Adult male C57BL/6N mice (Narabiotec Co., Ltd., Seoul, Republic of Korea, 8–10 weeks of age, 22–25 g body weight were purchased from Taconic Biosciences Inc. (Hudson, NY, USA). The mice were housed at a constant temperature of 23 ± 2°C with a 12-h light-dark cycle (lights on from 08:00 to 20:00) and provided with food and water ad libitum. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Kyung Hee University. Proper randomization of laboratory animals and handling of data were performed in a blinded manner in accordance with recent recommendations from an NIH workshop on preclinical models of neurological diseases (Landis et al., 2012).

SSC was obtained from Mungyeong-si Distribution Corporation (Mungyeong, Republic of Korea). The material was air-dried in a shaded lot and cut into small pieces (3–5 mm-thickness; Figure 1A). A voucher specimen (KHKM0030) was deposited at the Laboratory of Autoimmune and Neurodegenerative Diseases, Department of Convergence Science, College of Korean Medicine, Kyung Hee University. The SSC (200 g) was extracted with distilled water (2 L) for 1.5 h at room temperature, further extracted by continuous heating for 1.5 h under reflux conditions with hot water, and subsequently filtered by vacuum filtration. The filtrate was concentrated using a rotary vacuum evaporator (EYELAN-1200A, EYELA; Rikakikai Co. Ltd, Tokyo, Japan), lyophilized using a freeze drying system (OPR-FDA-8612; Operon Co. Ltd, Gimpo, Republic of Korea), and stored in a freezer (–80°C). The final yield of the SSCE was 10.55%.

Gomisin A, gomisin N, and Schizandrin were obtained from Wuhan ChemFaces Biochemical Co., Ltd. (Wuhan, China) and Sigma-Aldrich Co. (St. Louis, MO, USA). Quantitation of SSCE using HPLC-UV was accomplished by using a modification of a previously described protocol (Bae et al., 2012). Briefly, standard solutions containing each lignin were prepared in concentrations ranging from 5 to 250 ng/mL. One hundred milligrams SSCE was dissolved in 1 ml of distilled water. All standard and SSCE solutions were filtered through a 0.20 μm syringe filter (Merck Millipore Co., Schwalbach, Germany). The HPLC apparatus consisted of a model 1260 pump (Agilent Technologies, Santa Clara, CA, USA), a model G1367D autosampler, and a model G1315C DAD. Masshunter software (Agilent Technologies) was used. A YMC TRIART C18 column (50 × 2.0 ID-1.9 μm) was used for the HPLC analysis. The detection wavelength was set to 254 nm. The mobile phase contained acidified water with formic acid (0.1%, solvent A; Wako Pure Chemical Industries, Ltd., Osaka, Japan) and acidified acetonitrile (J.T. Baker Chemicals Co., Center Valley, NJ, USA) with formic acid (0.1%, solvent B). The gradient program consisted of 10% solvent B for 2 min, 50% solvent B for 20 min, and then was linearly increased to 80% solvent B for another 30 min. This linear gradient was followed by isocratic elution for 35 min and reconditioning steps to return to the initial mobile phase. The flow rate was 0.2 ml/min, and the injection volume of the standards and SSCE was 5 μL. A calibration curve was established using a methanol stock solution containing each dibenzocyclooctane lignan diluted to a specified concentration. The coefficient values (r²) were 0.999 and 1, demonstrating that the linearity in this range was sufficient to provide a highly accurate estimate of the content of gomisin A, gomisin N, and schizandrin in SSCE. Precision was determined using triplicate measurements of each standard, and the relative standard deviations (RSDs) were <2.2% (Figure 1).

The experiment was carried out in three stages. First, to determine the most effective dose and mechanism of administration of SSCE for pretreatment, mice were randomly divided into the following groups: sham (n = 10), 3-NPA (n = 16), 3-NPA + SSCE pretreatment (75, 150, and 300 mg/kg; n = 16 per each dose), and SSCE (n = 10). Second, to investigate the therapeutic time window of SSCE use, mice were randomly divided into sham (n = 4), 3-NPA (n = 9), 3-NPA + SSCE pre- (n = 9), onset- (n = 9), progression- (n = 9), and peak-treatment (n = 9), and SSCE (n = 4) groups. Third, to identify the active component(s) in SSCE, mice were randomly divided into sham (n = 4), 3-NPA (n = 7), 3-NPA + gomisin A, gomisin N, or schizandrin (n = 7 per each chemical), and gomisin A, gomisin N, or schizandrin alone (n = 4 per each chemical) groups. The group details are displayed in Table 1.

3-NPA was administered as previously described (Huang et al., 2006; Jang et al., 2013, 2014; Jang and Cho, 2016). Briefly, 3-NPA was dissolved in physiological saline to a concentration of 100 mg/ml (pH 7.4), passed through a 0.2 μm filter to remove any bacteria, and kept at –80°C until use. The 3-NPA solution was given intraperitoneally (i.p.) twice daily for 2 days at 12-h intervals (8:00 a.m. and 8:00 p.m.) at a dose of 60 mg/kg on the first day and 80 mg/kg on the second day (60–60–80–80 dose regimen). SSCE was prepared in physiological saline and administered at doses of 75, 150, or 300 mg/kg to determine the most effective dose. Subsequently, 300 mg/kg SSCE was given at the time of onset, progressive, and peak stages to investigate its therapeutic time window. Each experiment was repeated at least three times using the same protocol. Total daily dose of SSCE for mice was determined by formula for dose translation based on body surface area (Reagan-Shaw et al., 2008) after considering body weight of animals, final extract yield, and traditional dose in humans. Gomisin A (20 mg/kg), gomisin N (20 mg/kg), and schizandrin (45 mg/kg) were treated peritoneally once daily from 30 min before 3-NPA treatment.

To assess the severity of 3-NPA-induced neurological disorders, the previously described motor behavioral scale (Fernagut et al., 2002) and our standard for each subcriteria (Jang et al., 2014) were utilized. Briefly, the three-level (0, 1, and 2) scale was used to measure the severity of the following five items (maximal score = 10), which constituted the main motor symptoms observed: hindlimb clasping, global activity in a free-moving environment, hindlimb dystonia, truncal dystonia (kyphotic posture), and balance adjustment to a postural challenge. The neurological test was performed by an experimenter who was unaware of the experimental conditions and was done under constant temperature (23 ± 2°C) and humidity (55 ± 5%) in a quiet room, 24 h or 5 days after the final treatment.

We used our previous protocol to evaluate the histopathological changes in the striatum (Jang et al., 2013, 2014; Jang and Cho, 2016). Briefly, 24 h after the last 3-NPA treatment, the mice were anesthetized with ethyl ether and then perfused intracardially with saline and cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brains (n = 5 in the sham group, n = 5 in the 3-NPA group, n = 7 in the 3-NPA + SSCE 75 mg/kg group, n = 7 in the 3-NPA + SSCE 150 mg/kg group, n = 7 in the 3-NPA + SSCE 300 mg/kg group, and n = 5 in the SSCE group) were immediately removed, post-fixed overnight in the same fixative at 4°C, and serially cryoprotected in 10, 20, and 30% sucrose in PBS for 48 h at 4°C. Sequential coronal sections (30 μm thickness) were acquired on a model CM3050S freezing microtome (Leica Biosystems, Wetzlar, Germany), starting from the anterior aspect of the corpus callosum and proceeding through the entire striatum (bregma 1.40–1.30 mm), according to the mouse brain atlas (Franklin and Paxinos, 2008). For histological assessment, every tenth section (at intervals of 300 μm) was processed for cresyl violet staining, dehydrated, and coverslipped with Permount (Fisher Scientific, Waltham, MA, USA). The stained sections (n = 3 per brain) from the level of the mid-striatum were captured using a DP70 image analysis system (Olympus, Tokyo, Japan) and the level of 3-NPA-induced striatal damage was analyzed using the NIH Image J program (http://rsb.info.nih.gov/ij/).

A previously developed protocol (Jang et al., 2013, 2014; Jang and Cho, 2016) was used to investigate SDH activity in the striatum and the calf muscle. Briefly, fresh brains (n = 3 per group) were rapidly frozen in isopentane, sectioned at a thickness of 10 μm, and mounted on glass slides. The sections were incubated for 15 min in PBS at 37°C to activate the SDH. The three brain sections were washed in PBS and incubated in 0.05 M sodium succinate (Sigma-Aldrich), 0.3 mM nitroblue tetrazolium (Sigma-Aldrich), and 0.05 M PB (pH 7.6) for 20 min at 37°C. The sections were then rinsed in cold PBS for 5 min and dried at room temperature. Each section was digitally imaged using a DP70 image analysis system (Olympus) for quantitative microscopy. Quantification was performed as described previously using the Image J program. SDH activity was quantified in the cerebral cortex, and the striatum as regions of interest (ROIs) delineated on a digitized image. Extraction of color in each section was taken as the saturation value, and this value was used to represent SDH activity (% of sham group).

Immunohistochemical analysis of striatal sections was performed as previously described (Jang et al., 2013, 2014; Jang and Cho, 2016). Briefly, brain sections (30 μm thick) from each group (n = 5–7 per group) were incubated with 3% hydrogen peroxide in PBS and washed in PBS. The sections (n = 3 per brain) were then blocked with a solution containing 5% normal goat or horse serum, 2% bovine serum albumin, 2% fetal bovine serum, and 0.1% Triton X-100 for 2 h at room temperature. The sections were incubated overnight at 4°C with either rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1; 1:2,000; WAKO, Osaka, Japan), rabbit anti-glial fibrillary acidic protein (GFAP; 1:5,000; DAKO, Carpinteria, CA, USA), or rabbit anti-cleaved caspase-3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA). The sections were washed in PBS and then incubated with biotinylated rabbit/mouse IgG antibody (1:200; Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. After rinsing, the sections were incubated with avidin–biotinylated horseradish peroxidase (HRP) complex (1:200; Vector Laboratories) for 1 h at room temperature and visualized with 3,3′-diamino-benzidine (DAB). The sections were rinsed, dehydrated, and cover-slipped with Permount.

Western blot analysis was performed as previously described (Jang et al., 2013, 2014; Jang and Cho, 2016). Briefly, 24 h after the last injection of 3-NPA, mice (n = 3 per group) were anesthetized and the striatum was removed with lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, and protease inhibitor mixture). A total of 30 μg of tissue lysate from each sample was resolved by 10% SDS-PAGE. The proteins were transferred to polyvinylidene fluoride membranes and the membranes were blocked with 5% nonfat dry milk in Tween 20-containing Tris-buffered saline (TBST; 20 mM Tris, pH 7.4, 0.1% Tween 20, and 150 mM NaCl). The membranes were probed overnight with primary antibodies at 4°C, followed by incubation with HRP-conjugated secondary antibody at room temperature for 1 h prior to enhanced chemiluminescence analysis (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and exposure to X-ray film. The primary antibodies included mouse anti-β-III-tubulin and rabbit ant-iNOS (1:500; Sigma-Aldrich), rabbit anti-phospho (p)-c-JUN N-terminal kinase (JNK), p-extracellular signal-regulated kinase (ERK), p-p38, p-IκBα, p-NF-κB p65, and cleaved caspase-3 (1:1,000; Cell Signaling Technology), rabbit anti-interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and mouse anti-nicotinamide adenine dinucleotide phosphate dehydrogenase (quinone) 1 (NQO1; 1:1,000; Cell Signaling Technology), rabbit anti-Iba-1 (1:1,000; WAKO), rabbit anti-GFAP (1:1,000; DAKO), rabbit anti-Nrf2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-cyclooxygenase-2 (COX-2; 1:500; BD Biosciences, San Jose, CA, USA), and mouse anti-heme oxygenase-1 (HO-1; 1:1,000; Enzo Life Sciences, Farmingdale, NY, USA) antibodies. For normalization of the antibody signal, the membranes were stripped and reprobed with antibodies against JNK, ERK1/2, p38 (1:2,000; Cell Signaling Technology), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000; Cell Signaling Technology). After Western blot was performed several times, the density of each band was converted to a numerical value using the Photoshop CS2 program (Adobe, San Jose, CA, USA) after subtracting background values from an area of film immediately adjacent to the stained band. Data are expressed as the ratio of p-JNK, p-ERK, p-p38, p-IκBα, p-NF-κB, cleaved-caspase-3, β-III-tubulin, Iba-1, GFAP, IL-1β, IL-6, TNF-α, iNOS, COX-2, Nrf2, HO-1, and NQO-1 against total JNK, ERK 1/2, p38, or GAPDH for each sample.

The fragmentation of DNA was examined using an ApopTag® Peroxidase in situ Apoptosis Detection Kit (S7100) (Millipore) according to the manufacturer's instructions. Briefly, brain sections from each mouse (n = 5 per group) were subjected to enzymatic digestion with 20 μg/ml proteinase K for 5 min, treated with 5% hydrogen peroxide for 20 min to exhaust endogenous peroxidase activity, and washed with PBS. They were then immersed in ApopTag® equilibration buffer for 10 min to label the 3′-OH ends of fragmented DNA and incubated with terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) at 37°C for 1 h. After being washed with PBS, the sections were incubated with DAB to detect signs of apoptotic cell death.

To examine whether SSCE has a toxic effect when used over the long term in mice, SSCE (16, 80, 400, and 2,000 mg/kg/day) was orally administrated to normal, 9-week-old, male mice for 15 days. Body weight, food intake, and water intake were measured daily and serum was obtained at 24 h after the last administration of SSCE. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were measured using enzymatic or ultraviolet assays with an ALT, AST, or LDH detection kit (Roche, Basel, Switzerland) according to the manufacturer's instructions with a Cobas 8,000 modular analyzer (Roche) using a previously described protocol (Jang et al., 2014; Choi et al., 2015). General histological structure was evaluated by hematoxylin & eosin (H&E) staining as previously described.

Statistical analyses were performed using the SPSS 21.0 package (SPSS Inc, Chicago, IL, USA) for Windows. Two-sample comparisons were carried out using the Student's t-test and multiple comparisons were made using two-way ANOVA with Tukey's post hoc test. All data are presented as means ± S.E.M. and statistical difference was identified at the 5% level unless otherwise indicated.

3-NPA-induced neurological dysfunction results from neuronal death in the striatum (Fernagut et al., 2002; Jang et al., 2013, 2014; Jang and Cho, 2016). Appropriately, we investigated whether SSCE reduced striatal cell death. Twenty-four hours after the last (4th) 3-NPA treatment, brain slices including the striatum were stained with cresyl violet dye. Figure 3 shows representative striatal images from the sham, 3-NPA, and 3-NPA + SSCE (75, 150, and 300 mg/kg/day) groups. Fifty-five percent (n = 5/11) of the surviving 3-NPA-treated mice had visible bilateral striatal lesions (pale areas surrounded by dotted line), while the percentage of mice with striatal lesions was decreased to 36.3% (n = 4/11) and 20% (n = 2/10) in the groups pretreated with 75 and 150 mg/kg/day of SSCE, respectively (Figures 3A–G). Interestingly, visible striatal lesions were not detected in the group pretreated with 300 mg/kg/day of SSCE (0%; 0/10; Figures 3E,G). The ratio of the mean lesion area to the entire striatum was 28.7%, while the ratio was significantly reduced to 9.2% in the 150 mg/kg/day SSCE group (Figure 3H). Additionally, β-III-tubulin, a microtubule element of the tubulin family that is found almost exclusively in neurons, showed decreased expression in the striatum in the 3-NPA group (0.2 ± 0.01) compared with the striatum in the sham group (0.3 ± 0.01), whereas the decrease in expression was significantly rescued by pretreatment with SSCE (0.3 ± 0.01) (Figure 3I). The findings suggest that SSCE can reduce 3-NPA-induced striatal toxicity.

3-NPA induces striatal neuronal cell death by inhibiting mitochondrial SDH activity (Fernagut et al., 2002; Jang et al., 2013, 2014; Jang and Cho, 2016). Appropriately, changes in SDH activity were examined by histochemical analysis in the striatum 24 h after the last (4th) 3-NPA treatment. Figure 4 shows representative striatal images from the sham, 3-NPA, 3-NPA + SSCE (300 mg/kg/day), and SSCE groups (Figures 4A–D). When the regional difference in SDH activity was semi-quantified, a decreased of 78.2 ± 1.6% was evident as compared to activity in striatum from sham group (100 ± 0.3%). However, SDH activity was significantly increased to 90.6 ± 2.4% by SSCE pretreatment (Figure 4E). SSCE itself did not significantly affect SDH activity in the striatum (99.1 ± 0.4%). The findings suggest that SSCE may alleviate 3-NPA-induced striatal toxicity by blocking the decline of SDH activity.

Since the depletion of SDH activity in the striatum caused by 3-NPA leads to apoptosis in the striatum (Tunez et al., 2010; Jang et al., 2013, 2014; Jang and Cho, 2016), terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining (Figures 5A–D) and immunohistochemistry (Figures 5E–H) were used to determine whether SSCE reduces striatal apoptosis 24 h after the last (4th) 3-NPA treatment. The number of TUNEL-positive cells was significantly increased in the striatum in the 3-NPA group (Figures 5B,I) compared to the sham group (Figure 5A). But, the number of TUNEL-positive cells was significantly decreased in the 3-NPA + SSCE (300 mg/kg/day) group (Figures 5C,I). Also, the number of cleaved caspase-3-immunoreactive cells was increased in the striatum of the 3-NPA group (Figure 5F), as compared with the sham group (Figure 5E), while that number was significantly decreased in the 3-NPA + SSCE group (Figure 5G) as compared to the 3-NPA group, which corresponded to the expression pattern of cleaved caspase-3 protein as assessed by Western blot analysis (Figure 5J). SSCE itself did not induce apoptosis in the striatum as compared to the sham group. The results suggest that SSCE may reduce neurological dysfunction and striatal toxicity via inhibiting apoptosis in the striatum after 3-NPA treatment.

Microglia are activated in neurological diseases including HD, and activated microglia release pro- and anti-inflammatory cytokines (Pavese et al., 2006; Lobsiger and Cleveland, 2007; Ferger et al., 2010). Therefore, we examined whether SSCE could inhibit microglial activation in striatal lesions after 3-NPA treatment (Figure 6). In striatal tissue from the 3-NPA group, Iba-1 (a marker for microglia/macrophage lineage cells)-immunoreactive cells; (Ferger et al., 2010; Jang et al., 2013, 2014; Jang and Cho, 2016) displayed activated morphology with enlarged cell bodies and short and thick processes (Figure 6B) compared to the sham group (Figure 6A), which generally showed the typical forms of resting cells including small cell bodies and thin processes. However, the level of activation of Iba-1-immunoreactive cells was reduced in the striatum in the 3-NPA + SSCE group (Figure 6C) compared to the 3-NPA group (Figure 6B), corresponding to a change in protein expression of Iba-1 as measured by immunoblot analysis (Figure 6I). Microglia were not activated by treatment only with SSCE (300 mg/kg) (Figures 6D,I). Astrocytes may be activated by the debris from dead cells or by activated microglia within or around neurodegenerative lesions (Lobsiger and Cleveland, 2007). However, in the current study, GFAP-immunoreactive astrocytes were not significantly affected within or around the striatal lesions 24 h after the last (4th) 3-NPA treatment compared with those in the sham and SSCE groups (Figures 6E–H), which reflects a lack of change in GFAP protein expression (Figure 6J). The findings suggest that SSCE inhibits microglial activation, but not astroglial activation, and is associated with a reduction in neurological impairment and striatal cell death.

Since activated microglia within or around lesions secrete factors that can be either beneficial or detrimental to neuronal survival (Pavese et al., 2006; Lobsiger and Cleveland, 2007; Ferger et al., 2010), we examined whether the inhibition of microglial activation induced by SSCE (Figures 6A–D,I) is closely connected with the down-regulation of inflammatory factors in the striatum. Immunoblot analysis determined that protein expression levels of IL-1β, IL-6, TNF-α, iNOS, and COX-2 were markedly increased by 0.49-, 1.01-, 1.10-, 1.23-, and 0.98-fold, respectively, in the striatum 24 h after the last (4th) 3-NPA treatment, while their expression levels were significantly decreased by 36, 32, 34, 41, and 31%, respectively, in striatal tissue from the 3-NPA + SSCE group (300 mg/kg/day) compared with the 3-NPA group (Figures 6K–O). These results suggest that SSCE can mitigate the neurological dysfunction and striatal toxicity induced by 3-NPA treatment by suppressing the expression of inflammatory modulators in the striatum.

Schisandra chinensis and its components have antioxidant and neuroprotective effects through Nrf2 transcriptional activation (Chen et al., 2008; He et al., 2014; Giridharan et al., 2015). Therefore, we investigated the effect of SSCE on the Nrf2 pathway. Nrf2, HO-1, and NQO-1 protein expressions were significantly increased (0.50-, 0.57-, and 0.67-fold, respectively) by 3-NPA treatment compared to expression in the sham group, whereas it was further increased (0.85-, 0.93-, and 0.99-fold, respectively) by SSCE (300 mg/kg) pretreatment compared to expression in the 3-NPA group (Figures 7A–C). Collectively, these results indicate that SSCE exerts its antioxidant activity through stimulation of the Nrf2 pathway in the striatum.

Since MAPKs and the NF-κB pathway are activated in the striatum by 3-NPA treatment (Sugino et al., 2000; Khoshnan et al., 2004; Zheng et al., 2014), we tested the modulatory effect of SSCE on these pathways in the striatum after 3-NPA treatment. As expected, phosphorylation of ERK, JNK, and p38 proteins was significantly increased by 66.4-, 223.2-, and 42.9-fold, respectively, in the striatum 24 h after the last (4th) 3-NPA treatment, and by 59, 47, and 69%, respectively, compared with phosphorylation in the sham and SSCE alone groups, whereas the enhancement in activation was significantly reduced in the striatum in the 3-NPA + SSCE group (Figures 7D–F). We examined whether SSCE modulates the expression of NF-κB in the striatum after 3-NPA treatment. Protein expressions of p-NF-κB and p-IκBα were significantly increased by 68.0- and 49.4-fold, respectively, in the striatum 24 h after the final 3-NPA treatment, by 49 and 54%, respectively, compared to expression in the sham group, while the enhancement in activity was significantly decreased by SSCE treatment (300 mg/kg/day; Figures 7G,H). Activation of MAPKs or the NF-κB pathway was not increased or decreased by SSCE treatment alone (Figure 7). These results indicate that SSCE may reduce striatal toxicity and neurological dysfunction after 3-NPA treatment by suppressing MAPKs and the NF-κB pathways.

To identify the active neuroprotective components in SSCE, we examined the effects of certain representative components (gomisin A, gomisin N, and schizandrin) on 3-NPA-treated mice. Pretreatment with gomisin A (20 mg/kg, i.p.) and schizandrin (45 mg/kg, i.p.) alleviated neurological impairment (6.8 ± 0.4 and 5.6 ± 0.4, respectively) and increased the survival rate (71.4% in both) 24 h after the final (4th) 3-NPA treatment compared to neurological impairment (8.7 ± 0.6) and survival rate (42.9%) in the 3-NPA group (Figures 8A,B). Although gomisin N (20 mg/kg, i.p.) did not significantly reduce neurological impairment (7.1 ± 0.5; p = 0.0542), increased survival rate (57.1%) (Figures 8A,B). These results indicate that gomisin A, gomisin N, and schizandrin may be the active components responsible for the beneficial effect of SSCE on 3-NPA-induced striatal toxicity.

Finally, to examine if SSCE has toxicity, we orally treated to normal male mice (9-weeks-old) with SSCE (16–2,000 mg/kg/day) for 15 days and assessed toxicity. Daily body weight (Supplementary Data 1A), daily feed intake (Supplementary Data 1B), daily water intake (Supplementary Data 1C), histological structure of liver (Supplementary Data 1D–H) and kidney (Supplementary Data 1I–M), and mean serum levels (AST, ALT, and LDH; Supplementary Data 1N–P) were not affected by administration of SSCE. Results indicate that prolonged administration of SSCE does not induce toxicity.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer EC and handling Editor declared their shared affiliation.