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.

Isolation of Chlorella vulgaris and Its DNA Extraction Methods

[Objective] The aim of this study is to isolate Chlorella vulgaris(chlorella)and extract its genomic DNA.[Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples;the conditions for culturing chlorella were optimized and its genomic DNA was extracted by improved CTAB method and SDS method.[Result] The proper conditions for chlorella culture were as following:temperature 20-25 ℃,illumination 4.39-5.86 W/m2 and rotational speed 100-150r/min;improved CTAB method was suitable for extracting genomic DNA from chlorella.[Conclusion] The study is helpful to study the chlorella at molecular level and promote the exploitation and utilization of chlorella resources.

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.

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.

The Extraction,Isolation and Identification of Exudates from the Roots of Flaveria bidentis

Large amounts ofFlaveria bidentis＇s root culturing solution were obtained by using DFT （deep flow technique） equipment and these solution which was vacuum concentrated （10, 20 mg mL-~） can have a certain inhibition on Triticum aestivum, Cucumis sativus, Raphanus sativus, Amaranthus retroflexus, Setaria viridis, Chenopodium album, Echinochloa crusgalli and Chloris virgata. This outcome suggested some active compounds in the root exudates ofFIaveria bidentis can inhibit the germination, seedling elongation and root length. The dichloromethane extract of root exudates was identificated by GC-MS, and 29 kinds of compounds, including esters, hydrocarbons, ketones, thiazole, amines, etc. were obtained and the phthalate n-octyl ester, phthalate 2-ethylhexyl ester were proved to be allelochemicals. The culturing solution of root exudates was separated through the resin column and silica gel column and a component inhibiting seedling height, root length and fresh weight of wheat was got. There were 6 kinds of organic compounds in this component including dioctyl phthalate, 1,2-phthalate, mono（2-ethylhexyl） ester by GC-MS.

A simple method for the isolation and purification of resveratrol from Polygonum cuspidatum

Resveratrol, a polyphenol compound with strong biological activity, has been widely used in medicine, health products and cosmetic industries. It is also the main active component of Polygonurn cuspidatum, a well-known traditional Chinese medicine. We developed a simple and effective method for the preparation of resveratrol from P. cuspidatum. The whole preparative process consisted of reflux extraction, filtering, hydrolyzing, liquid-liquid extraction and eluting. Filtering is to remove non polar or less polar compounds and debris fragments from the extract. Hydrolyzing is to transform polydatin to resveratrol to improve the yield of resveratrol. Eluting is to remove impurities including strong acidic and water-soluble compounds. By acid hydrolysis of glycoside （polydatin）, the yield of resveratrol increased about 4-fold. The extraction recovery in different stages was high, and the content of resveratrol in the final product was over 73.8%. Compared with other methods reported, this technology is eco-friendly, easier to perform, and also has a lower cost.

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.

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.

Efficient Extraction and Isolation of Mangiferin from Mango Leaves by Ethyl Acetate Impurity Removal Method

[Objectives] To optimize the ethyl acetate impurity removal method for extracting and isolating mangiferin from mango leaves,and provide raw materials and technical support for development and use of mangiferin related products. [Methods]Five steps( material crushing→ ethyl acetate impurity removing → concentrated extract washing → extracting with methanol → crystallization and precipitation) were used.The single factor experiment and L9( 34) orthogonal experiment was carried out to optimize the process parameters including extraction time,ultrasonic power,extraction times,and extraction temperature.[Results] The optimum process of ethyl acetate impurity removal method for extracting and isolating mangiferin from mango leaves was as follows: the mango leaves were crushed and sieved; 3 m L/g of ethyl acetate was added,sealed and soaked for 4 h,ultrasonically shaken for 20 min( 50℃,350 W),filtered at room temperature,filtered with 100 mesh sieve,and extracted three times; added 100% methanol to the residue at 3 m L/g,extract by ultrasonic vibration for 20 min( 350 W,55℃)for four times,filtered with 100 mesh sieve when it was still hot; mixed the extract of each time,condensed by vacuum decompression to get the extract; added 100% methanol at 4 m L/g,mixed and washed for 5 min at room temperature,placed for 10 min,filtered with 100 mesh sieve,washed 3 times repeatedly,and dried the filter residue at 60℃ to obtain the crude mangiferin; added 100% methanol at 4 m L/g,mixed and washed at 50℃ for 5 min,placed at 6℃ for 8 h,dried the filter residue at 60℃,and repeatedly crystallized two times. According to the above process,crude and pure mangiferin products could be obtained,the purity of mangiferin of the crude product was higher than 64. 00%,the total recovery rate was 83. 90%,and the purity of mangiferin of the pure product was higher than 98. 00%,and the total recovery rate was about 66. 40%. [Conclusions] The optimized ethyl acetate impurity removal method is easy in operation,low in cost,and high in efficiency for extracting and isolating mangiferin,and can be applied for actual production of mangiferin.

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.

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.

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 and purification of TGFβ and its effect on the induction of apoptosis of hepatocytes

AIM To extract and purify the transforming growth factor β (TGF β), and to demonstrate its biological activity in vivo and induction of apoptosis of hepatocytes in vitro.METHODS TGF β was isolated from fresh bovine platelets by acid/ethanol extraction method and purified with ion exchange and gel chromatography. The extracted TGF β was injected subcutaneously to mice, and its biological activity in vivo was observed 72 hfs post-injection by HE staining. The morphological changes were observed by HE staining and the occurrence of apoptosis was detected by TUNEL method after the human normal hepatic cell line QZG was treated with 8 μg@L 1 TGFβ for 12 hrs in vitro.RESULTS The molecular mass 25 ku TGF β protein was successfully extracted. It was able to induce localized granulation tissue formation in vivo. TGF β-treated hepatocytes showed obvious apoptotic morphological changes, including the pyknosis and dense-stained nuclei and cytoplasm, the fragmentary, annular or crescent nuclei, and the "bubbling" cytoplasm. Moreover, its apoptotic rate was significantly higher than that of the control group (P＜0.05).CONCLUSION Biological active TGF β protein is extracted and purified successfully from bovine platelets, and it is able to induce the apoptosis of hepatocytes.

Apoptosis and oxidative injury of donor islets during isolation and purification

Objective To observe the changes of islet cell apoptosis and oxidation - antioxidation before transplantation,and to explore pathways of islet protection. Methods Fifteen human pancreases were perfused with Hanks solution containing collagenase,then digested and isolated.

A Simple Method for the Isolation and Purification of 2,4-Dihydroxy-7-Methoxy-2H-1,4-Benzoxazin-3(4H)-One (DIMBOA) from Maize (Zea mays L.) Seedlings

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3（4H）-one （DIMBOA）, the dominant benzoxazinoid hydroxamic acid in maize （Zea Mays L.）, serves as important factors of resistance against insects and microbial diseases, allelochemicals used in competition with other plants. In this paper, a novel and simple method for the isolation and purification of DIMBOA from maize seedlings was developed. Frozen shoots from 7-d-old maize seedlings （1 000×g） were firstly defrosted and then were directly homogenized and extracted with ethyl acetate. The macerate was allowed to stand at room temperature （25±2）°C for 1 h to allow enzymatic release of DIMBOA from DIMBOA-glucoside. Then the ethyl acetate phase was filtered, dried and evaporated to dryness. The resulting light-tan, semicrystalline residue was stored at -20°C for 24 h. Upon recrystallization from acetone-hexane, a relative higher yield （0.58 g） of pure DIMBOA crystals was obtained compared with the yield afforded by Woodward methodology （0.26 g）.