Systematic characterisation of the effective components of five Callicarpa species with UPLC-Q-TOF-MS and evaluation of their anti-hyperuricaemic activity

Abstract Callicarpa kwangtungensis (C. Kw), C. macrophylla (C. Ma), C. nudiflora (C. Nu), C. formosana (C. Fo), and C. kochiana (C. Ko) were medicinal plant resource in China. In this study, the UPLC/Q-TOF-MS analysis was performed and 151 compounds were identified. PCA analysis metabolic profiles of C. Nu, C. Ko and C. Kw leaves differ significantly from the other two Callicarpa species, while C. Fo and C. Ma share similar chemical constituents. OPLS-DA highlight with an S-plot indicated that there are 14 robust known chemical markers enabling the differentiation between these five Callicarpa plants. C. Ma, C. Nu, and C. Fo leaves extracts treatment effectively reversed the body weight loss, uric acid and creatinine content, hepatic XOD activity, kidney, liver, and ankle tissues injury and inflammation induced by potassium oxonate in hyperuricemia mice. While Ko and C. Kw leaves extracts treatment showed less improvement in hyperuricemia mice. Graphical Abstract


Introduction
The genus Callicarpa, belonging to the plant family Labiatae, comprises more than 190 species (Xu et al. 2015). Of those, 46 are found in China, including those used in Chinese traditional medicine: C. kwangtungensis (C. Kw), C. macrophylla (C. Ma), C. nudiflora (C. Nu), C. formosana (C. Fo), and C. kochiana (C. Ko) (Ding et al. 2015). They are cultivated widely in southern China and used clinically for haemostasis, anti-inflammation, alleviating pain and as anti-bacterials (Gao et al. 2018). Diterpenoids, flavonoids, phenylethanoids, lignans and volatile oils have been found in this genus (Mei et al. 2010;Ding et al. 2015;Wang et al. 2019;Yi et al. 2019;Bi et al. 2020;Lam et al. 2021), and these five Callicarpa plants are included in the Chinese Pharmacopoeia. There are 93 compounds reported in C. Nu (Feng et al. 2017a(Feng et al. , 2017b, 36 isolated and identified from C. Kw, 36 from C. Ma (Xu et al. 2015), 31 reported in C. Fo, and 17 isolated and identified from C. Ko (Lin et al. 2012). However, relative studies of C. Ma, C. Fo and C. Ko are limited and do not fully reflect the correlation and difference between their chemical components and pharmacological effects.
C. Nu and C. Fo have also been used to treat gout by the Hainan people. A clinical study reported that combined application of C. Nu extract and allopurinol showed a better anti-hyperuricaemic effect and reduced adverse reactions compared to allopurinol alone (Li 2015). Hyperuricaemia is a chronic metabolic disease caused by the disorder of purine metabolism (Bardin and Richette 2014). It has been regarded as the main risk factor for gout and is related to the occurrence of cardiovascular complications, chronic kidney disease, diabetes mellitus and coronary heart disease (Jin et al. 2012;Liu et al. 2015;Mortada 2017).
In this study, ultra-performance liquid chromatography with quadrupole time-offlight tandem mass spectrometry (UPLC-Q-TOF-MS/MS), combined with principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA) were performed to evaluate the similarities and differences between phytochemicals from these five Callicarpa species. In addition, some preliminary investigations were made to explore the anti-hyperuricaemic activity of C. Kw, C. Ma, C. Nu, C. Fo and C. Ko leaf extracts on potassium oxonate-induced hyperuricaemic mice.

Characterisation of chemical constituents of five Callicarpa species extracts by UPLC-Q-TOF-MS/MS
The total ion chromatograms (TICs) of C. Kw, C. Ma, C. Nu, C. Fo and C. Ko leaf extracts in negative-ion mode are illustrated in Figure S1. A total of 151 compounds were identified from these five Callicarpa species leaf extracts using UPLC-Q-TOF-MS/MS analysis by matching the fragmentation patterns with the standards, literature reports, Sci-finder and MassBank. Their detailed mass information and characterisation of each compound are shown in Table S2. Among them are 41 phenylpropanoids, 29 iridoids, 56 flavonoids, 13 triterpenoids and 12 other compound types. Their chemical structures are displayed in Figure S2 (phenylpropanoids), Figure S3 (iridoids), Figure S4 (flavonoids), Figure S5 (triterpenoids) and Figure S6 (other compound types). The detailed compound attributions are demonstrated in Figure S7B.

Marker compounds of the five Callicarpa species extracts
PCA was adopted to obtain a full-scale overview of the chemical differences between these five Callicarpa species extracts. As shown in Figure S7A, the PCA score plots showed an obvious separation between the extracts, suggesting that large metabolite differences exist between these five Callicarpa species. At the same time, C. Nu, C. Ko and C. Kw are far apart in the PCA scores plot, and the C. Fo and C. Ma extracts are clustered closely, indicating that the metabolic profiles of C. Nu, C. Ko and C. Kw differ significantly from the other two Callicarpa species, while the metabolic profiles of C. Fo and C. Ma share similar chemical constituents. Besides, the PCA scores plot ( Figure  S8B-F) shows that iridoids, phenylpropanoids, triterpenoids and other compound types identified could distinguish these five Callicarpa species (PC1 þ PC2 þ PC3 > 90), while the flavonoids could not separate them (PC1 þ PC2 þ PC3 < 90).
OPLS-DA highlight with an S-plot was used to identify compounds responsible for marking these five Callicarpa species extracts. A total of 14 robustly known chemical markers were marked and listed (Table S3; Figure S8G), their chemical structures are summarised in Figure S10 and their relative contents displayed in Figure S9. The box diagrams show that the contents of compounds decaffeoyl verbascoside (6), verminoside (21), 6-hydroxy-luteolin-7-O-b-D-glucoside (33), nudifloside (48), isoacteoside (62), parvifloroside B (65), luteolin-3 0 -O-b-D-glucopyranoside (87), and 6 0 -O-trans-p-coumaroyl-8-epiloganic acid (95) were higher in C. Nu leaf extract, which were the characteristic constituents of C. Nu and could be used to distinguish C. Nu from the other four species. While the contents of compounds luteolin-5-O-neohesperidoside (17) and hardwickiic acid (136) were higher in the C. Ko leaf extract and were characteristic for C. Ko compared to the others. C. Fo and C. Ma leaf extracts both contain higher amounts of b-hydroxy samioside (21), which were characteristic for C. Ma and C. Fa. Poliumoside (54), euscaphic acid (142), and forsythoside B (41) were the main components in C. Kw leaf extract and could be used to distinguish it from the other four. This is the first comprehensive screening analysis of the leaves of these five Callicarpa species, C. Fo, C. Ko, C. Ma, C. Kw and C. Nu, by UPLC-Q-TOF-MS/MS combined with the UNIFI platform. In the present study, 151 compounds were successfully screened. Phenylethanoid glycosides and flavonoids were the main components in all five plants followed by iridoids and triterpenoids (Wu et al. 2018;Fu et al. 2019). The content of flavone derivatives in C. Kw was the lowest among the species. Iridoids and phenylethanol glycoside always coexisted in one plant (Pia˛tczak et al. 2020); however, iridoids were not identified in C. Ko in this study. In addition, the contents of ursane and oleanolic-type triterpenoids were similar in all five Callicarpa species. Furthermore, 14 components were selected as biomarkers for the identification of different Callicarpa species.

Effect of the five Callicarpa species leaf extracts on potassium oxonate induced hyperuricaemic mice
In the present study, an acute hyperuricaemia mice model was established by injection of potassium oxonate, which is a selectively competitive uricase inhibitor and has produced hyperuricaemia in rodents in previous studies (Liang et al. 2019).Consistent with previous reports, the hyperuricaemia model mice exhibited significant body weight loss, increased uric acid and creatinine content, hepatic XOD activity, and injury and inflammation in kidney, liver and ankle tissues . Allopurinol, an XOD inhibitor, was used as a positive control in this study and is commonly used to inhibit the synthesis of uric acid (Fu et al. 2019). From the results of this study, C. Ma, C. Nu and C. Fo leaf extracts effectively reversed body weight loss, increased uric acid and creatinine content and hepatic XD activity, and reduced kidney, liver and ankle tissue injury and inflammation induced by potassium oxonate in hyperuricaemic mice, while C. Ko and C. Kw extracts showed less improvement ( Figure  S11-14). It is suggested that C. Ma, C. Nu and C. Fo leaf extracts have potent efficacy in ameliorating hyperuricaemia induced by potassium oxonate.

Conclusion
In summary, this study revealed that the structural diversity of secondary metabolites and similar patterns in the different germplasm resources of Callicarpa plants. It provided the basis for the clinical application of Callicarpa plants and the scientific basis for the identification of medicinal materials and mixed pseudo-medicinal materials. In addition, it identified new therapeutic medicines for treating hyperuricaemia, provided a new direction for the exploitation of Callicarpa species resources. However, further studies needed to explore the active compounds in C. Ma, C. Nu and C. Fo leaf extracts against hyperuricaemia.