Column on Quality Evaluation of Arnebiae Radix

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Editor: Invited Column Editor-in-chief：ZHENG Jian

Arnebiae Radix is a common medicinal herb used in ethnic minority areas, and is also a commonly used bulk Chinese medicine. At present, the annual demand for the domestic Chinese herbal medicine market is about 1,200 tons, but the annual output of Arnebiae Radix is less than 100 tons. Arnebiae Radix functions as clear heat, cool the blood, activate blood, remove toxin, promote eruption and resolve macule, and can be used for exuberant blood heat-toxin, manifested as dark purple macule and papule, unerupted measles, sore and ulcer, eczema, scald and burn. There are 122 Chinese patent medicine prescriptions and 195 Chinese medicine formulas containing Arnebiae Radix, indicating that Arnebiae Radix is widely used and has important medicinal value. In addition to Arnebia guttata Bunge and Arnebiae Radix adopted by the Pharmacopoeia of the People’s Republic of China (ChP), other species of Arnebiae Radix are common in the market. Lithospermum erythrorhizon is used in Hebei, Liaoning, and Jiangxi, among others. In Yunnan and other places, it is common to use Onosma paniculatum and Onosma exsertum Hemsl. In Sichuan and other places, it is common to use Onosma confertum. Potentilla chinensis Ser.（Commonly known as Northern Arnebiae Radix）of the Rosaceae family is commonly used in Guangxi and Guangdong. The insufficient supply of the species adopted by ChP and the inconsistent species used in local, and the imported species (the exact species are not yet clear) in the market lead to confused application of Arnebiae Radix. Currently, Arnebiae Radix, one of the species adopted by ChP, has been artificially cultivated. But the consistency between cultivated Arnebiae Radix and wild Arnebiae Radix needs to be examined. Therefore, it is a very important and urgent task to expand the resources of Arnebiae Radix to fill the gap of market demand.

In 2015, Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control, carried out a national sampling of Arnebiae Radix. It was concluded that the resource of Arnebiae Radix were scarce, and that of Arnebia guttata Bunge was on the verge of depletion in Inner Mongolia. Besides, a considerable proportion of Arnebiae Radix prepared slices from the market were unqualified. Therefore, the Institute for Control of Chinese Traditional Medicine and Ethnic Medicine initiated special research on Boraginaceous herbs and assisted in the cultivation of Arnebiae Radix. Now the herb has been successfully cultivated in many parts of the country and continues to expand its cultivation area, intending to solve the problem of the scarcity of resources for Arnebiae Radix. In 2022, the Institute for Control of Chinese Traditional Medicine and Ethnic Medicine performed the national drug sampling of Arnebiae Radix again, and the results showed that the qualification rate of Arnebiae Radix prepared slices increased from 43.9% in 2015 to 87.5%, a substantial increase in qualification rate.

In order to carry out in-depth research on the quality standard of Arnebiae Radix, this journal specializes in “Column on Quality Evaluation of Arnebiae Radix”. “Discussion on the quality status of Arnebiae Radix based on the national drug sampling and testing in 2015 and 2022” comprehensively analyzed the problems reflected in the two sampling tests of Arnebiae Radix. The results indicated that it was of great significance to improve the standards of the ChP of Arnebiae Radix, to establish scientific test items and to strengthen the construction of the quality control system for the supervision of Arnebiae Radix. “The ITS2 sequences’ characters study of imported Arnebiae Radix based on DNA barcoding and PCR-RFLP technologies” provided reference basis for quality control and authenticity identification of marketed Arnebiae Radix and its prepared slices. “The screening of identification primers for Arnebiae Radix based on nested PCR” could be used to efficiently amplify and identify the specific primers for the authenticity of Arnebiae Radix market samples, which provided a referable basis for the subsequent research and development of the identification methods of Arnebiae Radix.“Study on quality control of Arnebiae Radix based on ‘color discrimination grading’ ” compared the correlation between the color of Arnebiae Radix and the contents of six major purple pigment components, and evaluated the quality of Arnebiae Radix by the color of herbs. “The comparative study on the regulation effect of authentic and counterfeit Arnebiae Radix on intestinal flora in mice” compared the similarities and differences in the regulatory effects of authentic and counterfeit products of Arnebiae Radix market herbs on the intestinal flora of mice, relying on the method of macrogenomic sequencing. “Comparative study on the quality of wild and cultivated Arnebiae Radix” conducted a comparative study of the quality of wild and cultivated Arnebiae Radix by using trait comparisons and chemometrics to compare the dissimilar components of wild and cultivated Arnebiae Radix with those of cultivated products from three different regions to establish the optimal Arnebiae Radix cultivation site.

Through this research column, the journal hopes to provide new ideas and methods for Arnebiae Radix research, on the basis of which the depth and breadth of Arnebiae Radix research can be expanded, thus promoting the innovative development of Arnebiae Radix.

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Chinese Journal of Pharmaceutical Analysis (6)

Publication year

2024 (6)

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Column on Quality Evaluation of Arnebiae Radix (6)

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Column on Quality Evaluation of Arnebiae Radix
Discussion on the quality status of Arnebiae Radix based on the national drug sampling and testing in 2015 and 2022^*

DAI Sheng-yun, LIU Jie, YUN Su-ning, LIAN Chao-jie, QIAO Fei, ZAN Ke, GUO Li-nong, MA Shuang-cheng, ZHENG Jian

Chinese Journal of Pharmaceutical Analysis. 2024, 44(5): 740-749. https://doi.org/10.16155/j.0254-1793.2024.05.01

Abstract (67) PDF (64) Knowledge map Save

The national drug sampling and inspection project is an important way of drug quality supervision in China which providing strong support for drug supervision and standard improvement. This article summarizes the national drug sampling and inspection project of Arnebiae Radix completed by Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control in 2015 and 2022. The results illustrated that the qualification rate of Arnebiae Radix has increased from 43.9% in 2015 to 87.5%, and the qualification rate of Arnebiae Radix has significantly increased. The two nationwide inspections of Arnebiae Radix reflected the scarcity of Arnebiae Radix resources in Xinjiang and Inner Mongolia, resulting in a high market share of unqualified samples. The thin layer identification spots of the unqualified Arnebiae Radix sampled in 2015 were not consistent with the qualified samples. The thin layer identification of the unqualified samples sampled in 2022 was consistent with those of the qualified samples, but the depth of the spots were not consistent with those of the qualified samples, indicating that the current unqualified samples were adulterated samples, which posing greater challenges to the quality supervision of Arnebiae Radix. Through the exploratory research of twice National Drug Sampling and Inspection Project, it is preliminarily believed that it is of great significance to improve the standard test items of Arnebiae Radix, scientifically establish the limit value and strengthen the construction of the quality control system for the supervision.
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Column on Quality Evaluation of Arnebiae Radix
The ITS2 sequences’ characters study of imported Arnebiae Radix based on DNA barcoding and PCR-RFLP technologies^*

LIU Jie, DAI Sheng-yun, GU Hai-yuan, QIAO Fei, LIAN Chao-jie, GUO Li-nong, ZHENG Jian, MA Shuang-cheng, MI Jia

Chinese Journal of Pharmaceutical Analysis. 2024, 44(5): 750-755. https://doi.org/10.16155/j.0254-1793.2024.05.02

Abstract (34) PDF (19) Knowledge map Save

Objective: To provide reference for quality control and authenticity identification of Arnebiae Radix medicinal materials and decoction pieces in the market. By studied on the ITS2 sequences’ characters of imported Arnebiae Radix, based on DNA barcoding and PCR-RFLP technologies. Methods: The ITS2 region was selected as the DNA barcode sequence for comparison and identification of imported Arnebiae Radix and reference medicinal materials. The ITS2 sequences of imported Arnebiae Radix from different sources with reference medicinal materials were compared based on DNA barcoding and PCR-RFLP technologies. Results: After the restriction endonucliase AluI enzyme digestion, the agarose-gel electrophoresis results of 39 imported Arnebiae Radix samples showed that, only DH3 had bands at around 500 bp, and none bands between 100 bp and 300 bp. And the results of other imported Arnebiae Radix samples had two or three obvious bands between 100 bp and 300 bp. The ITS2 sequences of imported Arnebiae Radix samples were compared with the reference medicinal materials, among which DH3 had the largest differences of 15 bases compared to the reference medicinal materials, the ITS2 sequence of F2 was same to the reference medicinal materials, and other imported Arnebiae Radix samples had 1-9 bases difference compared to the reference medicinal materials. The clustering results showed that the imported Arnebiae Radix sample DH3 was clearly distinguished from other imported Arnebiae Radix samples and reference medicinal materials which was in a single branch. There were 14 samples, which were clustered together with the reference medicinal materials in one branch with support rate ≥50%. Conclusion: The ITS2 region is selected to compare the similarities and differences of ITS2 sequences between imported Arnebiae Radix samples and reference medicinal materials based on DNA barcode and PCR-RFLP technologies, which provids a reference for effective identification of Arnebiae Radix medicinal materials and decoction pieces, and a strong guarantee for market supervision of Arnebiae Radix medicinal materials.
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Column on Quality Evaluation of Arnebiae Radix
The screening of identification primers for Arnebiae Radix based on nested PCR^*

LIU Jie, GU Hai-yuan, DAI Sheng-yun, QIAO Fei, LIAN Chao-jie, ZHENG Jian, JIA Sha-er·SI Ha-ke

Chinese Journal of Pharmaceutical Analysis. 2024, 44(5): 756-765. https://doi.org/10.16155/j.0254-1793.2024.05.03

Abstract (32) PDF (20) Knowledge map Save

Objective: To design and screen specific primers for efficient amplification and identification of Arnebiae Radix from market based on the concept of nested PCR. Methods: Nested primers was designed using the software of Primer Premier 5 based on the ITS sequence of Arnebia euchroma and the ITS2 sequence of non-pharmacopoeial Arnebiae Radix. The amplification efficiency of genomic DNA by ITS2 universal primers PCR and nested PCR was compared. The genomic DNA of Arnebiae Radix was amplified directly by nested primers and was detected by agarose gel electrophoresis. The specific primers designed for Arnebiae Radix based on the fragment length and variation sites’ coverage of the amplified product was evaluated. Results: A total of 11 primers were selected for synthesis after the primers were designed by Primer Premier 5 software. The amplification efficiency of nested PCR was superior to ITS2 universal primers PCR in genomic DNA of Arnebiae Radix. The results of nested primers directly amplified genomic DNA of Arnebiae Radix by agarose gel electrophoresis were better than those of ITS2 primers, and showed a single band. Four pairs of primers, AE-9S/AE-2A, AE-4S/AE-10A, AE-12S/10A, AE-29S/AE-29A, were determined to be suitable for the identification of Arnebiae Radix. Conclusion: On the basis of DNA barcode identification and nested PCR technology, 4 pairs of specific primers are identified which can be used to effectively distinguish Arnebia euchroma from the mainstreamed non-pharmacopoeial Arnebiae Radix in the medicinal materials market, providing reference for the subsequent research and development of identification methods for Arnebiae Radix and other traditional Chinese medicines.
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Column on Quality Evaluation of Arnebiae Radix
Study on quality control of Arnebiae Radix based on “color discrimination grading”^*

LIAN Chao-jie, DAI Sheng-yun, LIU Jie, GUO Li-nong, QIAO Fei, YANG Rui, WANG Hui-juan, ZHENG Jian, LIU Jie

Chinese Journal of Pharmaceutical Analysis. 2024, 44(5): 766-771. https://doi.org/10.16155/j.0254-1793.2024.05.04

Abstract (36) PDF (17) Knowledge map Save

Objective: To determine the color of Arnebiae Radix, and the contents of six main purple pigment components (acetylshikonin, β-acetoxyisovalerylalkannin, deoxyshikonin, isobutylshikonin, β, β’-dimethylacrylalkannin and isovalerylshikonin) in Arnebiae Radix, and to study on the correlation between the color of Arnebiae Radix and the contents of six main purple pigment components. Methods: The L, a, and b values of the sample powder were determined using a spectrophotometer to characterize the color of Arnebiae Radix. The International Commission on Illumination (CIE) had developed a Lab color model, which was a digital description of human vision. A higher L value indicated greater brightness, a higher a value indicated redness and a lower a value indicates greenness, and a higher b value indicated yellowing and a lower b value indicates blueness. The contents of purple pigment components were determined using high performance liquid chromatography (HPLC), and the correlation between L, a, and b values and the contents of six main purple pigments was calculated using SPSS software. Results: The contents of acetylshikonin in 135 batches of samples ranged from 0.01% to 3.39%. The contents of β-acetoxyisovalerylalkannin ranged from 0.00% to 1.95%. The contents of deoxyshikonin ranged from 0.00% to 0.23%. The contents of isobutylshikonin ranged from 0.01% to 1.13%. And the contents of isovalerylshikonin ranged from 0.02% to 2.88%. The contents of β, β’-dimethylacrylalkannin ranged from 0.01% to 2.17%. There was a significant negative correlation between the contents of acetylshikonin, β-acetoxyisovalerylalkannin and isobutylshikonin and the L (black_white) chromaticity value of Arnebiae Radix, with a Spearman correlation coefficients between -0.138 and -0.222. The chromaticity value of a (red_green) was related to the five components other than acetylshikonin, which were β-acetoxyisovalerylalkannin, deoxyshikonin, isobutylshikonin, β, β’-dimethylacrylalkannin, and isovalerylshikonin, with a spearman correlation coefficients between 0.176 and 0.355; b (blue_yellow) chromaticity value was related to the five components other than β-acetoxyisovalerylalkannin, which were acetylshikonin, deoxyshikonin, and isobutylshikonin, β, β’-dimethylacrylalkannin. β, β’-dimethylacrylalkannin was positively correlated with a coefficient of 0.290, and negatively correlated with the other four components with a coefficients between -0.325 and -0.633. Conclusion: It is recommended that the assay limits of Arnebiae Radix be revised to β, β’-dimethylacrylalkannin not less than 0.30% and isovalerylshikonin not less than 0.29%.
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Column on Quality Evaluation of Arnebiae Radix
The comparative study on the regulation effect of authentic and counterfeit Arnebiae Radix on intestinal flora in mice^*

LIU Jie, DAI Sheng-yun, GU Hai-yuan, QIAO Fei, LIAN Chao-jie, GUO Li-nong, ZHENG Jian, MA Shuang-cheng, JIN Xiao-min

Chinese Journal of Pharmaceutical Analysis. 2024, 44(5): 772-782. https://doi.org/10.16155/j.0254-1793.2024.05.05

Abstract (39) PDF (42) Knowledge map Save

Objective: To compare the regulatory effects of authentic and counterfeit Arnebiae Radix on intestinal flora in mice based on metagenomic sequencing. Methods: Firstly, 24 clean grade female BLAB/C mice were randomly divided into 3 groups:blank control group, A1 (Arnebia euchroma) group and A2 (Arnebiae Radix whose origins were not included in Chinese Pharmacopoeia) group. After gavage arrived at the specified time, colon contents (feces), ileal contents (feces) and small intestinal contents (feces, except ileal parts) were extracted for intestinal flora analysis. Genomic DNA was extracted and amplified by PCR from the extracted mouse intestinal contents. The PCR products were mixed and purified. Then library was constructed and sequenced. After quality control of sequencing data and removal of chimera sequence, the final effective data was obtained. Operational taxonomic unit(OTU) clustering and species annotation were performed on the obtained valid data, and sample diversity analysis was conducted. Results: In this study, both A1 (Arnebia euchroma) group and A2 (Arnebiae Radix non-pharmacopoeia) group reduced the diversity of mice colon microbiota. At the phylum level, group A1 significantly increased the abundance of Firmicutes in the small intestine and ileum, and groups A1 and A2 significantly increased the relative abundance of Bacteroidetes in the colon. At the genus level, group A1 significantly increased the relative abundance of Lactobacillus in the small intestine of mice, and group A2 significantly increased the relative abundance of Lactobacillus in the ileum of mice. Group A1 increased the relative abundance of Lactobacillus, one of the dominant bacteria in the colon of mice, and group A2 increased the relative abundance of Bacteroides. Alistipes mainly existed in the colon, A2 group significantly reduced the relative abundance of Alistipes in the colon of mice, while Alistipes in the A1 group was cultivated. Conclusion: According to the results of the regulation effect of intestinal flora, the intestinal flora regulation effect in the two experimental groups of the authentic Arnebiae Radix and its confusion products from markets are not consistent, the results of this study can provide a theoretical basis for further exploring the mechanism of Arnebiae Radix.
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Column on Quality Evaluation of Arnebiae Radix
Comparative study on the quality of wild and cultivated Arnebiae Radix^*

HUANG Rui, DAI Sheng-yun, WU Dong-xue, MA Xiao-jun, LIU Jie, GUO Li-nong, Dao-er-jia-la, JING Song, MA Shuang-cheng, ZHENG Jian

Chinese Journal of Pharmaceutical Analysis. 2024, 44(5): 783-795. https://doi.org/10.16155/j.0254-1793.2024.05.06

Abstract (51) PDF (69) Knowledge map Save

Objective: To compare the quality of wild and cultivated Arnebiae Radix,using macroscopic investigation and chemometric analysis of the different components in wild and cultivated Arnebiae Radix from three different habitats. Methods: Wild and cultivated Arnebiae Radix were collected and their macroscopic features were compared. Using the ACQUITY UPLC BEH C₁₈(2.1 mm×100 mm, 1.7 μm) column, with acetonitrile-0.05% formic acid water as the mobile phase, the contents of D-shikonin, acetylshikonin, β-acetoxyisovalerylshikonin, isobutyrylshikonin, β,β’-dimethylacrylalkannin and isovalerylshikonin in 48 batches of wild and cultivated Arnebiae Radix were determined. The detection wavelength was 275 nm and the flow rate was 0.2 mL·min^-1. PCA and OPLS-DA were performed to reveal the differential components of wild and cultivated Arnebiae Radix. Results: There were great differences in macroscopic features of wild and cultivated Arnebiae Radix, and the linear relationship between the contents of six naphthoquinone components was good. The correlation coefficients were above 0.999, the average recovery rates were 93.4%-102.9%, and the RSDs were less than 3.0%. The contents of six components in different batches of wild and cultivated Arnebiae Radix were quite different, and the contents of D-shikonin and acetylshikonin in wild products were significantly higher than those in cultivated products, indicating that there were still certain differences between wild products and cultivated products. The PCA model established could distinguish wild products and cultivars, and two differentiating components in wild products and cultivars were revealed by OPLS-DA, namely isobutyryl shikonin, β,β’-dimethylacrylalkannin. Conclusion: By comparing the core size, cork curl degree and specific odor of wild and cultivated products, the two can be identified. The established content determination method is repeatable, specific, stable and feasible. The differential components in wild and cultivated Arnebiae Radix in three different regions are identified, which provides a basis for the quality control of Arnebiae Radix and provides ideas for expanding the source of Arnebiae Radix.

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