| Radix Aconiti Lateralis Praeparata（Fuzi）is a poisonous traditional Chinese medicine（TCM）with definite curative effect,as well a famous genuine medicinal material in Sichuan Province.Paofuzi,first published in Treatise on febrile diseases,is the earliest processed product of Fuzi,which is known as"the essential medicine to replenish the true fire of the life-gate".From the Han Dynasty to the Tang Dynasty,Paofuzi was the mainstream processed product of Fuzi,but it was gradually lost after the Ming Dynasty due to unstable fire source,difficult to control,low processing efficiency and other reasons.In recent years,with the rise of the development of classic famous prescriptions that pay attention to"original formula,dose,processed products and certificate",it is urgent to awaken the traditional Paofuzi and improve the processing technology of Paofuzi by using modern technological innovation for reference.Microwave technology is a new processing technology of TCM in recent years.It has the advantages of fast heating up,high processing efficiency and high degree of digitization.It is not only an innovative embodiment of modern fire method,but also an important means to restore the ancient Paofuzi.However,it is not very clear the early processing mechanism of toxicity reduction and survival effect of microwave Paofuzi.The adaptability of microwave industrial processing equipment is low so that the industrial production has not been fully realized.Therefore,this study abides by the processing essence of ancient Paofuzi,deeply analyze the regulations of composition-structure-activity of aconite under microwave conditions,clarify the main influencing factors of microwave processing,establish an intelligent industrial production line of microwave Paofuzi.It is expected to develop Paofuzi with stable quality and definite curative effect,promote the innovation and upgrading of aconite processing industry from industrialization,digitization and intelligence,and promote the application and transformation of microwave technology in the processing of TCM.The main research results and conclusions are as follows:1、Effect of microwave processing on hydrolysis law and detoxification of AconitineMicrowave processing has the advantages of rapid temperature rise,strong penetration and high selectivity.It can realize the heating,drying and dehydration of materials in a short time.Whether there is a special hydrolysis law of aconitine under microwave processing needs to be scientifically explained.The hydrolysis pathway and products of aconitine were calculated by quantum chemistry.It was found that the energy required for the hydrolysis of aconitine to produce benzoylaconitine（37.4kcal/mol）and pyroaconitine（37.8 kcal/mol）was the same,and the energy required for the conversion of benzoyl aconitine to pyroaconitine was higher（55.8 kcal/mol）,indicating that benzoylaconitine and pyroaconitine could not be transformed into each other.High resolution mass spectrometry analysis showed that there were six compounds in aconitine hydrolysate.Compared with the control,it was found that four compounds were aconitine（m/z 646.3226）,indaconitine（m/z 630.3273）,benzoylaconitine（m/z 604.3121）and aconine（m/z 500.2854）.The unknown compounds m/z 586.3016 and m/z 482.2751 were isolated and identified.The results showed that m/z 586.3016 had two configurations,respectivelyα-pyroaconitine andβ-pyroaconitine.m/z 482.2751,is 16-epi-pyroaconine.HPLC-MS/MS was used to further confirm that benzoylaconitine and pyroaconitine could not be transformed into each other,and it was proved that there were three independent pathways for aconitine hydrolysis.Zebrafish toxicity test was used to evaluate the toxicity of hydrolysates.It was found that indaconitine had the strongest toxicity(LC50was 2.8 mg·L-1),pyroaconitine had the weakest toxicity(LC50was 64.52 mg·L-1),and the toxicity of other hydrolysates was between them.The effects of traditional pyrolysis and microwave pyrolysis on aconitine hydrolysate were further compared.It was found that microwave could completely hydrolyze aconitine within 4 minutes,and the hydrolysate was mainly pyroaconitine.The traditional pyrolysis takes 60 minutes,and the product is mainly benzoylaconitine.The results of cardiac toxicity experiment showed that aconitine would not appear the poisoning of ventricular premature beat in rats with the maximum dose after being heated by microwave for 4 minutes,while it took 15 minutes to achieve the same detoxification effect.2、Effects on properties,structure and efficacy of aconite polysaccharide by of microwave processingFuzi polysaccharide is one of the main pharmacodynamic substances in aconite.Microwave may change the structure,physicochemical properties and biological activities of polysaccharides in the process of polysaccharide extraction.Therefore,it is necessary to study the effects of microwave processing on the properties,structure and efficacy of aconite polysaccharide.Polysaccharides from Paofuzi and Shengfupian were extracted by water extraction and alcohol precipitation.DE-52cellulose column and dextran gel G-100 column was used to separate these polysaccharides.The extraction rate of crude polysaccharides in Shengfupian was11.5%and the purity was 81.89%.The extraction rate of crude polysaccharides in Paofuzhi was 10%and the purity was 82.90%.Three polysaccharide fragments were isolated from the Shengfupian and Paofuzi polysaccharides respectively,and their water-soluble polysaccharide fragments were the mainstay,accounting for 15.26%and 18.83%of the crude polysaccharides.Through the in-depth analysis of the water-soluble fragments of the two polysaccharides,it was found that after microwave processing,the surface of aconite polysaccharide changed from rough to smooth,and there were circular pores and cracks,the whole was irregular and flaked,the degree of agglomeration was low.Its molecular weight changed from 7810 Da to 4753 Da,the composition of glucose increased from 83.636%to 94.831%,arabinose increased from 0.529%to 1.531%,and galactose decreased from 1.349%to 0.664%.By further analyzing the polysaccharide structure of Shengfupian and Paofuzi,it was found that both main chain structure of polysaccharides is formed→4)-α-D-Glcp-(1→,α-D-glcp-(1→terminal group connected to the main chain through O-6.However,the proton peaks of terminal group in these two polysaccharides are different inδ5.31,5.26 and 4.89 signal integrals.The integral ratio of Shengfupian polysaccharide was7.17:1.07:1 and Paofuzi polysaccharide was 4.29:1.16:1,indicating that the main chain of Paofuzi polysaccharide was significantly shorter.The results showed that Paofuzi polysaccharide(100 and 200μg·m L-1)and Shengfupian polysaccharide(200μg·m L-1)could significantly promote the characteristic marker CD206 expression of M2 macrophage（p<0.01）,thus promoting the polarization of macrophages to M2 and enhancing the anti-inflammatory activity of macrophages.In addition,the inhibitory effects of the two polysaccharides on the M1 polarization of macrophages induced by lipopolysaccharide were compared.Paofuzi polysaccharide(100 and 200μg·m L-1)could inhibit the M1 polarization of macrophages induced by lipopolysaccharide,but Shengfupian polysaccharide did not find this effect.3、Study on the main factors affecting the quality of microwave PaofuziTo clarify the main technological factors of microwave Paofuzi,so as to lay a foundation for the industrialized development of microwave Paofuzi.According to the early technology of microwave Paofuzi,a small-scale test was carried out in a household microwave oven,and the effects of thickness,water absorption,microwave power and time on the quality of microwave Paofuzi were investigated with diester and monoester alkaloids as indexes.The results show that the thickness of aconite is 5mm,the low-power microwave is difficult to penetrate the appendage,and the energy is easy to accumulate on the surface of the appendage,resulting in zooming on the outside and immature inside.Its total alkaloid content(about 249.89μg·g-1)is lower than that of the 3 mm Fuzi(about 315.52μg·g-1).So the thinner of Fuzi should be selected when the microwave power is small.The effects of water absorption of 40%,60%,80%and 100%on the quality of microwave Paofuzi were compared.This shows that the lower the water absorption,the easier it is to scorch the attached piece,the higher the water content of the attached piece,and the longer the processing time.The content of total alkaloids is 60%（about 574.42μg·g-1）>40%(about 355.27μg·g-1)>80%(312.52μg·g-1)≈100%(about 297.62μg·g-1),and the content of diester alkaloids is 60%(116.64μg·g-1)>100%(94.3μg·g-1)>80%(67.41μg·g-1)>40%(15.2μg·g-1).In order to ensure the safety and good appearance quality of Paofuzi,the moisture content of Fuzi should be controlled at 80%～100%.At the same processing time,compared with microwave power of 350W,the content of diester aconitine in Paofuzi under microwave power of 550W is lower(about 4.08μg·g-1)and the content of total alkaloids is higher(about 210.13μg·g-1).However,with the increase of processing time,Fuzi under high power condition is easier to be charred.Generally speaking,the domestic microwave oven can be used for a small amount of preparation of Fuzi,but it has small capacity,low processing efficiency,high requirements for the shape of medicinal materials,high waste rate and insufficient controllability of the preparation process,so it can not be used for the industrial production of microwave Paofuzi.4、Study on the industrial production of microwave PaofuziThe production line with controllable process and high intelligence is helpful to improve the quality and production efficiency of microwave Paofuzi,and promote the transformation and practical application of microwave Paofuzi.Based on the main influencing factors of microwave Paofuzi and the characteristics of microwave industrial equipment,we established a two-stage production process include microwave liquid sealing detoxification and microwave drying expansion.Then,taking diester alkaloids and monoester alkaloids as indexes,we investigated the effects of temperature,time,thickness and morphology of microwave Paofuzi and further optimized the industrialized production process of microwave Paofuzi.The results showed that the microwave liquid sealing stage was the main stage of detoxification in processing.The higher the temperature,the longer the time,and the more thorough the detoxification.The microwave drying expansion stage is mainly to complete the water removal and drying expansion of Paofuzi.Through single factor experiment,the optimum industrial production process of microwave Paofuzi was determined as follows:take Shengfupian and add an appropriate amount of water according to the weight of 1:1.2,microwave 90℃atmospheric pressure detoxification for 90 minutes,microwave vacuum frequency conversion drying at60℃until the water content of Fuzi was less than 12%.10 batches of Shengfupian from different sources and manufacturers were selected to prepare microwave Paofuzi according to the best production process and carry out relevant quality evaluation.The results showed that the content of monoester alkaloids in 10 batches of Paofuzi was0.071%～0.166%.The content of diester alkaloids was 0.004%～0.016%;The moisture content is 4.91%～11.82%;The ash content is 1.82%～3.11%;Acid insoluble ash is 0.04%～0.13%.The above results show that all 10 batches of Paofuzi comply with the relevant provisions under the Fuzi item of the standard for processing of TCM in Sichuan Province.The same batch of Fuzi was processed by microwave,traditional ancient method and modern sand frying respectively,and their appearance,texture and content of six alkaloids were compared.The results show that the microwave processed Fuzi is closer to the ancient processed Fuzi in terms of texture and appearance color.The results of alkaloid content showed that the total alkaloid content was microwave processing（0.181%）>ancient processing（0.108%）>modern sand frying（0.085%）,the content of diester alkaloid was ancient processing（0.051%）>modern sand frying（0.016%）≈microwave processing（0.015%）,and the content of monoester alkaloid was microwave processing（0.167%）>modern sand frying（0.070%）>ancient processing（0.052%）.It shows that microwave Paofuzi has fast and complete detoxification,high retention rate of effective components,and can highly restore ancient processed Fuzi.In addition,this study also uses raw Fuzi as material for microwave processing of Fuzi.The results show that the microwave processing time of raw Fuzi is shorter,the content of effective components is higher,and the content of monoester alkaloids is 0.20%～0.25%.The above research shows that microwave processing can be used for the rapid processing of Fuzi.5、Quality evaluation and pharmacological activity of microwave PaofuziThe core value of decoction pieces of traditional Chinese medicine is clinical efficacy.Microwave Paofuzi is a new processed product of Fuzi.Its characteristics and pharmacological effects need to be further studied.HPLC-MS/MS was used to compare the content of alkaloids between microwave processing and commercial Shengfupian,Heishunpian and Baifupian.It was found that the contents of diester alkaloids（0.114%～0.215%）,water-soluble alkaloids（0.009%～0.073%）and total alkaloids（0.155%～0.287%）in Shengfupian were the highest.After microwave processing,the contents of diester alkaloids decreased sharply（0.001%～0.012%）,the contents of monoester alkaloids（0.030%～0.140%）increased,and the contents of water-soluble alkaloids（0.008%～0.069%）and total alkaloids（0.050%～0.205%）decreased.The total alkaloid content of microwave Paofuzi is about 6-27 times that of Baifupian and Heishunpian,especially water-soluble alkaloids are about 8-69 times that of Baifupian and Heishunpian.Pharmacological activity experiments showed that microwave Paofuzi not only retained the anti-inflammatory and analgesic activities of Heishunpian and Baifupian,but also retained the cardiotonic activity of Shengfupian.At the same time,microwave Paofuzi and Shengfupian can significantly improve the white blood cells and lymphocytes of liver cancer chemotherapy mice.Microwave Paofuzi can also enhance the ratio of CD4/CD8 in spleen cells of liver cancer chemotherapy mice and regulate immunity（p<0.05）.It also can enhance the antitumor activity of chemotherapeutic drug cyclophosphamide,while Heishunpian and Baifupian tablets have weak effect and Shengfupian have no this effect.To sum up,the ancient Paofuzi was restored by microwave processing technology in this study.It analyzes the hydrolysis law of aconitine,a highly toxic component in Fuzi,under microwave conditions,reveals the potential mechanism of structure-activity change of Fuzi polysaccharide processed by microwave,clarifies the key influencing factors of microwave Paofuzi,establishes a production process of Fuzi with accurate and controllable process and high intelligence,and develops Fuzi with stable quality and definite curative effect.It realizes the synchronous improvement of productivity and environmental performance in the processing industry of Fuzi,and provides scientific support for the industrial application of microwave processing technology. |
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