Analysis of the biodistribution of natural products in mice by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Natural products originating from plants have various beneficial pharmacological effects, such as anticancer, antimicrobial, and anti-inflammatory activities, while being nontoxic. Therefore, tremendous efforts have been invested in understanding their bioactivities in the body to facilitate therapeutic target validation. However, such research is still challenging for certain natural products, such as flavonoids, which are rapidly metabolized in and eliminated from the human body. To investigate the bioactivities of such products, particularly in certain tissues, it is necessary to understand their biodistribution in vivo. In this respect, reliable analytical methods with simple and efficient procedures for the in vivo evaluation of natural small molecules are urgently required. In particular, mass spectrometry (MS) can be effectively used to analyze small molecules after tissue extraction, as MS has various advantages including accuracy, simplicity, and high sensitivity. Herein, we report the biodistribution of a natural small molecule by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). After intravenously injecting gomisin H into a mouse as a model natural product, it was extracted from each organ and then analyzed by MALDI-TOF MS. The analysis showed that gomisin H accumulated mainly in the liver and relatively large amounts of the product existed in the kidney and brain compared to those in other tissues.


Introduction
Natural products are small chemical molecules produced by living organisms, such as plants, microbes, and animals [1]. Notably, more than twenty thousand natural products can be obtained from plants, including medicinal plantderived chemical compounds called phytochemicals and their metabolites, which are widely and actively used as drugs in clinical medicine due to their diverse pharmacological activities, such as antioxidant, anti-inflammatory, anticancer, antimicrobial, and anti-viral activities with little to no toxicity [2][3][4][5][6]. Therefore, a multitude of research efforts are being undertaken to analyze their bioactivity in the body to facilitate therapeutic target validation [2]. In particular, understanding the biodistribution of natural products in the body is essential for investigating their bioactivities in certain tissues; such knowledge would prove useful in enhancing the therapeutic benefits of phytochemicals [7]. However, such investigation is still challenging for certain natural products, such as flavonoids, which are rapidly metabolized in and eliminated from the body, and therefore, only a few methods have been reported. Optical fluorescence imaging [8,9], high-performance liquid chromatography tandem mass spectrometry [10,11], and radioisotope-labeling methods [12][13][14] are examples of conventional methods used to monitor the Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13765-018-0353-9) contains supplementary material, which is available to authorized users.
& Woon-Seok Yeo wsyeo@konkuk.ac.kr accumulated target natural products in vivo. However, the general use of these methods is hindered by their disadvantageous features, including false-positive signals, high backgrounds, toxic and complicated steps, and/or changes in the physical and chemical properties of the natural products after labeling. In this context, reliable analytical methods for the in vivo evaluation of natural small molecules via simple and efficient procedures are urgently needed. In this study, we report a method for investigating the biodistribution of natural products using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which has several advantages over conventional methods, including high sensitivity, multiplexing capability, and label-free format.

Sample preparations and mass analysis
Briefly, gomisin H was intravenously injected to mice and the organ was extracted. The mouse tissues were homogenized and extracted with ethyl acetate, which were analyzed by MALDI-TOF MS in the presence of the internal standard. For details, see Supporting Information.
Results and discussion Figure 1 schematically illustrates our method for the qualitative and quantitative analysis of a natural small molecule in mouse organs using MALDI-TOF MS to determine its biodistribution after intravenous injection. Each isolated organ (the brain, heart, lung, kidney, spleen, liver, and pancreas) was extracted with an organic solvent 15 min after injection and the natural small molecule extracted from the harvested organs was analyzed by MALDI-TOF MS in the presence of an IS, which has a chemical structure similar to that of the analyte but a different molecular weight. Therefore, the mass spectrum can be used to derive quantitative information on the analyte by comparing the peak intensities of the analyte and IS. Gomisin H derived from Schisandra chinensis was used as a model compound in the current study. It is reported to be a lignan with various pharmacological effects, including anti-inflammatory, anti-hepatotoxic, antipyretic, and antiasthmatic properties [15,16]. We first constructed a calibration curve for the quantification of gomisin H extracted from mouse organs. Gomisin H at various concentrations in the presence of gomisin J as the IS was analyzed by MALDI-TOF MS using AuNPs as the matrix. In general, the use of an organic matrix hampers the analysis of small molecules due to the interference of a matrix in the low-mass region, and therefore, inorganic nanomaterials have been widely used such as AuNPs, one of the most popular inorganic matrices for small molecule analysis. Peaks at m/z values of 440.9 [M ? Na] ? and 410.8 [M ? Na] ? corresponding to gomisin H and the IS, respectively, could be clearly observed in the mass spectra (Fig. 2(A)). The calibration curve indicates a good linearity in the gomisin H concentration range of 10-1000 lM. Such linearity proves the feasibility of our strategy for the quantitative analysis of Fig. 1 Schematic representation of the investigative process used to analyze the biodistribution of gomisin H by MALDI-TOF MS the target molecule using MALDI-TOF MS, which has an inherent limitation for quantification.
We next verified the fidelity of our strategy by examining the limit of detection (LOD) and quantification of gomisin H in spiked samples. Gomisin H sample was prepared at various concentrations and analyzed with MALDI-TOF MS which gave LOD of 10 aM (S/N [ 3). We next measured MS intensity ratios between sample and IS (S/IS) against gomisin H spiked in liver tissue extracts of control mice. The quantification was performed using the constructed calibration curve. As shown in Table 1, recoveries of the known amount of gomisin H added in the liver extracts were obtained from 96 to 98%, and furthermore, we observed a coefficient of variance (CV) of 7.7 and 13.8%.
Next, we quantified the contents of gomisin H in the tissue extracts using the constructed calibration curve. Gomisin H in PBS containing 15% (v/v) each of Koliphor and DMSO was intravenously injected into ICR mice. In the control samples, PBS was injected into the mice. Fifteen minutes after the intravenous injection, the mice were killed and their organs were extracted. To evaluate the effects of organ extraction with and without a perfusion step, some of the mice were perfused with 0.9% saline to flush out blood. Subsequently, the mouse organs were collected, washed with PBS, weighed, and homogenized in PBS. The homogenates were then mixed with 10% SDS in PBS and EA, followed by sonication for 10 min. The supernatant was collected after centrifugation and extracted three times using equal volumes of EA. The combined organic layer was dried at room temperature. The dried extracts were dissolved in DMSO and analyzed using MALDI-TOF MS in the presence of the IS, gomisin J. Figure 3 shows the representative mass spectra of the tissue extracts with and without perfusion from a gomisin H-treated mouse (right panel) and a PBS-injected mouse (left panel). Peaks at m/z values of 410.8 [M ? Na] ? and 440.9 [M ? Na] ? corresponding to the IS (circles) and gomisin H (triangles), respectively, were clearly observed in the tissue extracts obtained from the gomisin H-treated mouse, whereas the tissues extracted from the PBS-injected control mouse exhibited the peak corresponding to IS only. This result proves that our strategy can be efficiently used to extract and analyze natural small molecules from complex biological samples, such as organs, without any complicated purification steps. Furthermore, in comparison with IS, the peak intensities of gomisin H without perfusion were higher than those obtained with perfusion; this observation can be attributed to the existence of the small molecules in blood. This result also indicates that our method has high sensitivity toward the amount of small molecules in the blood encapsulated in the organs. Later, we analyzed the biodistribution of gomisin H in mouse organs based on the quantitative results obtained using the calibration curve shown in Fig. 2(B). As shown in Fig. 4, the contents of gomisin H in the liver, kidney, brain, pancreas, lung, spleen, and heart samples after perfusion were (68.05 ± 5.08), (20.30 ± 3.07), (10.79 ± 0.39), (6.00 ± 1.01), (4.64 ± 1.33), (2.63 ± 0.11), and (1.80 ± 0.14) lg/g of organ, respectively. Without perfusion, the accumulated gomisin H contents were observed to slightly higher than those obtained after perfusion. The content of gomisin H in the liver was over 3 times greater than those in other organs. This is a reasonable observation because one of the main roles of the liver is the uptake and subsequent detoxification, metabolic transformation, and excretion of xenobiotics [17]. In addition, these results also illustrate the differences between the accumulated gomisin H contents in the organs with and without perfusion, stemming from the removal of gomisin H included in the blood. Notably, a significant decrease in the gomisin H content was observed in the liver, which might be attributed to the higher blood content in the liver compared to other organs.
In conclusion, we report a method for studying the biodistribution of natural products in mice using MALDI-  TOF MS. We successfully constructed a calibration curve, using which the accumulation of a natural product, gomisin H, in mouse organs was quantified; the small molecule contents were quantified by comparing the peak intensities of gomisin H and the IS. The results showed that a large proportion of the gomisin H intravenously injected into a mouse was accumulated in its liver due to its biological functions. Furthermore, we examined the effects of organ extraction with and without perfusion. We believe that our method can be practically used to study the in vivo biodistribution of natural compounds. In addition, the multiplexing capability of MS would enable our method to analyze pharmacokinetic drug-drug interactions.