Chemical Constituents Of Toona Sinensis And A New Method For Preparation Of Bioactive Components Of Chinese Medicine Based On Biphasic Hydrolysis

Toona sinensis?A.Juss.?Roemer?T.sinensis?,widely known as Chinese Toon or Red Toon,is an endemic species of Toona genus?Meliaceae family?in Angiosperm.As an easily accessible herbal material,the dried leaves of T.sinensis are used medicinally to treat enteritis,dysentery,diabetes,infection,halitosis,vomiting,and itch,without any significant irreversible side effects after long-term conventional therapy in clinics.In order to explore the material basis of medicinal materials deeply,and to provide scientific basis and spesific components for the quality control of red toon,the chemical constituents in the leaves and fruits of Toona sinensis were isolated and purified in this study.The chemical structures of these compounds were identified by physical and chemical methods in tandem with modern spectral techniques.In order to obtain glycosides or more active secondary glycosides in Chinese medicine,and to overcome the shortcomings of traditional acid hydrolysis or enzymatic hydrolysis,a new catalytic system was constructed using dioscorea saponins and primary glycosides of Epimedii Folium as model drugs,which was used to prepare the active components of traditional Chinese medicine efficiently and conveniently.Chapter 1 ReviewThe botanical characteristics,medicinal and edible value and the chemical constituents of the medicinal parts,including polyphenols,terpenoids,amphetamine,sulfur-containing compounds,etc.,were reviewed.In addition,the bioactivities of Toona sinensis extract and chemical constituents were described.The new approaches of preparing active components from Traditional Chinese Medicine were discussed,and the necessity of establishing new methods was put forward.On the basis of full investigation,the basis and design idea of the thesis are put forward,which lays a foundation for the development of the thesis work.Chapter 2 Study on the chemical constituents of Toona sinensisTo separate and purify the chemical constituents from fruits and leaves of Toona sinensis,organic solvent extraction,column chromatography and preparative HPLC were performed.A total of 23 compounds were obtained from them,whose structures were identified by physical and chemical methods and other modern spectral techniques including ultraviolet spectrophotometry,electrospray mass spectrometry,¹H-NMR spectrum and ¹³C-NMR spectrum.These compounds were n-dodecane?S1?,lignoceric acid?S2?,?-sitosterol?S3?,stigmasterol?S4?,vanillin?S5?,kaempferol?S6?,quercetin?S7?,methyl gallate?S8?,ethyl gallate?S9?,gallic acid?S10?,4-methyl-7-hydroxy coumarin?S11?,1,2,3,6-tetra-O-galloyl-?-D-glucopyranoside?L1?,1,2,3,4,6-penta-O-galloyl-?-D-glucopyranoside?L2?,?-?-epigallocatechin gallate?L3?,?+?-catechin?L4?,quercetin-3-O-?-D-glucopyranoside?L5?,quercetin-3-O-?-L-rhamnopyranoside?L6?,kaempferol-3-O-?-D-glucopyranoside?L7?,kaempferol-3-O-?-L-rhamnopyranoside?L8?,rutinoside?L9?,myricetin-3-O-?-L-rhamnopyranoside?L10?,quercetin-3-O-?-D-galactopyranoside?L11?,quercetin-3-O-?-L-arabinopyranoside?L12?.18 of these compounds are polyphenols.It was found that compounds S1,S2,S5,S8,S11,L3,L4and L11 were isolated from the plant for the first time,of which S1,S2,S11 and L11were isolated from the Toon genus for the first time.Chapter 3 Determination of polyphenols in the leaves of Toona sinensis1.Determination of gallic acid and methyl gallate in Toona sinensis leaves.A RP-HPLC-UV method for the determination of gallic acid and methyl gallate in Toona sinensis leaves?TSL?was established using the internal standard method,and the method was verified.The results showed that there was a good linear relationship between the peak area ratio and the concentration of gallic acid and methyl gallate within the range of 4.164?g/mL²08.2?g/mL and 4.972?g/mL²49.8?g/mL respectively.The correlation coefficient was 0.9998 and 0.9999,respectively.In the inter-day and intra-day precision test,the RSD of peak areas of componds was0.98%¹.74%,and the sample solution was stable within 24 hours.The RSD value of gallic acid content and methyl gallate content was 1.26%and 1.42%?n=6?respectively,which showed good repeatability of the method.The average recovery was in the range of 97.3%¹02.8%with the RSD value ranging from 2.16%to 4.26%?n=3?,verifying higher accuracy of the method.Using this method to analyze five batches of Red Toon leaves,the total amount of gallic acid and methyl gallate in TSL from Yantai?Shandong province?was much higher than the other four batches,while which from Zhenjiang?Jiangsu Province?was the lowest in these five batches.The results showed a geographical difference in total amout of these two components in TSL.2.Determination of Rutinoside in TSL by HPLC-UV.A method for the analysis of the content of rutinoside in TSL was established by HPLC-UV,and the analysis method was verified.The results showed that the linear relationship between the peak area and the concentration was good in the range of 10.44?g/mL²61.0?g/mL?r²=1.0000?.The RSD value of precision test was in the range of0.58%⁰.96%,and the sample solution was stable within 72 hours.The RSD value of rutinoside content is 1.63%?n=6?,which suggested that the method has good repeatability.The average recovery was in the range of 100.7%¹01.6%with the RSD value ranging from 2.03%to 3.53%?n=3?,which indicated higher accuracy of the method.Two batches of Red Toon leaves were analyzed by this method,and the contents of rutinoside were 0.673±0.010 mg/g and 0.498±0.005 mg/g respectively.Rutinosides have biological activities such as antioxidant activity and immunomodulatory,and can be used as index components of toona leaves.In additon,this method is simple and reliable,which is suitable for quality control and evaluation of TSL.3.Determination of flavonol glycosides in TSL of different origins and harvest months.A method was established to determine the content of eight flavonol glycosides simultaneously,named as L5 to L12 in TSL,and the changes of the contents of eight flavonol glycosides in different origins and harvest months were compared.The results showed that there were good linear relationships between the peak areas and the concentrations of these eight flavonol glycosides in the range of 0.9138?g/mL⁵8.48?g/mL?L5?,2.214?g/mL¹41.7?g/mL?L6?,0.3223?g/mL²0.62?g/mL?L7?,1.399?g/mL⁸9.52?g/mL?L8?,0.7725?g/mL⁴9.44?g/mL?L9?,0.08900?g/mL⁵.696?g/mL?L10?,0.09888?g/mL⁶.328?g/mL?L11?and 0.1394?g/mL⁸.924?g/mL?L12?,respectively.High correlation coefficients?r²>0.9990?of the calibration curves were obtained.The results of the intra-day and inter-day precision test showed the RSDs of the peak areas ranged from 0.45%to 1.86%?n=6?,indicating that the analysis of these eight flavonol glycosides was precise.The results of stability test indicated the test solution were stable within 72 hrs.The RSD of the contents of eight flavonol glycosides in Toon leaves was from 1.33%to 2.94%?n=6?,demonstrating agood repeatability of the developed analytical method.The results obtained from recovery test suggested that the percentage recoveries of these eight flavonol glycosides were within the range of 95.8%¹04.3%,with the RSD values ranging from 0.84%to4.61%,verifying the good accuracy of the proposed UPLC method.The contents of eight flavonol glycosides in TSL collected from ten different geographic origins were also determined.The results showed that the total amount of them in different origins was in the range of 3.667 mg/g¹3.824 mg/g.The content of flavonol glycosides L6 in these ten batchs all was the highest,with L5 and L8 slightly lower than it.The contents of eight beneficial flavonol glycosides differed largely in Red Toon harvested from continuous 10 months in the same origin.The total amount of them was in the range of 0.976 mg/g⁵.748 mg/g.The total amount of glycosides in Toon leaves began to accumulate quickly from March in the early half of the year.Afterwards,the content was maintained at a much higher level from April to June,followed by a sharp decrease and then fell down the lowest point until December.Considering that the shoots of Red Toon were mainly collected in March and April as edible vegetable,it is suitable to harvest a large number of TSL from May to June.Chapter 4 Extraction and determination of quercetin and kaempferol from TSL by biphasic acid hydrolysis and HPLC-UV method1.Extraction of quercetin and kaempferol from TSL by biphasic acid hydrolysis.A biphasic acid hydrolysis system was established to prepare flavonoid aglycones including quercetin and kaempferol from TSL.The effects of organic solvent,sulfuric acid concentration,hydrolysis temperature and hydrolysis time on the yield of quercetin and kaempferol were investigated and optimized by single factor experiment.After optimization of conditions,a biphase system was constructed with adding 50 mL of 4%sulfuric acid solution into the 60%ethanol extracted solution of TSL and then Xultrasound for 3 mins to make suspension solution.Hydrolysis reaction was performed in 80°C water bath for 1 h after 50 mL trichloromethane was added.Using this novel system for hydrolysis of TSL,the content of quercetin and kaempferol was the highest and reached to 0.228%and 0.091%,respectively.In addition,compared to conventional acid hydrolysis,the yields of quercetin and kaempferol were increased by39.0%and 44.4%respectively by developed biphasic acid hydrolysis system.Meanwhile,the concentration of sulfuric acid solution was greatly reduced by constructed biphasic acid hydrolysis system,and the operating procedures were simplified,suggesting it is a promising technology for preparation of flavonoid aglycones.2.Determination of quercetin and kaempferol from TSL by HPLC-UVA HPLC-UV method was established to determine the content of quercetin and kaempferol from TSL,and the linearity,precision,repeatability,stability and accuracy of the method were verified.The results showed that the peaks of quercetin,kaempferol and impurities in chromatogram were baseline separated and symmetrical,and the retention time was reasonable for analysis.There were strong linear relationships between the peak areas and the concentrations of these two flavonoid aglycones in the range of 0.6250?g/mL⁴0.00?g/mL with high correlation coefficients?r²=0.9999?.The results of precision test showed the RSD of the peak areas of flavonoid aglycones was less than 2%,indicating that the analysis of these two flavonoid aglycones was precise.The results of stability test indicated the test solution were stable within 48 hrs with the RSD value being less than 2%.The results obtained from recovery test suggested that the percentage recoveries of quercetin and kaempferol were within the range of 99.6%¹02.5%,with the RSD values ranging from 0.49%to 2.73%,verifying the good accuracy of the HPLC method.The contents of quercetin and kaempferol in TSL collected from ten different geographic origins were also determined and analyzed.The results showed that the amount of quercetin was obviously higher than kaempferol in all 10 origins.The maximum amount ratio of two flavonoid aglycones was 8.71,and were greater than 2in all 10 origins.The content of quercetin and total amount of two flavonoid aglycones in TSL collected from Heze city?Shandong province?were much higher than other places,while the content of kaempferol from Nanjing city?Jiangsu Province?was the highest.The content of two flavonoid aglycones in TSL from Qingdao city?Shandong province?were the lowest,0.212%and 0.023%respectively.Chapter 5 Preparation of diosgenin from Dioscorea nipponica Makino tubers by a pressurized liquid extraction method combined with pressurized biphasic acid hydrolysis1.Determination of diosgenin from Discorea nipponica Makino tubers by HPLC-UVA HPLC-UV method was established to accurately determine the content of diosgenin from Discorea nipponica Makino?DNM?tubers,and the linearity,precision,repeatability,stability and accuracy of the method were verified.The results showed that diosgenin could be well separated from interferences in test solution on baseline?R>1.5?and the peaks were symmetrical.Good linearity?r²=0.9999?was established between peak area and concentration of analyte,which was in the range of 0.01211mg/mL¹.550 mg/mL.The RSD of precision test was less than 2%and the RSD of diosgenin contents for repeatability was 1.86%,indicating the analysis method was precise and repeatable.The higher accuracy was supported with average recoveries ranging from 99.7%to 101.6%and RSD in the range of 1.07%³.63%.The sample solution prepared using the proposed method was stable for 72 h?RSD<2%?.Due to the high efficiency of sample preparation and high reliability of the HPLC method,it is feasible to use this method for routine analysis of diosgenin in the herb.2.Preparation of dioscorea saponin from DNM tubers by a pressurized liquid extraction methodA strategy was established to prepare dioscorea saponin from DNM tubers by pressurized liquid extraction.The effects of extraction temperature,solid/liquid ratio,EtOH concentration and sample amount on the yield of dioscorea saponin were investigated and optimized by single factor experiment.The optimized extracted condition was obtained when extraction temperature was 80°C,solid/liquid ratio was1:25 g/mL,EtOH concentration was 60%,and sample amount was 8.0 g.Compared to three conventional methods,the yield of dioscorea saponins extracted by proposed method was 9.1%,close to that of soxhlet extraction,and 5.3%and 10.2%higher than that of heating reflux method and ultrasonic extraction method,respectively.Total consumption of ethanol?mL/g?was reduced by 24.7%.In addition,total time consumption of pressurized liquid extraction was significantly decreased by 93.1%and44.5%,comparing to that of soxhlet extraction and heating reflux methods.Taking together,it is a more efficient and economical approach for extracting saponins from DNM tubers by pressurized liquid extraction.3.Preparation of diosgenin from dioscorea saponin by pressurized biphasic acid hydrolysisThe production of diosgenin from fresh DNM tubers was efficiently completed by pressurized biphase acid hydrolysis after extracting saponins by pressurized liquid extraction.The effects of four parameters,including the hydrolysis temperature,stirring speed,H₂SO₄ concentration and hydrolysis duration on the yield of diosgenin were investigated and optimized by single factor experiment.Based on the results of the single factor experiment,an orthogonal experiment design was used to further optimize and to investigate significance of the three factors on the diosgenin yield.The highest yield of Diosgenin was obtained under the optimal conditions,namely,150°C of hydrolysis temperature,100 rpm of stirring speed,4?L/mL of H₂SO₄ concentration and 2.0 h of hydrolysis duration.The results of orthogonal experiment indicated the influence on the yield of diosgenin decreased in the order as hydrolysis temperature?A?>sulfuric acid concentration?B?>hydrolysis duration?C?.Compared to three conventional methods,the yield of diosgenin for the new strategy was 1.86%,which was higher than the conventional methods including direct acid hydrolysis?53.7%?,acid hydrolysis after soxhlet extraction?37.8%?and two-phase combined acid hydrolysis?4.49%?,respectively.In addition,the H₂SO₄ consumption and the running cost was significantly reduced.Taking together,it is a more efficient and ecological approach for preparing diosgenin from DNM tubers by using pressurized biphasic acid hydrolysis after pressurized liquid extraction.Chapter 6 Preparation of baohuoside I and sagittatoside B from Epimedii Folium based on biphase enzymatic hydrolysisIn this chapter,a biphase enzymatic hydrolysis system was constructed to prepare rare secondary flavonol glycosides?baohuoside I and sagittatoside B?by hydrolyzing primary flavonol glycosides from Epimedii Folium.In addition,the conditions of enzyme hydrolysis reaction and the system of biphase enzymatic hydrolysis were optimized for constructing the key technology to conveniently and efficiently prepare the rare flavonol glycosides from Epimedii Folium.1.Preparation of baohuoside I from icariin by using biphase enzymatic hydrolysisA HPLC-UV method for analyzing icariin and baohuoside I was established.Then the effects of pH value,enzyme/substrate mass ratio,hydrolysis duration and temperature on the hydrolysis ratio of icariin in conventional enzyme hydrolysis were investigated and optimized by single factor experiment.After that,biphase enzymatic hydrolysis system was constructed to prepare baohuoside I from icariin,followed by optimization of the selected enzyme hydrolysis conditions such as organic solvent,substrate amount and EtOAc/buffer volume ratio.The sesults showed icariin and baohuoside I could be well separated from each other on baseline?R>1.5?under the chromatographic conditions and linearity was good within the range.As a result,for conventional enzyme hydrolysis of icariin,the optimal conditions were that,namely,the pH value of buffer was 4.0,hydrolysis duration was 6 h,enzyme/substrate mass ratio was 1?1 and temperature was 55°C.Under the conditions,icariin was completely hydrolyzed to baohuoside I by?-glucanase.The optimal conditions of biphase enzymatic hydrolysis after further experiment was ethyl acetate as the most appropriate solvent and a 5:1 ratio of EtOAc/buffer volume.Moreover,the recycling times of biphase enzymatic hydrolysis system was investigated.As a result,after four consecutive uses of enzyme solution and organic phase,the hydrolysis ratio of icariin was still higher than 80%.The results of 50-time scale-up experiments indicated the hydrolysis ratio of icariin was 88.6%and extraction ratio of baohuoside I was 95.2%when the novel system was reused by three times,with a 4957.50 yuan of the expected profit per 30 g of baohuoside I.Compared with conventional enzymatic hydrolysis,the hydrolysis rate of icariin could be increased by2.2%and had a double increase in substrate amount using biphase enzymatic hydrolysis system.Accordingly,the newly established method is a more efficient and convenient way to prepare baohuoside I from icariin.2.Preparation of sagittatoside B from epimedin B by biphase enzymatic hydrolysisA HPLC-UV method was established.to determine sagittatoside B and epimedin B.Then the effects of enzyme types,enzyme concentration,enzyme/epimedin B ratio,hydrolysis duration,reaction temperature,pH of buffer and metal ions on hydrolysis ratio of epimedin B in conventional enzyme hydrolysis were investigated and optimized by single factor experiment.After that,biphase enzymatic hydrolysis system was constructed to prepare sagittatoside B from epimedin B.Selected enzyme hydrolysis conditions such as organic solvent types,hydrolysis duration and propyl acetate/buffer volume ratio were optimized.The sesults showed sagittatoside B and epimedin B could be well separated from each other on baseline under the chromatographic conditions and linearity was good within the range?r²>0.9997?.As a result,for conventional enzyme hydrolysis of epimedin B,the optimal conditions were that,namely,the most suitable enzyme was?-glucanase,enzyme/substrate mass ratio was 1:2,hydrolysis duration was 40 mins,reaction temperature was 60°C and pH of buffer was 4.5.The optimal conditions of biphase enzymatic hydrolysis after further experiment was propyl acetate as the most appropriate organic solvent with a 1:1 ratio of propyl acetate/buffer volume and 1 h of hydrolysis duration.Furthermore,the recycling times of biphase enzymatic hydrolysis system was investigated.As a result,after seven consecutive uses of enzyme solution and organic phase,the hydrolysis ratio of epimedin B was still higher than 85%,suggesting the newly constructed system is a more efficient and convenient way to prepare sagittatoside B.