Fragrant secondary Metabolites of Acacia seyal, Combretum hartmannianum and Terminalia laxiflora Fungal Fermented Wood Extracts Used in Sudanese Cosmetics

Abstract

Acacia sayal(Fabacae), Combretum hartmannianum and Terminalia laxiflora(Combretaceae) are known for their medicinal uses in Africa and Asia. An ethnobotanical study of community-based segment of the women(100 women) for the three trees in Khartoum State - Sudan, with emphasis on three localities Khartoum, Khartoum North and Omdurman was carried out, the most common names of trees of A. seyal, C. hartmannianum and T. laxiflora used in these localities were: A. seyal was Talh (81%), while (19%) was Makntosh; C. hartmannianum was Habeel (100%) and T. laxiflora was Sobage (80%), Darot (12%) Kolit (8%), the common names of the fermented wood Nikhra of A. seyal, C. hartmannianum and T. laxiflora used in Khartoum state were: Nikhra (80%), Nukhara (10%) and Guur (10%). Analysis of 100 questionnaire showed that mostly married females (73%) use A. seyal, C. hartmannianum and T. laxiflora while only 27% single females used these plants. All the questionnaire responders agreed on the usage of the fermented would as a cosmetic, i.e. Dokhan purpose. In this regard there were no differences in their response irrespective of differences in their age, jobs and level of education. A. seyal was found be used for cosmetic Dokhan purpose by 68% of the respondents, followed by 25% Combretaceae for T. laxiflora and 20% for C. hartmannianum the study also revealed that the trees, fermented wood, were also used as Bakhour by 50% of the respondents for T. laxiflora followed by 22% for A. seyal and 1% for C. hartmannianum. Other uses include mosquitoes repellant; fuel and fodder were also reported. Fungi fermented heartwood A. seyal, C. hartmannianum and T. laxiflora, methanolic extracts percentage yielded where(2.99, 3.10 and 3.64) while the non fermented samples percentage yielded where (0.64, 0.92 and 2.27) respectively. All methanolic extracts were powder and fragrant, of dark and faint brown colors. Methanolic extracts of Nikhra of T. laxiflora, C. hartmannianum and A. seyal were fractioned using petroleum ether, ethyl acetate, chloroform and aqueous. Nikhra fractions of T. laxiflora were 0.39, 1.68, 0.16 and 1.71%, while the non fermented were 0.20, 1.31, 0.05, and 0.71%, respectively. Nikhra fractions of C. hartmannianum were 0.36, 0.35, 0.49, and 1.27%, while its non fermented fractions were 0.27, 0.21, 0.09, and 0.35%, respectively. Nikhra fractions of A.seyal were 0 .21, 0.97, 0.92, and 0.89%, while its non fermented fractions were 0.06, 0.36, 0.05, and 0.17% respectively. Organoliptic survey of fragrance for a group of women questionnaires in different fractions of A. seyal, C. hartmannianum and T. laxiflora Nikhra showed that petroleum ether and ethyl acetate fractions of the three plants studied were most fragrant. Questionnaires revealed that A. seyal fragrance is preferred 53% followed by T. laxiflora 47% and C. hartmannianum is not an aromatic plant it is mostly, used for medicinal purposes (89%). ANOVA statistic analysis for study ability of organic solvents (petroleum ether, ethyl acetate, chloroform and aqueous) to extract compounds from fermented and non fermented wood of A. seyal, T. laxiflora and C. hartmannianum, showed that weight of fractions of fermented wood of plants studied are bigger than non fermented wood of plants. On comparing f ratio and f table, f ratio is practically, bigger than f table. Hence fermentation and polarity of solvent have a clear impact on the weight of the extract. The minimum inhibitory concentration (MIC) of the ethyl acetate fraction of C. hartmannianum against (S. aureus, S. typhi and E.coli) were (0.04, 0.04, 0.07) mg/ml respectively, while ethyl acetate fraction of T. laxiflora against (S. aureus, S. typhi and E.coli) were (0.04, 1.25, 0.005) mg/ml respectively and ethyl acetate fraction of A. seyal were (0.005, 0.04, 0.15) mg/ml respectively. The MIC of the chloroform fraction of C. hartmannianum against C. albicans was 0.07 mg/ml, The MIC of both A. seyal and T. laxiflora was 0.15 mg/ml against C. albicans. The MIC of chloroform fraction of C. hartmannianum against A. flavus and A. niger were (0.3, 0.04) mg/ml respectively, The MIC of the chloroform fraction of T. laxiflora against both A. flavus and A. niger was 0.6 mg/ml. The MIC of the chloroform fraction of A. seyal against both A. flavus and A. niger was 0.6 mg/ml Fractions of ethyl acetate of the three plants were most active antioxidant by using the radical scavenging technique of DPPH we found antioxidant activity percentage of A. seyal (91±0.02), T. laxiflora (90±0.01), and C. hartmannianum (89±0.01) with half minimum inhibitory concentration IC50 of A. seyal (0.482±0.073), T. laxiflora (0.347±0.0260), C. hartmannianum (0.460±0.026) mg/ml. Fractions of the ethyl acetate of three plants which have most active antioxidant were so showed the highest phenolic compouds A. seyal (424.65) mg GAE/g, T. laxiflora (594.60) mg GAE/g, and C. hartmannianum (404.96) mg GAE/g. All fractions of three plants proved to be non toxic against A. salina expect ethyl acetate and chloroform fractions of A. seyal and chloroform fractions of C. hartmannianum which possessed slight toxicity. Phytochemical screning showed that alkaloids, flavonoids, cardiac glycosides, saponins, tannin and triterpenoid / steroid were present in all fractions of A. seyal, T. laxiflora and C. hartmannianum. Triterpenoid / steroid were absent in chloroform fraction of T. laxiflora, and it was also absent in both ethyl acetate, chloroform. Triterpenoid / steroid were absent in petroleum ether fractions of C. hartmannianum. Questionnaire showed that Nikhra fragrance was stronger than non fermented wood and by using (TLC) in three plants. Fragrance in the petroleum ether and, ethyl acetate fractions were also proved to be of different scents. Additionally, the ethyl acetate fraction especially that of A. seyal was fine textured with golden yellow color. Petroleum ether fractions were white resins in the contrast. Metabolites residing in the fragrant fractions of fermented and non fermented wood of the plants studied were analyzed using TLC with the aid of diagnostic reagents. They were found to be flavonoid, phenolic acids Natural product reagent(NPR), terpenoids(van H2SO4), catechins(van HCL) and lignans(van H3PO4). Polyphenolics and terpenoids were expected to be responsible for the fragrances in the petroleum ether and ethyl acetate fractions. Removal of polyphenols as tannins with the aids of 2% NaCl reduced the fragrance in ethyl acetate fractions proving the fact of their contribution to fragrance by using (TLC). GC/MS of petroleum ether fractions of T. laxiflora, A. seyal and C. hartmannianum revealed that they are mainly composed of phenolics (fragrance aromatics compounds) as major components Lup-20(29)-en-3-ol, acetate,(3. beta)(15.71%) and tetracosamethyl-cyclododecasiloxane(3.02%), and the main terpenoids compounds of petroleum ether fractions of T. laxiflora were Eicosamethylcyclodecasiloxane(2.69%). The phenolics of the petroleum ether fractions of A. seyal were petadecanoic acid(5.64%), and tetracosamethyl-cyclododecasiloxane(4.17%), and the main terponoids compounds of the petroleum ether fraction of A. seyal was octadecanoic acid(2.52%). The phenolic of the petroleum ether fractions of C. hartmannianum was 2-tert-Butyl-5-(hydroxtmethyl)-4-formylfuran(7.73%), and the main terponoids compounds was Octadecanoic acid(2.36%).