| The Aconitum, one of the toxic traditional Chinese medicine, is known as its expelling cold, regulating meridian and relieving pain. Because of its high toxicity, Aconitum can cause intoxication or life-threatening in improper use. The major reason of death is a serious arrhythmia. Therefore, the processed drug is needed and is first decocted in the clinical use. The common processing method is under damp and heat conditions, which also cause poisoning or greatly reduce the efficacy if the processing is not appropriate. This article adopts the technology of bi-direction solid state fermentation (SSF) and liquid state fermentation (LSF), and the fermentation processing of Aconitum is under the mild conditions. The fungus substance can be obtained, which can reduce the incidence of arrhythmias and can enhance efficacy that is to be further studied. Main methods and results are as follows.1. Based on bi-direction SSF technology, screening19fungi were inoculated on Aconitum kusnezoffii Reichb. From the perspective of the biological characteristics and the change of the main alkaloids, the relation of the conversion rate of the matrix and the growth of fungi is negative correlation. According to the changes of the content and the peak of monoester diterpenoid alkaloidss, the most suitable fungi of bi-direction SSF are optimal selected, namely Ganoderma lucidum (Curt, ex Fr.) Karst, Trametes cinnabarina (Jacq.) Fr., Poria cocos (Schw.) Wolf, and Panellus stypticus (Bull, ex Fr.) Karst. Whose corresponding fermentation products are Lingwu FS, Shuanwu FS, Fuwu FS and Shanwu FS.2. From the point of reducing toxicity and enhancing efficacy, the four fungi medicine combination are selected, which are used to comparative study by acute toxicity, arrhythmia and the effects of analgesic, anti-inflammatory. The results of four FS toxicity test, reducing the incidence of arrhythmia, analgesic and anti-inflammatory are as follows:(1) The maximum tolerated dose (equivalent to original medicinal16g/kg) test showed that at the same doses, the weight of each treatment group was significantly lower than the normal control group after7days administration, while the weight of Lingwu FS increases rapidly, and was close to the normal control group after14days of administration;(2) The results of the arrhythmia effects in rats showed that after consecutive administration7days, Shuanwu FS、Shanwu FS and Lingwu FS had significant difference to reduce the occurrence of the rat cardiac arrhythmia (P<0.05) comparedwith the processed product group by damp-’heat processing method;(3) The experimental studies of acetic acid-induced writhing times, xylene-induced mice ear edema in mice showed the analgesic and anti-inflammatory effects of Lingwu FS had significant difference (P<0.05) and the analgesic effect of Shanwu FS was enhanced (P<0.05) compared with the processed products by damp-heat processing method.3. Fermentation by Panellus stypticus (Bull, ex Fr.) Karst. From the matrix conversion rate and the content of alkaloids, it was comprehensively explored at the different time as research object, terminal point of SSF was determined about35to40days. Compared with the control, diester diterpenoid alkaloids decreased and the content of monoester diterpenoid alkaloids increased in a greater degree.In experiments of acetic acid-induced writhing, the mouse’s auricle swelling that is caused by xylene and rat’s voix pedis swelling that be caused by carrageenin, the experimental results showed that each dose group had varying degrees of enhancement compared with the normal group. Compared with the processed product group by damp-heat processing method, the effects of anti-inflammatory and analgesic were significantly increased (P<0.05) in high dose group.Based on single factor (Carbon and nitrogen sources) and orthogonal experiments, the optimize culture condition was that:carbon source the mixture glucose and corn meal,nitrogen source cake bean powder and liquid volume of100mL(a250mL flask),by adding0.3power, inoculated two evenly mycelia pellet, culture28degrees Celsius for five days.Based on the optimize liquid fermentation culture, the contents of six ester diterpenoid alkaloids were analyzed, the results showed that the content of the high toxic diester diterpenoid alkaloids decreased and the content of low toxic monoester diterpenoid alkaloids increased compared with the control [only Aconitum and liquid fermentation culture, not inoculated by Panellus stypticus (Bull, ex Fr.) Karst.].Based on the optimize liquid fermentation culture, the contents of ester diterpenoid alkaloids were analyzed, the results showed that the content of the high toxic diester diterpenoid alkaloids decreased and the content of low toxic monoester diterpenoid alkaloids increased compared with the total alkaloid control [only the total diterpenoid alkaloids and liquid fermentation culture, not inoculated by Panellus stypticus (Bull, ex Fr.) Karst.].Based on the optimize liquid fermentation culture, the contents of six aconitum alkaloids were analyzed, the results showed that the content of the high toxic diester-aconitum alkaloids decreased and the content of low toxic mono-ester aconitum alkaloids increased compared with the single component control (only the the single component and liquid fermentation culture, not inoculated by Panellus stypticus (Bull, ex Fr.) Karst.).4. Fermentation by Ganoderma lucidum (Curt, ex Fr.) Karst. From the matrix conversion rate and the content of alkaloids, it was comprehensively explored at the different time as research object, the terminal point of fermentation was determined about35to45days. Compared with the control, diester diterpenoid alkaloids decreased and the content of monoester diterpenoid alkaloids increased in a greater degree. In experiments of acetic acid-induced writhing, xylene-induced mice ear edema in mice and carrageenan-induced hind paw swelling in rats, the experimental results showed that each dose group had varying degrees of enhancement compared with the model group. Compared with the processed product group by damp-heat processing method, the effects of anti-inflammatory and analgesic were significantly increased (P<0.05) in high dose group.5. Partial correlation analysis on three variables which were efficacy and toxicity was studied by SPSS17.0; four fungal substances were selected as research objects, which were Shuanwu FS, Fuwu FS, Shanwu FS and Lingwu FS. The result revealed that the toxic component was Aconitine, Mesaconitine and Hypaconitine.In summary, the method used the medicinal fungi to ferment Aconitum kusnezoffii Reichb. can be used to significantly reduce the contents of diester diterpenoid alkaloids in Aconitum which have seriously toxic, while significantly increased the contents of monoester diterpenoid alkaloids which have little toxic. Compared with the processed product group by damp-heat processing method, Lingwu FS and Shanwu FS have the significant function of reducing toxicity and increase efficacy. The research of this paper scientifically interprets the principle of bi-direction solid state fermentation. |
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