Study On Factors Influenced Pharmaceutical Effects And Yield Of Introduced Artemisia Annua In High Latitude Area And Analytical Methodology Of Artemisinin Content

In this dissertation,the modified extraction and analytical method of artemisinin is proposed,evaluating the value of wild Artemisia annua resource in Heilongjiang Province on the basis.Some high quality provenances of A.annua from Yunnan,Guangxi,Sichuan and Hunanmain in northern latitude 23～30⁰ were introduced in the different areas over northern latitude 46⁰ of Heilongjiang Province in 2006,2007and 2008,for selecting the one which adapted to the environment and climate of the cultivated area and has high artemisinin content. The technique of introduced A.annua cultivation is established.The main factors which affect A.annua's artemisinin content and medicinal biomass yield are studied in detail and major of them are researched to establish fertilization technique of high A.annua yield by orthogonal design test.It provides theoretical basis for introducing A.annua and promoting a large-scale planting production in Heilongjiang Province,of which the concrete results are as below:1.The modified extraction method of constant-temperature oscillating digestion is proposed,of which the appropriate technical conditions are established as follows:drying the powder sample in a baker(40℃) to constant weight,weighing 1.000 g and puting it in a reactor,adding petroleum ether(60～90℃) 20 mL,oscillating digestion(120 r/min) at constant temperature of 30℃for 24 h.Do the same thing but using petroleum ether(60～90℃) 10mL to the residue,repeatedly 3 times.Put the extract of the four times together(50mL in total) as the artemisinin extract.This extraction,which performed with simple apparatus under easy operation,uses economical,low-toxicity,recyclable and easy-recovering solvent.It also provides extraction of a relatively high pureness,with which all the above makes it fit for mass production.2.The artemisinin content detecting method of the newly modified HPLC-UV with Postcolumn Derivation is proposed.Doing the test according to the references showed unstable results and very complex operation.According to our researches and experiments,the main factors that affect the results of HPLC-UV with postcolumn derivation are the amount of 95% ethanol,which is used as solvent for artemisinin standard sample(or epurated artemisinin),and that of NaOH,HAc used during alkali-treating and acid-treating process,by which the newly modified method was proposed.3.Evaluated the developing value of wild A.annua resource in Heilongjiang Province. After detecting the artemisinin content of A.annua in different areas of Heilongjiang Province, we found that the artemisinin contents are lower than 0.1%,which is far lower than the medicinal criterion of artemisinin content≥0.5%.It means that the wild A.annua in Heilongjiang is useless. 4.The technique of introducing A.annua cultivation is established.Comparing with the methods and results of sowing seedling in fields and greenhouse,the later,can prolong the growth period about 45 days.What's more,the integrated plate-type paper containers,which require very little soil and enable easy transplanting,is quite economical.If we harvest A. annua in different areas at the same time,the earlier we grow,the higher yield of artemisinin we have.The nutritious growth period can prolong to the beginning of September for introduced A.annua,which is nearly 170 days.Above all,great medicinal biomass can be obtained.After 20^th of August,the nutritious growth period of A.annua transfers to breeding growth period in succession,and in this period,high artemisinin content can be obtained because the local climate is at high temperature and strong illumination.It is that the growth period can be lengthened if growing seedlings and transplanting are done earlier.Therefore, not only high artemisinin content can be accumulated,but also great medicinal biomass can be obtained at the month of high temperature.Thus,artemisinin production is increased.5.Through field planting and outdoor pot experiments,we studied most of the essential nutritious factors for plants growth,adversity stress,irradiating the seeds of A.annua,sunshine, temperature difference between day and night,planting density,sowing seedling and transplanting time,which affect A.annua's artemisinin content and medicinal biomass.The results are as below:Each factor of mineral nutrition has its own influence on the artemisinin production, generalizing the results,the sequence ranked:NPK > K > Ca > Fe > Mg > B > P > Mn > Cu > N > Mo > Zn > ck(control).With proper fertilization,artemisinin content can be increased by 49.3%,medicinal biomass by 55.4%,artemisinin production by 111.8%as a maximum, compared with the control.Each factor of hormone analogue can influence artemisinin production differently.As for the effect of each factor,the sequence ranked:gibberellins(GA₃)> naphthlcetic acid (NAA)>animo acid micro fertilizer>B₉>6-furifuryl-aminopurine(KT).When used properly, artemisinin content can be increased by 36.9%,medicinal biomass by 93.3%,artemisinin production by 125.5%as a maximum,compared with the control.Planting density also can influence artemisinin content but not greatly.However, relatively high planting density leads to high artimisinin production(mainly due to high medicinal biomass).The fertilization technique of planting A.annua in field is as follow:N is 720 kg/ha,P is 1080 kg/ha,K is 1080 kg/ha,Ca is 360 kg/ha.Besides,animo acid micro fertilizer is diluted to 225 times,and GA₃,NAA is 40 mg/L and 5 mg/L,respectively.The highest artemisinin content is 1.0484%,and the lowest one is 0.5598%.6.The drifts of artemisinin content of introduced A.annua and local wild ones are the same,but their levels differ greatly.It means artemisinin content is mainly controlled by their genetic backgrounds and secondly controlled by the natural circumstance.The highest artemisinin content is 1.0484%,and the lowest one is 0.5598%of introduced A.annua,which are far higher than the medicinal criterion of artemisinin content≥0.5%.While,with artemisinin content reaching industrial standard and medicinal biomass exceeding that of their origin,introduced A.annua has showed great adaptation to the local natural conditions.It means introduced A.annua get great succeed.So considering A.annua's advantage of great adaptation,starting mass planting production in wasteland,abandoned land,uncultivated land and wasteland with some debris will be a nice forestry and agriculture-benifiting project.