Methodological Studies on Genomic DNA Extraction and Purification from Plant Drug Materials

This paper reports the rationale and methods of DNA extraction and purification from nine species of Compositae and four commercial drugs of corresponding plant Elephantopus scaber. The comparison of three methods: CsCl gradient, CTAB/CsCl gradient and CTAB miniprep extraction by yield, purity and factors affecting PCR was carried out. In conclusion, CTAB miniprep method provides a rapid, effective, economic approach for isolating genomic DNA for Chinese drug identification by genomic fingerprints.

The Extraction and Purification of Stevioside from Stevia

tevioside is a natural sweetener extracted from stevia,which is widely used in the food industry and the pharmacy.Centre on the extraction and purification of stevioside,using ADS-7 resin to extract stevioside through one single step,greatly shorten the process and solve the problems of lower yield and higher raw materials cost existed in the general process.

Extraction, purification and antioxidant activity of polysaccharides from bamboo leaves

Ultrasonic extraction （UE） was employed for the extraction of bamboo leaf polysaccharides （BLP）. The influential parameters of UE procedure including extraction time, ultrasonic power and solid/liquid ratio were optimized by orthogonal experiments. DEAE-cellulose col- umn chromatography was applied to purify BLP and then the radical scavenging activity of BLP was also evaluated. Optimal extraction con- ditions were： extraction time .of 15 min, ultrasonic power of 300 W, and solid/liquid ratio of 1：15. Four kinds of polysaccharides were obtained by DEAE-cellulose colunm chromatography; the maximum superoxide radical scavenging rate （20.4%） of BLP was inferior to that of vitamin C （Vc, the control） and the hydroxyl radical scavenging rate （50%） was equivalent to that of Vc.

CTAB-silica Method for DNA Extraction and Purification from Castanea mollissima and Ginkgo biloba

A new method CTAB-silica for DNA extraction and purification from the leaves and buds of Castanea mollissima and Ginkgo biloba was tested. The method is based on the silica-based purification protocol developed by Boom et al. (1990). By modifying the protocol, plant genome DNA could be extracted easily from dormant buds, mature leaves, and other parts of plant. Our results showed that the purified DNA was of high purity and could be analyzed by PCR. Furthermore, this CTAB-silica method took much less time for a successful DNA purification process compared to the traditional methods (CTAB and SDS). By our method, the suitable DNA can be extracted and purified from over 10 plant samples by one person in an hour.

Comparisons of extraction and purification methods of soil microorganism DNA from rhizosphere soil

Microorganism DNA of rhizosphere soil from Pinus koraiensis and Pinus sylvestriformis were extracted by proteinase K based on SDS method, CTAB method, PVP （polyvinylpolypyrrolidone） method, and freezing and thawing method and the crude DNA from rhizosphere soil were purified by dialysis method, silver beads absorption method, and squeezing DNA gel method. The results of different extracting and purifying methods were compared and evaluated. Results indicated that the best method of extraction for microorganism DNA in rhizosphere soil was proteinse K based on SDS method with high salt concentration of 1.0% （w/v） NaCl, which could effectively eliminate humic acids and other impurities. The dialysis method was suitable to purify DNA from rhizosphere soil because of effectively removing brown matters and humic acids and the purified products were suited to PCR amplification. Squeezing DNA gel method was also a good purification method with the advantage of inexpensive in cost and efficient in use.

Extraction and purification of the polysaccharides in Hippohaere rhamnoides L.

The different extraction technology and purification technology ofHippohpae rhamoides polysaccharides were researched in the paper. The best method of papain extraction were obtained, the ratio of papain 2%, pH at 5.5, temperature at 45℃ and extraction time of 20 min were suitable for papain extraction. The highest content of Hippohpae rhamoides polysaccharides was 44.28 mg·g^-1. The optimum process of ultrasonic extraction were obtained, namely extracted for 55 min at 480 W with the material ratio of 1：20. The highest content of Hippohpae rhamoides polysaccharides was 48.63 mg·g^-1. The results showed that the ultrasonic and papain extraction together was the best method, the content was 54.30 mg·g^-1. After the removing protein, pigment and dialysis. Two fraction were separated from the purified Hippohpae rhamoides by DEAE-cellulose chromatography, the main fraction was collected finally. The fraction was identified by Sepharose CL-4B gel filtration. Ultraviolet spectrometry, freeze-thawing analysis showed that fraction was purified. Its molecular weight was probably 109.4 ku.

Effects of Different Extraction and Purification Methods on the Acquisition of Phycoerythrin from Porphyridium purpureum

Phycoerythrin, as the main light-harvesting antenna in Porphyridium purpureum, exists at the outermost end of the phycobilisome. It has advantages of good fluorescence intensity, anti-oxidation, scavenging free radicals, and high chroma, so it has been widely used in food, cosmetics, pharmaceuticals, and other industries. In this study, the effects of different extraction(ultrasonic breaking method, bead grinding method, liquid nitrogen grinding method, and freezing-thawing method) and purification methods(salting out method, ultrafiltration method, and combination of salting out and ultrafiltration method) on the acquisition of phycoerythrin from P. purpureum were studied, and the characteristics of phycoerythrin in the P. purpureum were identified. The results showed that the freezing-thawing method could extract phycoerythrin from the powder of P. purpureum to the utmost extent, and the concentration of the extracted phycoerythrin was up to 0.036 g/L. The salting out method could most effectively purify phycoerythrin, and the purity index was 2.216. The identification of phycoerythrin by ultraviolet absorption spectroscopy and fluorescence spectroscopy indicated that the phycoerythrin had the maximum absorption peak at 545 nm, and the maximum Stokes shift was up to 79 nm. Due to its high fluorescence characteristics, it can be used as a fluorescent marker in the fields of molecular biology and clinical medicine, and can also be used as a good photosensitizer in tumor therapy.

Application of response surface methodology to the modeling of cellulase purification by solvent extraction

Central composite design (CCD)sp. JS14 in a solvent extraction was established with Response surface methodology (RSM). Solvent concentration, pH, temperature and retention time were selected as process variables to evaluate the purification impact factor in solvent precipitation, including the purification fold and % recovery. An experimental space with 13 purification fold and 23 recovery percentage recovery is achieved through the optimized condition based on the model. The molecular weight of the purified enzyme was estimated to be 32.5 KDa. Optimum activity of purified enzyme was at pH and temperature 6.5℃ and 40℃ respectively. Enzyme showed maximum activity with carboxymethyl cellulose as substrate with compare to rice husk, wheat straw and sucrose. The purified cellulase activity was inhibited by Na+, Cl- Mg2+ Tween 80 and EDTA.

Extraction and Purification Process Optimization and Antioxidant Activity in vitro of Flavonoids in Amaranthus caudatus L.

[Objectives]To explore the optimal extraction and purification process of the flavonoids in Amaranthus caudatus L.and to study the antioxidant activity in vitro of the flavonoids in A.caudatus.[Methods]Taking A.caudatus as the raw material,flavonoids were extracted by alcohol extraction method,and AB-8 macroporous adsorption resin was selected for purification.The hydroxyl radical scavenging ability,DPPH radical scavenging ability,and O^2-radical scavenging ability were used as evaluation indicators,to explore the antioxidant activity in vitro of the flavonoids in A.caudatus.[Results]The optimal extraction process conditions of flavonoids in A.caudatus are:liquid-to-material ratio 40:1,extraction temperature 60℃,ethanol concentration 60%,ultrasonic power 320 W,extraction time 50 min.Under these conditions,the extraction yield of flavonoids in A.caudatus is(1.35±0.01)%.The optimal purification process conditions of flavonoids in A.caudatus are 2.5 g AB-8 macroporous adsorption resin,sample volume 5 mL,mass concentration of adsorption solution 1.60 mg/mL,pH value of adsorption solution 3.0,sample flow rate 3 BV/h,ethanol concentration in desorption process is 70%and the desorption flow rate is 3 BV/h.Under these conditions,the recovery rate reaches 88.35%±0.68%.[Conclusions]A.caudatus has a high content of flavonoids and has excellent free radical scavenging ability in vitro.This study is intended to provide important technical support for the research of flavonoid activity of A.caudatus and the development of functional products.

Beneficiation of coal using supercritical water and carbon dioxide extraction:sulfur removal

This work explores the use of carbon dioxide,water,and their mixtures as solvent for the precombustion beneficiation of raw coal without using any toxic mineral acids in the temperature range of 200-400℃.The fluid polarity,ionic constant,and supercritical point can be adjusted by H_(2)O/CO_(2)ratio and temperature.Adding carbon dioxide to hydrothermal fluid also increases the ionization by forming carbonic acid.Extractions with supercritical fluids have several benefits including enhanced mass transport,ease of separation and recycle,wide range of extractive capability and tunability,better inherent safety,and in the case of carbon dioxide and water-low cost.A semi-continuous extraction system was designed and built in which pressure,temperature and the relative flow rates of CO_(2)and H_(2)O can be controlled.Coal powder is kept in a packed bed and the extraction is carried out at 143 bar pressure.Using sulfur as a model heteroatom,extractive efficiency is examined as a function of the temperature,fluid composition,fluid flow,and extraction time.The results indicate that carbon dioxide,water,and supercritical water-carbon dioxide(ScWC)all can effectively extract about 50%of total sulfur from bituminous coal in 1 h.Extraction above 350℃decreased effectiveness,and extraction above the supercritical point of pure water caused hydrothermal carbonization.ScWC extraction may provide necessary control to prevent organic dissolution while removing sulfur.

Isolation and Purification of Unstable Iridoid Glucosides from Traditional Chinese Medicine by Preparative High Performance Liquid Chromatography Coupled with Solid-phase Extraction

An efficient preparative method was successfully developed for isolation and purification of unstable components from medicinal plant extracts, using a combined method of preparative high performance liquid chro-matography(HPLC) and solid-phase extraction(SPE). The aim of this study was to obtain an effective method with high preparative efficiency and importantly to avoid the transformation of unstable compounds. The preparative HPLC system was based on an LC/MS controlled four-channel autopurification system. The SPE method was performed with a C18 packing material to trap the target compounds and to remove the acidic additive derived from the mobile phase. Using this method, the unstable iridoid glucosides(IGs) as model compounds were successfully isolated and purified from the extract of Hedyotis diffusa Willd. Six IGs(including one new minor IG) and one nucleotide compound were simultaneously obtained, each with a purity of 91% as determined by HPLC. The structures of the isolated compounds were identified by UPLC/Q-TOF MS, UV, 1D and/or 2D NMR. It was demonstrated that the combination of preparative HPLC with SPE is a versatile tool for preparative purification of unstable compounds from complex natural products.

Extraction,Purification,and Applications of Hemicellulose

In addition to cellulose and lignin,hemicellulose is an important biomass material.Recently,hemicellulose and its derivatives and materials have attracted increasing attention owing to their unique structures,improved properties,and promising application potential,and many reports on the extraction,isolation,and modification of hemicellulose are currently available.We summarized the recent developments of hemicellulose and its derivatives and materials by focusing on the extraction,purification,and modification of hemicellulose.The synthesis of hemicellulose-based derivatives and materials was also reviewed.Various methods of extracting,isolating,and modifying hemicellulose were discussed.Remaining challenges related to hemicellulose extraction,purification,and application were mentioned,and directions for further research on hemicellulose were proposed.

Advances in Researches of Extraction, Separation, and Purification Technologies for Total Saponins of Panax notoginseng

Total saponins of Panax notoginseng have the functions of promoting blood circulation and removing phlegm, thus they have high medicinal value. There are many different extraction methods in the extraction and separation of total saponins of P. notoginseng . The extraction methods of total saponins of P. notoginseng are mainly divided into traditional extraction methods, modern extraction methods and compound extraction methods.

Improvements on environmental DNA extraction and purification procedures for matagenomic analysis

Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which interferes downstream molecular analyses. To remedy this situation, two DNA extraction buffers （PIPES and Tris-HCl） and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX, Omega and Promega were evaluated with diverse soil samples. The PIPES buffer （pH 6.5） is found to be more effective for removing the humic substances, but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HC1 buffer （pH 8.0）. Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit, and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above. Considering all results together, two alternative methods for DNA extraction and purification are proposed： one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern, and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs. Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes. It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.

Modeling of Liquid-Liquid Extraction Process for Glycerol Purification from Biodiesel Production

This paper shows some generalities about the glycerin byproduct obtained from biodiesel production process, presents an analysis of the ternary equilibrium between methanol, water and glycerol, and shows the influence that temperature has on the balance. This phase diagram illustrates ternary equilibrium at 10, 20 and 50 ℃, keeping the pressure constant at 1 atm （atmosphere） to standardize the analysis. The purpose of it is to establish the best temperature for the purification of glycerol by liquid-liquid extraction method under the ＂extraction in several stages cross flow＂ taking an initial mixture of glycerol with composition 15 wt.% water, 25 wt.% methanol and 60 wt.% glycerol. Water was used as liquid-liquid extraction solvent in order to remove as much methanol as possible from the initial solution due to the existence of a zone of insolubility between the glycerol and water in the ternary equilibrium. By this reason, two solutions are obtained, one consisting of water and methanol containing a trace of glycerin and the other consisting of water, glycerine and traces of methanol, which contain only 4.62% of the total methanol which enter in the process of liquid-liquid extraction, with 60.62% of the total glycerol, which is sent to a fractional distillation process to purify glycerin up to 98% by weight.

THE STUDY ON THE WATER SOLUBLE PEPTIDES IN PAPAVER SOMNIFERUM POLLEN EXTRACTION,PURIFICATION,SEQUENCE DETERMINATION AND SYNTHESIS

Four peptides PSPP1,PSPP2,PSPP3 and PSPP4 were purified from the water-extract of Papaver somniferum pollen.Their sequences,with 21,17,13 and 16 amino acid residues respectively,have been determined by Edman degradation-N-terminal dansylation.PSPP2, PSPP3 and PSPP4 were synthesized using solid phase method.The immunopromotive activities of PSPP1,PSPP2,PSPP3,PSPP4 and the initially separated sample PSPP have been also observed by the methods of counting erythrocyte rosette forming cells(ERFC) and T-lymphocyte transformation test in vitro.

Efficient and selective extraction of sinomenine by deep eutectic solvents

Sinomenine is the main bio-active ingredient of Sinomenii Caulis and usually produced by solventextraction techniques. However, the extraction of sinomenine suffers from the lack of highly efficient and environmentally-benign solvents. In this work, deep eutectic solvents(DESs) based on fragrances were synthesized, hydrogen-bond donors(HBDs) and hydrogen-bond acceptors(HBAs) components of DESs were identified and their extraction ability for sinomenine was evaluated and the extraction conditions were optimized by single-factor and orthogonal design experiments. It was found that the hydrogen-bonding interaction between sinomenine and DESs was the main extraction driving force and there was no explicit relationship between the extraction ability and the hydrophobicity of the DESs. The DESs could be recycled and sinomenine could be recovered quantitatively via backextraction. High-purity sinomenine((95.0 ± 2.3)%) could be produced. These findings suggest that DESs are highly-effective solvents for the isolation of sinomenine and exhibit great potential for the extraction of other bio-active compounds.

Extraction and Refining Process of Polysaccharide from Tricholoma matsutake and Verification of Its Whitening Effect

[Objectives]The paper was to determine the extraction and refining process of polysaccharide from Tricholoma matsutake,and to verify its whitening effect.[Methods]With T.matsutake as the research object,the effects of eluent concentration,loading amount and diameter-to-height ratio on the refining process of T.matsutake polysaccharide were explored by orthogonal test,to optimize the dynamic elution conditions of T.matsutake polysaccharide.The survival rate of mouse melanoma cells(B16),inhibition of melanin synthesis and inhibition of tyrosinase activity of crude extract A1 and refined extracts A2-A4 were tested to verify the whitening effect of T.matsutake.[Results]The best extraction and refining process for T.matsutake polysaccharide was:eluent 40%ethanol(volume fraction),loading amount 30 mg/g(polysaccharide-resin),diameter-to-height ratio 1∶8.Under this condition,the extraction rate of T.matsutake can reach up to 33%(A3).The whitening efficacy test showed that crude extract A1 and refined extracts A2-A4 had good whitening effects,and the whitening effects were A3≥A4>A1>A2.[Conclusions]The study provides a reference for the application of T.matsutake polysaccharide in cosmetics.

Purification method of rohitukine from the stem bark of Dysoxylum binectariferum

To develop a simple and rapid purification method of rohitukine from the stem bark of Dysoxylum binectariferum. A L9 （34） orthogonal test was designed to optimize the extraction condition. Rohitukine in the plant extract was purified by using solvent-solvent partition and cation exchange resion （CER）. Five different types of packing materials, including XAD-2 resin, polyamide, Sephadex LH-20, ODS and CER, were compared and CER showed the best capacity for rohitukine separation. The purification procedure was optimized as follows： the plant material powder was extracted with 70% ethanol （v/m = 60） by ultrasonic agitation for 60 min, then the 70% ethanol extract was dissolved in aqueous solution （pH 1, adjusted with 0.5 mol/L HCl） and extracted with equal volume of n-butanol. The aqueous layer was retained and the pH was adjusted to 10 with 25% aqueous ammonia and a solventsolvent partition was performed with equal volume of n-butanol. The obtained n-butanol extract was dissolved in aqueous solution （pH 1, adjusted with 0.5 mol/L HCl）, and purified by a CER column eluting with H2O and 70% ethanol （pH 10, adjusted with 25% aqueous ammonia）, successively. Rohitukine existed in 70% ethanol eluate, with a purity up to 53.3%. The method developed in this study provides a simple and rapid approach for the preparation of rohitukine from the stem bark ofD. binectariferum.

Determination of Iprobenfos Residue in Rice by GC-FTD using Two-dim Ensional Purification

[Objective] This study aimed to establish a determination method for iprobenfos residue in rice straw and husked rice. [Method] The rice straw and husked rice samples were extracted by acetone-ethyl acetate mixed solvent. The extracts were purified using SPE C18 column and SPE NH2 column, and the iprobenfos residues were determined by GC-FTD. [Result] In the concentration range of 0.005-5.0 mg/kg, iprobenfos concentration showed a good linear relationship with peak area （r=0.999 8）. When the iprobenfos concentrations were 0.01, 0.1 and 1.0 mg/kg respectively, the recoveries of added iprobenfos from rice straw ranged from 72.6% to 99.7% with relative standard deviation ranging from 5.65% to 8.48%; the recoveries of added iprobenfos from husked rice ranged from 81.6% to 97.6% with relative standard deviation ranging from 3.74% to 7.63%. The minimum detectable quantity of iprobenfos was 5×10^-12 g, and the minimum detectable concentrations of iprobenfos in rice straw and husked rice samples were 2.0 and 0.5 μg/kg, respec- tively. [Conclusion] The established determination method is characterized by low de- termination limit, high sensitivity, good reproducibility and high operability, which all meet the requirements by Guideline on Pesticide Residue Trials of the Ministry of Agriculture.