HPLC fingerprint spectra and determination of four alkaloids content in different regions of Fu Mao iron rod hammer
Aconitum flavum Hand. – Mazz. belongs to the Aconitum genus of the Ranunculaceae family. It is also known as Duanchongcao, Wuyao, and Yizhihao, and is a traditional Chinese medicinal herb and folk herb. Its roots and seedlings are used as medicine, and its dried roots are commonly known as “iron rod hammer”. The Shaanxi Chinese Herbal Medicine records that the Fumao Iron Rod Hammer has the effects of promoting blood circulation, removing blood stasis, relieving pain, dispelling wind and dampness, reducing swelling and toxins, stopping bleeding, treating injuries caused by falls, rheumatoid arthritis, and lower back and leg pain.

According to literature reports, alkaloids are the main active ingredients of the Chinese medicinal herb, Aconitum carmichaelii, and aconitine is the main chemical component with a relatively high content. The main components of the difference in content between Fumao Iron Hammer and Iron Hammer are 12 epiaconitine and aconitine. Fumao Iron Hammer has a higher content of 12 epiaconitine and aconitine than Iron Hammer. Modern pharmacological experimental studies have shown that aconitine, 12 epiaconitine, sophocarpine, and 3-acetylaconitine have analgesic, anti-inflammatory, and anticancer activities.

At present, there are few reports on the quality standards of Fumao Iron Hammer. The fingerprint spectrum of traditional Chinese medicine is widely used for quality evaluation, which can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine. Therefore, this study established HPLC fingerprint chromatograms for 15 batches of different origins of Fumao Tiebang Hammer medicinal materials, and determined the contents of four alkaloids, including 12 epiaconitine, Song Guoling, aconitine, and 3-acetylaconitine; Secondly, the Chinese medicine chromatographic fingerprint similarity evaluation system (2012A version) was used for similarity evaluation, and SPSS software was used for cluster analysis and principal component analysis, in order to provide scientific basis for the screening of indicator components and the comprehensive control of the quality of the Chinese medicine Fumao Tiebang Hammer.

 

 

Determination of chromatographic conditions. The maximum absorption wavelength of 12 aconitine and Song Guoling is 210nm, and the maximum absorption wavelength of aconitine and 3-acetylaconitine is 235nm. 210, 215, 220, and 230nm were investigated to balance the four peaks and achieve the highest response value. Finally, 215nm was selected as the detection wavelength. We investigated Agilent Eclipse Plus C18 (50mm × 4.6mm, 5 μ m), Unitary C18 (250mm × 4.6mm, 5 μ m), JADE-PAK ODS (250mm × 4.6mm, 5 μ m), and ACE Excel C18PFP (250mm × 4.6mm, 5 μ m). By comparing the symmetry factor, resolution, and peak shape, we ultimately chose the ACE Excel C18 PFP chromatographic column. We investigated the addition of 2.0, 2.5, and 3.0 mL of triethylamine to the aqueous phase, and found that the addition of 2.5 mL of triethylamine resulted in better chromatographic peak shapes and gradient elution effects. Therefore, we added 2.5 mL of triethylamine to the aqueous phase. According to the pharmacopoeia and literature, mobile phases such as acetonitrile: 0.1% ammonium acetate, acetonitrile: 0.2% glacial acetic acid (adjusted to pH=6.2 with triethylamine), acetonitrile and water, and acetonitrile: 0.1% phosphoric acid were investigated. Finally, a gradient elution of acetonitrile: 0.1% phosphoric acid aqueous solution was selected, resulting in a smooth baseline, uniform peak distribution, and good separation effect.

Fingerprint spectrum and content determination analysis. There are differences in the number of chromatographic peaks, retention time, and peak area at the same retention time in the fingerprint spectrum of samples from different origins of Fu Mao iron rod hammer; The similarity evaluation results of the fingerprint spectra of 15 batches of medicinal materials ranged from 0.769 to 0.952; Cluster analysis classified S1 and S2 samples as Class I; Samples S3~S5 are classified as Class II; S6~S13 are classified as Class III; S14 and S15 are classified as Class IV. Consistent with the results of principal component analysis. Principal component analysis was used to screen out common characteristic components, identify determining factors, and provide reference for the quality evaluation of Fumao Iron Rod Hammer medicinal materials. Principal component analysis uses eigenvalues greater than 1.2 as the extraction criterion, and when the cumulative variance contribution rate is greater than 80%, five principal components are obtained, which can represent most of the information of the common peaks in the fingerprint spectrum of the Fu Mao iron rod hammer. According to the content determination results, it can be seen that the contents of 12 epiaconitine, Song Guoling, and aconitine are relatively high, and previous literature reviews have found that they all have anti-inflammatory and analgesic activities. However, the content of 3-acetylaconitine is generally low in various samples and is not used as a quality control indicator for Fuzhong Iron Hammer. Therefore, based on the results of content determination and principal component analysis, this experiment simultaneously used six characteristic components, namely 2, 3, 4 (12 epiaconitine), 5 (Song Guoling), 6, and 15 (aconitine), which contribute significantly to the five principal components and have large peak areas. Among them, peaks 2, 3, and 6 are unknown and can be further confirmed by mass spectrometry to study whether they can be used as quality control indicators for the later stage of the Fu Mao Iron Hammer.

The content determination results indicate that Yushu (S3), Jiuzhi (S4), and Maqin (S5) in Qinghai production area have higher alkaloid content; Wang Rong et al. determined the alkaloid content of the Fumao Iron Hammer in Guoluo, Yushu, Haidong, and Guyuan, Ningxia. The results showed that the alkaloid content was highest in the Fumao Iron Hammer in Guoluo Banma and Yushu, which is consistent with the results of this study. The total amount of aconitine is the highest among the four components, while the total amount of 3-acetylaconitine is the lowest, which is consistent with the research results of Zhao Meiyu. The production areas with the highest content of 12 aconitine, Song Guoling, aconitine, and 3-acetylaconitine are Jiangxi Forest Farm in Yushu, Qinghai, Maqin Airport in Qinghai, Aba in Sichuan, and Guinan in Qinghai; The total amount of four alkaloids in the samples from Jiangxi Forest Farm and Maqin Airport was also the highest. There are differences in the quality of medicinal materials such as Fu Mao Tie Ban Hammer in different regions, which may be due to factors such as the growth environment, climate, soil, collection time, and growth years of the medicinal materials, which affect the accumulation of secondary metabolites in the medicinal materials, leading to differences in the similarity and content of the medicinal materials.

The Chinese Pharmacopoeia does not cover the HPLC quality control of the medicinal herb Fu Mao Tie Ban Zhong. Therefore, in this study, HPLC-DAD method was used to establish the fingerprint spectrum of Fu Mao Tie Ban Zhong, and the content of four alkaloid components was determined. At the same time, similarity evaluation, cluster analysis, and principal component analysis were used to comprehensively evaluate the quality of Fu Mao Tie Ban Zhong from different origins. The three methods were mutually verified and supplemented, avoiding the disadvantage of evaluating the quality of medicinal herbs based on the content of a single component, and providing reference for the quality evaluation of Fu Mao Tie Ban Zhong medicinal herbs.