Improving the position specificity of Themomyces lanuginosus lipase based on semi-rational design / 生物工程学报

Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.

Optimisation of asarone removal from Piper sarmentosum Roxburgh leaves using supercritical carbon dioxide extraction: the Box-Behnken design

Abstract Piper sarmentosum is a herbaceous shrub with numerous pharmacological benefits. However, the presence of two toxic phenylpropanoids (α- and β-asarone) limits the medicinal usage of the plant. In this study, the extraction of three asarone isomers, namely α-, β-, and -asarone was optimised using supercritical carbon dioxide extraction (SC-CO2) combined with Box-Behnken experimental design. Comparison of asarone contents in different conventional solvent extracts of P. sarmentosum leaves prior to and after SC-CO2 extraction was performed. The SC-CO2 method successfully maximised the extraction of α-, β-, and ɣ-asarone at P = 81.16 bar, T = 50.11°C, and t = 80.90 min, yielding 13.91% α-asarone, 3.43% β-asarone, and 14.95% ɣ-asarone. The SC-CO2 residue of the leaves re-extracted with conventional solvents showed a significant decrease of asarone ranging from 45% to 100% (p<0.001) compared to their counterparts without SC-CO2 treatment. α-, β-, and ɣ-asarone were completely removed in the ethanol extract of the residue. These findings suggested that the optimised SC-CO2 extraction parameters may serve as a quick treatment step for the selective removal of asarone from P. sarmentosum to develop safer extracts for the food and nutraceutical industries applications.

Simultaneous enantioseparation and simulation studies of atenolol,metoprolol and propranolol on Chiralpak(R) IG column using supercritical fluid chromatography / 药物分析学报

Enantioseparation of three β-blockers,i.e.,atenolol,metoprolol and propranolol,was studied on amylose tris(3-chloro-5-methylphenylcarbamate) immobilized chiral stationary phase using supercritical fluid chromatography (SFC).The effect of organic modifiers (methanol,isopropanol and their mixture),col-umn temperature and back pressure on chiral separation of β-blockers was evaluated.Optimum chro-matographic separation with respect to resolution,retention,and analysis time was achieved using a mixture of CO2 and 0.1％ isopropyl amine in isopropanol:methanol (50:50,V/V),in 75:25 (V/V) ratio.Under the optimized conditions,the resolution factors (Rs) and separation factors (α) were greater than 3.0 and 1.5,respectively.Further,with increase in temperature (25-45 ℃) and pressure (100-150 bars)there was corresponding decrease in retention factors (k),α and Rs.However,a reverse trend (α and Rs)was observed for atenolol with increase in temperature.The thermodynamic data from van't Hoff plots revealed that the enantioseparation was enthalpy driven for metoprolol and propranolol while entropy driven for atenolol.To understand the mechanism of chiral recognition and the elution behavior of the enantiomers,molecular docking studies were performed.The binding energies obtained from simulation studies were in good agreement with the elution order found experimentally and also with the free energy values.The method was validated in the concentration range of 0.5-10 μg/mL for all the enan-tiomers.The limit of detection and limit of quantitation ranged from 0.126 to 0.137 μg/mL and 0.376-0.414 μg/mL,respectively.The method was used successfully to analyze these drugs in pharmaceutical preparations.

Comparison of Different Extraction Techniques of Zingiber officinale Essential Oil

Abstract This study aims to find the best conditions for the extraction of Zingiber officinale essential oil using the supercritical fluid extraction (SFE), steam distillation (SD) and hydrodistillation (HD) techniques, regarding the maximum oil yield. For the HD technique is evaluated the best ratio between plant mass and water volume and for SFE and SD the pressure condition was investigated. Principal Component Analysis (PCA) was used to evaluate the similarity between the composition of the essential oil in different pressures and extraction methods. The experimental extraction curve was plotted and three different mathematical models were used to fit the data for SD and SFE methods, obtaining the relevant mass transfer parameters. The essential oil compounds were identified by gas chromatography coupled with mass spectrometry (GC-MS), being α-zingiberene the main component with different contents (from 11.9 to 28.9%). The best condition for the SFE was 100 bar, 40 °C (0.0508 goil/gplant) with 19.34% of α-zingiberene; for the SD, 3 bar (133 °C) (0.00616 goil/gplant) with 28.9% of α-zingiberene; and HD, the volume of 750 mL (0.006988 goil/gplant) with 15.70% of α-zingiberene, all measured on a dry basis.

Study on the Extraction Technology of Fructus Gleditisae Abnormalis and GC-MS Analysis of Extracted Com- ponents / 中国药房

OBJECTIVE：To establish and optimize a extraction method of Fructus Gleditsiae Abnormallis ，and to analyze and identify chemical components of the extract simultaneously. METHODS ：Fructus Gleditsiae Abnormallis was extracted with CO 2 supercritical fluid extraction （SFE）method. Based on single factor tests ，using extraction yield as index ，extraction temperature ， extraction pressure and extraction time as investigation factors ，SFE technology was optimized with orthogonal test ，and validation test was performed. Chemical components in the extract were identified by GC-MS. Relative percentage of each component was calculated with area normalization method. RESULTS ：The optimal SFE extraction technology of Fructus Gleditsiae Abnormallis was extraction temperature of 60 ℃，extraction pressure of 300 MPa and pression time of 15 min. Average extraction of 3 times of validation tests was 1.73％（RSD＝1.78％，n＝3）. The 48 components in the extracts of Fructus Gleditsiae Abnormallis were identified，which accounted for 98.31％ of the total amount of the extracts. The extracts of Fructus Gleditisae Abnormalis mainly included organic acids ，accounting for 36.99％，followed by alkaloids ，accounting for 12.59％ in total. Main components were palmitic acid （16.62％），oleic acid （14.12％），N-aminotetrahydropyrrole（9.79％），2，6-dimethyloctane-1，7-dien-3-ol（5.95％）， tetrahydropyran（3.83％），vanillin（3.39％），etc. CONCLUSIONS ：SFE method of Fructus Gleditisae Abnormalis is established successfully，and the extract is mainly organic acids.

Comparative study on volatile oil in Rhizoma chuanxiong by steam distillation and supercritical fluid extraction / 药学实践杂志

Objective To clarify the influence of the extraction process on its active ingredients by comparing the volatile oils of Rhizoma Chuanxiong obtained under different processes. Methods The volatile oil of Rhizoma Chuanxiong was extracted by supercritical CO2 extraction (SFE) and steam distillation. The main chemical components and relative contents were identified by gas chromatography-mass spectrometry (GC-MS). Results A total of 18 common components were identified in the volatile oil samples of Chuanxiong from the two methods. In steam distillation samples, main components included phthalides (61%), monoterpenoids (25%) and sesquiterpenes (10%). In SFE samples, phthalides (97%) were major components, followed by monoterpenoids (1%),sesquiterpenes (0.4%) and other minor components. Conclusion The steam distillation retains highly volatile components in Rhizoma chuanxiong such as monoterpenoids and sesquiterpenes. For SFE approach, the phthalides were extracted more efficiently compared with other components. The effect of the extraction process on the active ingredients should be fully considered in obtained products of Rhizoma chuanxiong since the difference in constituents may result in varied effects.

Solubility enhancement of nimodipine through preparation of Soluplus® dispersions

The present work aims to enhance the water solubility of nimodipine, a hydrophobic drug, using a solid dispersion(SD) technique. Soluplus® as a novel hydrophilic polymeric carrier was used. Nimodipine-Soluplus® SDs (1:10) wereprepared by impregnation technique using supercritical fluid technology (SCF) and compared with the ones whichwere prepared by conventional hot-melt (HM) method. The solubility and the in vitro release study of the raw drug,solid dispersions, and the corresponding physical mixtures were characterized and compared. The prepared SD bySCF technology showed 77-fold increase in nimodipine solubility, in comparison to 48-fold increase when preparedby HM and 7.7-fold when physically mixed. Moreover, they showed the highest percentage of nimodipine cumulativerelease within the studied period. The results were confirmed the amorphous transfer of the drug into the polymermatrix which was assured by the powder X-ray diffraction and the thermal analysis. In addition to the hydrogen bondformation between nimodipine and Soluplus®, which was evident in the FTIR spectra; A weakening of peak related tonimodipine N–H stretching and C=O of the ester group. Nimodipine solid dispersion with Soluplus® using the SCFtechnology might represent a promising formulation for nimodipine to enhance its oral bioavailability

Loading and evaluation of meloxicam and atorvastatin in carrageenan microspherical aerogels particles

In this work, the potential of using microspherical aerogel particles based on commercial carrageenan as a drug vehiclewas evaluated. Carrageenan hydrogel microparticles were prepared following the emulsion gelation approach. Afterthe successive solvent exchange, supercritical CO2 drying procedure was employed to obtain aerogel microsphericalparticles. Meloxicam and atorvastatin (class II drug) were loaded into the aerogel matrix by adsorption from theircorresponding supercritical CO2 solution. All preparations were characterized by their physicochemical properties. Invitro drug released was investigated for the drug-aerogel formulation to assist the effect of aerogel technology on therelease profile of the targeted drug. Meloxicam and atorvastatin model drugs maintained their crystalline structure.Significant enhancement in the release profile of meloxicam after loading in the carrageenan aerogel can be related tode-aggregation of meloxicam inside the particle, while no enhancement in atorvastatin release was observed. Resultswere indicative of a failure in the loading of atorvastatin inside the carrageenan particle at the selected experimentalprocessing parameters.

Applications of new methods in extraction of flavonoids from Chinese materia medica / 中草药

Flavonoids are widely found in medicinal plants, which have important medical properties. Flavonoids were proved to have many pharmacological activities, such as anti-oxidation, antitumor, antimutation, anti-inflammatory, antibacterial and anti-aging. The extraction of flavonoids is the crucial link in their clinical applications. In recent years, many emerging Chinese medicine extraction methods have also been widely used in the extraction of flavonoids. This paper reviews the current application of new methods for flavonoid extraction, in order to provide references for the extraction, development and utilization of flavonoids. These new extraction methods include supercritical fluid extraction (SFE), ultrasonic assisted extraction (UAE), microwave assisted extraction (MAE), pressurized liquid extraction (PLE), pulsed electric field (PEF) assisted extraction, enzyme assisted extraction (EAE), green solvent extraction, steam explosion assisted extraction, dynamic high pressure microfluidization (DHPM) assisted extraction, etc.

Preparation Technology Study on Oridonin A Oral Liposomes Based on Supercritical Fluid Solution-enhanced Dispersion / 中国药房

OBJECTIVE： To optimize the preparation technology of Oridonin A oral liposomes （ORI-LIP） by using supercritical fluidsolution-enhanced dispersion （SEDS） technology， and to investigate its advantage with routine liposome preparation technologies. METHODS： Using particle size as evaluation index， orthogonal design was employed to investigate the influence of pressure， temperature and flow rate on the preparation technology of ORI-LIP by SEDS. At the same time， thin film dispersion and reverse evaporation method were used to prepare ORI liposomes. The particle size， encapsulation efficiency， drug loading amount and stability （accelerated test for 6 months） were compared among 3 methods. Moreover， the difference in dissolution behavior in vitro of ORI crude drug and 3 kinds of liposomes was evaluated. RESULTS： The optimized preparation condition of ORI liposomes by SEDS included temperature of 50 ℃， pressure of 18 MPa， flow rate of 1 mL/min. Compared with thin film dispersion and reverse evaporation method， the liposomes prepared by the SEDS method exhibited smaller particle size [（147.4±4.8）nm]， better encapsulation efficiency （67.8％）， drug-loading amount （7.8％） and stability （particle size increased slightly， encapsulation efficiency decreased only by 4.4％）. Results of in vitro dissolution test showed that compared with crude drug， release rate of each liposome was slow and persistent， and the cumulative release rate was higher. The accumulative release rate of ORI-LIP prepared by SEDS could achieve to 67.2％， and reached to dissolution equilibrium at 24 h. CONCLUSIONS： ORI-LIP prepared by SEDS has smaller particle size， higher encapsulation efficiency， drug loading amount and stability， which can improve the in vitro release of ORI. Compared with conventional methods， SEDS technology has certain advantages.

Separation and Purification of Costunolide and Dehydrocostus Lactone in Aucklandiae Radix by Supercritical Fluid Chromatography / 中国实验方剂学杂志

Objective:To establish a supercritical fluid chromatography(SFC) method for separating and purifying costunolide and dehydrocostus lactone in Aucklandiae Radix. Method:With supercritical carbon dioxide as the mobile phase,the effect of six factors, such as type of chromatographic columns,modifiers and modifiers ratio, flow rate of mobile phase,pressure and temperature, on the separation process of supercritical fluid chromatography were explored. The target components were separated and prepared by semi-preparative supercritical fluid chromatography. High performance liquid chromatography and nuclear magnetic resonance were used to analyze the components and study the thermodynamic regularity of the chromatographic process. Result:C18 column (10 mm×250 mm,5 μm) was adopted, with supercritical fluid dioxide as the mobile phase,the ratio of methanol was 0.13%,the flow rate was 12 mL·min-1,column pressure was 13 MPa,column temperature was 318℃, and detection wavelength was 225 nm. The sample was injected for 20 times,crude extract was 4 mg,and each target component was collected according to the chromatogram. Its purity was determined to be more than 99%by HPLC,and its structure was determined as costunolide and dehydrocostus lactone by NMR. Under this condition,the SFC separation process was normal-phase chromatography. Conclusion:The method can be used to prepare effective components of Aucklandiae Radix with a high purity and low solvent residue.

Simultaneous determination of amino acids in different teas using supercritical fluid chromatography coupled with single quadrupole mass spectrometry / 药物分析学报

Tea is a widely consumed beverage and has many important physiological properties and potential health benefits. In this study, a novel method based on supercritical fluid chromatography coupled with mass spectrometry (SFC-MS) was developed to simultaneously determine 11 amino acids in different types of tea (green teas, Oolong tea, black tea and Pu-erh tea). The separation conditions for the analysis of the selected amino acids including the column type, temperature and backpressure as well as the type of additive, were carefully optimized. The best separation of the 11 amino acids was obtained by adding water (5％, v/v) and trifluoroacetic acid (0.4％, v/v) to the organic modifier (methanol). Finally, the developed SFC-MS method was fully validated and successfully applied to the determination of these amino acids in six different tea samples. Good linearity (r ! 0.993), precision (RSDs 2.99％), accuracy (91.95％e107.09％) as well as good sample stability were observed. The limits of detection ranged from 1.42 to 14.69 ng/mL, while the limits of quantification were between 4.53 and 47.0 ng/mL. The results indicate that the contents of the 11 amino acids in the six different tea samples are greatly influenced by the degree of fermentation. The proposed SFC-MS method shows a great potential for further investi-gation of tea varieties.

Advances of supercritical fluid chromatography in lipid profiling / 药物分析学报

Supercritical fluid chromatography (SFC) meets with great favor due to its high efficiency, low organic solvent consumption, and the specialty for the identification of the isomeric species. This review de-scribes the advances of SFC in targeted and untargeted lipid profiling. The advancement of the SFC in-struments and the stationary phases are summarized. Typical applications of SFC to the targeted and untargeted lipid profiling are discussed in detail. Moreover, the perspectives of SFC in the lipid profiling are also proposed. As a useful and promising tool for investigating lipids in vitro and in vivo, SFC will predictably obtain further development.

Determination of related substances in cinnarizine by supercritical fluid chromatography / 中国药学杂志

OBJECTIVE: To develop a gradient supercritical fluid chromatography method for the separation of cinnarizine and its four related substances. METHODS: Cinnarizine and its four related substances were separated on a Torus DIOL column (3.0 mm×100 mm,1.7 μm) maintained at 40 ℃ with the mobile phase consisting of CO2 and methanol with 0.1% TFA-0.1% TEA at 1.5 mL•min-1, the detection wavelength was set at 230 nm and the back pressure was set at 1.38×107 Pa. RESULTS: Cinnarizine and its four related substances were separated in 4.5 min with satisfying resolutions. Good linear relationships were established between the peak response and the concentration in the range of 2-20 μg•mL-1 for each related substance (r>0.999 9) and the detection limits were 0.7-1.3 ng(S/N≥3). Good linear relationships were established between the peak response and the concentration in the range of 0.05-1.0 μg•mL-1 for cinnarizine (r=1.000 0). The spiked recovery of four related substances of cinnarizine was 98.0%-106.7%, and the RSD was 2.57%-4.44%(n=9). CONCLUSION: The established method can be applied in the simultaneous determination of the related substances of cinnarizine and provide reference for the quality control.

Chiral Separation of Atorvastatin Calcium and Its Enantiomeric Impurity by Supercritical Fluid Chromatography / 中国药学杂志

OBJECTIVE: To develop a supercritical fluid chromatography method for the separation of atorvastatin calcium and its enantiomer, meanwhile assaying the enantiomer. METHODS: Atorvastatin calcium and its enantiomer were separated on a ACQUITY UPC2 Trefoil CEL2 column(3.0 mm×150 mm, 2.5 μm) maintained at 45 ℃ with the mobile phase containing a mixture of CO2 and methanol with 0.1% TFA(78∶22, V/V) at 1.5 mL·min-1, and the detection wavelength was set at 244 nm. The back pressure was set at 13.8 MPa. RESULTS: The enantiomer and atorvastatin calcium were separated successfully in 5 min with a resolution factor of 4.1. Good linear relationship was established between the peak response and the concentration in the range of 2.5-50 μg·mL-1 for enantiomer(r2=0.999 9, n=6), the quantitative limit(S/N=10) was 2.5 μg·mL-1, and the detection limit(S/N=3) was 1.0 μg·mL-1. The spiked recovery of the enantiomer was 100.40%(n=9). CONCLUSION: The proposed method shows high accuracy, repeatability and stability. It can be employed for the quality control and stability research of the enantiomer of atorvastatin calcium.

Advances in particle engineering techniques for pulmonary drug delivery / 国际药学研究杂志

In recent years, pulmonary drug delivery as an attractive non-invasive administration way has been gaining intensive attention. The market for inhalable therapy has constantly grown over past years. Various particle engineering techniques have been employed to exploit the drug particles or drug-loaded particles used for pulmonary delivery with suitable physical properties. Those tailor-made inhalable particles offer the possibility of efficient delivering to lungs and the most optimal therapeutic outcomes. This review highlights the deposition mechanism of particles in lungs and several particle engineering techniques for pulmonary drug delivery, in particular two novel techniques PulmoSphere™ and TechnoSphere™, which have been used in the marketed products.

Optimization of Supercritical Fluid Extraction and Rapid Resolution LC-MS/ESI Identification of Chromones from Saposhnikoviae Radix through Orthogonal Array Design / 中草药·英文版

Objective To establish a rapid and effective supercritical fluid extraction (SFE) and rapid resolution liquid chromatography method coupled with diode-array detector (RRLC-DAD) to quantify the chromones in a species. Methods The effects of four parameters including ethanol concentration (50%–90%), pressure (25–45 MPa), temperature (40–60 oC), and time (30–90 min) on the chromones yields, namely prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside, and sec-O-gluco-sylhamaudol, were investigated using SFE system with orthogonal array design (OAD). Furthermore, the extracts were analyzed using rapid resolution liquid chromatography coupled with diode-array detector (RRLC-DAD) system to confirm the results. Results Under the optimized conditions, i. e., 35 MPa of pressure, 60 °C of temperature, 70% ethanol, and 60 min of time, the yields of prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside, sec-O-glucosylhamaudol, and total chromones were 3.514, 0.132, 6.242, 0.342, and 10.231 mg/g, respectively. In comparison with ultrasonic assisted extraction (UAE), SFE was able to yield a 20.7% increase in the total chromones from Saposhnikoviae Radix. Conclusion SFE is an alternative and promising method to extract chromones from this species, and the established RRLC-DAD method could serve as a rapid and effective method for the identification of chromones from Saposhnikoviae Radix.

Application of supercritical fluid precipitation technology for preparation of poorly water-soluble drugs / 中国药学杂志

To improve the dissolution and bioavailability of poorly water-soluble drugs have been being emphasis and difficulty in pharmaceutical research. Supercritical fluid precipitation(SFP) technology has an extensive prospect in new drug delivery systems of the poorly water-soluble drugs base on its advantages, such as green, friendliness to the environment and it is possible to achieve industrial-scale production. In this paper, related literatures were retrieved to summarize the application of SFP for preparation of poorly water-soluble drugs in recent years.

Chemical constituents isolation of dendrobium officinale based on supercritical fluid extraction and simulated moving bed / 中国药学杂志

OBJECTIVE: To isolate the chemical constituents and screen out the activity index of Dendrobium officinale, and establish a reliable method for isolation and determination. METHODS: A supercritical fluid extraction (SFE) combined with simulated moving bed (SMB) chromatography method was established for the chemical composition extraction of D. officinale. RESULTS: Under the optimal SFE condition 46 g crude extract and 30 mg naringenin could be obtained from 1 kg of dried D. officinale. Naringenin and peak X could be fully extracted and isolation by the optimal supercritical fluid simulated moving bed (SF-SMB) operation. The content of naringenin increased from 1 199 mg to 2 400 mg in 1 kg of crude extract and the percentage content of peak X increased from 4.99% to 20.44%. The pure peak X was isolation by SMB and identified as 2, 6 -dimethoxy-4-(2-propen-1-yl)-phenol using GC-MS, 1H-NMR, and IR. CONCLUSION: The technology of SFE combined with SMB is stable and excellent for extraction and isolation of the active ingredients from D. officinale, and it provides a new idea for the industrial extraction.

Extraction and purification process of total fatty acid in Brassica campestris pollen / 中国中药杂志

This study was aimed to optimize the extraction (CO2 supercritical extraction) process and purification (ethanol washing and decontaminating) process of total fatty acid in Brassica campestris pollen. With the extraction yield of total fatty acid as index, CO2 supercritical extraction of total fatty acid in B. campestris polle was optimized by central composite design-response surface methodology. Ethanol washing and decontaminating was done for the extract through orthogonal design with content of total fatty acid (linolenic acid amide, linolenic acid glyceride, linolenic acid and palmitic acid) as the indexes. The optimum parameters of CO2 supercritical extraction technology were as follows: extraction pressure of 35 MPa, extraction temperature of 60 ℃and extraction time of 3 h. When extract of supercritical fluid was purified by 50 times of 80% ethanol for 1.5 h, the content of total fatty acid can reach 60%. In addition, this process was stable and steady, provided reliable basis for production.