Optimization of the Ethanol Extraction Method of Monoester Alkaloids

Changkai ZHOU Rui ZHANG Xiuling SHANG Lihua ZHOU Zukang ZHANG Jing GAO Hongxia YU Hongyan JI Xiaomin XING Qie GUO Donghua LIU Fangang MENG Ji

Changkai ZHOU  Rui ZHANG  Xiuling SHANG  Lihua ZHOU  Zukang ZHANG Jing GAO  Hongxia YU  Hongyan JI  Xiaomin XING Qie GUO  Donghua LIU  Fangang MENG Jingchun YAO Na ZHANG Yongjun LIU  Fanbo JING

Abstract ［Objectives］ This study was conducted to optimize the ethanol extraction technology of monoester alkaloids from Radix Aconiti Preparata .

［Methods］ On the basis of defined extraction times, ethanol concentration, ethanol times and extraction time were investigated by HPLC-MS combined with orthogonal test to optimize extraction process using the content of monoester alkaloids (the sum of benzoyl neoaconitine, benzoyl hypoaconitine and benzoyl aconitine) as an index.

［Results］ The optimum ethanol extraction technology was as follows: 75% ethanol, ethanol amount 25 times of the medicinal material, and each extraction for 1.5 h.

［Conclusions］ The optimal extraction technology is simple, feasible, stable and reliable. It can provide reference for the industrial production and quality control of monoester alkaloids from Radix Aconiti Preparata .

Key words Radix aconiti preparata; monoester alkaloid; HPLC-MS; orthogonal test

Received: January 21, 2020Accepted: March 13, 2020

Supported by Traditional Chinese Medicine Science and Technology Development Program of Shandong Province (2017-198; 2019-0400); Major Science and Technology Innovation Project of Shandong Province (2018CXGC1304).

Changkai ZHOU (1987-), male, P. R. China, pharmacist in charge, devoted to research about new formulations and technologies of traditional Chinese medicine.

*Corresponding author. E-mail: jingbf178@sina.com.

Radix aconiti refers to the dried mother roots of Aconitum carmichaelii in Ranthaceae, and Radix aconiti preparata is a processed product of Radix aconiti, which is spicy, bitter, hot, and poisonous. It acts through the heart, liver, kidney, and spleen meridians with the effects of expelling wind and dampness and warming the meridian to relieve pain, and has high medicinal value. Modern studies have confirmed that Radix aconiti has pharmacological effects such as anti-inflammatory, antitumor, immunity regulating and analgesic［1-4］, but due to its toxicity, Radix aconiti餾 clinical application is greatly limited. Diester alkaloids are the main toxic components of Radix aconiti, which could be converted into monoester alkaloids after processing, the toxicity of which is 0.002-0.005 of diester alkaloids［5-7］, so Radix aconiti is safe after processing while maintaining pharmacological activity and thus has broad application prospects. However, at present, there are no preparations related to Radix aconiti extract, and the research on the extraction of monoester alkaloids is extremely scarce. This study was intended to optimize the ethanol extraction process for Radix aconiti preparata monoester alkaloids by orthogonal test, so as to provide data support for large-scale industrial production and research and development of new preparations.

Experimental Materials

The used instruments included API4000 + triple quadrupole tandem mass spectrometry system and Agilent 1290II high performance liquid chromatography. Benzoyl neoaconitine, benzoyl hypoaconitine and benzoyl aconitine standards were provided by Shanghai Yilin Biotechnology Co., Ltd. Radix aconiti preparata decoction pieces were purchased from the medicinal materials market. The used methanol and acetonitrile were chromatographically pure, and other reagents were analytically pure.

Methods and Results

LC conditions

Chromatographic column: Ultimate AQ-C18 column (3.0 μm, 2.1 mm×100 mm, Shanghai Yuexu Technology Co., Ltd.); column temperature: 40 ℃; flow rate: 0.4 ml/min; mobile phase∶0.1% formic acid-methanol=55∶45.

MS conditions

The MS detection was performed with an electrospray ionization ion source at a source voltage of 5.5 kV, a capillary temperature of 550 ℃ and a sheath gas flow rate of 40 in positive mode. The mass analyzer was quadrupole, and MRM mode detection was adopted. The detection conditions and MRM diagrams are shown in Table 1 and Fig. 1.

Preparation of test solution

Radix aconiti preparata powder (10 g) was added in a round-bottom flask. Then, 250 ml of 75% ethanol was added, and the mixture was heated and refluxed for 1 h, obtaining an extract, which was diluted to 250 ml and filtered, giving a test solution.

Preparation of reference solution

Benzoyl neoaconitine, benzoyl hypoconitine and benzoyl aconitine standards (10 mg each) were precisely weighed and added into 10 ml volumetric flasks, respectively. Then, methanol was added into each flask to dissolve the standard, obtaining a solution, which was diluted to constant volume and shaken well.

Methodological investigation

Linear relation investigation

The benzoyl neoaconitine (1 mg/ml), benzoyl hypoconitine (1 mg/ml) and benzoyl aconitine (1 mg/ml) were diluted to 1 000, 2 000, 5 000, 10 000, 20 000 and 50 000 times and injected into an LC-MS instrument in sequence, and the peak area was recorded. The standard curves were drawn with the injection amount (ng) as the abscissa and the peak area as the ordinate. The regression equation of benzoyl neoconconine was Y=2.92 ×103X+1.31×102, r=0.999 5 ; the regression equation of benzoyl hypoconicine was Y=4.37×103X+1.55×102, r=0.999 8  ; and the regression equation of benzoyl aconitine was Y=1.12×103X-1.05×102, r=0.999 6 . The results showed that benzoyl neoaconitine, benzoyl hypoconitine and benzoyl aconitine were linear in the range of 20-1 000 ng.

Precision test

The same reference solution (10 μl) was accurately pipetted, and six consecutive injections were performed to record the peak area. The results showed that the RSD values of the peak areas of benzoyl neoaconitine, benzoyl hypoconitine and benzoyl aconitine were 1.46%, 1.27% and 1.19%, respectively, indicating that the precision of the instrument was good.

Stability test

The test solution (10 μl) prepared under "Preparation of test solution" was injected at 0, 2, 4, 6, 8 and 10 h, and the peak area was recorded. The results showed that the RSD values of the peak areas of benzoyl neoaconitine, benzoyl hypoconitine and benzoyl aconitine were 1.14%, 1.35% and 1.27% ( n =6), respectively, indicating that the test solution had good stability within 10 h.

Reproducibility test

Six parts of test solutions (10 μl) were prepared in parallel under "Preparation of test solution", and injected precisely and determined. The RSD values of the contents of benzoyl neoaconitine, benzoyl hypoconitine and benzoyl aconitine in the test product were 1.26%, 1.32% and 1.13%, respectively, indicating that the method had good reproducibility.

Recovery test

Nine parts of the test product with a known content were weighed precisely, about 1.00 g each. Each of the weighed test product was added with the three reference substances in an amount equivalent to 80%, 100% and 120% of the amount of each reference substance, respectively, and in three repetitions for each level in parallel. Test solutions were prepared according to the method under "Preparation of test solution" and determined according to "LC conditions" and "MS conditions". The peak area was recorded, followed by the calculation of the content of monoester alkaloids (based on the sum of benzoyl neoaconitine, benzoyl hypoconitine and benzoyl aconitine), sample recovery and RSD values, as shown in Table 2. The results showed that the average recovery values for the three concentrations were 100.9%, 101.5%, and 101.9%, respectively, and the RSD values were 1.21% , 1.86%, and 2.50%, respectively.

Investigation of quantitation and detection limits

An appropriate amount of each reference solution prepared under "Preparation of reference solution" was diluted by a factor of two, and measured 6 times in succession according to "LC conditions" and "MS conditions", and the peak area was recorded. Based on the principles of minimum quantitation limit S/N =10 and detection limit S/N =3, the calculated limits of quantification and detection were 0.020 0 and 0.006 5 μg/ml, respectively.

Single factor test

Effect of extraction times on monoester alkaloid content

The medium-fine powder of Radix aconiti preparata (10 g) was added in a round-bottom flask, followed by the addition of 250 ml of 75% ethanol. The mixture was extracted under reflux for 1 h. The volume of the extract was adjusted to 250 ml and filtered. The residue was extracted twice referring to the first time of extraction. The monoester alkaloid contents in the three samples were measured, and the results are shown in Fig. 2.

Changkai ZHOU et al. Optimization of the Ethanol Extraction Method of Monoester Alkaloids from Radix Aconiti Preparata Based on HPLC-MS and Orthogonal Test

Effect of particle size on monoester alkaloid content

Each of Radix aconiti preparata decoction pieces, coarse powder, medium-fine powder and fine powder (10 g) was placed in a round-bottomed flask, and added with 250 ml of 75% ethanol. Each mixture was extracted under reflux for 1 h. The volume of the extract was adjusted to 250 ml and filtered, obtaining a filtrate, which was determined for the monoester alkaloid content. The results are shown in Fig. 3.

Effect of ethanol concentration on monoester alkaloid content

The medium-fine powder of Radix aconiti preparata (10 g) was added in round-bottom flasks, into which 250 ml of 35%, 55%, 75% and 95% ethanol were added, respectively. Each mixture was extracted under reflux for 1 h. The volume of the extract was adjusted to 250 ml and filtered, obtaining a filtrate, which was determined for the monoester alkaloid content. The results are shown in Fig. 4.

Effect of ethanol times on monoester alkaloid content

The medium-fine powder of Radix aconiti preparata (10 g) was added in round-bottom flasks, into which 50, 150, 250, 350 and 450 ml of 75% ethanol were added, respectively. Each mixture was extracted under reflux for 1 h. The volume of the extract was adjusted to 500 ml and filtered, obtaining a filtrate, which was determined for the monoester alkaloid content. The results are shown in Fig. 5.

Effect of extraction time on monoester alkaloid content

The medium-fine powder of Radix aconiti preparata (10 g) was added in round-bottom flasks, into which 250 ml of 75% ethanol was added, respectively. The mixtures were extracted under reflux for 0.5, 1, 1.5, 2 and 2.5 h, respectively. The volume of the extract was adjusted to 500 ml and filtered, obtaining a filtrate, which was determined for the monoester alkaloid content. The results are shown in Fig. 6.

Orthogonal test

Orthogonal test table

According to the results of the single factor test, the target components could be completely extracted after two times of extraction, and the amount extracted by the third time only accounted for 0.86% of the sum of the previous two times, so the extraction times were fixed at two. In terms of particle size, the content of the target components from the fine powder was only 0.93% higher than that of the medium-fine powder. Considering the production operability and cost, the particle size was selected as the medium-fine powder. For ethanol concentration, with the increase of the ethanol mass fraction, the content of the components gradually increased, and was the highest at 75%. The target components had high solubility at this concentration, and the content decreased significantly at 95%, so the adjacent contents of 65%, 75% and 85% were selected as the orthogonal test factor levels. In terms of solvent times, the content of the components gradually increased with the increase of the extraction volume. The extraction was sufficient at 25 times (250 ml), and the content did not increase significantly at 35 and 45 times. Therefore, 15, 25 and 35 times were selected as the orthogonal test factor level. As to extraction time, the component content was highest when it was extracted for 1.5 h, and the extraction was complete. The content began to decline at 2 h, and prolonged heating might cause further hydrolysis of the monoester alkaloids. Therefore, 1, 1.5 and 2 h were selected as the orthogonal test factor levels.

Results of orthogonal test

It can be known from Table 4 that the ranges ranked as RA>RC>RB , that is, the primary and secondary order of the influencing factors was ethanol concentration>extraction time>ethanol times. The analysis of variance from Table 5 showed that for the extraction of target components, the concentration of ethanol had a significant effect, and the extraction time and the amount of ethanol had certain effects. According to the analysis of the k value, the optimal process was A2B2C3, but the single extraction time of C3 was 2 h, which was a long time, and the extraction effect was not significantly better than the level of C2. Considering the actual production and environmental protection factors, A2B2C2 was determined as the best process, i.e. , 75% ethanol, ethanol amount 25 times of the medicinal material, and each extraction for 1.5 h.

Process validation test

In order to verify the feasibility of the obtained combined process, three batches of validation tests were performed, and extraction was performed according to the preferred process to determine the contents of benzoyl neoaconitine, benzoyl hypoconitine and benzoyl aconitine, which are shown in Table 6. It could be seen that the mean of the validation test was 12.87 and the RSD was 1.91% ( n =3), indicating that the optimal conditions for the orthogonal test were stable and feasible, and could be used as an extraction process for the preparation of Radix aconiti preparata monoester alkaloids.

Discussion

The diterpenoid alkaloids in Radix aconiti are its main chemical components and the main medicinal components of Radix aconiti. They are mainly composed of C-19 diterpenoid alkaloids, including toxic diester-type alkaloids such as aconitine, hypoconitine and aconitine, which can be processed into low-toxic monoester-type alkaloids such as benzoyl aconitine, benzoyl hypoconitine, benzoyl neoaconitine, etc. , thereby reaching the goals of enhancing efficiency and reducing toxicity and greatly improving the clinical safety of Radix aconiti preparata. However, the research on the extraction, pharmacology and mechanism of Radix aconiti preparata monoester alkaloids is relatively scarce, so that Radix aconiti preparata preparations are still not available in the market. Therefore, the development and creation of new Radix aconiti preparata preparations have a broad market space.

In the selection of the target components for the orthogonal test, Radix aconiti preparata mainly contains monoester alkaloids, and the content of diester alkaloids is relatively low. The 2015 edition of Chinese Pharmacopoeia set the upper limit of the sum of such three types of diester alkaloids as aconitine, hypaconitine and neoaconitine to not exceed 0.04%. Referring to the "Content Determination" in Chinese Pharmacopoeia , the characteristic components of the three monoester alkaloids, namely benzoyl neoaconitine, benzoyl hypoaconitine and benzoyl aconitine, were selected as index components, and the sum of the contents was recorded as the monoester alkaloid content, which served as a preferred index of process. In terms of extraction methods, we have found through pre-experiments that the ethanol extraction method used in this study has a higher extraction rate of index components than the pharmacopoeial extraction method without adjusting the pH, and the ethanol extraction method is also more suitable for industrial production. In addition, during the extraction process, it was found that the target components had a low content, and the contents of the three components were very different. The content of benzoyl aconitine was extremely low, which was once not detected in the pre-xperiments, but after the optimization of the process, its content was relatively stable, and the extraction process was simple and reliable after optimization. This study has certain reference significance for the further development of Radix aconiti preparata in the future and the industrial production of new monoester alkaloid preparations.

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