Determination and comparison of alkaloids and triterpenes among tissues after oral administration of crude and processed Phellodendri Chinensis Cortex by UPLC-QqQ-MS

Abstract Phellodendri Chinensis Cortex is widely used in the clinic of traditional Chinese medicine. In order to enlarge the range of application, it is necessary to processed with honey, salt-water, and rice-wine, respectively. We hope to elucidate the connotation of processing, an UPLC-QqQ-MS method was used for determination and comparison the tissue distribution of alkaloids and triterpenes after oral administration water-extracts of crude and processed products. The results showed that the berberine, phellodendrine, magnoflorine, limonin, and obacunone in crude and processed products were distributed in all tissues, especially in the small intestine and stomach. In this study, we can provide a scientific basis for explaining the processing connotation of Phellodendri Chinensis Cortex processed with salt-water and rice-wine, respectively. Graphical Abstract


Introduction
Phellodendri Chinensis Cortex, the dried bark of Phellodendron chinense Schneid., commonly called Chuan Huang Bo (CHB), is widely used for clearing heat, drying dampness, purging fire, relieving steaming, and removing toxin to treat sore in the clinic of traditional Chinese medicine (TCM) (Jia 2008;Chinese Pharmacopoeia Committee 2015). In TCM, processing is a necessary and significant part in preparation of decoction pieces before clinical use. There are three mainly processed CHB products and methods documented, including processing with rice-wine (RCHB), salt-water (SCHB), and honey (HCHB). The CHB processed with rice-wine, salt-water, and honey were mostly made for relieving the property of bitter-cold, nourishing yin and downbearing fire, and treating middle energizer and tonifying qi, respectively (Jia 2008). Chemical studies have been reported that CHB contains rich of alkaloids, triterpenes and isocoumarin, which are the primary active constituents (Miyake et al. 1992;Ida et al. 1993;Cui et al. 2003). The modern experimental studies have shown that alkaloids and triterpenes possess pharmacological effects including anti-inflammatory, antitumor, hypoglycemic, and antioxidant (Li et al. 2013;Choi et al. 2014;Li et al. 2014). Besides, with the advantage of high selectivity and sensitivity, UPLC-QqQ-MS is mainly applied to elucidate the inherent components in herbal medicines so that it can explain the processing mechanism and provide a scientific basis for processing of TCM (Yi et al. 2013;Wang et al. 2015;Ji et al. 2017). So, in this paper we intend to determinate and compare the concentration of alkaloids and triterpenes in different rat tissues after oral administration of crude and processed Phellodendri Chinensis Cortex, and hope it can help evaluate the connotation of processing technologies and methods on CHB.

Results and discussion
The tissue concentrations of berberine, phellodendrine, magnoflorine, limonin, and obacunone determined at 30, 60, and 180 min after oral administration crude and processed CHB at a dose of 8 g/kg are shown in Tables S2-S6 and Figures S2-S6 (supplementary material).
In the CHB, berberine, jatrorrhizine, and palmatine are belong to berberine-type alkaloids. Phellodendrine and magnoflorine are belong to isoaporphine-type alkaloids. Limonin and obacunone are belong to limonin-type triterpenes. So, we studied the tissue distribution regulations of the three types of compounds after oral administration water-extracts of crude and processed CHB products in rats. Nevertheless, jatrorrhizine, and palmatine were not detected due to low levels. The results show that all of the five compounds were distributed in all tissues after oral administration water-extracts of crude and processed CHB products. In the small intestine and stomach, the concentration of berberine, phellodendrine, and magnoflorine were higher than other tissues of crude. Thus, it can be inferred that berberine and phellodendrine were mainly absorbed and distributed in the small intestine and stomach. The concentration of limonin and obacunone were higher in the stomach than other tissues. So, we can see that limonin and obacunone were mainly absorbed and distributed in the stomach ( Figures S1-S5). Compared CHB with HCHB, the concentration of berberine and phellodendrine were increased in the stomach of HCHB, the magnolia and obacunone were decreased in 30-minute, and the limonin unchanged. Thus, the CHB processed with honey promoted the absorption of berberine and phellodendrine in the stomach, which also promoted the distribution in tissues else ( Figures S1 and S2), and the HCHB reduced the absorption of magnolia and obacunone, but has no effect on the limonin (Figures S3-S5). Respectively, the CHB after processing with salt-water and rice-wine, the concentrations of five analytes were significantly decreased in the stomach (30-minute). In previous studies, our team found that some compounds reduced (quantitative and/or qualitative changed) after processing as the result of heating in the SCHB and RCHB. Such as the berberine transformed into berberrubine (Jiang et al. 2011). The alkaloids and limonins taste bitter, and as the five compounds in the RCHB have the lowest absorption in the stomach. Thereby, the CHB after processing with rice-wine could alleviate the bitter-cold. The data (30-minute) displayed that the concentrations of limonin and obacunone were higher in the SCHB than crude in the kidney, large intestine, and small intestine. And the concentrations of phellodendrine, limonin and obacunone in the RCHB were higher than crude in the heart. Respectively, these results may be contribute to account for the effect of acting in lower and upper of CHB processed with salt-water and rice-wine in accordance with TCM theory, and the further research is needed for the reason ( Figures S2, S4 and S5). In addition, the IS we tested nitidine chloride and carbamazepine. Ultimately, we chosen carbamazepine as IS, because of the better stability and no interference peaks from internal elements.

Experimental
The detailed experimentation and parameters are shown in supplementary material (Tian et al. 2014;Liu et al. 2015;Li et al. 2016;Liu et al. 2017;Yuan et al. 2017).

Conclusion
In this study, it is the first time to developed and validated a UPLC-QqQ-MS method for determination and comparison the tissue distribution of alkaloids and triterpenes in different tissues after oral administration water-extracts of crude and processed CHB products. Berberine, phellodendrine, magnoflorine, limonin, and obacunone were distributed in all tissues, mainly in the small intestine and stomach. The results of phellodendrine, limonin and obacunone from SCHB and RCHB would provide a reference for revealing connotation of processing technique with salt-water and rice-wine, respectively.

Disclosure statement
No conflict of interest was reported by the authors.