Phytochemical profile of S. horneri extract and its cell viability assay
The approximate composition of the dried S. horneri samples was as follows: 14.19% moisture, 45.99% carbohydrates, 20.93% ash, 4.37% crude fat, and 14.52% crude protein. In this study, a 70% EtOH extract fraction was obtained from S. horneri dried powder, and samples of this extract were sequentially subdivided with equal volumes of n-BuOH, n-Hexane, EtOAc, CH2Cl2, and water. After vacuum evaporator of these solvents, the sequentially partitioned subfractions were collected, 6.9 g of n-Hexane (9.8%), 8.6 g of CH2Cl2 (12.1%), 0.25 g of EtOAc (0.3%), 5.1 g of n-BuOH (7.2%), and 47.15 g of water (67.3%) as shown in Fig. 1a. Thereafter, we evaluated the cytotoxicity of S. horneri 70% EtOH extract and its subfractions using RAW264.7 cells via the MTT assay. After being seeded in microplates, the macrophages were treated with 25–200 µg mL− 1 concentrations of S. horneri 70% EtOH extract and its subfractions. The results indicate that treatment with concentrations of up to 100 µg mL− 1 of the 70% EtOH extract as well as its Hexane-, CH2Cl2-, and EtOAc-soluble subfractions exerted no toxic effect on cell viability. Concentrations of the BuOH- and water-soluble subfractions up to 200 µg mL− 1 had no toxic effect on cell viability (Fig. 1b). Therefore, non-toxic ranges were selected for the subsequent evaluation of in vitro anti-inflammatory effect.
Among all the prepared fractions, the 70% EtOH extract was used to identify potential bioactive compounds in S. horneri using an HPLC analysis UV-chromatogram at a 210 nm absorption wavelength, and fucosterol was detected after a retention time of approximately 20.6 min. The fucosterol peak area for the S. horneri extract in the HPLC chromatogram was substituted with a fucosterol calibration curve to analyze the contents. The S. horneri 70% EtOH extract contained approximately 34.4 ± 0.75 µg mg− 1 of fucosterol contents (C29H48O, tR 20.5, MW 412.7) are shown in Fig. 1c, 2a.
Moreover, ultra-performance liquid chromatography–tandem quadrupole time-of-flight–high-resolution mass spectrometry (UPLC-QTOF-HRMS) analysis of the positive and negative modes of the UPLC-MS analysis demonstrated significant bioactive compound diversity, as shown in Fig. 1(d-e). The ion peaks intensity chromatograms in positive mode and MS information of the compounds were analyzed using Elements Viewer (version 2.1). The base peak (b) in Fig. 1d, benzophenone (C6H5COC6H5, tR 1.67, m/z 205.06 [M + Na]+) (PubChem CID:3102), peak (c) (2S,3S)-4-benzyloxy-1-(tert-butyldimethylsi1yloxy)-3-methylbutan-2-ol (C18H32O3Si, tR 25.34, m/z 325.22 [M + H]+), peak (d) denatonium (C21H29N2O+, tR 25.38, m/z 325.23 [M + H]+) (PubChem CID:15488), peak (e) D-(+)-Cellobiose octaacetate (C28H38O19, tR 30.68, m/z 701.18 [M + Na]+) (PubChem CID:107429), peak (f) 1-hydroxy Vitamin D3 (C27H44O2, tR 46.07, m/z 383.33 [M + H-H2O]+) (PubChem CID:5283731), peak (g) deoxykhivorin (C32H42O9, tR 46.10, m/z 593.27 [M + Na]+) (PubChem CID:6708722), and peak (h) pheophytin A (C55H74N4O5, tR 51.92, m/z 871.57 [M + H]+) (PubChem CID:135398712). The peak intensity chromatograms of S. horneri dried sample in negative UPLC-MS ionization mode (Fig. 1e, 2i-l), peak (i) 5-methoxysalicylic acid sulfate (C8H8O7S, tR 3.19, m/z 246.99 [M-H]–), peak (j) palmitoleoyl 3-carbacyclic phosphatidic acid (C20H39O5P, tR 25.26, m/z 369.22 [M-H-H2O]–) (PubChem CID:10178491), peak (k) 3-Hydroxybenzoic acid sulfate (C7H6O6S, tR 5.14, m/z 216.98 [M-H-H2O]–) and peak (l) Asn-Thr-Lys (C14H27N5O6, tR 43.81, m/z 721.39 [2M-H]–) (PubChem CID:145454267) was identified.
S. horneri in vitro antioxidant activity
The antioxidant activity of the S. horneri 70% EtOH extract and its subfractions was evaluated by scavenging DPPH and ABTS radicals. This method is simple and widely used to ascertain S. horneri’s antioxidant properties. The results indicate that DPPH and ABTS radical-scavenging activities gradually increased with increasing concentrations of the S. horneri EtOH extract and its subfractions, as shown in Table 2. In particular, the CH2Cl2 subfraction yielded a relatively low IC50 value (0.28–0.44), exhibiting comparatively high scavenging ability for both DPPH and ABTS radicals. The EtOAc-soluble fraction also displayed superior potency for scavenging DPPH and ABTS radicals, yielding a relatively low IC50 value of 0.3–0.47. Overall, the CH2Cl2-subfraction exerted the most remarkable effect compared with the other fractions.
Table 2
Total yield (%) and antioxidant properties (IC50 values) of the S. horneri 70% EtOH extract and its subfractions
S. horneri fractions | Yield (%) | DPPH (IC50 mg mL− 1) | ABTS (IC50 mg mL− 1) |
70% EtOH extract | 19.7 | 1.5 | 1.5 |
n-Hexane fraction | 9.8 | 0.75 | 1.13 |
CH2Cl2 fraction | 12.1 | 0.28 | 0.44 |
EtOAc fraction | 0.3 | 0.34 | 0.47 |
BuOH fraction | 7.2 | 0.62 | 1.06 |
Water fraction | 67.3 | 3.5 | 2.75 |
DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); IC50: half maximal inhibitory concentration.
Impact of the S. horneri extract and its subfractions on inflammatory cytokines production in LPS-treated RAW264.7 cells
NO is a short-lived free radical, and excessive NO can induce pro-inflammatory responses and inflammatory disorders (Ren et al. 2020). Therefore, NO production inhibition may be of therapeutic benefit for diseases related to NO overproduction. In this study, the macrophages were pretreated with different concentrations (25–200 µg mL− 1) of the S. horneri 70% EtOH extract and its subfractions and subsequently stimulated for 24 h with LPS (1 µg mL− 1). The results reveal that NO levels increased in LPS-induced RAW264.7 macrophages, and they were subsequently attenuated by increasing the concentrations of the S. horneri 70% EtOH extract and its subfractions (Fig. 3a). These observations indicate that S. horneri’s bioactive compounds, fucosterol, may significantly reduce NO production in macrophages by inhibiting iNOS expression in a dose-related manner.
To determine whether a reduction in NO can regulate the inflammatory response, subsequent experiments were performed to analyze TNF-α, IL-6, and PGE2 levels in LPS-stimulated macrophages using the ELISA method. During this process, the cells were pretreated with S. horneri extract and its CH2Cl2- (12.1%) and water-soluble (67.3%) subfractions owing to their high yield percentage; thereafter, they were treated with LPS for 24 h. The results clarify that LPS treatment significantly induced IL-6, TNF-α, and PGE2 production, while pre-treatment with the S. horneri 70% EtOH extract and its CH2Cl2- and water-soluble subfractions significantly (p < 0.001) reduced PGE2 levels (Fig. 3b). In addition, LPS-induced pro-inflammatory cytokine (TNF-α and IL-6) expression was significantly suppressed (p < 0.001) by the 70% EtOH extract and its CH2Cl2- and water-soluble subfractions dose-dependently (Fig. 3c, d).
To determine whether the S. horneri 70% EtOH extract and its CH2Cl2- and water-soluble subfractions reduced NO and PGE2 production via protein expressions regulation, we evaluated the iNOS and COX-2 expression levels in RAW264.7 macrophage cells via Western blot analysis. Upon inflammation, vast qualities of these two inflammatory mediators were produced. These results reveal that LPS treatment stimulated iNOS and COX-2 expression in macrophages compared with the control, while pretreatment with the S. horneri 70% EtOH extract and its CH2Cl2- and water-soluble subfractions inhibited NO and PGE2 production via iNOS and COX-2 expression downregulation in a dose-dependent manner (Fig. 3e, f).
Effects of the S. horneri EtOH extract and its subfractions on NF-κB activation in LPS-treated RAW264.7 macrophages
To examine the anti-inflammatory of the S. horneri 70% EtOH extract and its CH2Cl2- and water-soluble subfractions, we analyzed NF-κB-p65 subunit protein expression and its DNA binding activity. In this study, we prepared RAW264.7 macrophages with or without the S. horneri 70% EtOH extract and its subfractions for 3 h and subsequently treated them with 1 µg mL− 1 of LPS treatment for 24 h. The results indicate that pretreatment with the 70% EtOH extract (100 µg mL− 1) and its CH2Cl2- (100 µg mL− 1) and water-soluble (200 µg mL− 1) subfractions, inhibited p65 translocation into the nucleus (Fig. 4a). Furthermore, LPS-stimulated NF-κB DNA-binding activity (4.1 ± 0.3 fold) was impaired by 100 µg mL− 1 of the S. horneri 70% EtOH extract (2.38 ± 0.2-fold), 100 µg mL− 1 of its CH2Cl2-soluble subfraction (1.94 ± 0.27-fold), and 200 µg mL− 1 of its water-soluble subfraction (2.35 ± 0.28-fold), as shown in Fig. 4b.
In addition, we ascertained whether the anti-inflammatory effects of the S. horneri 70% EtOH extract (100 µg mL− 1) and its subfractions (100 or 200 µg mL− 1) are mediated by the NF-κB signaling pathway. In this study, we observed the nuclear localization of NF-κB p65 transcription factors in LPS-stimulated (1 µg mL− 1) RAW264.7 cells using an FITC-labeled anti-p65 secondary antibody via immunofluorescence with DAPI as the nuclear stain. The results indicate that the control or non-stimulated RAW264.7 cells exhibited p65 expression in the cytosol, suggesting low constitutive nuclear translocation of NF-κB. However, after 24 h LPS stimulation, green-colored fluorescence signals increased in response to both DAPI and p65 staining owing to p65 accumulation in the nuclei, as shown in the merged images of Fig. 4(c-e). These results indicate that NF-κB p65 accumulated in the nucleus because of translocation from the cytoplasm, thus activating RAW264.7 macrophage immunomodulation and promoting the NO, TNF-α, and IL-6 production. Three-hour pretreatment with the S. horneri 70% EtOH extract and its subfractions before LPS stimulation resulted in markedly weaker green fluorescence in the macrophages compared with that in the individual LPS-stimulated cells; this might have resulted from p65 expression throughout the cytosol. Therefore, the S. horneri extract and its subfractions presumably regulated iNOS and COX-2 protein expressions by preventing NF-κB and IκB-α phosphorylation in RAW264.7 cells.
Effects of the S. horneri EtOH extract and its subfractions on HO-1 protein expression and Nrf2 nuclear translocation in RAW264.7 macrophages
We conducted western blotting analysis to determine whether the S. horneri 70% EtOH extract (100 µg mL− 1) and its n-Hexane- (100 µg mL− 1), CH2Cl2- (100 µg mL− 1), and water-soluble (200 µg mL− 1) subfractions exert an anti-inflammatory effect by regulating HO-1 protein expression in LPS-treated RAW264.7 macrophages. In this study, we used cobalt protoporphyrin (CoPP) to induce HO-1 expression as a positive control. The results reveal that the CH2Cl2-soluble subfraction of S. horneri treatment significantly increased HO-1 protein expression compared with other S. horneri extracts (Fig. 5a). This indicates that the S. horneri CH2Cl2-soluble subfraction potentially suppresses the inflammatory response in macrophages by initiating the Nrf2/HO-1 pathway. Accordingly, we examined the effect of CH2Cl2-soluble subfraction treatment on the activation of Nrf2 nuclear translocation. During this process, RAW264.7 macrophages were treated with the CH2Cl2-soluble subfraction (100 µg mL− 1) over different time durations (0.5–1.5 h), and the Nrf2 protein expression levels were subsequently analyzed via western blotting. The results shown in Fig. 5b indicate that CH2Cl2-soluble subfraction treatment induced Nrf2 nuclear translocation in a time-dependent manner.
Furthermore, we determined whether the S. horneri CH2Cl2-soluble subfraction has an anti-inflammatory effect by stimulating the Nrf2/HO-1 pathway. Accordingly, NO and TNF-α concentrations and the NF-κB-binding activity were examined in the presence of tin protoporphyrin (SnPP) as an HO-1 inhibitor. In this study, the cells were treated with the CH2Cl2-soluble subfraction (100 µg mL− 1) for 3 h, with or without SnPP (50 µM), and subsequently stimulated with 1 µg mL− 1 LPS treatment for 24 h. The results in Fig. 5c-e indicate that exclusive treatment with the S. horneri CH2Cl2-soluble subfraction significantly inhibited NO and TNF-α production, including NF-κB DNA-binding activity, in LPS-treated RAW264.7 macrophages. However, these inhibitory effects attenuated after SnPP addition. In contrast, SnPP treatment did not affect the NO and TNF-α levels and NF-κB DNA-binding activity of LPS-stimulated macrophages. These results suggest that the S. horneri CH2Cl2-soluble subfraction exhibited the anti-inflammatory effects of LPS-treated macrophages by inducing HO-1 expression.
Effects of S. horneri on physiological, serological, and histological examination in HFD-fed obese mice
We assessed the food efficiency ratio (FER) and body weight (BW) of ICR mice across all experimental groups in order to investigate the impact of S. horneri on body weight increase. Compared to the other groups, the obese CON group fed a high-fat diet (HFD) gained more weight (Fig. 6a, b). However, when compared to the HFD-induced obese CON group, there was a substantial (p < 0.05) reduction in body weight gain of 4.59% and 8.28% in the S. horneri treated SH1 and SH2 groups, respectively. There was no significant difference in food intake among the HFD-fed groups, despite a significant difference in food intake between the NOR and HFD-fed groups. However, a similar trend in the FER was noted in conjunction with the increase in body weight (Fig. 6b). Furthermore, compared to the NOR group, the CON group's weight of the liver and all other adipose tissues (perirenal, retroperitoneal, mesenteric, and epididymal) increased significantly. Additionally, the S. horneri therapy significantly decreased the weight of the liver and fat tissues in a dose-dependent manner. Figure 6c-d shows that the weight changes in the kidney and heart were not statistically significant (p < 0.05). The HFD-fed obese mice showed signs of hyperlipidemia, as seen by the higher levels of blood TC, TG, and LDL cholesterols as well as the decreased HDL-cholesterol in the CON group of mice. Furthermore, in HFD-fed obese mice, S. horneri treatment (15 and 30 g kg− 1 body weight) markedly decreased the serum cholesterols (TC, TG, and LDL), and elevated HDL levels (Fig. 6e-f). Moreover, S. horneri treatment significantly lowered the arteriosclerosis index (AI), and cardiovascular risk factor (CVRF) values in HFD-fed obese mice (Fig. 6f).
The histological alterations in the liver and adipose tissue of HFD-fed mice treated with S. horneri are summarized in Fig. 6g-i. The liver tissues of NOR and obese mice were stained with H&E, and any structural damage was then observed. The obese CON group exhibited signs of inflammation, hepatocyte cytoplasmic vacuoles, and severe micro- and macro-steatosis with hepatocyte atrophy. Supplementation with 1.5% and 3% S. horneri reduced or eliminated fat droplet production in the liver tissues of HFD-fed mice. The hepatocyte architecture and hepatic tissue morphology of mice treated with S. horneri were similar to those of mice in the NOR group. The treatment of S. horneri reduced these histological changes in the liver tissues of the HFD group in a dose-dependent manner (Fig. 6g). The adipocytes in the CON obesity group were significantly larger, according to histological alterations of the epididymal tissues. Nevertheless, compared to the CON group, the adipocytes in the groups of ICR mice treated with S. horneri (at low and high dosages) were much smaller (Fig. 6h, i). In comparison to the NOR group, the CON group's total adipocyte area also rise. However, the overall adipocyte area was dramatically reduced in a dose-dependent manner after the S. horneri therapy (Fig. 6i).
By examining biochemical markers in serum samples from experimental animals given S. horneri treatment, we assessed liver and kidney functioning (Table 3). The NOR group's protein and albumin levels were noticeably higher than those of the HFD-fed animals. When compared to the CON group, the activity of the marker enzymes ALT and AST was considerably lower in the S. horneri-treated groups. No significant change was noted in the BUN levels as an indicator of renal function across the HFD-diet groups, while creatinine levels in the NOR group were not substantially different from those in the S. horneri treated SH1 and SH2 groups.
Table 3
Changes in the serum biomarkers of S. horneri-fed mice over 6 weeks
| Groups1) |
NOR | CON | SH1 | SH2 |
Protein (g dL− 1) | 5.59 ± 0.22a | 5.22 ± 0.16ab | 4.91 ± 0.13ab | 4.83 ± 0.12b |
Albumin (g dL− 1) | 3.04 ± 0.09a | 2.64 ± 0.06b | 2.54 ± 0.05b | 2.52 ± 0.05b |
AST (U L− 1) | 131.50 ± 35.51b | 156.67 ± 37.43a | 100.33 ± 22bc | 96.33 ± 10.74c |
ALT (U L− 1) | 30.00 ± 4.04b | 50.67 ± 8.84a | 32.67 ± 7.31bc | 35.67 ± 16.19bc |
BUN (mg dL− 1) | 18.45 ± 1.18b | 15.57 ± 0.59a | 14.90 ± 1.21a | 14.20 ± 1.85a |
Creatinine (mg dL− 1) | 0.09 ± 0.01b | 0.15 ± 0.02a | 0.11 ± 0.01ab | 0.11 ± 0.01ab |
1) The experimental diet groups were as follows: NOR, a normal group; CON, a high-fat diet induced obese mice; SH1, a high fat diet with S. horneri (1.5%); and SH2, a high fat diet with S. horneri (3%).
Changes in the activities of antioxidant enzymes in liver tissues
The purpose of this work is to determine whether administering S. horneri to HFD-fed ICR mice increases the activity of liver antioxidant enzymes. The GPx, GST, GR, CAT, SOD enzyme activities and GSH levels were reduced in the obesity CON group as compared to the NOR group. Figure 7 illustrates the increased activity of these antioxidant enzymes in the groups supplemented with S. horneri powder. The NOR, CON, and SH1 groups did not significantly differ in their GST antioxidant activity; however, the GST antioxidant activity was highest in the SH2 group. A high-fat diet has been shown to reduce the antioxidant activity of the liver. These findings imply that adding S. horneri to a supplement increases antioxidant activity, which in turn inhibits free radicals.
S. horneri activates AMPK signaling pathway in liver tissues
We investigated whether S. horneri affects the expression of genes and proteins involved in the metabolism of fatty acids in the liver tissues of obese mice. Western blot analysis results showed that, in comparison to control HFD-fed obese mice, S. horneri therapy significantly enhanced the AMPK levels. Furthermore, in a dose-dependent way, S. horneri treatment significantly suppressed the protein expression of transcription factors SREBP1, FAS, and ACC (Fig. 8a-b). These findings revealed that S. horneri inhibits the activity of lipogenic enzymes such SREBP1, FAS, and ACC by upregulating the AMPK pathway, which in turn has an anti-obesity impact. Additionally, S. horneri treatment markedly raised the expression of genes involved in fatty acid oxidation (Fig. 8f; CPT) and dramatically decreased the expression of genes involved in fatty acid synthesis (Fig. 8c-e; FAS, ACC, and SCD).