AND METHODS: The extract was fractionated using various chromatography techniques and the structures of the isolated compounds were determined based on their chemical and spectral data. The antioxidant activity was assessed using multiple models, including 2,2-diphenyl-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC) and ß-carotene bleaching assays. The anti-skin aging effect was evaluated using different relevant enzymatic assays. The antinociceptive activity was investigated using acetic acid-induced writhing, hot plate test, and formalin-induced paw licking in mice models. The antidiarrheal activity was gauge using the castor oil induced diarrhea, enter pooling and gastrointestinal motility in vivo models. RESULTS: Diverse polyphenols, including quercetin-3-O-ß-D-glucuronopyranoside (1), kaempferol-3-O-ß-D-glucuronopyranoside (2), strictinin (3), quercetin-3-O-(2``-O-galloyl)-ß-D-glucuronopyranoside (4), afzelin (5), di-galloyl glucose (6), mono-galloyl glucose (7), acacetin (8), apigenin-6,7-dimethyl ether (9), kaempferol trimethyl ether (10), dimethoxy chrysin (11), quercetin (12), kaempferol (13), methyl gallate (14), and gallic acid (15) were identified. The extract exhibited as significant antioxidant activity even better than that of Trolox or BHT. Moreover, it exerts elastase, tyrosinase, and collagenase inhibition activities, in addition to the significant peripheral and central analgesic activity in a dose-dependent manner (P < 0.0001). In castor oil induced diarrhea model, AME significantly prolonged the diarrhea onset, decreased the frequency of defecation, and weight of feces. Likewise, it exhibited a significant reduction in the gastrointestinal motility in charcoal meal model (P < 0.0001) and a considerable inhibitory effect on gastrointestinal transit and peristaltic index with all investigated doses (P < 0.0001). CONCLUSION: Ethnobotanicals are versatile resources for the management of various ailments by indigenous people and the experimental research is utmost to validate and uncover their pharmacological relevance. C. sabulatus leaves have strong antioxidant, analgesic, anti-skin aging, and antidiarrheal activities which are validated for the first time by various in vitro and in vivo models. The metabolic profile of the unprecedented AME of C. sabulatus leaves compromises a wide array of bioactive polyphenolic metabolites including, flavonoids, tannins, and phenolic acids that are correlated to the observed bioactivities. Altogether, ethnobotanicals with high and diverse contents of polyphenols are potential candidates for the management of various human aliments including neuropathies, GIT disorders, and skin aging conditions.