A method for rapid screening of quality control indicators of Ramulus Loranthi based on UPLC-MS / MS and multivariate statistics

Abstract

This invention discloses a method for rapid screening of quality control indicators for *Taxillus chinensis* (mulberry mistletoe) based on UPLC-MS / MS and multivariate statistics, belonging to the field of quality control technology for traditional Chinese medicinal materials. The method includes the following steps: S1, determining the components of secondary metabolites in mixtures of *Taxillus chinensis* stems and leaves from different origins and hosts using UPLC-MS / MS to obtain raw data on the components of each group; S2, using the coefficient of variation (CV) evaluation system method combined with the principal component contribution in principal component analysis (PCA) to screen indicative components; and using orthogonal partial least squares discriminant analysis (OPLS-DA) to verify inter-group differences and evaluate model stability. This invention can rapidly screen various indicative components for quality evaluation based on the CV evaluation system method, building upon existing large-scale component group analysis, while simultaneously clarifying the differences between *Taxillus chinensis* from different hosts and origins. The CV evaluation system method has the characteristics of horizontal comparison and intuitive readability, providing an innovative and rapid method for processing complex and large-scale data and for quality control and evaluation of *Taxillus chinensis* from different origins and hosts.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology of traditional Chinese medicinal materials, specifically involving a method for rapid screening of quality control indicators of Loranthus parasiticus based on UPLC-MS / MS and multivariate statistics. Background Technology

[0002] Taxillus chinensis (DC.) Danser, also known as mulberry mistletoe, is a semi-parasitic shrub belonging to the genus Taxillus in the family Loranthaceae. It is listed as a superior herb in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), which states: "Sangjisheng (mulberry mistletoe) is bitter and sweet in taste, and neutral in nature. It treats lower back pain, stiff back in children, carbuncles, and promotes fetal development, nourishes the skin, strengthens hair and teeth, and promotes the growth of eyebrows and beard." The stems and branches with leaves are used medicinally, possessing the effects of dispelling wind and dampness, tonifying the liver and kidneys, strengthening muscles and bones, and promoting fetal development. It primarily parasitizes various plants such as mulberry, peach, Masson pine, and sweetgum in evergreen broad-leaved forests in plains or low mountains at altitudes of 200–400 meters, mainly distributed in Guangxi, Guangdong, and Fujian provinces.

[0003] Although named after the mulberry tree, *Taxillus chinensis* (mulberry mistletoe) is not limited to mulberry trees. According to ancient herbal records and modern research, its hosts encompass more than 50 species from 29 families. Surveys in Guangxi and other regions have even identified over 150 species from 36 families. Therefore, this host diversity not only reflects its ecological adaptability but also raises complex issues regarding the safety of its medicinal use.

[0004] High-throughput detection technology, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), can reveal the actual metabolic state of organisms under the influence of genetic and environmental factors. It is a core support for metabolomics research, enabling rapid and accurate analysis of hundreds to thousands of metabolites. This comprehensively reveals the complex composition of traditional Chinese medicine (TCM) and the relative content of each component, making it particularly suitable for analyzing mulberry mistletoe-like medicinal materials with numerous hosts and diverse growth environments. The content of components is the core of TCM quality evaluation, and secondary metabolites directly reflect the biological characteristics and environmental adaptability of TCM. While comprehensively revealing secondary metabolites, metabolomics research also generates massive amounts of component information, such as mass spectrometry peak information and ion current spectral data. The multivariate values ​​within these data require further analytical models for judgment, processing, and analysis.

[0005] The quality of mulberry mistletoe (Sangjisheng) is affected by various factors such as host, environment, and geographical location. Different hosts alter the composition, efficacy, and medicinal properties of mulberry mistletoe. Currently, the 2025 edition of the Chinese Pharmacopoeia uses only a single qualitative evaluation index, and the lack of clarity in many quality control indicators makes it difficult to effectively control the quality of mulberry mistletoe. UPLC-MS / MS technology can rapidly identify multiple metabolic components in complex samples. Based on this, using a simple and rapid analytical method to find multiple indicator components suitable for the quality evaluation of traditional Chinese medicine is of great significance. Summary of the Invention

[0006] To address the lack of various indicator components in existing quality control methods for mulberry mistletoe, this invention provides a method for rapid screening of quality control indicators for mulberry mistletoe based on UPLC-MS / MS and multivariate statistics. This method can screen specific, common, and differential indicator components for quality markers of mulberry mistletoe. Specific indicator components refer to characteristic components that can distinguish mulberry mistletoe from different hosts or production areas; common indicator components refer to qualitative indicators that can be used in the quality evaluation of mulberry mistletoe; and differential indicator components refer to quantitative indicators that can be used to evaluate the quality of mulberry mistletoe. This invention provides a scientific and effective method for the quality control and material basis research of mulberry mistletoe.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] (1) Fresh stems and leaves of mulberry mistletoe growing on mulberry trees in four different production areas (Wuzhou, Yulin, Chongzuo and Nanning) and mulberry mistletoe with three different hosts (mulberry, sweetgum and Masson pine) in Wuzhou were mixed at a ratio of 1:2 and analyzed by UPLC-MS / MS.

[0009] (2) The peak areas of the original data of different groups of Loranthus parasiticus were sorted and analyzed to obtain their average value, standard deviation and coefficient of variation (CV).

[0010] The component analysis method for *Taxillus chinensis* described above uses the coefficient of variation (CV) as the basis for analysis. The CV value reflects the degree of difference; the larger the CV value, the greater the difference between groups. The peak area of ​​each component in each group is divided into three categories: ① CV < 0.2, indicating small differences in component content within each group, making it more suitable for screening qualitative index components for *Taxillus chinensis* quality evaluation; ② 0.2 ≤ CV < 0.5, indicating differences in component content between groups, making it more suitable for screening quantitative index components; ③ CV > 0.5, indicating large differences in component content between groups, making it more suitable for screening specific index components.

[0011] Furthermore, the method for rapid screening of quality control indicators of mulberry mistletoe based on UPLC-MS / MS and multivariate statistics provided by this invention is very suitable for mulberry mistletoe medicinal materials from complex sources.

[0012] The beneficial effects of this invention are as follows:

[0013] The content of various components in mulberry mistletoe medicinal materials varies under different parasitic conditions. The quality of mulberry mistletoe medicinal materials is the result of the synergistic effect of multiple factors such as host characteristics and environmental interaction. This invention experimentally studied the effects of different origins and hosts on the quality and composition of *Mallotus officinalis*. Through research on the differences in component content among groups, components with high content and a CV (volume response factor) less than 0.2 were ultimately selected as qualitative indicators for evaluating the quality of *Mallotus officinalis*, such as catechins, quercetin, and isoquercetin. Components with high content in most groups but low content in a few groups are suitable as quantitative indicators for evaluating the quality of *Mallotus officinalis*, such as gallic acid and proanthocyanidins B5. The CV values ​​of these components are mostly between 0.2 and 0.5. Components with high content in a few groups but low content in others are suitable as specific indicators. For example, cyanidin-3-O-glucoside can be used to identify *Mallotus officinalis* from Wuzhou with mulberry as the host; isonaringin can be used to identify *Mallotus officinalis* from Wuzhou with *Pinus massoniana* as the host; and cymbidium can be used to identify *Mallotus officinalis* from Wuzhou with *Liquidambar formosana* as the host. The CV values ​​of these components are all greater than 0.5. Based on the above patterns, it was found that components with high content and CV values ​​greater than 0.2 are suitable as indicative components for evaluating the quality of mulberry mistletoe. By combining principal component analysis, components with high contribution were selected. These components are suitable as indicative components for evaluating quality and identifying varieties, such as parasitic glycosides and renosides.

[0014] This invention employs a CV value discrimination method and multivariate statistical analysis in the rapid screening of indicative components for the quality evaluation of *Taxillus chinensis* from different origins and hosts based on UPLC-MS / MS. Within the groups defined by CV values, it is easier to identify common and differential indicative components of traditional Chinese medicine. This method allows for cross-sectional comparison of components at different content levels, enabling faster screening of indicative components for quality evaluation. This research provides comprehensive component information for the quality control of *Taxillus chinensis* and contributes to the development of the field of traditional Chinese medicine quality. Detailed Implementation

[0015] Example 1

[0016] A method for rapid screening of quality control indicators for *Mistletoe* based on UPLC-MS / MS and multivariate statistics includes the following steps:

[0017] 1 Experimental Methods

[0018] Medicinal materials: Mistletoe with mulberry as host was collected from Yulin, Nanning, Chongzuo, Wuzhou, and other regions. The samples included Mistletoe with mulberry as host, Mistletoe with Liquidambar formosana as host, and Mistletoe with Pinus massoniana as host. Information on all samples is shown in Table 1. The stems and leaves from the middle part of the branches were taken. The samples from Wuzhou were grown in the same environment and were collected in June 2024.

[0019] Table 1 Information on Mistletoe Samples

[0020]

[0021] The processing method is as follows: After collecting fresh medicinal materials, quickly collect them into 6 groups of samples using centrifuge tubes (50mL), with each sample in 3 replicates. Then, quickly place them in liquid nitrogen for a moment, and then transfer them to an ultra-low temperature freezer (-80℃) for storage for later use. Eighteen samples of *Taxillus chinensis* (mulberry mistletoe) stored at ultra-low temperatures were taken out and dried in a freeze dryer. The completely dried herbs were mixed at a stem-to-leaf ratio of 1:2 and ground into powder using a grinder. 50 mg of the sample powder was weighed using an electronic balance and 1200 μL of pre-cooled 70% methanol-water internal standard extract (for samples less than 50 mg, 1200 μL of extract was added per 50 mg sample). The internal standard extract was prepared by dissolving 1 mg of standard in 1 mL of 70% methanol-water to prepare a 1000 μg / mL stock solution, which was then further diluted with 70% methanol-water solution to prepare a 250 μg / mL internal standard solution. The solution was vortexed every 30 minutes for 30 seconds, for a total of 6 vortexes. After centrifugation (12000 rpm, 3 minutes), the supernatant was collected and filtered through a microporous membrane (0.22 μm pore size). The sample (size) was filtered and stored in a vial for UPLC-MS / MS analysis.

[0022] 2 Experimental Results

[0023] A total of 519 secondary metabolites were identified from *Malva oleracea* plants grown in different locations and on different hosts. These included 162 flavonoids (31.21%), 78 phenolic acids (15.03%), 6 quinones (1.16%), 60 lignans and coumarins (11.56%), 85 alkaloids (16.38%), 36 terpenes (6.94%), 24 tannins (4.62%), and 78 other compounds (13.10%).

[0024] Using peak area as the evaluation criterion, the average value and standard deviation of each component's content in all samples were calculated, and the coefficient of variation (CV) value was further calculated. The CV value was used as the judgment criterion. The smaller the CV value, the smaller the difference in the content of the component among the sample groups, and the more suitable it is as a qualitative indicator component in the quality evaluation of mulberry mistletoe. The larger the CV value, the greater the difference in the content of the component among the groups. When comparing 6 groups and the CV is >2.44, it can be said that the component is a specific indicator component of a certain group.

[0025] To further observe the overall differences in mulberry mistletoe among the groups, principal component analysis was performed on the mulberry mistletoe samples. The results are as follows: Figure 1As shown in (c).

[0026] Overall differences in mulberry mistletoe species from different production areas, with mulberry trees as the host, are as follows: Figure 1 As shown in (a), using principal component analysis, the contribution rates of principal component 1 (PC1) are 26.96%, principal component 2 (PC2) is 18.14%, principal component 3 (PC3) is 11.39%, and principal component 4 (PC4) is 8.85%, with a cumulative contribution of 65.34% for the four principal components; the results are shown in Table 2.

[0027] Table 2. Analysis of the host components of mulberry trees from different origins.

[0028]

[0029] The overall differences in mulberry mistletoe from different host plants in Wuzhou, including mulberry, sweetgum, and Masson pine, are as follows: Figure 1 As shown in (b), using principal component analysis, the contribution rate of principal component 1 (PC1) is 29.61%, the contribution rate of principal component 2 (PC2) is 20.47%, and the contribution rate of principal component 3 (PC3) is 13.49%, with a cumulative contribution of 63.57% for the three principal components; the results are shown in Table 3.

[0030] Table 3. Principal component analysis of different host plants of Mistletoe in Wuzhou.

[0031]

[0032] Based on the contribution of principal components, components with high contribution and high content in different hosts and origins of Loranthus parasiticus, such as renoside and parasitic glycoside, were screened out as the main compounds that may affect the differences in Loranthus parasiticus among different origins and different hosts; the results are shown in Tables 4 and 5.

[0033] Table 4. Top 10 components with the highest contribution rates of PC1, PC2, and PC3 in mulberry mistletoe from different mulberry host plants in different origins.

[0034]

[0035] Table 5. Top 10 components with the highest contribution rates of PC1, PC2, and PC3 in different host plants of *Taxillus chinensis* from Wuzhou.

[0036]

[0037] The evaluation parameter index (R) of orthogonal partial least squares discriminant analysis (OPLS-DA) is used. 2 Y = 0.989, Q 2 =0.796) indicates that the model has good stability and high predictive ability, and is reliable without overfitting. For example... Figure 2 As shown in Figure 3, the results of the OPLS-DA model indicate that both origin and host are factors that cause differences in the content of chemical components in Loranthus parasiticus.

[0038] Based on PCA and OPLS-DA results, among the common components with high content, in *Mistletoe* from different origins with mulberry as the host, renoside, gallic acid-3-O-(6′-O-galloyl)glucoside, gentianin, luteolin, kaempferol-4′-O-glucoside, and cypermethrin flavonoid glycoside are likely the main compounds affecting the differences between different origins of *Mistletoe*. In *Mistletoe* from Wuzhou with mulberry, *Liquidambar formosana*, and *Pinus massoniana* as hosts, kaempferol-3-O-neohesperidin, kaempferol-3-O-glucoside-7-O-rhamnoside, Myrciaphenone B, cyanidin-3-O-galactoside, and cyanidin-3-O-glucoside are likely the main compounds affecting the differences between different hosts of *Mistletoe*. These indicator components can be used to evaluate the quality of *Mistletoe* or to identify its origin and host.

[0039] 3. Summary and Discussion

[0040] Based on peak area, this method uses CV value grouping and multivariate statistical analysis to find that components with higher peak area and lower CV value, i.e., higher content and smaller differences between groups, are suitable as qualitative indicator components in the quality evaluation of mulberry mistletoe, such as quercetin, isoquercetin, catechin, rutin, hyperoside, etc. The CV values ​​of these components are all less than 0.2, and the differences between groups are small. The results are shown in Table 6.

[0041] Table 6. Components suitable as qualitative indicators in the quality evaluation of Mistletoe.

[0042]

[0043] Some components, such as gallic acid, proanthocyanidin B5, and luteolin, are suitable as quantitative indicator components in the quality evaluation of mulberry mistletoe, which are present in high concentrations in many groups and low concentrations in a few groups. The CV values ​​of these components are mostly between 0.2 and 0.5. Components, such as cyanidin-3-O-glucoside, which can be used to identify mulberry mistletoe from Wuzhou with mulberry as the host, isonaringin, which can be used to identify mulberry mistletoe from Wuzhou with pine as the host, and cymbidium, which can be used to identify mulberry mistletoe from Wuzhou with sweetgum as the host, all have CV values ​​greater than 0.5. Based on the above patterns, components with high content and CV values ​​greater than 0.2 are suitable for screening as differential indicator components for evaluating the quality of *Mallotus officinalis*, such as *Salvia miltiorrhiza* flavonoid glycosides, astragaloside, and vanillin glucoside. Furthermore, combined with principal component analysis, components with high content, significant inter-group differences, and large contributions are suitable as indicator components for evaluating quality and identifying varieties, such as renoside, parasitic glycoside, gallic acid-3-O-(6′-O-galloyl)glucoside, luteolin glycoside, kaempferol-4′-O-glucoside, *Salvia miltiorrhiza* flavonoid glycosides, kaempferol-3-O-neohesperidin, kaempferol-3-O-glucoside-7-O-rhamnoside, myrciaphenone B, cyanidin-3-O-galactoside, and cyanidin-3-O-glucoside. Table 7 shows some of the suitable indicator components for evaluating the quality of *Mallotus officinalis*.

[0044] Table 7 shows suitable candidates for screening as indicative components in the quality evaluation of Loranthus parasiticus.

[0045]

[0046] In summary, the CV value grading evaluation system is convenient and quick, balancing the dimensions of components with higher and lower content to jointly analyze inter-group differences. Therefore, the coefficient of variation is suitable for comparing multiple groups and components with significant differences in content. It is more intuitive and can be combined with other mathematical analysis models to jointly screen various indicator components in quality evaluation. When comparing six groups using the coefficient of variation method, components such as catechins, quercetin, hyperoside, and proanthocyanidins B4 have CV values ​​below 0.2, indicating small inter-group differences, and are more suitable as qualitative indicator components in the quality evaluation of Loranthus parasiticus. Components with differences of 0.2 < CV < 2.0 are suitable for screening as differential indicator components, such as gallic acid, parasitic glycoside, and renoside. Components with differences of CV ≥ 2.0 can be used as host identification markers; for example, crystallized glycoside is only found in Loranthus parasiticus growing on Liquidambar formosana and is a specific metabolite of this host. Attached Figure Description

[0047] Figure 1 Principal component analysis diagrams (a), (b), and (c) of mulberry mistletoe from different origins but with the same host. Figure 2 OPLS-DA score plots for mistletoe from different origins and with different hosts; Figure 3 OPLS-DA verification diagram for mistletoe from different origins and different hosts.

Claims

1. A method for rapid screening of quality control indicators for *Mistletoe* based on UPLC-MS / MS and multivariate statistics, characterized in that... Includes the following steps: S1. Fresh samples to be tested were mixed at a stem-to-leaf ratio of 1:2 and extracted with methanol. The samples were then detected by ultra-high performance liquid chromatography-tandem mass spectrometry to obtain the chromatograms. The samples to be tested were fresh stems and leaves of mulberry mistletoe from Yulin, Nanning, Chongzuo and Wuzhou, with mulberry trees as the host, and mulberry mistletoe from Wuzhou with sweetgum and Masson pine as the host. S2. Based on metabolomics, distinguish and screen the chemical phenotypes of mulberry mistletoe from different origins and hosts, and screen the indicative components for quality evaluation: Based on the peak area, the components are subjected to the coefficient of variation evaluation system method, principal component analysis method, and orthogonal partial least squares discriminant analysis in sequence to quickly screen out various indicative components for mulberry mistletoe quality evaluation.

2. The method for rapid screening of quality control index components of Loranthus parasiticus according to claim 1, characterized in that, In S2, the coefficient of variation (CV) evaluation method calculates the CV value of each component content. The larger the CV value, the greater the difference in the content of each component. Component differences are grouped by CV < 0.2, 0.2 ≤ CV < 0.5, and CV ≥ 0.

5. Combined with principal component analysis, the contribution of principal components is used as the basis. The higher the contribution, the more suitable it is as an indicator component in the quality evaluation of mulberry mistletoe. This method can quickly screen out differential indicator components, common indicator components, and specific indicator components for the quality evaluation of mulberry mistletoe.

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