Yongheng WEI,Beiyue ZHANG,Jie Liu,Jinli SHI

Beijing University of Chinese Medicine，Beijing 100102,China

Sterilization of traditional Chinese medicinal materials has always been an important content related to safety in clinical use of traditional Chinese medicines.Due to the characteristics of short cultivation period,high curing requirement and easy discoloration of medicinal flowers,common sterilization techniques for traditional Chinese medicinal materials are not suitable for medicinal flowers completely.However,the sterilization technology of medicinal flowers has not been discussed in detail yet.This paper reviewed the latest progress on the sterilization technology suitable for medicinal flowers,and analyzed the application prospect.

Chemical sterilization,autoclave sterilization, irradiation sterilization,light radiation sterilization and gas sterilization are common sterilization methods for medicinal flowers.Some new sterilization methods reported in literatures are high electrostatic voltage sterilization,light pulse sterilization,electric pulse sterilization,ohmic heating sterilization,electron beam sterilization and electron ray sterilization techniques.

Common Sterilization Techniques of Medicinal Flowers and Application

Chemical sterilization

Ethylene oxide fumigation methodEthylene oxide can kill various bacterial reproductive bodies,spores,viruses and fungal sporesbynon-specific alkylation of DNA,RNA and proteins of microorganisms.It has a low boiling point only of 10.8℃,and it is suitable for heat-sensitive medicinal materials.However,it has been banned according to the international environment protection treaty,the Montreal Protocol,due to the pollution to environment and medicinal materials[1].

Ethanol sterilizationEthanol sterilization is another conventional sterilization method.Xue[2]adopted ethanol with a certain concentration to sterilize pollen,and the result indicated that the sterilization effect was excellent.She adopted the ethanolconcentration calculated according to following equation:

in which,X% is the ethanol concentration used for sterilization,and A% is the water content in pollen grains before sterilization.

Propolis sterilizationPropolis sterilization is performed by adding some propolis in 75% -85% ethanol and soaking or spraying pollen with the obtained 0.01% propolis solution,and the effect is better than that of ethanol sterilization[3].

Autoclave sterilization

Dry heat sterilizationThe dry heat method kills microorganisms by causing oxidation, denaturation, carbonization and electrolyte concentration toxication of protein by dehydration.The method is performed by adding raw medicinal powder sterilized into a proper container,heating in an electro oven or a calandria at 120℃for 30 min,and cooling.Tang[4]sterilizedLonicera japonicawith different sterilization methods,examined the sterilized medicinal material with a microbial test method,and determined the changes in various component contents by HPLC,and it was found that the dry heat method exhibited an undesirable effect and obviously-decreased effective components.

Steam sterilization method(circulating steam sterilization)The steam sterilization kills microorganisms mainly by solidifying proteins of microorganisms.In steam sterilization,raw medicinal powder is putted into cloth bags which are added into a sterilizer(or vulcanizer)and heated with circulating steam,timing begins when the temperature reaches 100℃,steam supply is stopped when the sterilization is finished,and examination is conducted after cooling.Lin[5]sterilized six medicinal materials includingPanax pseudoginsengwith dry heat sterilization and steam sterilization,and found that,steam sterilization was obviously better than dry heat sterilization,and the microbiological contamination was greatly reduced by the two methods,both of which satisfied the requirement of sterilization.However,steam sterilization lead to more loss of volatile oils in medicinal materials,and thus is not suitable for medicinal flowers containing effective components of volatile oils.

Ethanol vapor sterilizationThe mechanism of ethanol sterilization is that it permeates into cells of microorganism reproductive bodies,denaturizes proteins and kills the reproductive bodies.Certain vacuum degree can improve the penetration power of ethanol to medicinal decoction pieces and spores,and enable the ethanol vapor sterilization for traditional Chinese medicines.Wang et al.[6]selected six kinds of main medicinal decoction pieces in Chinese patent medicines,sterilized them with ethanolvapor sterilization,and found that the quantities of bacteria,fungi,coliform and specified microorganisms were reduced remarkably.

Irradiation sterilization

60Co-γ ray irradiation sterilization

60Co-γ ray irradiation sterilization is a cold sterilization method which uses the penetrating power of γ ray to medicinal materials for killing microorganisms and pests in medicinal materials.Jin[7]performed irradiation sterilization onErigeron breviscapusandPanax pseudoginsengand analyzed the sterilization effect and changes in the contents of effective components underdifferentirradiation dosages,founding that γ ray radiation had no obvious effect on the contents of effective components inErigeron breviscapusandPanax pseudoginsengpowders.

Fanet al.[8]irradiated ten medicinal materials includingDatura metelL.with 4.0,6.0 and 10.0 KGy of60C0-γ rays,the result of detection showed that the amount of bacteria in medicinal materials greatly reduced,the survival rate of other bacteria decreased with increasing irradiation dose,the amount of bacteria inDatura metelL.was reduced after the irradiation by 6.0 KGy of60C0-γ rays to a value within the range of national drug standards(bacterial quantity less than 104 bacteria/g,fungus quantity less than 103 fungi/g),and 10.0 KGy could kill bacteria thoroughly.

Mouet al.[9]selected two60Co-γ ray irradiation doses of 5 KGy and 10 KGy to perform irradiation,and found that theCarthamustinctoriusL.reached the hygienic standard after being irradiated with a dose of 5 KGy,the content of hydroxysafflor yellow as an effective component had no obvious change,while the hydroxysafflor yellow A contained inCarthamus tinctoriusL.subjected to 10 KGy of irradiation decreased by 34%.

Yanget al.[10]sterilized chrysanthemum by60Co-γ ray irradiation and determined the chlorogenic acid contents in irradiated chrysanthemum medicinal material and powder and unsterilized chrysanthemum medicinal material by HPLC,it was found that chlorogenic acid content was reduced remarkably,indicating that irradiation sterilization was not suitable for chrysanthemum medicinal material.

Myung-Woo Byunet al.[11]sterilized 21 medicinal materials includingAgrimonia pilosaLedeb.andPoria cocos,with 10 KGy of60Co-γ rays,compared with before irradiation,the result indicated that there were no significant differences in their antioxidant.anticomplement functions and nitrite scavenging effect.

Light wave radiation sterilization

Ultraviolet light radiationWhen microorganisms are irradiated by ultraviolet light,partial amino acids and nucleic acids in cells absorb ultraviolet light and suffer photochemical effect which causes chemical changes in components in cells,especially chemical changes in nucleic acids,protoplasmic proteins and esters,resulted in denaturation of cytoplasm leading to death of microorganisms.

Microwave sterilizationMicrowave sterilization is the result caused by microwave thermal effect and non-thermal effect,among which thermal effect mainlykillsmicroorganismsbyincreasing temperature rapidly,while non-thermal effect changes proteins and physiologically-active substances in microorganisms and thereby inactivates orkills microorganism cells.Tang[4]treatedLonicera japonicawith microwave sterilization,60Co-γ ray irradiation sterilization and dry heat sterilization,respectively,and found that both microwave sterilization and60Co-γ ray irradiation sterilization achieved a good sterilization effect,but microwave sterilization was considered to be a better choice forLonicera japonicasterilization in enterprises due to the fact that radiation sterilization was affected by the factors including enterprise conditions.

Infrared radiationThe sterilization effect of infrared light is similar to that of dry heat,with a high temperature up to 820℃.The thermal effect of infrared irradiation can only be produced on the surfaces to which the infrared light is incidence,and therefore,infrared radiation cannot heat objects uniformly.As a result,the influence on medicinal materials is also similar to that of dry heat,and there is certain influence on material characters,effective components as well as on the generation of harmful substance[12].However,this method is free of naked fire and not affected by wind,having good safety.This technique is widely applied to biosafety cabinets,clean work stations,exhaust fans and on mobile vehicles.

Far infrared sterilizationThis technique uses the far infrared rays re-flected by far infrared radiation elements,which are absorbed by heated medicinal materials in which molecular resonance happened and causes vibration and rotation of molecules and atoms,which are then converted into heat energy for sterilization.YAMANO Togo from Japan carried out sterilization experiment by putting bacteria,yeasts and fungus suspensions into plastic bags,and the result showed that far infrared irradiation could kill all heat-sensitive bacteria and reduce the quantity of heat-tolerant bacteria,and all fungi could be killed under the irradiation power above 8 kW.Kim W Wet al.[13]found that the methanol extract ofChrysanthemum indicumcould significantly reduce nitrite,TN F-protein,COX-2 protein and PGE2,and theChrysanthemum indicumsubjected to far infrared treatment had an inhibition effectobviously strongerthan the product dried in the shade.

Gas sterilization

Ozone sterilizationThe mechanism and process of ozone sterilization belong to biochemical reaction.Ozone can oxidize glucose oxidase which is necessary to oxidation of glucose in bacteria.It can directly act with bacteria and viruses to destroy their organelles and ribonucleic acids and decompose DNA,RNA,proteins,lipids and macromolecular polymers including polysaccharides,thus damaging material metabolism and reproductive process of bacteria.Ozone also can penetrate into cell membranes of bacteria to oxidize phospholipids and proteins in cell membranes,resulting in lysis without residues[13].

Sterilization by other gasesOther frequently-used gases are ethylene oxide,phosphorane,methyl bromide,bromoformaldehyde, epoxypropane,etc.The sterilization principle lies in alkylation used for inerting of substances necessary to sustain life.

New Sterilization Techniques for Medicinal Flowers[15-16]

Light pulse sterilization equipment and technique

The equipment and technique are developed by America PPT food engineering corporation.The adopted wide-spectrum light pulses with short duration and high illumination intensity can effectively kill microorganisms including bacteria and spores,fungi and spores and without damaging food cells and deep structure by irradiating a food surface after reflection and double focus,and have the advantages of short time and high efficiency.This technique has passed the authentication by America FDA.

Electric pulse sterilization equipment and technique

The equipment and technique are developed by SANYO Electric Co.,Ltd.of Japan.The adopted supply voltage is in the range of several thousand voltages to tens of thousands of voltages,the electric field intensity is 5 kV/cm-100 kV/cm,and the pulse width is 1 μs-100 μs to numerous ms.When performing sterilization with electric pulses,a sterilization effect is generated similar to pasteurization with the damage to medium quality less than that by heat sterilization.The technique can kill fungi and other bacteria on the surface of rice with a killing rate up to 99%.The equipment has the advantages of low cost,no pesticide residue,and rapid sterilization speed.

Ohimic heating sterilization equipment and technique

The equipment and technique are developed by Japan.During application,an alternating current passes food to heat the food using its own electrical impedance.This technique is used for the sterile processing of fluid food containing particles,and solves the problem of uneven heating for sterilization between liquid and solid particles.Compared with other methods with a thermal energy efficiency of 45% -50%,this method has a thermal energy conversion rate up to 90%,greatly saving energy.For sterilization of traditional Chinese medicinal materials,the technique is suitable for continuous sterilization of medicinal flowers containing serous fluid and used in fresh form.

Electron beam sterilization equipment and technique

The equipment and technique are developed by America.The technique uses a magnetic-induction linear accelerator to generate electron beam for sterilization ofmedicinalmaterials.Compared with other irradiation sterilization methods,it has the advantages of easy control,high output capacity and high-speed continuously treatment.In addition,a linear induction electron accelerator is developed to an ionization irradiation technique,which can change,inactivate and kill microorganism cells,with the advantages of higher sterilization energy and simple operation.

Electron ray sterilization equipment and technique

The equipment and technique are developed by Japan.With heating by an electron ray source or white hot filament,a cathode generates electrons under vacuum,the speed of which is accelerated when passing a vacuum electric field,resulting in high energy and strong penetrating power.By one electron ray irradiation device,the whole face of a conveyed container is irradiated with electron rays for sterilization.This technique can efficiently kill microorganisms with a high speed.

Conclusions

Chemical sterilization is limited and it is prone to bad consequences including solvent residues and decrease in quality of medicinal materials.Autoclave sterilization has the advantages of simple equipment and operation process and good sterilization effect,but it is not suitable for heatsensitive medicinal flowers due to the fact that high temperature greatly influences the contents of effective components.For medicinal flowers,radiation sterilization and microwave sterilization have the advantages of low sterilization temperature and good effect,but they need more investment and have higher operation requirement,in addition,these techniques are not mature and only suitable for some valuable medicinal flowers.Compared with other sterilization techniques,ozone sterilization can be performed at room temperature and ordinary pressure with the advantages of short sterilization time,low resource consumption,low cost and environment friendliness,and it can realize linked operation and automatic control in largescale production of medicinal flowers,and suitable for production lines in enterprises for producing ordinary medicinalflowers.Therefore,a suitable sterilization method for medicinal flowers should be selected according to the characteristics of medicinal materials,processing requirements indexes of component contents as well as enterprise conditions.In addition,the application ofnew sterilization techniques need the further study and import,which will help to improve the quality index ofmicrobial limitfor medicinal flowers,and accelerate the internationalization process of traditional Chinese medicines.

[1]NI YR(倪贇榮),ZHANG ZD(章振東),LI FG (李福高),et al.Application and progress of pollen sterilization technology(花粉滅菌技術應用與進展)[J].Journal of Bee(蜜蜂雜志),2007,7(7):13-15.

[2]XUE YM(薛育梅).Ethanol sterilization method for pollen(花粉的乙醇滅菌方法)[P].CN:1596588,2004.

[3]LIU JT(劉敬濤).Storage and sterilization of pollen(花粉的貯存與滅菌)[J].Rural Practical Technology(農(nóng)村實用技術),2005(5):38.

[4]TANG ZW(湯宗武).Discussion of sterilization methods forLonicera japonica(金銀花藥材滅菌方法的探討)[J].Strait Pharmaceutical Journal(海峽藥學),2014,26(4):45-47.

[5]LIN YJ(林藝娟).Comparison between the effects of dry heat sterilization and steam sterilization for medicinal materials(中藥干熱滅菌法和濕熱滅菌法的效果比較)[J].Journal of Sichuan Continuing Education College of Medical Sciences(四川省衛(wèi)生管理干部學院學報),2000,19(2):133.

[6]WANG LP(王麗萍),YANG JH(楊金華),CHEN TC(陳天朝).Discussion of sterilization method of tcm decoction pieces by ethanol vapor(乙醇蒸氣滅菌中藥飲片的方法探討)[J].China Pharmacy(中國藥房),2012,23(35):3308-3309.

[7]JIN H(金輝),WANG BZ(王保忠),LIU ZB(劉智斌).Influence of radiation on sterilization and quality of special traditional Chinese medicine in Yunnan(輻射對云南特色中藥的滅菌效果及品質(zhì)影響研究)[J].Chinese Pharmaceutical Journal(中國藥學雜志),2006,41(22):1700-1702.

[8]FAN TL(范鐵林),LI YW(李耀維),LIU ZG(劉振國),et al.Influences of irradiation sterilization on effective component contents in 10 traditionalChinese medicines(輻照滅菌對10種中藥有效成分含量的影響)[J].Journal of Shanxi Agricultural University(山西農(nóng)業(yè)大學學報),1999,19(4):337-339.

[9]MOU XW(牟學文),TIAN XL(田喜蓮).Influence of60Co-γ ray radiant sterilization on the hydroxylsafflor yellow a in carthamus flower and ethanol extracts from carthamus flower(60Co-γ輻照對紅花藥材及醇提取物羥基紅花黃色素A的影響)[J].Chinese Pharmaceutical Affairs(中國藥事),2012,26(3):232-234.

[10]YANG ZJ(楊振俊),ZHAO L(趙琳),ZHAO HJ(趙煥君),et al.Influences of irradiation sterilization on chlorogenic acid in chrysanthemum(輻照滅菌對菊花中氯原酸含量的影響)[J].China Medical Engineering(中國醫(yī)學工程),2012,20(8):125-127.

[11]MYUNG-WOO BYUNA,HONG-SUN YOOKA,KYONG-SU KIMB,et al.Effects of gamma irradiation on physiological effectiveness of Korean medicinal herbs[J].Radiation Physics and Chemistry,1999(54):291–300.

[12]XU YQ(徐永群),HUANG H(黃昊),ZHOU Q(周群),et al.The application offingerprintinfrared spectra and clustering analysis in the discrimination of geographical origin of Paeonia lactiflora Pall(紅外指紋圖譜和聚類分析法在赤芍產(chǎn)域分類鑒別中的應用)[J].Chinese Journal of Analytical Chemistry(分析化學),2003,31(1):5-9.

[13]KIM WW,GHIMERAY K,WU JC,et al.Effect of far infrared drying on antioxidant property,anti-inflammatory activity,and inhibitory activity in A549 cells of gamguk(Chrysanthemum indicumL.)flower[J].FoodSciBiotechnol,2012,21(1):261-265.

[14]WANG LC(王立成),ZHOU XJ(周興軍).Medicine sterilization method and its influences on traditional Chinese medicines(藥物滅菌技術及其對中藥的影響)[J].Practical Pharmacy and Clinical Remedies(實用藥物及臨床),2008,11(6):375-376.

[15]WANG YY(王云陽),YUE TL(岳田利),ZHANG L(張麗),et al.New sterilization technology in food engineering(食品殺菌新技術)[J].Journal of Northwest Sci-Tech University of Agriculture and Forestry:Natural Science Edition(西北農(nóng)林科技大學學報:自然科學版),2002,(sl):99-102.

[16]CHEN SP(陳少平).Comparison of common sterilization method for traditional Chinese medicines and introduction of new techniques(常用中藥滅菌技術比較及其新技術介紹)[J].Chin J Ethnomed Ethnopharm(中國民族民間醫(yī)藥),2013,3(6):104.