Extraction, analysis, and biological screening of Callitris species essential oils

The natural products have provided considerable value to the pharmaceutical industry over the past century and its demand is steadily increasing. This is mainly attributed to several factors; unachieved therapeutic needs, demand for bulk supplies and great impact of herbal remedies in the global market. Essential oils or essences have an extraordinary range of pharmacological activities including antiallergic, antiinflarnmatory, antimutagenic and antimicrobial activities. Essential oils find uses in pharmaceutics, cosmaceutics and aromatherapy. Essential oils can be obtained by cold press extraction, steam distillation, supercritical fluid extraction and solvent extraction. The objectives of the study were to characterise the composition of essential oils from different plant parts of Tasmanian Callitris spp C. rhomboidea and C. oblonga, to identify some of the major unknown constituents in the root oil, to investigate the composition of the solvent extracts, to investigate the biological activities of the C. rhomboidea and C. oblonga SD oils and extracts against a range of bacteria and fungi, to investigate the release of essential oils from C. rhomboidea roots in situ, and to investigate the allelopathic activities of C. rhomboidea and C. oblonga root and leaf oils. Volatile fractions from the roots, leaves, bark and fruits of Callitris oblonga and Callitris rhomboidea were obtained by steam distillation (SD) and solvent extraction with petroleum ether (PE) and dichloromethane (DCM). Essential oil composition was analysed by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionisation detection (GC-FID). Thirty six compounds, representing 85% of the steam distillate from C. rhomboidea roots, and 45 compounds representing 90% of the steam distillate from the C. oblonga roots were identified. Sesquiterpene hydrocarbons constituted the major portion of Callitris spp. root oils. The main identified constituents found in the SD root oil of C. rhomboidea were longiborneol (23%) and longifolene (5%); the main constituent from SD root oil of C. oblonga was columellarin (30%). In the SD fraction of the C. rhomboidea leaf ˜í¬±-pinene (42%), geranyl acetate (12%), citronellyl acetate (7%) and neryl acetate (6%) were the major constituents. C. oblonga SD leaf oil contained significant amount of ˜í¬±-pinene (45%), isopulegol (6%) and pulegol (2%). The major components in the fruit and bark oil of the Callitris spp. were monoterpenes such as ˜í¬±- and ˜í‚â§-pinenes. Solvent extracts contained a high percentage of diterpenes. However the major compounds analysed by GC-MS from the SD fractions of the respective plant organs were also present in their respective solvent extracts. Experiments were performed on collected roots and leaves ('in vial' experiments) and on intact roots of Callitris plants to investigate the release of volatiles utilising solid phase microextraction (SPME). A soil probe designed and manufactured inhouse was used for the study of volatiles evolved from the intact roots. Model experiments using known quantities of volatiles adsorbed onto sand were performed to determine the efficiency of the soil probe. Analysis of 'in vial' SPME sample experiments of roots demonstrated a release pattern of volatiles characterstic of the respective root oils; while leaf sample 'in vial' analysis did not show a characterstic pattern of their respective leaf oils. In situ samplings were done from 2 trees growing at different locations. GC-MS analysis of both samples demonstrated the presence of petrochemical-like hydrocarbons. The samples collected from the second site demonstrated the presence of monoterpene hydrocarbons. These monoterpenes were present in the same ratios as in the steam distilled root oils. Antimicrobial screening of Callitris essential oils, leaf oil fractions and extracts of different organs of Callitris spp. was performed against the bacteria Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermis, Dermatophilus congolensis, Proteus mirabilis, Escherichia coli and Pseudomonas aeruginosa, and the fungi Epidermophyton floccosum, Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton soudanese, Candida albicans and Phytopthora infestans. Oils were screend by a disk diffusion assay technique and the most potent essential oils against a particular organism were tested by a broth microdilution assay. None of the Callitris spp. essential oils exhibited potent activity against bacterial organisms. C. rhomboidea leaf oil exhibited comparatively high activity against M canis. The oil was fractionated and screened by a broth microdilution assay. The constituents mainly responsible for the antifungal activity were geraniol and citronellol, with MIC values of 0.13-0.06% v/v and 2.0- 1.0% v/v respectively. Antioxidant assays were performed on essential oils and methanolic extracts by the 2,2-diphenyl picrylhydrazyl (DPPH) radical scavenging assay. The oils and extracts were compared against gallic acid standards. The tested Callitris essential oils showed very weak antioxidant activity. Lettuce seeds were used to study the allelopathic effects of essential oils. Callitris oils at a concentration of 20, 4, and 1 micro litre per 20 ml, exhibited strong allelopathic effects on the growth of lettuce plants. The major unknown constituent of the C. oblonga root oil was identified as Columellarin. The composition of the solvent extracts were analysed and established. The SPME experiments demonstrated that the C. rhomboidea roots released volatiles insitu. Root oil novel to the Cupressaceae family displayed little or no activity against the tested fungal and bacterial organisms. C. rhomboidea leaf oil exhibited useful activity against Microsporum canis; the antifungal activity was primarily due to the presence of geraniol and citronellol. Lettuce seed bioassay demonstrated that Callitris oils possess allelpathic activity against the gowth of plants.