Differentiating Curculigo Orchioides Rhizoma and Curculigo glabrescens Rhizoma by a Unique New Compound


 Background: Market research found that Curculigo glabrescens Rhizoma (CGR) is the major counterfeit of the Curculigo orchioides Rhizoma (COR). C. orchioides Gaertn and C. glabrescens (Ridl.) Merr. belong to the same family and genus, with close plant relationships and high genetic similarity, and rhizomes of the herbs part have less distinguished characteristics, which make the identification difficult.Methods: In order to distinguish COR from CGR more accurately and conveniently, HPLC method was used to establish a characteristic chromatogram of the two herbs, and a unique component in CGR was discovered for first time. Based on that, the characteristic component was systematically separated and purified.Results: The unique component was a new neolignans and named glabrescenin, which could specifically distinguish COR from CGR. The HPLC method we used could establish a characteristic chromatography of CGR.Conclusion: This study was conducive to the quality control of Curculigo, and could promote the correct application of genuine COR in clinical practice.

leaves. However, the appearance of the CGR is similar to the COR. Thus, it is necessary to establish a quick and accurate method to distinguish the two medicines.
The 2020 edition of the PPRC only includes COR, and stipulates that the content of curculigoside is not less than 0.10%. Preliminary research found that the existing standards of the PPRC could not accurately distinguish CGR and COR through microscopic characteristics, TLC, and curculigoside content. A chemical ngerprint/characteristic de nes an unique pattern of a herb and re ects the presence of multiple chemical constituents. This approach follows the fundamental holistic theory of traditional Chinese medicines as major and minor chemical constituents are analyzed simultaneously in herbal sample [15,16]. This method may contribute to evaluate herbal medicine, quality, safety and effectiveness. High performance liquid chromatography (HPLC) is the most popular analytical method in separation science and is regarded as one of the gold standard in the authentication of pharmaceutics and herbal medicines due to its good precision, sensitivity and reproducibility [17]. Compared with traditional morphological and microscopic identi cation methods, the HPLC method is relatively faster and more accurate.
Based on that, in this study, in order to distinguish COR and CGR more accurately and conveniently, HPLC method was used to analyze their components. A characteristic component from CGR was discovered for the rst time, and was systematically separated and puri ed. The structure was identi ed as 5-(3',4'dihydroxyphenyl)-1-(4''-hydroxyphenyl) pentane-1,4-dione, it was a new neolignans and named glabrescenin (Fig. 3). Therefore, the HPLC method we used could establish a characteristic chromatography of CGR, which can distinguish between COR and CGR. The novel compound and characteristic chromatography discovered in this study could effectively identify CGR, and provided technical reference and support for the correct medication, market norms and healthy development of COR.  . 1). The collected samples were authenticated by comparing their macroscopic and microscopic characteristics with the descriptions mentioned in the PPRC. Three samples were authenticated as COR, whereas three samples were authenticated as CGR by Prof. Minru Jia (Chengdu University of Traditional Chinese Medicine). Voucher specimens were kept at the Sichuan Institute for Food and Drug Control. HPLC analysis 1.0 g COR and CGR powder was accurately weighed respectively, accurately added 50 mL of methanol, weighed it, heated to re ux for 2 h. The mixture was cooled down to room temperature and weighed it again, methanol were added to make up the lost weight, shook and ltered through lter paper. Took 20 mL of the ltrate and evaporated to dryness, dissolved the residue with methanol, transferred to a 10 mL volumetric ask, then added methanol to the mark to obtain the sample solution. The solution was ltered through a 0.22 µm membrane lter before injection into the HPLC system.

Methods And
The sample solution of COR and CGR were analyzed by Agilent 1260 High Performance Liquid Chromatography (Agilent Technologies, CA, USA) equipped with a Zorbax Eclipse Plus C18 analytical column (4.6 mm × 150 mm, 5 µm) and a guard column. The temperature was set at 30 ℃, the injection volume was 10 µL, and the detection wavelength was set to 285 nm. A binary elution at a ow rate of 1.0 mL/min was employed using an aqueous phase of 0.1% formic acid as solvent A and acetonitrile as solvent B, the gradient elution procedure was A:B = 21:79, detection time was 18 minutes.

HPLC characteristic peak
The chemical feature can describe and evaluate the medicine materials as a whole. The HPLC method has good precision, sensitivity and reproducibility, it can quickly and speci cally identify different herbs based on the overall chemical composition. The HPLC chromatograms of the COR and CGR are illustrated in Fig. 2. The identities of the peaks were con rmed with the retention time and ultra-violet spectra (285 nm) of the chemical markers. The main chemical composition of COR and CGR were similar.
The overall peak area of CGR were signi cantly lower than that of COR. As anticipated, curculigoside (peak 1),the indicator component in COR was signi cantly higher than that in CGR, too. Notably, CGR has a unique compound (peak 2) through the HPLC chromatograms, which was not found in COR. Therefore, this unique compound was speci cally separated, puri ed and the structure was identi ed by modern spectroscopy techniques.

Structure elucidation of new compound
As shown in Table 1 Table 1). Compared with the 1 H and 13 C NMR spectrum data of compound breviscapin C [18], compound 1 was supposed to be the same type. In order to further con rm the structure of compound 1, a 2D NMR experiment was performed (Fig. 4).
The chemical shift assignments of the carbon atoms were established from direct 1 H- 13  ' correlated with one carbon at δ C 196.6 (C-1) (Fig. 4). From the above mentioned evidence,

Declarations Acknowledgement
We express our great appreciation to Sichuan Guoqiang Traditional Chinese Medicine Co., Ltd., Sichuan Institute for Food and Drug Control and Sichuan Academy of Chinese Medicine Sciences for providing samples and technical assistance.

Discussion
Correct species identi cation is crucial for ensuing the quality, safety and e cacy of a medicinal herb. The substitution and wrong identi cation occurs in clinical practice, when the medicinal herbs have similar morphological characteristics or names [19]. In the market, CGR is the major counterfeit of COR. Nevertheless, it is di cult to identify COR and CGR accurately. C. orchioides Gaertn and C. glabrescens (Ridl.) Merr. belong to the same family and genus, with close plant relationships and high genetic similarity, and the appearance of the CGR is similar to the COR. In our study, based on the HPLC characteristic chromatograms of COR and CGR, the new neolignans compound, glabrescenin, was separated and puri ed from CGR for the rst time. It could speci cally identify COR and CGR, providing a simple and friendly idea for the identi cation and quality control of Curculigo herbs, which is conducive to the market standard of COR. Through the analysis, we suggested that PPRC could add this check item and stipulate glabrescenin shall not be detected in COR, so as to promote the correct application of genuine COR in clinical practice. However, there is no systematic study on the chemical composition of CGR, and there are some controversies in the botanical classi cation of Curculigo, warranting further investigation.

Conclusion
The novel compound and characteristic chromatography discovered in this study could effectively identify CGR, and provided technical reference and support for the correct medication, market norms and healthy development of COR. Availability of data and materials

Abbreviations
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Not applicable.