Purification And Partial Characterization Of Lectins Form Alocasia Cucullata

Background:Alocasia cucullata belongs to Alocasia cucullata(Lour.)Schott(family Araceae),widely distributed in Yunnan,Sichuan,Guangdong,Guangxi,Guizhou and other provinces.As one of the poisonous plants in Chinese Plant Mapping Database,it has been a traditional Zhuang medicine.Alocasia cucullata relieves internal heat or feve,detumescence analgesic effect.It is used to treat a wide variety of tumors,influenza,high fever,leptospirosis,snake bites,poisonous wasps sting,chronic bronchitis etc.Little attention to its medicinal value at present,most of the medicinal efficacy is limited to civil use and records,lack of system thorough pharmacological study,also influence its further development and utilization.Our research group uses traditional Chinese medicine decoction method of this water extract pharmacodynamic studies,the researchers found that although Alocasia cucullata direct inhibition of tumor cells and damage is relatively weak,by strengthening spleen cells,peritoneal macrophages,increased serum IL-2,TNF-?,INF-? and other immune factors to curb tumor-burdened bone tumor growth in mice,and can extend the lifetime of melanoma in mice.It is illustrated that Alocasia cucullata extracts by non-specific stimulate and enhance the body's immune function,make the body produce antitumor immune response,so as to control and kill tumor cells.In vitro,it induces monocyte to macrophage differentiation,releasing of TNF-?,IL-1? and other strong anti-tumor activity cytokines,suggesting that the use of civil anti-tumor effect may be Alocasia cucullata enhance the patients' own immune function.Lectins are a class of proteins or glycoproteins having a sugar binding specificity,can promote cell aggregation.Lectins widely exists in animals and plants,it acts as a kind of important recognition molecules in cell,between cells or between cells and tissue that playing an important physiological function,can be used in blood type identification,cell signal transduction,malignant tumor cell inhibition,immune response,and drug target orientation of research.Although plant lectins as early as 1888 was discovered by Hermann Stillmark,but,due to low extraction,difficult purification and high cost,limits the application of lectins.so the development of a variety of extraction methods,the comparison and analysis is still one of lectins have been the focus of the study.Objectives:The traditional method of lectin extration is soaked in buffer overnight,but long extraction time and low efficiency.This subject for Alocasia cucullata lectin,suggested that using ultrasonic treatment to extract,exploring the impact of this method on extraction efficiency,and optimizing the extraction process of Alocasia cucullata Iectin.Chromatography is biochemistry workers in the separation,purification and identification of biological macromolecules,one of the most commonly used techniques so use chromatography for separation and purification of Alocasia cucullata lectin then determine the purification efficiency and relative molecular mass of the lectin.Finally,the Finally,the part pointed tail taro lectin properties were studied.lectin properties were studied.In this paper,not only the Alocasia cucullata lectin has carried on the extraction,separation and purification,but also carries on the preliminary study on some of its basic properties,which can provide theoretical basis for functional development of the Alocasia cucullata lectin.Methods:1 Alocasia cucullata lectin extraction process optimization research1.1 Extraction method of choice Make fresh roots of Alocasia cucullata pare off the skin,washed chopped,at 60? oven to dry,crushed into meal.Take Alocasia cucullata thick powder 10 g,a total of 9,with 50 mL of 0.01 mol/L phosphate buffer solution(PBS,pH 7.4)immerse homogenate,placed 1 h after processing respectively:(1)take 7 sets(100 W)with ultrasonic treatment respectively 0 min,10 min,20 min,30 min,40 min,50 min,60 min;2(2)take placed under 4?for the night(24 h).With several layers of gauze processing fluid filtration,the filtrate of high-speed centrifugal,take the supernatant,Alocasia cucullata lectin crude extract 1-1-1-7 and 2-1-2-2.In the crude extract,by salting-out precipitation,dialysis desalination,adding water volume,get lectins extract 1-1?1-7,2-1-2-2.Storage.Detection of the above the clotting activity and protein content of extract,compute than vitality.Than vitality as indicators to judge the above two extraction methods,to determine the best extraction method.1.2 The choice of the buffer Take Alocasia cucullata roots thick powder 10 g,a total of 8,divided into four groups,each group to join the following 50 mL different buffer for 1 h,buffer system as follows:0.01 mol/L phosphate buffer(PB,pH 7.4);0.01 mol/L phosphate buffer solution(PBS,pH 7.4);0.05 mol/L Tris HCI buffer(TB,pH 7.4);0.05 mol/L Tris HCl salt buffer(TBS,pH 7.4).After soaking by ultrasonic treatment,filtration,the filtrate through high-speed centrifugal treatment,take the supernatant,lectin crude extract,through salting-out precipitation,dialysis desalination,the capacity,the water storage and set aside.Test each extract of clotting activity and protein content,calculate the ratio.Choice is higher than activity of extract of buffer as the experiment of extraction buffer.1.3 Extraction process on single factor experiments Selection of ultrasonic power,ultrasonic time,solid-liquid ratio,ammonium sulfate saturation four factors in single factor experiment.Test samples of extract blood clotting activity and protein content,than vitality as indicators,determine the extraction factors on the pointed tail taro lectin extraction effect.1.4 Optimization of the extraction process According to the results of single factor experiments,the selection of Alocasia cucullata lectin levels extraction effect and influential factors,than vitality as evaluation index,using the orthogonal experiment design method of four factors(three level),the single factor experiment with the extraction and purification process flow process,bleeding from coagulation effect is the best extraction conditions.2 The separation and purification of Alocasia cucullata lectin2.1 The extraction of raw Alocasia cucullata lectin Take Alocasia cucullata with 100 g,crushed into meal,"1.4" to be the optimal solution was used to extract its tail taro lectin extract,vacuum freeze drying for a quick lectin crude products.2.2 DEAE-52 anion exchange chromatography DEAE-52 fiber after activation of the wet packing column.Get a 10 mg of lectin coarse product with the capacity to 2 ml,the distilled water samples.With distilled water and increasing the concentration of NaCl solution elution in turn.By uv and agglutination activity detection,collect with clotting activity protein peak,dialysis desalination,freeze-dried,lectin and half is tasted.2.3 Sephadex G-75 molecular sieve chromatography Let Sephadex G-75 packed column after activation.Take lectin half is tasted 20 mg and the capacity to 1 ml,the distilled water samples.Elution with distilled water,by uv and agglutination activity detection,collect with clotting activity protein peak,dialysis desalination,freeze-dried,lectins is tasted.2.4 Alocasia cucullata lectin coagulation dynamic detection of the purified lectin clotting activity assay.2.5 SDS-PAGE gel electrophoresis separation Gel concentration of 10%purity detection,concentrated gum concentration was 3%,the point sample of 40 mu g,60 V electrophoresis about 30 min,after the sample into the separation of glue,adjustable voltage to 120 V,for bromophenol blue to stop the electrophoresis gel 1 cm at the bottom edge.Carefully stripping gel,staining with coomassie brilliant blue R-250,reoccupy decoloring liquid gel background faded to colorless,protein bands is clearly visible.3 Properties research of Alocasia cucullata lectin3.1 Thermal stability test Respectively in the Alocasia cucullata lectin solution temperature of 30??100? water bath process,determine the treatment fluid and blood coagulation.3.2 Acid-base stability test Configuration,respectively,of the buffer pH range of 2?13,solution under different pH,Alocasia cucullata lectin and clotting.3.3 Gucose inhibition test Respectively makes a certain content of monosaccharides and disaccharides solution,the determination of the sugar solution environment pointed tail taro lectin clotting vitality.3.4 Alocasia cucullata lectin ultraviolet absorption spectrum The measured pointed tail cone area in the uv absorbance of the lectins solution,record and draw out the ultraviolet absorption spectrum.Results:1.Alocasia cucullata lectin extraction process optimization researchIt is already determined the extraction method for ultrasonic treatment,extraction solvent is 0.01 moL/L phosphate buffer solution(PBS,pH 7.4);Single factor experimental result shows that the ultrasonic power,ultrasonic time,solid-liquid ratio,the ammonium sulfate saturation were have effects on extraction Alocasia cucullata lectins,select the appropriate scope of various factors according to the experiment and orthogonal experiment was carried out.The orthogonal test results showed that the optimum extraction conditions for Alocasia cucullata lectin as follows:ultrasonic power 240 W,ultrasonic time 10 min,solid-liquid ratio of 1:8(g/mL),ammonium sulfate saturation of 90%,which solid-liquid ratio on the extraction of a significant effect(P<0.05).2.The separation and purification of Alocasia cucullata lectinAfter the crude Alocasia cucullata lectin purified by DEAE-52 column chromatography,it got ?,?,? three elution peak,the elution peak ?agglutination activity,collect peak ? eluent,freeze drying.Column chromatography with Sephadex G-75 for further purification,collected with strong agglutination activity peak 1 eluent,dialysis desalination,freeze drying,got the pure Alocasia cucullata lectin.After the testing,the lectin clotting activity was 28.Alocasia cucullata lectin by SDS-PAGE electrophoresis,showed a single band,calculated molecular weight based on the mobility of about 60 kDa.3.Properties of Alocasia cucullata lectinThermally stable experimental results show that the Alocasia cucullata lectin heat resistance is poor,when the temperature above 60?,the lectin begin inactivation.When 90?,the lectin agglutination activity is complete loss.The acid and alkali stability experiments results show that Alocasia cucullata lectin is sensitive to acid,alkali,only on the environment of pH7?8,agglutination activity remained unchangeable.Sugar inhibits the experimental results show that the n-acetyl glucosamine can strong restrain hemagglutination activity of Alocasia cucullata lectin.Ultraviolet absorption spectra showed that the characteristic absorption peaks of the lectins is 237 nm,276 nm.Conclusion:1.Optimized the Alocasia cucullata lectin extraction method by research,it is proposed to use ultrasonic treatment for extracting method,as opposed to the traditional terms of the immersion method,greatly improving the extraction efficiency.Research by single factor experiment and orthogonal experiment,the lectin can be screened effectively improve the extraction efficiency of extraction conditions:ultrasonic power 240 W,10 min,ultrasonic time and liquid ratio 1:8(g/mL),90%ammonium sulfate saturation.2.The crude Alocasia cucullata lectin has successively through the DEAE-52 ion exchange column,Sephadex G-75 gel column,can isolate pure Alocasia cucullata lectin with strong agglutination activity.3.The relative molecular mass of Alocasia cucullata lectin is about 60 kDa,clotting vitality is 28.The lectin heat resistance is poor,should be stored under 60?.With the pH7?8 environment,agglutination activity remained.N-acetyl glucosamine is Alocasia cucullata lectin agglutination activity inhibitor.Ultraviolet absorption spectra showed that the characteristic absorption peaks of the lectins is 237 nm.276 nm.