Microencapsulation of citronella oil for mosquito-repellent application: formulation and in vitro permeation studies.

Citronella oil (CO) has been reported to possess a mosquito-repellent action. However, its application in topical preparations is limited due to its rapid volatility. The objective of this study was therefore to reduce the rate of evaporation of the oil via microencapsulation. Microcapsules (MCs) were prepared using gelatin simple coacervation method and sodium sulfate (20%) as a coacervating agent. The MCs were hardened with a cross-linking agent, formaldehyde (37%). The effects of three variables, stirring rate, oil loading and the amount of cross-linking agent, on encapsulation efficiency (EE, %) were studied. Response surface methodology was employed to optimize the EE (%), and a polynomial regression model equation was generated. The effect of the amount of cross-linker was insignificant on EE (%). The response surface plot constructed for the polynomial equation provided an optimum area. The MCs under the optimized conditions provided EE of 60%. The optimized MCs were observed to have a sustained in vitro release profile (70% of the content was released at the 10th hour of the study) with minimum initial burst effect. Topical formulations of the microencapsulated oil and non-microencapsulated oil were prepared with different bases, white petrolatum, wool wax alcohol, hydrophilic ointment (USP) and PEG ointment (USP). In vitro membrane permeation of CO from the ointments was evaluated in Franz diffusion cells using cellulose acetate membrane at 32 °C, with the receptor compartment containing a water-ethanol solution (50:50). The receptor phase samples were analyzed with GC/MS, using citronellal as a reference standard. The results showed that microencapsulation decreased membrane permeation of the CO by at least 50%. The amount of CO permeated was dependent on the type of ointment base used; PEG base exhibited the highest degree of release. Therefore, microencapsulation reduces membrane permeation of CO while maintaining a constant supply of the oil.

Identification and quantification of hepatotoxic pyrrolizidine alkaloids in the Ethiopian medicinal plant Solanecio gigas (Asteraceae).

The pyrrolizidine alkaloid content of Solanecio gigas (Vatke) C. Jeffrey (Asteraceae), an Ethiopian medicinal plant widely used for the treatment of colic, diarrhea, gout, otitis media, typhoid fever, and noted for its wound dressing and antiabortifacient activities was studied. The flower and leaf extracts contained 0.19% and 0.14% alkaloids (dry weight), respectively. GLC-MS analysis indicated that all the alkaloids in the flowers are pyrrolizidine alkaloids (PAs), whereas the leaves contain other type of alkaloids with PAs occurring in low concentrations. Roughly, 80% and 90% of the total PAs in the flowers and the leaves, respectively, were shown to occur as N-oxides. Eighteen alkaloids were detected in the flower extract with the retronecine type twelve-membered macrocyclic diesters integerrimine, senecionine and usaramine comprising 82% of the total PA content. Analysis of the PA profile of the leaves indicated that it has a simpler pattern than the one observed for the flowers. Only five PAs were detected in the leaves with integerrimine making up about 50% of the total PAs. Quantification of the PA content by GLC showed that the flowers and leaves contain 3321.21 and 84.84 microg per 10 g of dried plant material, respectively. These results indicate that users of this herb are at high risk of poisoning since the most toxic twelve membered macrocyclics of the retronecine type are the dominant PAs in the plant.

Knipholone, a selective inhibitor of leukotriene metabolism.

Inhibition of leukotriene formation is one of the approaches to the treatment of asthma and other inflammatory diseases. We have investigated knipholone, isolated from the roots of Kniphofia foliosa, Hochst (Asphodelaceae), for inhibition of leukotriene biosynthesis in an ex vivo bioassay using activated human neutrophile granulocytes. Moreover, activities on 12-lipoxygenase from human platelets and cycloxygenase (COX)-1 and -2 from sheep cotyledons and seminal vesicles, respectively, have been evaluated. Knipholone was found to be a selective inhibitor of leukotriene metabolism in a human blood assay with an IC(50) value of 4.2microM. However, at a concentration of 10microg/ml, the compound showed weak inhibition of 12(S)-HETE production in human platelets and at a concentration of 50microM it produced no inhibition of COX-1 and -2. In our attempt to explain the mechanism of inhibition, we examined the antioxidant activity of knipholone using various in vitro assay systems including free radical scavenging, non-enzymatic lipid peroxidation, and metal chelation. Knipholone was found to be a weak dose-independent free radical scavenger and lipid peroxidation inhibitor, but not a metal chelator. Therefore, the leukotriene biosynthesis inhibitory effect of knipholone was evident by its ability either to inhibit the 5-lipoxygenase activating protein (FLAP) or as a competitive (non-redox) inhibitor of the enzyme. Cytotoxicity results also provided evidence that knipholone exhibits less toxicity for a mammalian host cell.

Anti-inflammatory activity of extracts and a saponin isolated from Melilotus elegans.

The crude methanol extract of Melilotus elegans Ser. (Fabaceae), a plant widely used in Ethiopian traditional medicine for the treatment of asthma, haemorrhoid and lacerated wounds showed a significant anti-inflammatory activity against carrageenin-induced rat paw oedema. At a dose corresponding to 333.3 mg per kg body weight of dry plant material, the methanol extract displayed a strong inhibitory effect that was comparable to the inhibitory effect of 1 mg/kg of indomethacin in the same test system. Bioassay guided fractionation of the alcoholic extract led to the isolation of an oleanene-type triterpene saponin identified as azukisaponin V (1) ((3-O-[alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl(1 --> 2)-beta-D-glucuronopyranosyl]-soyasapogenol B). The structure of the compound was identified by using MS and extensive one- and two-dimensional NMR experiments (1H, 13C, COSY, HMQC, HMBC and NOESY). One hour after injection of carrageenin, inhibition of oedema exerted by 1 was approximately ten times higher than that of indomethacin on a molar basis.

Crown gall -- a plant tumour with biological activities.

Petroleum ether, acetone, 80% MeOH and water extracts of crown gall, a plant tumour, obtained from Eucalyptus globulus tree were screened for cytotoxic, antioxidant, antiinflammatory, embryotoxic, antitumour-promoting and antimicrobial activities. In terms of bioactivity the 80% MeOH extract was most effective followed by the acetone extract. The petroleum ether extract showed weak to moderate cytotoxic activity in dose-dependent manner against PC12 cells, mouse L fibroblasts and 1321N1 glia cells, whereas the hydroalcohol extract had no or a weak cytotoxic effect. The 80% MeOH extract exhibited strong antioxidant activity. Based on the in vitro HET-CAM assay all the extracts were effective against inflammation. The extracts did not show any embryotoxic effect at the concentrations tested. Antitumour-promoting activity (100% inhibition; 100 microg/mL) was observed in the 80% MeOH and acetone extracts. In the antimicrobial screening all extracts displayed predominantly antifungal activity against Candida sp. The extracts also showed various levels of antibacterial activity against E. faecalis, Ps. aeruginosa, Bac. subtilis and Staph. epidermidis. From the results of the investigations it can be concluded that crown gall is a valuable plant tumour tissue having interesting biological activities.

In vitro antiprotozoal activity of extract and compounds from the stem bark of Combretum molle.

The antiprotozoal activity of the Ethiopian medicinal plant Combretum molle (R. Br. ex G. Don.) Engl & Diels (Combretaceae) was evaluated by in vitro testing against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania donovani. The acetone fraction of the stem bark of this plant prepared by soxhlet extraction was inactive against the intracellular amastigotes of L. donovani and T. cruzi in murine peritoneal macrophages but showed significant activity against extracellular T. b. rhodesiense blood stream form trypomastigotes and trophozoites of P. falciparum with IC(50) values of 2.19 and 8.17 microg/mL, respectively. Phytochemical examination of the bioactive fraction resulted in the isolation of two tannins and two oleanane-type pentacyclic triterpene glycosides. One of the tannins was identified as the ellagitannin, punicalagin, whilst the structure of the other (CM-A) has not yet been fully elucidated. The saponins that were characterized as arjunglucoside (also called 4-epi-sericoside) and sericoside displayed no activity against any of the four species of protozoa tested. On the other hand, punicalagin and CM-A had IC(50) values of 1.75 and 1.50 microM, respectively, against T. b. rhodesiense and were relatively less toxic to KB cells (cytotoxic/antiprotozoal ratios of 70 and 48, respectively). The tannins also showed intermediate activity against P. falciparum, although their selectivity against these parasites was less favourable than the above. It appears that our findings are the first report of hydrolysable tannins exhibiting antitrypanosomal and antiplasmodial activities.

Investigations on antimycobacterial activity of some Ethiopian medicinal plants.

Fifteen crude extracts prepared from seven Ethiopian medicinal plants used to treat various infectious diseases were assessed for their ability to inhibit the growth of Mycobacterium tuberculosis. A preliminary screening of the crude extracts against M. tuberculosis typus humanus (ATCC 27294) was done by dilution assay using Löwenstein-Jensen medium. None of the tested extracts except the acetone fraction obtained from the stem bark of Combretum molle (R. Br. ex G. Don.) Engl & Diels (Combretaceae) showed significant inhibitory action against this strain. The acetone fraction of the stem bark of C. molle caused complete inhibition at concentrations higher than 1 mg/mL. Phytochemical analysis of the bioactive fraction led to the isolation of a major tannin and two oleanane-type pentacyclic triterpene glycosides. The tannin was identified as the ellagitannin, punicalagin, whilst the saponins were characterized as arjunglucoside (also called 4-epi-sericoside) and sericoside. All the pure compounds were further tested against the ATCC strain. Punicalagin was found to inhibit totally growth of the ATCC and also of a patient strain, which was fully sensitive to the standard antituberculosis drugs, at concentrations higher than 600 microg/mL and 1.2 mg/mL, respectively. On the other hand, the saponins failed to show any action on the ATCC strain. It appears that our findings are the first report of tannins exhibiting antimycobacterial activity.

The antioxidant activity of the essential oils of Artemisia afra, Artemisia abyssinica and Juniperus procera.

The essential oils of Artemisia afra Jacq., Artemisia abyssinica Schultz-Bip. and Juniperus procera Hoechst ex Endl. were examined for their potential radical scavenging activities. First a rapid evaluation of antioxidants was made using a TLC screening method. The abilities of the volatile oils to act as nonspecific donors for hydrogen atoms or electrons were checked in the diphenylpicrylhydrazyl assay. Oils from all three species showed positive results and were examined further. The oils of A. afra and J. procera were also effective hydroxyl radical scavenging agents when assessed in the deoxyribose degradation assay, whilst oils from A. abyssinica exhibited a paradoxical effect. In the in vitro assay for non-enzymatic lipid peroxidation in liposomes, the oils of A. afra and J. procera also displayed antioxidant potential. It was not possible to measure the effect of A. abyssinica oil in this system because certain components, e.g. alk-2-enals, interfered with the assay. The compounds that contribute to the radical scavenging activities of A. afra and J. procera were identified and then assessed for their effects in the various test systems. Finally, the qualitative and quantitative compositions of the essential oils were studied by GC-MS.

Antiviral activity against human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) of ethnobotanically selected Ethiopian medicinal plants.

Ethiopian medicinal plants used for the treatment of a variety of ailments including infectious diseases were screened for activity against human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). Seventy-one polar and nonpolar extracts derived from 21 plants belonging to 14 families were tested for inhibition of viral replication using HIV-1 (III(B)) and HIV-2 (ROD) strains. Selective inhibition of viral growth was assessed by the simultaneous determination of the in vitro cytotoxicity of each of the extracts against MT-4 cells. Six extracts made from the root bark of Bersama abyssinica Fresen, the leaves of Combretum paniculatum Vent., and Dodonaea angustifolia L.f., and the stem bark of Ximenia americana L. displayed antiviral activity at concentrations that were nontoxic to MT-4 cells. The highest selective inhibition of HIV-1 replication was observed with the acetone fraction of C. paniculatum and the methanol fraction of D. angustifolia which showed selectivity indices (ratio of 50% cytotoxic concentration to 50% effective antiviral concentration) of 6.4 and 4.9, and afforded cell protection of viral induced cytopathic effect of 100% and 99%, respectively, when compared with control samples. The greatest degree of antiviral activity against HIV-2 was achieved with the acetone extract of C. paniculatum (EC(50): 3 microg/mL), which also showed the highest selectivity index (32). The 50% cytotoxic concentration ranged from 0.5 microg/mL for the hexane extract of D. angustifolia L.f., the most cytotoxic of the extracts tested, to >250 microg/mL for some extracts such as the methanol fraction of Alcea rosea L., the least toxic tested. Only the polar extracts that were obtained by extraction with hydroalcohol, methanol or acetone exhibited inhibition of viral growth at subtoxic concentrations. The results obtained in this study enable the selection of extracts which show some specificity of action and support the further investigation of these extracts for their potential as new lead antiretroviral compounds.

Terpenoid composition of the wound-induced bark exudate of Commiphora tenuis from Ethiopia.

The bark of Commiphora tenuis Vollensen exudes a translucent, free-flowing odoriferous liquid upon wounding which was analysed by capillary GLC and GLC-MS. 42 mono- and sesquiterpenes were detected and 37 identified. The main components of the monoterpenoid fraction were alpha-pinene (60.8%), beta-pinene (8.8%), sabinene (6.3%), alpha-thujene (8.9%), limonene (5.5%), 3-carene (3.7%), beta-myrcene (1.8%), and beta-elemene (1.1%) constituting 97% of the oil. Identified sesquiterpenoid components constituted approximately 1.6% of the oil. Oleanolic acid acetate was isolated and identified as the main triterpene from the resin by 1H- and 13C-NMR. Three other triterpenes of the olean-12-ene group were also detected using GC-MS. The essential oil exhibited antibacterial activities against Staphylococcus aureus. Proteus mirabilis and E. coli with MIC between 0.5 and 1%.

The in vitro activity of Vernonia amygdalina on Leishmania aethiopica.

Anti-leishmanial activity of chloroform and methanol extracts of Vernonia amygdalina, a plant widely used in Ethiopia for the treatment of parasitic infections, has been assessed in vitro on Leishmania aethiopica. Amastigotes were more sensitive to V. amygdalina than promastigotes. The chloroform extract had a stronger parasiticidal activity, with median effective doses (ED50) of 18.5 micrograms/ml and 13.3 micrograms/ml for promastigotes and amastigotes, than the methanol extract with ED50 of 74.4 micrograms/ml and 45.8 micrograms/ml respectively. Cytotoxicity caused by V. amygdalina to host cells, the human leukaemia monocyte THP-1 cell line, as determined by the methyl tetrazolium assay, resulted in a median lethal dose (LD50) of 19.6 micrograms/ml for the chloroform extract and 243.4 micrograms/ml for the methanol extract. In comparison, the ED50 and LD50 of pentamidine, a standard anti-leishmanial drug, were 0.5 micrograms/ml and 1.4 micrograms/ml respectively. These results indicate that V. amygdalina displays potent anti-leishmanial activities and warrants further investigation.

Two novel minor alkaloids from Ethiopian Calpurnia aurea Ssp. aurea.

Two novel minor quinolizidine alkaloids 4 beta-hydroxy-13alpha- O-(2'-pyrrolylcarbonyl)-lupanine (digittine) and its amino alcohol, 4 beta, 13alpha-dihydroxylupanine, have been isolated from Ethiopian Calpurnia aurea ssp. aurea. The structures of these alkaloids were determined by chemical transformation and by means of spectroscopic techniques (UV, IR, CD, MS, (1)H-NMR and (13)C-NMR) including two dimensional NMR.