| Loquat leaf(Eriobotrya japonica) is a traditional Chinese medicinal plant with a promising source of plant bioactive compounds. Loquat leaf extract contain pentacyclic triterpenoids(PTTs) especially corosolic acid(CA) and ursolic acid(UA) which have been shown to exhibit pharmaceutical activities such as anticancer, anti-inflammation and hypoglycemic effects. CA and UA are naturally occurring PTTs in plants which have attracted much attention due their usefulness to deal with global health problems including obesity and diabetes mellitus.Previous PTTs was extracted with conventional extraction(CE) and Soxhlet extraction(SE) methods, however these methods are time consuming, laborious, less efficient and use organic solvents which are costly, less selective, or even toxic to consumers and environments. Therefore, the objective of this research project was to extract the bioactive compounds with the central focus on extraction of PTTs with a reported human health potential from Chinese loquat dry leaves by using novel extraction techniques such as ultrasonic/microwave assisted extraction(UMAE) and sub-critical water extraction(SWE). Furthermore, the development of UMAE and SWE methods to obtain PTTs from loquat dry leaves was carried out to expand their utility as a novel extraction techniques in food discipline. Nevertheless, these new methods have to demonstrate their ability to match the extraction capabilities of conventional extraction methods. In the view of these requirements CE and SE were used to authenticate the newly developed extraction techniques(UMAE and SWE).In the first study, classic extraction techniques(CE and SE) were compared with UMAE using 90% ethanol solvent on the extraction efficiency of PTTs. It was observed that the yields of PTTs were significant different(p < 0.05) between extraction techniques. The extraction yields of PTTs obtained by SE(0.39 % for UA and 0.26 % for CA) were greater than those obtained by CE(0.33 ± 0.02 % for UA and 0.20 ± 0.01 % for CA), but were less than UMAE(0.57 ± 0.01 % for UA and 0.34 ± 0.02 % for CA). In addition, the volume of solvent used by UMAE was the lowest(100 m L of 90 % ethanol) compared to SE(150 m L of the same solvent) and CE(400 m L of the same solvent). Also, UMAE used the shortest extraction time(7.5 min per cycle) compared to the time used by CE(4 h) and SE(24 h). UMAE used the shortest time because ultrasound and microwave radiation could enhance the extraction efficiency of PTTs.The second study was carried in an effort to develop the SWE technique which used only pure water for the selective extraction of PTTs from dry loquat leaves in order to reduce the time and replace organic solvent with pure water. The temperature, pressure, time, flow rate, static and /or dynamic extraction modes were investigated to assess the effect of experimental conditions on the performance of SWE to extract PTTs from dry loquat leaves. The CE was employed in order to compare with SWE performances. SWE were performed in a sub-critical water condition at a system pressure 4- 10 MPa with the flow rate 33.33- 50.00 m L/min, temperatures 100- 250 °C, and extraction time 5- 90 min. The static mode, dynamic mode and static-dynamic mode for the extraction of CA and UA were optimized separately. LC/MS was employed for the quantification of total PTTs. The results showed high yields of PTTs(25.02 ± 0.71 mg/g) obtained at SWE conditions of 200 °C, 10 MPa, 41.67 m L/min and extraction time of 30 min(10 min static mode and 20 min dynamic mode) compared to 4 h extraction with CE(14.39 ± 1.12 mg/g). CA(9.63 ± 0.33 mg/g) and UA(15.04 ± 0.81 mg/g) were the main PTTs obtained from dry loquat leaves at 180 °C and 200 °C, respectively using static-dynamic mode. Findings from this study suggest that SWE at 150 to 200 °C, 10 MPa and 30 to 60 min might be a potential green technology to obtain PTTs from dry loquat leaves.The third study was carried out to apply the efficient and selective SWE technique operated at static-dynamic mode to compare and characterize the triterpene acids yields obtained from Chinese and Tanzanian dry loquat leaf extracts. When the extraction temperature increased from 100 to 150 °C, there was an increase of PTTs extraction efficiency from both countries, but was not significant different(p > 0.05) with exception of UA obtained from Tanzania dry loquat leaves. PTTs extraction efficiency was significant(p < 0.05) higher from temperatures 180 to 200 °C, than the lower temperatures. The highest yield of CA and UA from both materials were obtained at temperature of 180 °C and 200 °C, respectively. The total PTTs yields were 25.02 ± 0.71 mg/g and 26.05 ± 1.74 mg/g for China and Tanzania respectively. At 250 °C, the extraction efficiency decreased to about 50%. The observed decrease on extraction efficiency at temperature higher than 200 °C could be due to thermal degradation of extracts.Therefore, 180 °C and 200 °C were selected as the most efficient extraction temperatures for CA and UA, respectively. These results suggest that sub-critical water extraction technique could be considered as an alternative green extraction technology to obtain important phytochemicals from plant from different geographical regions.The fourth study was carried out to quantify and characterize major triterpenes in the loquat leaf extracts obtained by SWE compared to other extraction techniques. The crude extracts obtained by both SWE technique and traditional extraction methods(CE and SE) mainly consisted of polyphenols, flavonoids as well as triterpenes. The extraction efficiency of triterpenes using SWE was found to be about 1.7 times higher than those obtained using the traditional extraction methods. In addition, the main structural patterns of the cured extracts obtained by SWE technique were comparable to extracts obtained using the conventional methods and the only extract obtained by SWE, UA and OA were able to separate completely using LC/MS.The last study was carried out to investigate the extraction kinetics of PTTs and their extraction yield results were modeled by using the second order kinetic equation. The second order kinetics model provided yield predictions of 5.28 ± 0.04 mg/g CA at 180 °C and 7.20 ± 0.09 mg/g UA at 200 °C, these predicted values were consistent with their respective experimental data 5.38 ± 0.12 mg/g and 7.20 ± 0.11 mg/g for CA and UA respectively. The mean relative percent deviation varied from 2.2 to 4.1%, with R2 equals to 0.9798. Therefore, the novelty of SWE extraction technique identified in this dissertation in conjunction with loquat extracts rich in PTTs have great potential for application in functional food industry. |
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