Chemical Species, Micromorphology, and XRD Fingerprint Analysis of Tibetan Medicine Zuotai Containing Mercury

Zuotai (gTso thal) is one of the famous drugs containing mercury in Tibetan medicine. However, little is known about the chemical substance basis of its pharmacodynamics and the intrinsic link of different samples sources so far. Given this, energy dispersive spectrometry of X-ray (EDX), scanning electron microscopy (SEM), atomic force microscopy (AFM), and powder X-ray diffraction (XRD) were used to assay the elements, micromorphology, and phase composition of nine Zuotai samples from different regions, respectively; the XRD fingerprint features of Zuotai were analyzed by multivariate statistical analysis. EDX result shows that Zuotai contains Hg, S, O, Fe, Al, Cu, and other elements. SEM and AFM observations suggest that Zuotai is a kind of ancient nanodrug. Its particles are mainly in the range of 100–800 nm, which commonly further aggregate into 1–30 μm loosely amorphous particles. XRD test shows that β-HgS, S8, and α-HgS are its main phase compositions. XRD fingerprint analysis indicates that the similarity degrees of nine samples are very high, and the results of multivariate statistical analysis are broadly consistent with sample sources. The present research has revealed the physicochemical characteristics of Zuotai, and it would play a positive role in interpreting this mysterious Tibetan drug.

As known, heavy metals could pose potentially serious hazard to human health.So, the biological safety and efficacy of Zuotai containing mercury need urgently to be reevaluated from a modern medical science perspective.Surprisingly, its toxicity tests suggest that Zuotai has no apparently adverse effects on animal subjects under clinical equivalent dose and clinical medication cycle [11,[13][14][15][16].Besides, pharmacological experiments show that Zuotai not only can tranquilize and allay excitement, promote sleep, and produce antipyretic effect [17,18], but also can enhance immunity, have antiinflammatory effects, extend the life of fruit flies, and inhibit the expression of caspase-3, along with other effects [18][19][20][21].It is confusing why this Tibetan drug containing mercury shows tiny/nontoxicity and various efficacies in animal tests.Biological effects of heavy metals not only relate to doses, but also have closer relationship with their chemical forms; for example, Cr +3 is an essential element for mammals with a role in maintaining proper carbohydrate and lipid metabolism; on the contrary, Cr +6 has carcinogenic toxicity and genotoxicity [22][23][24][25].So, it is crucial to reveal the chemical species of heavy metals in Zuotai for elucidating its pharmacological functions and mechanisms.
Until now, only a few literatures about physicochemical characterization of Zuotai are available.Yan [26,27], Lan et al. [28], and Zhao et al. [29] reported that Zuotai was mainly an inorganic mixture of HgS, sulfur, graphite, and other tiny organic molecules, and it was composed of nanoparticles, which further aggregated into microsized particles.However, there still lacks more information about the chemical structure, micromorphology, and XRD fingerprint features of Zuotai from large sample sizes.Therefore, in the present research, nine Zuotai samples were collected from four major Tibetan regions (Tibet, Qinghai, Gansu, and Sichuan) in China.The element compositions, phase structures, and micromorphology of these samples are analyzed by energy dispersive spectrometry of X-ray (EDX), powder X-ray diffraction (XRD), scanning electron microscopy (SEM), and atomic force microscopy (AFM), respectively.Additionally, the powder XRD fingerprint features of Zuotai were analyzed by applying multivariate statistical analysis methods (similarity calculation, systemic cluster analysis, and principal component analysis).

Materials.
Nine Zuotai samples were kindly provided from the four Tibetan medicine organizations of four major Tibetan regions in China, respectively.The sources of Zuotai samples were listed in Table 1.The main technology that processes these samples was well recorded and protected by the patent (number 88107006.8) in China [8].Before characterization, these samples were sealed in glass bottles and covered by blue cloths according to their traditional storage method.

Measurement of Physicochemical Characteristics.
The elements in nine Zuotai samples were detected by energy dispersive spectrometry of X-ray (EDX, INCA, Oxford Instruments, Inc.).EDX is a kind of semiquantitative elemental analysis method.The working conditions of EDX are operating voltage of 20 kV, beam spot size of 55 nm, working distance of 8 mm, and hit rates of 2500 cps.
The phase compositions of samples were characterized by powder X-ray diffraction (XRD, X'Pert Pro, PANalytical Company).The working conditions of XRD are Cu target, wavelength of 0.15406 nm, K (1) energy of 40 keV, current intensity of 30 mA, scanning speed of 2 ∘ /min, and 2 angle scan range of 5 ∘ -80 ∘ .Powder X-ray diffraction data were acquired by X'Pert Data Collector and analyzed by X'Pert HighScore Plus using PDF2-2004 of the International Centre for Diffraction Data.Powder XRD spectra were drawn by Origin 8.0.The crystal structure graphs of phase compositions in Zuotai were acquired by Diamond 3.2 and FindIt 2009.

Elemental Compositions of Zuotai.
A large amount of mercury (Hg), sulfur (S), and oxygen (O) was found in nine Zuotai samples by EDX analysis.Besides, a small amount of other elements has also been found, such as ferrum (Fe), aluminum (Al), and cuprum (Cu).
The results are shown in Table 2 and Supplementary Figure 1 (in Supplementary Material available online at http://dx.doi.org/10.1155/2016/7010519).However, we should note that the oxygen (O) in Zuotai may be interfered with inevitably to a certain extent by the residual oxygen from air in the sample testing chamber of EDX.So, whether Zuotai samples contain such high level oxygen element needs further study.

Micromorphology of Zuotai.
Nine Zuotai samples display a variety of micromorphologies, which are composed of a large number of irregular nano-microparticles by scanning electron microscopy (SEM) and atomic force microscopy (AFM), as shown in Figures 1 and 2, respectively.The diameters of these particles vary mostly in the range of 100-800 nm, some even less than 100 nm.And these nanomicroparticles commonly further aggregate into 1-30 m loosely amorphous particles.There are a number of literature reports in both the basic science and pharmaceutics which scientifically define dimensions of nanoparticles ranging in size from 1 to 1000 nm [30][31][32][33][34].According to this, Zuotai should be a kind of ancient nanodrug in traditional Tibetan medicine.

Phase Compositions of Zuotai.
Powder XRD analysis found that the common major phase compositions are cubic crystal mercuric sulfide (-HgS) and orthorhombic crystal elemental sulfur (S 8 ) in nine Zuotai samples (Figure 3, Table 3, and Figure 4).The spacing group of -HgS in Zuotai is F-43m  This result indicates that the chemical species of mercury in Zuotai are cubic crystal mercuric sulfide (-HgS) and hexagonal mercuric sulfide (-HgS) without elemental mercury.Both mercuric sulfides are typical covalent insoluble sulfides.The solubility product constant of -HgS is 1.6 × 10 −52 ; the solubility product constant of -HgS is 4.0 × 10 −53 [35].So far, there is no definitive evidence showing that the two chemical forms of mercury are toxic in the human body [36,37].

Powder XRD Fingerprint Analysis of Zuotai
3.4.1.Establishing XRD Fingerprint.The means of diffraction intensity at different 2 angles were acquired by overlaying the diffraction topology spectra of nine samples firstly and then averaging.According to these means, the powder XRD fingerprint (average) of Zuotai was drawn by Origin 8.0 (Figure 5).Besides, according to the medians of diffraction intensity of nine samples at different 2 angles, the powder XRD fingerprint (median) was also drawn by Origin 8.0 (Figure 6).

Similarity Calculation of Nine Zuotai Samples.
The XRD fingerprints (average and median) of Zuotai were considered as controls; the similarity degrees (cosine and correlation coefficient) of nine Zuotai samples were calculated with SPSS 22.0.The results show that the similarity degrees of the nine samples are very high (all cosines and correlation coefficients > 0.9950) in Table 4.

Principal Component Analysis (PCA).
Principal component analysis (PCA) is a kind of reducing dimensions method, in that data are projectedfrom high-dimensional  space onto low-dimensional space, namely, using a smaller number of new variables instead of a huge number of original variables with minimal information loss [38][39][40][41].In the process of PCA, the absolute intensity of X-ray diffraction can be used.The absolute intensities (I) of 25 common peaks of nine Zuotai samples were analyzed with PCA by SPSS 22.0, and then the initial eigenvalues (Table 5) and load matrix (Supplementary Table 2) of principal components are acquired.
In Table 5, the initial eigenvalues of the first three principal components are all greater than 2, and the cumulative contribution rate of variance is 83.35 (>80%).Therefore, the first three components (denoted as F1, F2, and F3, resp.) can well explain the inherent information of the original 25 characteristic peaks of nine Zuotai samples.
And the variables coefficients of principal component linear equation, namely, standardized eigenvector, can be calculated from the load values of principal components.  of sample).According to this equation, standardized eigenvectors (shown in Supplementary Table 3) of the first three principal components (F1, F2, and F3) are calculated.The linear equations of the first three principal components (F1, F2, and F3) are obtained through multiplying the standardized eigenvectors with the corresponding normalized peak intensity ().The equations of F1, F2, and F3 of Zuotai are as follows:  ( According to the above linear equations of Zuotai, the principal component scores of nine Zuotai samples were calculated, and the 3D space scatter diagram (Figure 8) of these samples is drawn by SPSS 22.0.
The PCA result shows that nine Zuotai samples can be classed into three groups: #1, #2, #6, #7, and #8 are clustered together; #3, #4, and #5 samples are clustered together; #9 sample is clustered as a single group.Moreover, the distance between the first two groups is closer than with the third group on F1 coordinate axis.The result of PCA is consistent broadly with systemic cluster analysis.

Discussion
Tibetan medicine has been playing an important role in the health, reproduction, and development of Tibetan people.Zuotai is the key raw material of many precious compound preparations in Tibetan medicine, with tonic effects, reducing toxicity, increasing efficacy, replenishing blood, promoting blood flow, promoting tissue regeneration, invigorating the spleen, and prolonging one's life, among other effects [4][5][6][7][8].Zuotai is the common name of "Renqing Ouqu Zuozhu Qinmu" in Tibetan medicine."Zuo" means refining, burning, smelting, and cooking; "Tai" means ashes, powder, and gray powder; "Zuotai" means the powder of burning, burning into powder, and calcining mercury into ash [4,11,19,20,26,27,[42][43][44].Therefore, Zuotai not only is the name of processing product, but also is the name of processing technology [4,5,11].The technology of mercury refining into Zuotai was recorded in "Sibu Yidian" (Four Medical Codes), a Tibetan medicine classic, in the AC 8th century, and was improved by U rgyan pa Rin chen dpal, a famous Tibetan medicine practitioner, in AC 13th century [12,45].This technology has had profound influence on the sciences of Tibetan medicine processing, medical formulae, and life cultivation.Almost the alchemy of Chinese Taoist and the mercury praeparatum technology of Indian Tantra had already gone away with history, but only the processing technology of Zuotai survives and flourishes till today; this may have been because Zuotai has been used as a core adjuvant in many precious Tibetan medicine component preparations [45,46].Meanwhile, this conserves a kind of unique ancient technology for traditional Tibetan culture.In 2006, the processing technology of Zuotai has been collected in the Chinese Intangible Cultural Heritage List [47].
Nanometer is not only a concept of spatial scale, but also a new way of thinking.The present research found that Zuotai particles are mainly in 100-800 nm range, and some are less than 100 nm, which commonly aggregate into about 1-30 m loosely amorphous particles.This is consistent with the reports of Yan et al. [26], Lan et al. [28], and Zhao et al. [29].In the pharmaceutical field, the particle size of a nanodrug is generally defined in the range from 1 nm to 1000 nm [30][31][32][33][34]. So, this indicates that Zuotai is a kind of ancient nanodrug.There are three main absorption ways of oral nanoparticles in the intestinal tract [48,49]: (1) paracellular passageparticles "kneading" between intestinal epithelial cells due to their extremely small size (<50 nm); (2) endocytotic uptake-particles absorbed by intestinal enterocytes through endocytosis (particles size < 500 nm); (3) lymphatic uptakeparticles adsorbed by M cells of Peyer's patches (particle size < 5 m).It is surprising to find that the scale of Zuotai particles coincides with modern nanodrug concept.This finding greatly arouses our interests in this ancient nanodrug.According to the above absorption ways of nanodrugs in the intestine, Zuotai particles may mainly be absorbed through the intestinal epithelial cells and the M cells of Peyer's patches.
Accurate characterization for the chemical species of heavy metals in Zuotai is the prerequisite of understanding its peculiarly biological effects.The present study suggests that Zuotai contains cubic -HgS (F-43m, 216), orthorhombic S 8 (Fddd, 70), and hexagonal -HgS (P3221, 154).Besides, there are still many unknown very weak X-ray diffraction signals, which are difficult to analyze.According to the processing technology of Zuotai, we can know that it is obtained through mixing the burned Nengchi Eight Eshes and the burned Nengchi Eight Mineral Ashes into the prepared mercury powder at 3.3% rate, respectively.The chemical components of Nengchi Eight Eshes and Nengchi Eight Mineral Ashes have been reported [50,51] by some authors of the present paper.The other phase compositions of Zuotai can be deduced from the Nengchi Eight Eshes and the refined Nengchi Eight Mineral Ashes.Therefore, except for the main phase compositions above, Zuotai also should contain minor or trace quadratic AuPb 2 (I4/mcm, 144), quadratic PbO (P4/nmm, 129), orthorhombic PbO (Pbma, 57), cubic Pb (Fm-3m, 225), monoclinic Ag 2 S (P21/n, 14), quadratic Cu  [45,47].Besides, Yan et al. [26,27] and Lan et al. [28] reported that Zuotai may also contain some amount of FeC, -Fe 2 O 3 , (S 4 N 3 )Cl, C, and trace organics.As seen from the above, the chemical components and crystal structures of Zuotai are extremely complex, and this may be the substance basis of its peculiar curative effects.
In summary, the present research suggests that mercury (Hg) and sulfur (S) are the major elements, and ferrum (Fe), aluminum (Al), and cuprum (Cu) are the minor metal elements in Zuotai.The chemical species of mercury in Zuotai are insoluble cubic mercuric sulfide (-HgS) and minor hexagonal mercuric sulfide (-HgS).And orthorhombic sulfur (S 8 ) is also the main phase composition in Zuotai.Moreover, Zuotai is a kind of ancient nanodrug in traditional Tibetan medicine.Its particles are mainly in the range from 100 nm to 800 nm and further aggregate into 1-30 m loosely amorphous particles.Besides, the intrinsic relationship among samples from different sources is revealed through multivariate statistical analysis, according to the established powder XRD fingerprint of Zuotai.As an important drug containing heavy metals in Tibetan medicine, Zuotai has many mysteries, which are worth exploring.Science always has its historical limitation, and if the knowledge of contemporary science cannot explain it, we cannot abandon it blindly, but it needs to be handed down, waiting for the science of the future to explain it.

Figure 1 :
Figure 1: Micron size morphologies of nine Zuotai samples by scanning electron microscopy (SEM).Note: #1 is the sample of Aba Prefecture Tibetan Medicine Hospital; #2 is the sample of Gan'na Prefecture Tibetan Medicine Hospital; #3, #4, and #5 are the samples of the Company of Tibetan Medicine of Tibetan Traditional Medical College; #6, #7, and #8 are the samples of Qinghai Province Tibetan Medicine Hospital; #9 is the sample of the Company of Tibetan Medicine of Tibetan Autonomous Region.

Figure 2 :Figure 3 :
Figure 2: Nanometer size morphologies of nine Zuotai samples by atomic force microscopy (AFM).Note: #1 is the sample of Aba Prefecture Tibetan Medicine Hospital; #2 is the sample of Gan'na Prefecture Tibetan Medicine Hospital; #3, #4, and #5 are the samples of the Company of Tibetan Medicine of Tibetan Traditional Medical College; #6, #7, and #8 are the samples of Qinghai Province Tibetan Medicine Hospital; #9 is the sample of the Company of Tibetan Medicine of Tibetan Autonomous Region.

Figure 7 :
Figure 7: The cluster analysis result of nine Zuotai samples.Note: #1 is the sample of Aba Prefecture Tibetan Medicine Hospital; #2 is the sample of Gan'na Prefecture Tibetan Medicine Hospital; #3, #4, and #5 are the samples of the Company of Tibetan Medicine of Tibetan Traditional Medical College; #6, #7, and #8 are the samples of Qinghai Province Tibetan Medicine Hospital; #9 is the sample of the Company of Tibetan Medicine of Tibetan Autonomous Region.

Figure 8 :
Figure 8: The PCA result of XRD fingerprint of nine Zuotai samples.Note: #1 is the sample of Aba Prefecture Tibetan Medicine Hospital; #2 is the sample of Gan'na Prefecture Tibetan Medicine Hospital; #3, #4, and #5 are the samples of the Company of Tibetan Medicine of Tibetan Traditional Medical College; #6, #7, and #8 are the samples of Qinghai Province Tibetan Medicine Hospital; #9 is the sample of the Company of Tibetan Medicine of Tibetan Autonomous Region.

Table 2 :
Elemental contents in nine Zuotai samples by energy dispersive spectrometry of X-ray (EDX).

Table 3 :
Phase compositions in nine Zuotai samples by powder XRD.
Note: #1 is the sample of Aba Prefecture Tibetan Medicine Hospital; #2 is the sample of Gan'na Prefecture Tibetan Medicine Hospital; #3, #4, and #5 are the samples of the Company of Tibetan Medicine of Tibetan Traditional Medical College; #6, #7, and #8 are the samples of Qinghai Province Tibetan Hospital; #9 is the sample of the Company of Tibetan Medicine of Tibetan Autonomous Region.

Table 4 :
The similarity of XRD fingerprints of nine Zuotai samples.
Medical College; #6, #7, and are the samples of Qinghai Province Tibetan Medicine Hospital; #9 is the sample of the Company of Tibetan Medicine of Tibetan Autonomous Region.