Study On Extraction, Purification And Activitives Of Effective Ingredients From Toona Sinensis (A.Juss.) Roem

In order to utilize effectively nature source of Toona sinensis (A.Juss.) Roem leaves, extraction methods of volatile oil from Toona sinensis sprouts and rapid determination of volatile compounds in Toona sinensis sprouts and leaves were studied. The essential oil components and their activities of different parts, producing areaes and seasons of Toona sinensis were studied; the chemical composition of the ethyl acetate extracts was isolated and identified by tracking the activitives of different polar fractions of Toona sinensis leaf extracts. Trace elements from Toona sinensis were determined. The different determination, extraction and purification methods of total flavonoids in Toona sinensis leaves were compared. Combined extracting methods of Toona sinensis sprouts volatile oil and total flavonoids were studied. The main results were as follows:(1) Supercritical CO2 extraction, ultrasonic-assisted extraction with ether, microwave-assisted extraction with ether and microwave-water vapour distillation were employed to extract the volatile oil from Toona sinensis sprouts and its compositions were analyzed by gas chromatography-mass spectrometry(GC-MS). Results showed that the chemical compositions, yield, appearance of volatile oil were extacted by four methods are different; the yield of essential oils extracted by supercritical fluid CO2 is the highest (13.3 mL·kg-1), and the color and fragrance of the oil is only inferior to that of essential oils extracted by microwave distillation extraction (yield 3.1 mL·kg-1).(2) The volatile compounds from Toona sinensis sprouts and leaves were analyzed by headspace-solid phase microextraction(HS-SPME) /GC-MS,26 and 46 kinds volatile compounds were identified and accounted for 74.86% and 91.20% of total volatile compounds, respectively (mass fraction, same as below), the major volatile compound from sprouts and leaves isβ-Caryophyllene, which accounts for 10.12% and 46.87%, respectively. HS-SPME/GC-MS is a simple, time-saving and solvent-free method, and it is a potential analytic tool for the determination of the volatile compounds of Toona sinensis.(3) Essential oils were extracted from different parts of Toona sinensis, Toona sinensis leaves in different sensons and different producing areaes by simultaneous distillation extraction (SDE)/GC-MS method were studied. The results showed that the yield of essential oil is different, The yield from Guangxi Nandan in August is the highest (7.33 mL·kg-1); the chemical compositions of all essential oils are different, but the main components are sesquiterpenoids.(4) Essential oils were extracted from Toona sinensis leaves, leaf axis and bark grown in Nanning, Guangxi in November by steam distillation (HD) and the volatile comounds were identified by GC-MS. The results showed that the chemical compositions of essential oil are different, however each kinds of essential oil contains nine kinds same compounds (sesquiterpenes oxide), but each content is different.(5) According to the results of tracking the activitives of different polar fractions of Toona sinensis leaf extracts, the ethyl acetate extracts were selected to isolate and purify chemical composition. Seven compounds were obtained by isolated and purified through normal pressure silica gel, SephadexLH-20 column chromatography and recrystallization. The structures of these compounds were elucidated by spectroscopic methods (including IR, UV, LC-ESI-MS,1H-NMR,13C-NMR) and compared with reported data. They were identified as scopoletin (1), kaempferol (2), ethyl gallate (3), quercetin (4), gallic acid (5), rutin (6), quercetin-3-O-α-L rhamnoside (7), respectively.(6) A method for the simultaneous determination of elements Fe, Mn, Cu, Zn, Ca, Mg, Na, K, Al, Pb, Cd, and Ni in Toona sinensis (A Juss.) Roem by ICP-AES was studied. The results showed that Toona sinensis (A Juss.) Roem is rich in Ca, Mg, Fe, Zn and relatively rich in K, Na, Mn, Cu, and the elements content changes with seasons and the parts in plants.(7) AICI3 spectrophotometric method and NaNO2-Al(NO3)3 spectrophotometric method for determination of flavonoids in the extracts of Toona sinensis leaves (FETs) were established and compared, the results showed that the former is more suitable for determination of flavonoids content of Toona sinensis leaf extracts.(8) A spectrophotomctric method was determined for determining 2,2-Diphenyl-1-picrylhydrazyl (DPPH·) scavenging activity of Toona sinensis leaves extracts. The evaluating criterion of DPPH·scavenging activity is regarded as "IM50", its physical meaning is:the DPPH·quality removed by the unit mass extracts when DPPH·scavenging rate is 50%.(9) Supercritical CO2 extraction, microwave-assisted extraction, ultrasound-assisted extraction, enzyme-assisted extraction, semi-bionic extraction, conventional solvent extraction, these six process conditions were studied and compared by extractive yield and IM50 of scavenging DPPH·of total flavonoids as index. Results showed that: microwave-assisted extraction is the best, it is efficient, saving time, especially suitable for extraction of Toona sinensis leaf flavonoids(ETsF). Enzyme-assisted extraction is the second. Mild with high extraction yield, it is also suitable for ETsF. Ultrasound-assisted extraction and conventional solvent extraction(there are little difference between their effects) are inferior to microwave-assisted extraction and enzyme-assisted extraction, but still could be used to ETsF. While extraction yield of supercritical carbon dioxide extraction and semi-bionic extraction is small, these two methods are unsuitable for ETsF. Optimization extraction process of the microwave-assisted extraction and enzyme-assisted extraction are determined as follows:Microwave-assisted extraction process is:temperature 80℃, each extraction time 20 min,70% EtOH as extracting solvent, stock ratio 1:12 (g·mL-1, mass:volume), microwave extracting for four times. The extractive yield is 99.08%. IM50 of scavenging DPPH·of total flavonoid is 37.1073 g DPPH··g-1 flavonoids.Enzyme-assisted extraction process is:enzyme dosage 7 mg·g-1 Toona sinensis leaves, enzymatic hydrolysis temperature 50℃, enzymatic hydrolysis time 2 h, enzymatic hydrolysis pH=4.0, extracting temperature 80℃, each extraction time 1 h,65% EtOH as extracting solvent, stock ratio 1:8 (g·mL-1, mass:volume), extracting for five times, the extractive yield was 95.00%, IM50 of scavenging DPPH·of total flavonoid was 28.0907 g DPPH··g-1 flavonoids.(10) Toona sinensis sprouts were extracted by supercritical CO2 to get volatile oil (yield 13.3 mL·kg-1) and then microwave was used to extract total flavonoids. The optimum conditions are:extraction temperature 70℃, ethanol volume (used to extract 1 g Toona sinensis sprouts) 12 mL,50% ethanol as extraction solvent, each extraction time 15 min and microwave extraction for three to four times. The mass of total flavonoids extracted from Toona sinensis sprouts is 65.1140 mg·g-1 to 72.9344 mg·g-1.(11) X-5 macroporous resin adsorption, polyethylene glycol ammonium sulfate aqueous two-phase extraction, ultrafiltration, three purification process conditions were studied and compared, results showed that:the former two motheds workes well, but SCM300 cup ultrafilter's ultrafiltration, purification has little effects. The results of better purification process showed as follows:X-5 resin separation and purification process conditions are: adsorption:temperature 15℃or so, flowing speed 3 BV/h, solution treatment capacity 6 BV, pH value 5-6. Desorption:total flavonoids adsorbted can be desorbed by 70% alcohol at flowing speed 3 BV/h, dosage 6 BV. Compared to the 7.2% of total flavonoids in crude extracts, the recovery and purity of total flavonoids are 95.5% and 43.5%, respectively. when the fresh resin is used. The recovery and purity of total flavonoids are over 80% and 20%, respectively when the resin is used five times.Polyethylene glycol ammonium sulfate aqueous two-phase extraction for enrichment and separation purification process conditions are:2.5 g PEG (MW600) and 2.5 g ammonium sulfate are added to 8 mL crude extracts, the contents are mixed thoroughly by shaking at 40℃and pH value 5.35 (the inherent pH of extracts system) for equilibration and are allowed for phase separation for 3 h. On the above-mentioned appropriate conditions, the crude extracts flavonoids concentration is enhanced from 3238.011 mg·L-1 to 4024.036 mg·L-1 in top phase, the flavonoids yield reaches 99.92%, the distribution coefficient was 1406.564.(12) Different polar fractions of ethanol (φ=90%) extracts from Toona sinensis leaves and leaf axis exhibit certain growth inhibitory activity against bacteria Escherichia coli and Bacillus subtilis, but has weak growth inhibitory activity against fungi Penicillium citrinum and Colletotrichum gloeosporioides. Among them, ethyl acetate fractions show the strongest antibacterial activity with the minimal inhibitory concentration(MIC) and the minimal bactericidal concentration(MBC) for Escherichia coli 0.3 mg·mL-1 (MIC),0.6 mg·mL-1 (MBC) and for Bacillus subtilis 0.6 mg·mL-1 (MIC),0.6 mg·mL-1 (MBC). S-8 fractions exhibit the second strongest antibacterial activity against bacteria Escherichia coli and Bacillus subtilis, MIC and MBC are both 0.8 mg·mL-1. N-butanol fractions show the most remarkable activity against fungi Penicillium citrinum and Colletotrichum gloeosporioides, MIC and MBC are both 7.1 mg·mL-1.Different polar fractions of ethanol (φ=90%) extracts from Toona sinensis leaves and leaf axis were used to test insecticidal activity against Plutella xylostella L., Spodoptera litura Fab. and Myzus persicae(Sulzer) when the total solid concentration was 10 mg·mL-1 by spray method and dipping method. Results showed that all fractions exhibited weak insecticidal activity against tested insects. All fractions have no insecticidal effect against Plutella xylostella L.; ethyl acetate fraction of leaves show the strongest insecticidal activitiy against Spodoptera litura Fab. with the mortality rate 44% after treated 72 hours; n-butanol fractions of leaves showes the strongest insecticidal activity against Myzus persicae(Sulzer) with the mortality rate 23.6% after treated 48 hours.Toona sinensis leaf or leaf axis extracts and different polar fractions exhibit DPPH·scavenging activity, but they are different. Ethyl acetate extraction fractions of Toona sinensis leaves and leaf-axis show the strongest DPPH·scavenging activity, followed by 90% ethanol extracts, and then followed by n-butanol extraction fractions. DPPH·scavenging activity of these three extraction fractions from the leaf axis are stronger than that from the leaves. (13) Essential oils of Toona sinensis leaves from Guangxi Nandan and Dongnan in August, essential oils of Toona sinensis leaf axis from Guangxi Dongnan in August exhibit significant antibacterial activity against bacteria Escherichia coli and Bacillus subtilis and fungi Penicillium citrinum and Colletotrichum gloeosporioides. The diameter of inhibition zone of essential oils are 26 mm-60 mm against bacteria and 11 mm-50 mm against fungi. MIC and MBC against Escherichia coli and Bacillus subtilis are less than 0.03125%; MIC and MBC against Penicillium citrinum is 0.2500% and 0.5000%; MIC and MBC against Colletotrichum gloeosporioides. is 0.06250% and 0.2500% for all essential oils samples.Essential oils from Toona sinensis leaves exhibit weak DPPH·scavenging activity.