Effects of the Salt-Processing Method on the Pharmacokinetics and Tissue Distribution of Orally Administered Morinda officinalis How. Extract

Salt processing, which involves steaming with salt water, directs herbs into the kidney channel. After being salt processed, kidney invigorating effects occur. However, the underlying mechanism of this method remains elusive. The compounds monotropein, rubiadin, and rubiadin 1-methyl ether are the major effective components of Morinda officinalis How. To clarify the pharmacokinetics and tissue distribution of these three compounds, we employed liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to determine the contents of the three components in rat plasma and tissues. Separation was achieved on an Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm, Waters). Formic acid aqueous solution (0.1%; A) and acetonitrile (containing 0.1% formic acid; B) were used as the mobile phase system with a programmed elution of 0∼5 min with 70% A and then 5∼7 min with 60% A. All analytes were measured with optimized multiple reaction monitoring (MRM) in negative ion mode. Geniposide and 1,8-dihydroxyanthraquinone were used as the internal standards (IS). The linear ranges were 1.2∼190, 1.3∼510, and 0.047∼37.5 μg/mL, respectively. Compared with the Morinda officinalis without wood (MO) group, the Cmax and AUC0-t parameters of rubiadin and rubiadin 1-methyl ether elevated remarkably for the salt-processed Morinda officinalis (SMO) groups, which indicates that steaming by salt could increase the bioavailability of rubiadin and rubiadin 1-methyl ether. The Tmax for monotropein is shorter (0.5 h) in SMO groups than that in MO group, which means that monotropein was quickly absorbed in the SMO extract. Moreover, the contents of three compounds in the small intestine were the highest.

Chinese medicinal herb processing is a unique pharmaceutical technique, which can turn Chinese medicinal materials into decoction pieces using processing methods like stir-frying, steaming, and frying with sand or wheat bran [10]. Decocting pieces are the main form prescribed in TCM clinics [1]. e pharmacological actions and energy properties (nature, flavor, and channel tropism) might be changed after processing, side effects and disagreeable odors can be eliminated. It is essential to use the proper processing method to ensure the quality and safety of traditional Chinese medicine decocting pieces. Steaming or stir-frying with salt can change the activity direction of Chinese herbal medicines and can also improve their efficacy [11][12][13][14]. e commercially available products of Morinda officinalis in herbal markets are M. officinalis without wood (MO) and salt-processed products (SMO). It was revealed that MO extract exerts tonifying kidney and supporting yang effects by regulating the functions of the hypothalamus-pituitaryadrenal axis [15,16]. e medicinal efficacy of MO and SMO is different because of their different processing methods. After being salt processed, the effects of strengthening kidney-yang can be dramatically enhanced. e chemical composition changes during processing, for example, glycosides can be hydrolyzed into aglycone or converted into other constituents, the contents of toxic ingredients can be decreased or converted into other constituents [17,18]. Monotropein, which possess anti-inflammatory, analgesia, and antiosteoporotic activities, is the main iridoid glycoside isolated from M. officinalis [19,20]. Anthraquinones, especially alizarin-anthraquinones, like rubiadin and rubiadin 1-methyl ether, can inhibit osteoclastic bone resorption in vitro and invigorate the kidney-yang [21][22][23][24].
erefore, in the present study, we chose monotropein, rubiadin, and rubiadin 1-methyl ether as the representative compounds to explain the effects of the different processing methods on the pharmacokinetics of M. officinalis.
ere have been reports on the pharmacokinetics or tissue distribution of iridoids and oligosaccharides using HPC-DAD and LC-MS [25][26][27][28]. However, the abovementioned studies paid more attention to pure iridoid, inulin-type fructo-oligosaccharides and neglected interactions among iridoids and other compounds. ere still has been no report about the pharmacokinetics and tissue distribution characteristics of alizarin-anthraquinones in M. officinalis. Also, the effect of processing methods on the pharmacokinetics, bioavailability, and tissue distribution characteristics of iridoids and anthraquinones has barely been reported.
In this study, we developed a selective and sensitive UPLC-MS/MS method for simultaneous determination of monotropein, rubiadin, and rubiadin 1-methyl ether in rat plasma and tissues. e analysis method was successfully applied to the pharmacokinetics study of MO and SMO extracts.

LC-MS Analytical Conditions.
For LC-MS/MS analysis, data acquisition and instrument control were performed using MassLynx 4.1 software (Waters Corp., Milford, MA, USA). e analysis column was an Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm, Waters) with a temperature of 40°C. e mobile phase was acetonitrile containing 0.1% formic acid (A) and 0.1% formic acid aqueous solution (B). e elution gradient was 0.00-0.50 min with 2% B; 0.51-5.00 min with 2%-30% B; and 5.01-7.00 min with 40% B. e flow rate was 0.2 mL/min. e Waters triple quadruple mass spectrometer (Xevo TQD, Waters Corp., Milford, MA, USA) equipped with an electrospray ionization source (ESI) was used in negative ion mode. e desolvation gas was nitrogen with a flow rate of 500 L/h at a temperature of 250°C. All detected compounds were measured in multiple reaction monitoring (MRM) mode. Figure

Preparation of Reference Substances.
Monotropein, rubiadin, and rubiadin 1-methyl ether were dissolved in methanol to prepare a stock solution with a concentration of 0.5 mg/mL. e stock solution was diluted with methanol to get the appropriate concentrations for the working standard solutions. e IS stock solution (1 mg/mL) and IS working solution (200 ng/mL) were also prepared in methanol, as described above. All prepared solutions were stored at 4°C before use. e preparation of calibration standards was done as follows: e appropriate working solution was spiked into blank plasma or tissues to obtain concentrations of 75∼0.047 μg/mL for monotropein, 396∼1.54 μg/mL for rubiadin, and 596∼1.16 μg/mL for rubiadin 1-methyl ether. e QC concentrations for monotropein were 0.14 μg/mL, 2 μg/mL, and 28.1 μg/mL; for rubiadin, they were 1.6 μg/mL, 50.6 μg/mL, and 712.5 μg/mL; and for rubiadin 1-methyl ether, they were 3.9 μg/mL, 54.6 μg/mL, and 765 μg/mL. e QC samples used for the recovery, matrix effect, intra-and interday accuracy, precision, and stability studies were prepared in the same way as that of the calibration standards. e solutions were stored for one week at − 20°C.

Preparation of MO and SMO Extract.
We obtained the products of M. officinalis without wood and salt-processed M. officinalis by following processing methods.
MO: steam the clean Morindae officinalis Radix for 0.5 h, remove the woody cores while hot, cut into sections, and dry. SMO: steam the clean Morindae officinalis Radix with salt water for 4.0 h, until it is steamed thoroughly, remove the woody cores while hot, cut into sections and dry.
en, MO and SMO were refluxed with 80% ethanol three times for 1 h each to make a combined filtrate. en, the extracts were concentrated to 4 g·mL − 1 .
e contents of the three constituents in the MO and SMO extracts were detected by the same UPLC-MS/MS method, including the same column, mobile phase system, and column temperature as used for the pharmacokinetics and tissue distribution study. e contents of monotropein, rubiadin, and rubiadin 1-methyl ether in the MO and SMO extracts were 6.44, 0.088, and 0.174 and 3.95, 0.091, and 0.178 mg/g, respectively.
e concentrated extract was redissolved in distilled water.

Preparation of Biological
Samples. 10 μL of IS working solution and 400 μL of methanol were added to 100 μL plasma.
e solution was vortexed for 10 min and then centrifuged at 12,000 rpm for 5 min. e supernatant was shifted to another centrifugation tube and dried with nitrogen at 37°C. e residue was redissolved in 100 μL acetonitrile and centrifuged for 5 min at 12,000 rpm. Five microliters of supernatant was drawn and analyzed by UPLC-MS/MS. e whole tissue was cut into pieces and homogenized with ice-cold 0.9% (m/v) sodium chloride solution (1 : 9, w/v). Using 50 μL of tissue homogenate, the tissue samples were prepared in the same way as the plasma samples.

Method Validation
2.7.1. Specificity. Six individual sources of blank plasma were used to measure the specificity of this analysis method. Meanwhile, we also analyzed the lower limit of quantification (LLOQ) of samples, the IS in blank plasma samples, and plasma samples within 2 h of being subjected to the MO extract (40 g/kg).

Calibration Curve and Sensitivity.
e peak-area ratio of the compounds to the IS was plotted to obtain the linearity.
e weighted least-squares linear regression (weighting factor 1/X 2 ) was applied to determine the regression equations. e lowest concentration in the calibration curve was determined as the LLOQ with signal-tonoise of ≥10.

Precision, Accuracy, and Stability.
rough evaluating three levels of QC samples (n � 5) after one day and then for three days in a row, we obtained the intra-and interday precision, along with the accuracy data. e permitted range was within 15%.

Extraction Recovery and Matrix Effect.
ree different QC sample concentrations were used for the recovery experiment. e absolute extraction recovery was measured as the ratio of the QC sample concentration extracted from the blank plasma/tissue to those in the QC samples. e peak areas of the biosamples extracted in blank plasma/tissue versus those dissolved in methanol solution were measured to obtain the matrix effect.

Analysis of Pharmacokinetics.
e SD rats (6, male) were housed at 22 ± 2°C and fasted for 12 h with drinking water available ad libitum. MO and SMO ethanol extract were orally administered at a dose of 40 g/kg (equivalent to 6.44 and 3.95 mg/kg of monotropein, 0.088 and 0.091 mg/kg of rubiadin, and 0.174 and 0.178 mg/kg of rubiadin 1-methyl ether). Blood samples (0.5 mL) were collected from the venous plexus of the eye socket at 0.083, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 4.0, 6.0, 12.0, and 24 h under anesthesia. en, the blood samples were centrifuged at 4500 rpm for 10 min, and then the plasma was immediately transferred to new tubes and stored at − 80°C until analysis. e pharmacokinetic parameters were calculated using DAS software (3.0 version, China Food and Drug Administration) based on noncompartmental method. All data were recorded as mean ± SD.    was orally administered to SD rats (weight, 180-220 g, n � 6), at 0.25, 1, 3, and 6 h, respectively. Heart, liver, spleen, stomach, kidney, brain, and small intestine tissues were extracted, washed with saline, blotted with filter paper, weighed, and then stored at − 80°C.

Liquid Chromatography Optimization.
We tested mobile phase and gradient elution programs to determine the best chromatographic performance. With the mobile phase of water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid), the responses of the three components and two ISs were considerably better. To reach a better and more rapid separation effect, we optimized the gradient elution program.

Mass Spectrometry Optimization.
We carried out both positive and negative modes in mass spectrometry. ree components and the ISs all had high responses under the negative mode. e MS/MS transitions and parameters are given in Table 1.

Extraction Procedure Optimization.
Protein precipitation (PP) was used for the pretreatment of biological samples. We applied methanol and acetonitrile for PP. e samples had better peak shapes and recovery after being treated by acetonitrile, so we chose acetonitrile for further PP in this study.

Method Validation
3.4.1. Specificity. e MRM chromatograms of three components and the ISs are shown in Figure 2. e peak separation was better for the three components and the ISs under the established UPLC-MS/MS conditions, with no significant interference and no cross interference.

Linearity and Sensitivity.
e internal standard method was applied for the establishment of calibration curves, which showed good linearity (r 2 > 0.9907) in the linear ranges. e regression equations, correlation coefficients, linear ranges, and LLOQs of the three components in the plasma and tissues are listed in Table 2.

Precision, Accuracy, and Stability.
e range of intra and interday precisions was 0.21% to 5.96%, and the accuracy range was from − 8.06% to 1.33% for QC samples in plasma and tissues. ese values are in the acceptable range, and the results are shown in Table 3. Table 4 shows the results for the stability of the plasma and tissue samples in different conditions. ey are also in the acceptable range.

Extraction Recovery and Matrix Effect.
e range of extraction recoveries was 80.07∼98.64% for the three QC sample levels ( Table 5). ese data indicate that the sample treatment method is reasonable. No significant effect of endogenous substances was identified.

Pharmacokinetics Study.
After oral administration of MO and SMO extract (40.0 g/kg), we successfully determined the concentrations of monotropein, rubiadin, and rubiadin 1methyl ether in rat plasma using the established method. Figure 3 shows the mean plasma concentration-time profiles. Table 6 displays the pharmacokinetic parameters. e concentration of monotropein in SMO reached C max in 0.5 h, whereas the T max of monotropein in the MO groups was 1.0 h, which indicates that monotropein was quickly adsorbed into the blood after oral administration. e T max for rubiadin was 1.5 h, longer than that for rubiadin 1methyl ether, whereas the C max for rubiadin 1-methyl ether was the highest, especially in the SMO groups. In the plasma concentration-time curves of monotropein, an obvious double-peak phenomenon was found, which is related to enterohepatic recirculation. e pharmacokinetics properties shown in the present assay could be helpful for further studies on the pharmacokinetics of MO extract and further applications in different processing methods.

Tissue Distribution.
e concentrations of monotropein, rubiadin, and rubiadin 1-methyl ether in the liver, kidney, lung, and small intestine were determined at 0.25, 1, 3, and 6 h after administration. e results are shown in Figure 4. All compounds were detected in tissues, except for monotropein, which could not be quantified in the lung tissue.
is result indicates that monotropein might be rapidly transformed into its metabolites in the lungs, or the content of monotropein in the lung was lower. Meanwhile, all three components had higher concentrations in the small intestine, especially rubiadin and rubiadin 1-methyl ether in the SMO groups.

Conclusion
In this study, we established an efficient and accurate UPLC-MS/MS method for the determination of three components from MO and SMO in plasma and tissues after oral administration in rats. Meanwhile, this is the first simultaneous determination of monotropein, rubiadin, and rubiadin-1methyl ether in rat plasma and tissues. e study examined the pharmacokinetics and tissue distribution. e information found might partially illustrate their metabolic mechanisms in vivo, as well as provide a scientific basis for the strengthening of kidney-yang using the salt-processed principle of M. officinalis because of better bioavailability.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare no conflicts of interest.