Materials

The Nelumbo Seed materials used in this study were purchased by KOBAYASHI Pharmaceutical Co., Ltd. from a wholesaler. Because the amounts of materials were limited, the materials used for experiments, the results of which are presented in Figs. 1–3, were different. Information on Nelumbo Seed No.1 to 5 used in the rat excised bladder smooth muscle contraction inhibitory activity test and LC/MS chemical profiling is presented in Table 1. Information on Nelumbo Seed No. 6 to 9 used for neferine (Nef) quantification is presented in Table 2. The Nelumbo Seed materials used in the experiments on frequent urination model rats were purchased separately. Nelumbo Seed mentioned in Tables 2, 3 and Figs. 2, 3 were from a production site in Hunan Province, China.

Fig. 1

Inhibitory activity assay of bladder smooth muscle contraction and LC/MS metabolic profiling of Nelumbo Seed extract. a Photographs of the samples used in this study. The numbers are the same as those in Table 1. b Assay of the inhibitory activity of the extract on rat-excised bladder smooth muscle contraction. 0 and 100% of inhibitory activity indicate that the contraction length when the excised bladder was treated simultaneously with the Nelumbo Seed extract and carbamylcholine was the same as when treated with carbamylcholine alone, and was not observed, respectively. SRI: Seishinrenshiin; Tol: tolterodine (100 ng/mL, positive control). c LC/MS total ion current chromatograms of Nelumbo Seed extracts. d, e Results of OPLS between LC/MS peaks and rat-excised bladder smooth muscle contraction inhibitory activity. d Y fit, e VIP. f Mass chromatograms of m/z 625 from Nelumbo Seed extracts. g Area of m/z 625 peak at 10.1 min, assigned to neferine. The value on the vertical axis is an arbitrary value. h Scatter plot between neferine peak area and rat-excised bladder smooth muscle contraction inhibitory activity of Nelumbo Seed extracts. i Rat-excised bladder smooth muscle contraction inhibitory activity of neferine standard

Table 1 Nelumbo Seed materials used for the assay of inhibitory activity on excised rat bladder smooth muscle contraction and LC/MS metabolic profilingTable 2 Nelumbo Seed materials used for neferine quantitationTable 3 Neferine concentration in the plasma of rats administered the Nelumbo Seed extractFig. 2

Localization of neferine accumulation in Nelumbo Seed. a Photograph of Nelumbo Seed. Left, slice of seed; arrow-head shows the embryo. Middle, outside of the seed slice without the endocarp; Right, Nelumbo Seed without any treatment. b Photographs of the materials used in study on neferine accumulation in Nelumbo Seeds. c HPLC chromatograms of Nelumbo Seed extract (upper) and neferine standard solution (lower). Arrow indicates neferine

Fig. 3

Effects of Nelumbo Seed extract on frequent-urination model rat. a LC/MS mass chromatogram of blank (upper) and Nelumbo Seed extract-administered rat (lower). MRM transition are 625.77 > 206.5. Peak i, dauricine (internal standard), ii, neferine. b Neferine concentrations in Nelumbo Seed extract-administered rat plasma. c Raw data of urinary pressure and interval measurement. d, e Maximum bladder contraction pressure (d) and bladder contraction interval (e) in frequent-urination model rats administered a different type of Nelumbo Seed extract. Values above the bars indicate P values obtained using the one-tailed Dunnett’s test in comparison with a control group, which was administered normal feed and subjected to pelvic congestion

Preparation of Nelumbo Seed extracts for ex vivo and in vivo assays and LC/MS analysis

The extracts were prepared by an extract manufacturing company commissioned by KOBAYASHI Pharmaceutical Co., Ltd. Nelumbo Seeds (dry weight, 150–300 g) were crushed in a mill until they were reduced to approximately half of their original size. Five times the amount of distilled water was added and extraction was carried out at 95 °C for 1 h. The extract was filtered through a wire mesh with 0.109 mm openings and concentrated under a reduced pressure of 20 kPa or less at 60 °C or lower temperature using an evaporator. The concentrated liquid was then freeze-dried and the extract was powdered.

Reagents

LC/MS grade acetonitrile, distilled water, and formic acid used for LC/MS analysis were purchased from Fujifilm Wako Pure Chemical Industries (Osaka, Japan). HPLC grade methanol, distilled water, trifluoroacetate, and ammonium acetate, used for HPLC analysis, were obtained from Nacalai Tesque (Kyoto, Japan). Neferine, used for the activity test, was purchased from Sigma-Aldrich (now, Merck KGaA, Darmstadt, Germany).

Evaluation of bladder smooth muscle contraction inhibitory activity using excised rat bladder tissue

This test was commissioned by KOBAYASHI Pharmaceutical Co., Ltd. to the Shiga Research Institute of Nissei Baylis Co., Ltd. The experiment was approved by the Animal Experiment Committee of the testing facility (Approval number: 1610-21).

The Nelumbo Seed extracts used in this test were prepared by dissolving 300 mg of powdered extracts in 3 mL of water for injection (Fuso Pharmaceutical Industries, Ltd., Osaka, Japan).

Eight-week-old male Slc:SD rats (SPF grade; Japan SLC Co., Ltd.) were used for the experiment. After a 6-day quarantine period from receipt at the facility, the rats were acclimatized and used for the experiment. The rats were euthanized using excessive isoflurane anesthesia, and their abdomen was opened to remove the bladder. The bladder was cut vertically into strips, 2–3 mm wide and 5–10 mm long, to prepare the bladder specimens, with four specimens prepared from each animal. An organ bath (volume 10 mL, temperature 37 ± 1 °C) was filled with Krebs–Henseleit solution with a mixture of 95% O2 and 5% CO2 and suspended with a load of approximately 1 g. After equilibrating the specimen for 60 min, 50 μL of 2 mmol/L carbamylcholine solution (solvent: water for injection Fuso Pharmaceutical Industries, Ltd.) was added to the organ bath to get a final concentration of 10 μmol/L and the contractile response was recorded. After maximum contraction was achieved, the specimen was washed with the Krebs–Henseleit solution and allowed to rest for 15 min. During the rest period, the Krebs–Henseleit solution was replaced twice at 5 min intervals. Thereafter, the contractile response was recorded using the same procedure as followed for the first addition of carbamylcholine, and the specimen was washed with the Krebs–Henseleit solution and rested for 15 min. These procedures were repeated, and the contractile response to carbamylcholine was recorded for a total of three times. After recording the contractile response to the third carbamylcholine addition, the specimen was washed with the Krebs–Henseleit solution and rested for approximately 15 min. After the pause, 1 mL of each sample extract was added to the organ bath to obtain a final concentration of 10 mg/mL and incubated for 10 min. Carbamylcholine was then added, and the contractile response was recorded. The contraction width (mm) was measured using a ruler from the baseline immediately before the addition of carbamylcholine to maximum contraction width after the addition. The activity of each sample extract was defined as the decrease in contraction width upon addition of carbamylcholine after treatment with the Nelumbo Seed extract compared to the contraction width upon addition of carbamylcholine and without treatment with the Nelumbo Seed extract. Five samples of each extract were tested, and the average activity and standard deviation were calculated.

Chemical profiling of Nelumbo Seed extract using liquid chromatography/mass spectrometry

Nelumbo Seed extract powder was dissolved in acetonitrile, adjusted to 1 mg/mL, and then filtrated through a Millex-LG filter (disc diameter: 13 mm; pore size: 0.2 μm; Merck Life Science, Darmstadt, Germany).

LC/MS analysis was performed using an Acquity UPLC system (Waters Co., Milford, MA, USA) and a Quattro-Premier XE (Waters). LC separation was carried out at 40 °C using a TSKgel-Amide 80 column (5 μm particle size, 2.0 mm i.d. × 150 mm; Tosoh Co., Tokyo, Japan). The mobile phase was delivered at a flow rate of 0.250 mL/min. A gradient elution profile consisting of solvent A (0.1% formic acid in distilled water) and solvent B (acetonitrile) was employed. The initial composition of the binary solvent was 0% solvent A, which was increased to 95% over 20 min. The solvent A was maintained at 95% for 5 min. Ten microliter of the sample solution was injected into the column. The mass spectrometer was set to MS scan mode and operated with an electrospray ionization source in positive ion mode. The capillary and cone voltages were set to 4.5 kV and 35 V, respectively. The scanning was performed over an m/z range of 100–1,000. The scan and interval times were set at 0.95 and 0.05 s, respectively. The MassLynx software Ver 4.1 (Waters) was used for the operation of the LC–MS system and data acquisition.

Data analysis of LC/MS profile

The raw LC/MS data were converted to the netCDF format and uploaded to the MZmine 2.26 software [23]. The following operations were carried out using this software: i) scan by scan filtering (method: Savitzky–Golay filter; number of datapoints: 11), ii) mass detection (method: recursive threshold mass detector; noise level: 1.0 E5; minimum m/z peak width: 0.8; maximum m/z peak width: 1.5), iii) chromatogram builder (minimum time span: 0.05 min; minimum height: 3.0 E5; m/z tolerance: 0.5 or 500 ppm), iv) chromatogram deconvolution (method: local minimum search; chromatographic threshold: 1.0%; search minimum in RT range: 0.05 min; minimum relative height: 1.0%; minimum absolute height: 5.0 E5; minimum ratio of peak top/edge: 1; peak duration range: 0.0–15.0 min), v) isotopic peak grouper (m/z tolerance: 0.5 m/z or 500 ppm; retention time tolerance: 0.05 absolute min; monotonic shape: no; maximum charge: 2; representative isotope: lowest m/z), vi) alignment (method: join aligner; m/z tolerance: 0.5 m/z or 500 ppm; weight for m/z: 1; retention time tolerance: 0.05 min absolute; weight for RT: 1; isotope m/z tolerance: 0.5 m/z or 500 ppm; minimum absolute intensity: 1.0E4; minimum score: 70%), vii) export to csv format. This file contained information on 1,474 peaks. The exported data were opened in Microsoft Excel, and peaks that were detected in only one sample were deleted, leaving a total of 98 peaks. The excised rat bladder smooth muscle contraction inhibitory activity (%) was added to these data as an objective variable, and the peak IDs were modified for easy identification. These data were imported into SIMCA 13 (Sartorius Stedim Biotech, Goettingen, Germany), and orthogonal-projection to latent structure by means of partial least square (OPLS) was performed. The unit variance (UV) was used for scaling.

Quantification of neferine in Nelumbo Seed materials

Nelumbo Seed No. 5–8 were ground using a Wonder Blender (Osaka Chemical Co., Ltd., Osaka, Japan). The powdered sample (200 mg) was weighted and 3 mL of 1.0 mol/L ammonium acetate (pH 10.0)/methanol (1/99, v/v) was added. After shaking for 15 min, the mixture was centrifuged at 5000 × g for 5 min and the supernatant was collected. This procedure was repeated twice, and the resulting extracts were mixed and made up to 10 mL with the extraction solvent. This sample was filtered through a Millex-LG filer (disc diameter: 4 mm; pore size: 0.2 μm; Merck Life Science) and subjected to HPLC.

The HPLC pump, column oven, autosampler, and fluorescence detector were L-2100, L-2300, L-2200, and L-2485, respectively (Hitachi High-Tech Corporation, Tokyo, Japan). The column used was an InertSustain Phenyl column (5 μm particle size, 4.6 mm i.d. × 250 mm length; GL Sciences Inc., Tokyo, Japan). The column oven temperature was set at 40 °C. The eluents used were 0.1% trifluoroacetic acid in distilled water as solvent A, and methanol as solvent B and autosampler solvent. The flow rate was 1.0 mL/min, and the proportion of solvent B was 40% from 0 to 18 min. The sample volume was 10 μL. The excitation and emission wavelengths for fluorescence detection were set to 284 and 326 nm, respectively, based on the results for a Nef standard solution analyzed using a HITACHI F-2500 fluorescence spectrophotometer (Hitachi High-Tech). The data were acquired using a D-2000 Elite (Hitachi, High-Tech).

Effects of neferine in the Nelumbo Seed extract on frequent-urination model rat

The experiment was conducted at Southern Knights’ Laboratory, Co., Ltd. The experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of the Ryukyus (Approval number: A2017012, A2017189). A rat model of frequent urination was prepared as follows [24]: SD rats were subjected to laparotomy under isoflurane inhalation anesthesia, and both common iliac veins and both uterine veins were ligated, and 50 mg of cefamezin was administered subcutaneously. In the sham group, both common iliac veins were not ligated, but were only dissected from the common iliac artery. After suturing, the rats other than those in the sham group were divided into the following five groups that were fed different feeds: i) ‘Control’: normal feed, ii) ‘Used in KOBAYASHI Pharmaceutical’s Product’: feed contained 0.25% extract of Nelumbo Seed, which is used in KOBAYASHI Pharmaceutical’s SRI formulation, with embryo not completely removed, iii) ‘Removed Embryo’: feed contained 0.25% extract of Nelumbo Seed, with embryo removed completely, iv) ‘Sekirenshi’: feed contained 0.25% extract of ‘Sekirenshi’, Nelumbo Seed in which the endocarp and embryo were not removed at all, v) ‘solifenacin’: feed contained 0.01% solifenacin. The rats were raised for two weeks. Thereafter, a catheter was inserted into the bladder through the urethra, while the rats were under light urethane anesthesia (0.6 g/kg, subcutaneous injection) and restrained. One hour after the anesthetic effect appeared, continuous cystometric measurements were performed while saline was injected through the indwelling bladder catheter at a rate of 3 mL/h. The maximum bladder contraction pressure and bladder contraction interval were used as evaluation indices [24, 25].

Quantification of neferine in the plasma of model rats

To 490 μL of rat plasma, 10 μL of 500 ng/mL dauricine (PhytoLab GmbH & Co., KG, Vestenbergsgreuth, Germany) in methanol solution was added, followed by 500 μL of ethyl acetate. After shaking for 10 min, the mixture was centrifuged at 15,000 × g for 5 min and the supernatant was collected. Ethyl acetate (500 μL) was added to the remaining plasma layer and the mixture was shaken and centrifuged as described above. The collected supernatant was dried under a nitrogen stream. After removing the solvent, the residue was dissolved in 100 μL of acetonitrile/methanol (90/10, v/v) and filtered through a Millex-LG filter (4 mm, pore size 0.2 μm; Merck Millipore). LC/MS analysis was performed using an Acquity UPLC and a Quattro-Premier XE. LC separation was carried out at 40 °C using an Acquity UPLC BEH Amide column (1.7 μm particle size, 2.1 mm i.d. × 50 mm length; Waters). The mobile phase was delivered at a flow rate of 0.5 mL/min using a gradient elution profile comprising solvent A (0.1% formic acid, 10 mmol/L ammonium formate in methanol) and solvent B (0.1% formic acid, 10 mmol/L ammonium formate in acetonitrile/methanol (90/10, v/v). The composition of solvent B was 100% (0–0.5 min), 100–90% (0.5–1.5 min), 90–10% (1.5–2.0 min), 10% (2.0– 3.0 min), 10–100% (3.0–3.1 min), and 100% (3.1–6.0 min). The sample volume injected into the column was 10 μL. The mass spectrometer was set to the multiple reaction monitoring (MRM) mode and operated with an electrospray ionization source in the positive ion mode. The capillary voltage was set at 4.5 kV. MRM transitions (m/z of precursor ion > m/z of product ion) for Nef and dauricine measurement were 625.8 > 206.5 (for quantification), 625.8 > 121.3 (for confirmation), and 625.8 > 489.7 (for confirmation). The cone voltage was 56 V, and the collision energies were 34 (for 625.8 > 206.5), 68 (for 625.8 > 121.3), and 36 (for 625.8 > 489.7) eV, respectively. The dwell time for each MRM transition was 0.1 s. The MassLynx software Ver 4.1 (Waters) was used for the operation of LC–MS and data acquisition.

Statistical analysis

The statistical significance of differences in the maximum bladder contraction pressure and bladder contraction interval between rats administered the Nelumbo Seed extract and control rats, which consumed normal feed and were subjected to pelvic congestion, was assessed with one-tailed Dunnett’s test performed using the R software.