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In order to reveal the chemical substance basis of pharmacodynamic effects of Zuotai, energy dispersive spectrometry of X-ray (EDX), X-ray fluorescence spectroscopy (XRF), synchrotron radiation X-ray absorption fine structure (SR-XAFS), X-ray diffraction (XRD), scanning electron microscope (SEM) and atomic force microscope (AFM) were used to analyze the elements, the chemical valence and local structure of mercury, and the chemical phase composition and micro-morphology of Zuotai. EDX and XRF analysis shows that the main elements in Zuotai are Hg and S, with some other minor elements, such as 0, Fe, Al, Cu, K, Ag, Ca, Mg etc. SR-XAFS analysis shows that: the oxidation state of mercury in Zuotai is divalence, its neighbor atoms are S, and its coordination number is four. XRD assay found that β-HgS (cubic, F-43m 216) and S8 (orthorhombic, Fddd 70) are the main phase compositions in Zuotai. Besides, it also has a small amount of C (hexagonal, P63/mmc 194), Fel.05 S0.95 (hexagonal, P63/mmc 194), Cu6S6 (hexagonal, P63/mmc 194), Cu1.8 S (cubic, F-43m 216) and so on. And it was found that the crystallinity of Zuotai is about 59%, and the amorphous morphology substance in it is about 41%. SEM and AFM detection suggests that Zuotai is a kind of ancient micro-nano drug, and its particle size is mainly in the range of 100-600 nm, even less than 100 nm, which commonly further aggregate into several to 30 µm loose amorphous particles. In summary, the present study elucidated physicochemical characterization(elements composition, coordination information of mercury, phase composition and micro-morphology) of Zuotai, and it will play a positive role in promoting the interpretation of this mysterious drug.;

Zuotai, a famous Tibetan medicinal mixture containing β-HgS, has been used to combine with herbal remedies for treating diseases for more than 1 300 years. The target organ for inorganic mercury toxicity is generally considered to be the kidney. Therefore, it is crucial to reveal the chemical speciation, spatial distribution and potential nephrotoxicity of mercury from Zuotai in kidney. To date, this remains poorly understood. We used X-ray absorption spectroscopy (XAS) and micro X-ray fluorescence (μ-XRF) imaging based on synchrotron radiation to study mercury chemical forms and mercury special distribution in kidney after mice were treated orally with Zuotai, β-HgS or HgCl2. Meanwhile, the histopathology of kidney was observed. Mice exposed with Zuotai showed kidney with significant proportion of mercury ions bound to sulfydryl biomolecules (e.g. Cys-S-Hg-S-Cys) plus some of unknown species, but without methylmercury cysteine, which is the same as β-HgS and HgCl2. The mercury is mainly deposited in renal cortex in mouse treated with Zuotai, β-HgS or HgCl2, but with a low level of mercury in medulla. The total mercury in kidney of mice treated with HgCl2 was much higher than that of β-HgS, and the later was higher than that of Zuotai. And, HgCl2 cause severe impairments in mouse kidney, but that was not observed in the Zuotai and β-HgS groups. Meanwhile, the bio-metals (Ca, Zn, Fe and Cu) micro-distributions in kidney were also revealed. These findings elucidated the chemical nature, spatial distribution and toxicity difference of mercury from Zuotai, β-HgS and HgCl2 in mouse kidney, and provide new insights into the appropriate methods for biological monitoring.