Zhou et al. Chin Med (2021) 16:85 https://doi.org/10.1186/s13020-021-00494-3 Chinese Medicine RESEARCH Open Access A novel multidimensional strategy to evaluate Belamcanda chinensis (L) DC and Iris tectorum Maxim based on plant metabolomics, digital reference standard analyzer and biological activities evaluation Hongxu Zhou1,2†, Yi Zhang2†, Hui Liang1, Huijie Song1, Jiaming Zhao1, Li Liu1, Jun Zeng2, Lei Sun3, Shuangcheng Ma3* and Dali Meng1* Abstract Background: Belamcanda chinensis (L.) DC. (BC) belongs to the family of Iridaceae and is widely cultivated and used in many Chinese patent medicine and Chinese medicinal formulae. However, due to the high similarities in appear- ance such as color and shape to Iris tectorum Maxim (ITM), another plant from the same family, BC is often confused or even misused with ITM. Methods: Therefore, in order to distinguish the chemical constituents, qualities and biological activities of BC and ITM, multiple technologies including plant metabolomics, digital reference standard (DRS) analyzer and biological activities assay were employed to provide a sufcient basis for their practical applications. Results: In plant metabolomics, the PCA and OPLS-DA score plot indicated the obvious diferences in chemical profling between BC and ITM and 6 compounds were successfully identifed to contribute to the diferences. In DRS study, the fngerprints of 10 and 8 compounds in BC and ITM were developed based on DRS analyzer, respectively, involving relative retention time (RRT) method and linear calibration using two reference substances (LCTRS) tech- nique. The DRS analyzer also accurately identifed 10 and 8 compounds from BC and ITM, respectively, by using only two reference standards. In biological activities assay, BC had a better anticancer efect than ITM due to the high abundance of irigenin, while ITM showed stronger hepatoprotective activity than BC because of the high abundance of tectoridin. Conclusions: Therefore, due to the signifcant diferences of B. chinensis and I. dichotoma in chemical composition and biological activities, the current studies strongly proved that these two medicinal plants could not be mixed in industrial production and clinical medication. *Correspondence: [email protected]; [email protected] †Hongxu Zhou and Yi Zhang contributed equally 1 School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China 3 National Institutes for Food and Drug Control, Beijing 100050, People’s Republic of China Full list of author information is available at the end of the article © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhou et al. Chin Med (2021) 16:85 Page 2 of 18 Keywords: Belamcanda chinensis (L.) DC, Iris tectorum Maxim, Plant metabolomics, Digital reference standard, Biological activities Background were used for qualitative studies to distinguish BC and Belamcanda chinensis (L.) DC. (BC), a perennial her- ITM. baceous plant whose rhizome is named as She-gan in a Quality control analysis of TCM is important for safe traditional Chinese medicine (TCM) belongs to the fam- and efective use. Te reference standard is the most ily of Iridaceae and is widely cultivated in China, Korea, important role for qualitative and quantitative analysis Japan, India and eastern Russia as an economic medici- of TCM. Tere is an increasing demand for reference nal plant. She-gan has been used in many Chinese patent standards with the development of TCM quality control. medicine and Chinese medicinal formulae such as Xiaoer At the same time, some TCM compounds are difcult to Qingre granules, Xiaoer Qingfei oral liquid, and Shengan be extracted, isolated, and purifed, led to a signifcant Liyan oral liquid, etc., for the treatment coughing and increase in the cost of TCM analysis. Linear calibration pharyngitis. using two reference substances (LCTRS) approaches, a However, Chuan-she-gan, the rhizome of Iris tecto- substitute reference standard method, can deal with the rum Maxim (ITM), another medicinal plant comes from above problems efectively [10–12]. LCTRS is a method the family of Iridacae mainly distributing in Sichuan of for the qualitative determination of several compounds China, is also used for the treatment of asthma, cough, to be measured by two reference standards by using sev- tonsillitis and pharyngitis. Actually, both of these two eral constant eigenvalues and algorithms. Te principle medicinal plants are rich in isofavonoids, stilbenes, xan- of LCTRS is that there is a linear relationship between thones, and simple phenols [1–3]. Among them, isofa- the retention time (tR) of the compounds on two difer- vones, such as tectoridin, iridin, tectorigenin, irigenin, ent HPLC systems (including chromatographs and col- irisforentin and so on [3–5], are the major bioactive con- umns). Te method has been successfully developed for stituents of two medicines that have shown a wide range the quality analysis of Salvia miltiorrhiza, Paris poly- of biological activity, such as anticancer, hepatoprotec- phylla, Rheum ofcinale [10–12]. Relative retention time tive, antiatherosclerosis, antiosteoporosis and antihyper- (RRT) technique, a qualitative substitute reference stand- lipidemic, etc. [1, 6–8]. ard method, can qualitative determination of several Consequently, in the medicinal market, as well as in compounds to be measured by one reference standard. the pharmaceutical industry, because of the high simi- Finally, we introduced the concept of the digital reference larities in appearance such as color and shape between standard (DRS), which supports the chromatographic BC and ITM, they were often confused or even mixed algorithm methods of RRT and LCTRS. In the present with each other when used for the treatments of cough- study, quality control methods of fngerprint involving 10 ing and pharyngitis. Terefore, it is very necessary to compounds of BC and 8 compounds of ITM respectively distinguish these two plants from their chemical con- were developed based on DRS method. stituents and biological activities by multiple technolo- Recently, several studies on the quality control of BC gies and approaches, so as to provide a sufcient basis were reported. Li et al. [13] evaluated the quality of BC and guidance for their industrial production and clinical by the establishment of chromatographic fngerprinting medication. profle employing HPLC–DAD-MS method and simul- Metabolomics, genomics, and proteomics are impor- taneous determination of seven phenol compounds. tant components of system biology. As an important Chen et al. [14] reported the spatial chemical profles of branch of metabolomics, plant metabolomics describes BC at diferent growth ages from various origins through the alterations in the content and composition of dif- qualitative and quantitative analyses by using UHPLC-Q/ ferent plant phytochemicals [9]. Currently, the most TOF–MS and UHPLC-QqQ-MS. Wen et al. [15] used a promising technique in terms of metabolome coverage chemical profling method to evaluate the quality of BC is ultra-high-performance liquid chromatography-high and compare the chemical compositions by HPLC analy- resolution tandem mass spectrometry (UHPLC-HRMS/ sis combining with multivariate data analysis. However, MS), which is a powerful analytical tool for the analysis of these methods only focused on one or several marker the known compounds and elucidation of unknown com- compounds and failed to distinguish these two plants pounds in herbal medicines. Here, UHPLC-Q Exactive- from their chemical constituents and biological activities. HRMS/MS based untargeted metabolomics techniques Herein, the systematic studies on the chemical constitu- ents, qualities and biological activities of BC and ITM Zhou et al. Chin Med (2021) 16:85 Page 3 of 18 Table 1 The sorts of Belamcanda chinensis (L) DC and Iris Table 3 The gradient elution conditions of the mobile phase tectorum Maxim Time (min) Phase A (%) Phase B (%) Phase C (%) No. Species Location Collection time 0–15 5–17 0–83 95–0 BC01 B. chineses Anguo, Hebei April 2020 15–24 17–20 83–80 0 BC02 B. chineses Baoding, Hebei April 2020 24–48 20–24 80–76 0 BC03 B. chineses Yuncheng, Henan April 2020 48–52 24–28 76–72 0 BC04 B. chineses Qinhuangdao, Hebei April 2020 52–60 28–31 72–69 0 BC05 B. chineses Nangong, Hebei May 2020 60–65 31–35 69–65 0 BC06 B. chineses Yuncheng, Shanxi May 2020 65–80 35–70 65–30 0 ITM01 I. tectorum Chengdu, Sichuan April 2020 80–81 70–5 30–0 0–95 ITM02 I. tectorum Chengdu, Sichuan April 2020 81–90 5 0 95 ITM03 I. tectorum Chengdu, Sichuan May 2020 ITM04 I. tectorum Chengdu, Sichuan May 2020 ITM05 I. tectorum Baoding, Hebei May 2020 ITM06 I. tectorum Guiyang, Guizhou April 2020 Professor Yi Zhang. Teir sample number, species, habi- ITM07 I. tectorum Bozhou, Anhui April 2020 tats and collection time were listed in Table 1. Chemicals and reagents were carried out by plant metabolomics, digital reference Chromatography-grade acetonitrile and methanol were standard analyzer and biological activities assay.