Synergistic modulation of proinflammatory mediators and cytokines in lipopolysaccharide-activated RAW 264.7 macrophages: The therapeutic potential of Elephantopus scaber and Sauropus androgynus ethanol extract.

Background and Aim: Elephantopus scaber (ES) and Sauropus androgynus (SA) have broad biological effects and have long been used in traditional medicine. However, the anti-inflammatory properties of the combination of ES and SA have not yet been fully explored. This study aimed to investigate the anti-inflammatory activities of the combination of ES and SA ethanol extract on lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cell lines by inhibiting proinflammatory mediators and cytokines. Materials and Methods: Nitric oxide (NO) production in RAW 264.7 cells was assessed using the Griess protocol. The effects of the combination of ES and SA ethanol extract on RAW 264.7 cell viability were determined using WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitro-phenyl)-2H-5-tetrazolio]-1,3-benzene sulfonate) assay. The levels of proinflammatory cytokines, including interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-ß), as well as the production of inducible nitric oxide synthase (iNOS), were assessed using flow cytometry. Results: This study demonstrated that ES and SA have excellent NO, iNOS, and proinflammatory inhibitory activities on LPS-induced RAW 264.7 macrophages. The formula ratio of 2ES:1SA showed the best NO inhibitory activity without any cytotoxicity, whereas the higher dose of SA (1ES:2SA) showed the best suppression of iNOS and proinflammatory cytokines IL-1ß, IFN-γ, and TNF-α. Conclusion: The combination of ES and SA ethanol extract could be an alternative agent for reducing excessive inflammation in inflammatory diseases.

The combination of Elephantopus scaber and Phaleria macrocarpa leaves extract promotes anticancer activity via downregulation of ER-α, Nrf2 and PI3K/AKT/mTOR pathway.

BACKGROUND: Elephantopus scaber and Phaleria macrocarpa have recently been interested as novel anticancer agents. However, there was no scientific evaluation of the anticancer effect of both plant combinations. OBJECTIVE: This study investigated the potential anticancer effects of combined E. scaber and P. macrocarpa leaves extract on human breast cancer cells lines. MATERIALS AND METHODS: T47D cells were treated with the combination of E. scaber and each part of P. macrocarpa (leaves/EL, mesocarp/EM, seed/ES and pericarp/EP). T47D cells were then exposed to three ratios (1:1, 2:1, and 1:2) of the best combination for 24, 48, and 72 h. The cell viability of T47D and TIG-1 cells was assessed using WST-1 assay. The apoptotic hallmarks were determined using FITC Annexin V-PI staining and DNA fragmentation assay. The cell proliferation and cell cycle profiles were analyzed using CFSE (carboxyfluorescein succinimidyl ester) and Propidium iodide-flowcytometry assays. The relative number of p-ERα, p-Nrf2, p-PI3K, p-AKT, and p-mTOR were assessed using flow cytometry. The molecular docking analysis was also performed to confirm the mechanism of the extract in silico. RESULTS: The combination of E. scaber and P. macrocarpa leaves (EL) possessed strong cytotoxic activity (p < 0.05) than other combination groups and cisplatin. EL showed selective killing only to T47D cells. EL at a ratio of 2:1 potentially suppressed the cell viability and cell division, induced apoptosis, and arrested the cell cycle of T47D cells by triple inhibiting the p-Nrf2, p-ERα, and p-PI3K/AKT/mTOR signaling pathway. Molecular docking analysis confirmed that the possible mechanism of EL to reduce T47D cell growth was by inhibiting ERα and Nrf2-complex, resulting in the reduction in the crosstalk effect of Nrf2, ERα and PI3K/AKT/mTOR pathways. CONCLUSION: The combination of leaf extracts from E. scaber and P. macrocarpa caused cell death in breast cancer cells T47D and not in normal cells TIG-1; hence has the potential to show anticancer efficacy in preclinical and clinical trials.

Comparative Study of Antiproliferative Activity in Different Plant Parts of Phaleria macrocarpa and the Underlying Mechanism of Action.

Phaleria macrocarpa is a medicinal plant widely used in Indonesian folk medicine to treat several diseases, including cancer. However, the comparative evaluation of various plant parts of P. macrocarpa has not been studied for their anticancer properties on breast cancer. The study aimed to assess the antiproliferative activity of the ethanol extract of various parts of Phaleria macrocarpa against T47D human breast cancer cell lines. Several parts of P. macrocarpa, including pericarp, mesocarp, seed, and leaf, were used to determine the most potent plant part to inhibit the growth of T47D cells. The cytotoxic effects of each plant part were evaluated by WST-1 assay. The apoptotic level of T47D cells was determined by annexin V-FITC-PI and DNA fragmentation assay. Propidium iodide staining and the CFSE assay were used to examine the effect of each extract on cell cycle distribution and cell division, respectively. The relative number of caspase-3, Bax, and Bcl-2 was analyzed by flow cytometry technique. WST-1 assay revealed that P. macrocarpa leaves exhibited the most potent antiproliferative activity (p < 0.05) compared to other plant parts with selectivity only to T47D cells. P. macrocarpa leaves extract effectively induced apoptosis, inhibited proliferation, and arrested the cell cycle of T47D cells. The relative number of caspase-3 was significantly (p < 0.05) increased after being treated with P. macrocarpa leaf extract. P. macrocarpa leaf extract also leads to the dose-dependent accumulation in the Bax/Bcl-2 ratio due to upregulation of Bax and downregulation of Bcl-2. The overall results indicated that P. macrocarpa leaves could inhibit the proliferation of T47D cells and trigger apoptosis through caspase-3 activation and Bax/Bcl regulation. Therefore, P. macrocarpa leaves can be used for breast cancer therapy.

Single-bulb garlic oil regulates toll-like receptors and Nrf2 cross-talk and IL-17 production in mice fed with high-fat diet.

The present study aimed to evaluate the effect of single-bulb garlic oil (SGO) on toll-like receptors 3 and 4 (TLR3 and TLR 4) and nuclear erythroid factor-like 2 (Nrf2) signaling pathway resulted from a high-fat diet and its underlying mechanism. Twenty-four Balb/c mice allocated into six groups: 1) N: mice fed with standard chow; 2) HFD: mice fed a high-fat diet for 45 days without any treatment; 3) HFD + Simv: mice fed a high-fat diet for 45 days and treated with simvastatin; 4-6) HFD + SGO 100, 200, 400 (mice fed a high-fat diet for 45 days and treated with single-bulb garlic oil at dose: 100, 200, and 400 mg/kg body weight for 30 days), respectively. At the end of treatment, spleen and hepar were isolated. The flow cytometry analysis was performed to analyze the relative number of nrf2, superoxide dismutase (SOD), malondialdehyde (MDA), TLR3, TLR4 and interleukin (IL-17). The results showed that HFD induction significantly reduced Nrf-2 and antioxidant enzyme levels. Furthermore, HFD induction increased TLR3 and TLR4 signaling and IL-17 production. Interestingly, 200 mg/kg BW of SGO increased the relative number Nrf-2 followed by SOD and HO-1 elevation at a dose of 100 mg/kg BW. SGO100 notably decrease the relative number of TLR3 (CD11b+TLR3+) and TLR4 (CD11b+TLR4+). The production of IL-17 by CD4 and CD8 were also reduced after receiving SGO at 200 mg/kg BW. This study suggests that the protective effect of SGO treatment on HFD mice was achieved by modulating TLR-Nrf2 cross-talks and decreasing IL-17 production. Our findings support a potential beneficial role of SGO for treating metabolic disease caused by a high-fat diet.

The combination of Elephantopus scaber and Sauropus androgynus promotes erythroid lineages and modulates follicle-stimulating hormone and luteinizing hormone levels in pregnant mice infected with Escherichia coli.

BACKGROUND AND AIM: Escherichia coli infection produces an adverse effect on the erythrocyte lineage and hormone levels during pregnancy. This study aimed to evaluate the effects of Elephantopus scaber (ES) and Sauropus androgynus (SA) in combination on circulating follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels and erythropoiesis changes in E. coli-infected pregnant mice. MATERIALS AND METHODS: Female Balb/c mice were mated with normal male mice and pregnancies were identified by the formation of vaginal plugs. Twenty-eight pregnant mice were divided randomly into seven groups: A control group (N), E. coli-infected pregnant mice (K+), and infected pregnant mice received the following five treatments: (1) Only ES; (2) ESSA1 (75:25); (3) ESSA2 (50:50); (4) ESSA3 (25:75); and (5) only SA, beginning from the 1st to the 16th day of pregnancy. Pregnant mice were infected with 107 CFU/mL of E. coli on day 4. Blood serum was collected on days 8, 12, and 16 of pregnancy and LH and FSH levels were measured by enzyme-linked immunosorbent assay. Bone marrow was isolated to determine the relative number of TER-119+VLA4+ and TER-119+CD34+ using flow cytometry. RESULTS: The ESSA1 and SA groups exhibited a marked increase in LH levels. The combination of ES and SA administered at a 25:75 ratio (ESSA3) altered FSH levels and the relative number of TER-119+VLA4+ in infected pregnant mice. Combined with SA at an equal ratio (50:50), ESSA2 group exhibited a significant increase in the expression of TER119+CD34+ compared with the other treatment groups. CONCLUSION: ES and SA combined at a ratio of 25:75 exhibited optimal results in altering hormonal and erythropoiesis in infected pregnant mice.

Marsilea crenata ethanol extract prevents monosodium glutamate adverse effects on the serum levels of reproductive hormones, sperm quality, and testis histology in male rats.

BACKGROUND AND AIM: Marsilea crenata is an aquatic plant that contains high antioxidants level and could prevent cell damages caused by free radicals. The present study aimed to investigate the effect of M. crenata ethanol extract on luteinizing hormone (LH), testosterone levels, sperm quality, and testis histology of adult male rats induced by monosodium glutamate (MSG). MATERIALS AND METHODS: This study randomly divided 48 male rats into eight groups (n=6): control group; MSG group (4 mg/g body weight [b.w.] for 30 days); MS1, MS2, and MS3 groups (4 mg/g b.w. MSG and M. crenata ethanol extract at dose 0.216, 0.432, and 0.648 mg/g b.w., respectively, for 30 days); and S1, S2, and S3 groups (M. crenata ethanol extract at dose of 0.216, 0.432, and 0.648 mg/g b.w., respectively, for 30 days). The blood sample was collected on days 0 and 30 to determine the LH and testosterone levels. The animals were dissected on day 30, and the testes were isolated for morphometric, histology (spermatogenic cell number), and malondialdehyde (MDA) examination. Moreover, semen was collected to determine the sperm quality parameter. RESULTS: The LH and testosterone levels significantly increased (p<0.05) after M. crenata administration at all doses. The higher dose of M. crenata ethanol extract demonstrated a high decrease in MDA level in MSG-treated rat testis; increase of spermatogonia, spermatocytes, spermatids, and Leydig cells number; and increase of seminiferous tubular diameter and germinal epithelium thickness. CONCLUSION: The ethanol extract of M. crenata can improve the levels of LH, testosterone, sperm quality, number of testis morphometric, spermatogenic, and Leydig cells in MSG-treated male rats.

Hormone-balancing and protective effect of combined extract of Sauropus androgynus and Elephantopus scaber against Escherichia coli-induced renal and hepatic necrosis in pregnant mice.

BACKGROUND: Elephantopus scaber (ES) and Sauropus androgynous (SA) have been frequently reported to possess antibacterial activity through in vitro, but in vivo studies about the protective effect of combined ES and SA have acquired less attention. OBJECTIVES: To evaluate protective effect of combined ethanol extract of ES and SA on hormone imbalance and renal and hepatic necrosis formation in Escherichia coli-infected pregnant mice. MATERIALS AND METHODS: A total of 28 pregnant Balb/c mice were divided into seven groups (n = 4): control, E. coli-infected pregnant mice, infected pregnant mice received 200 mg/kg ES, infected pregnant mice received combined 150 mg/kg ES and 37.5 mg/kg SA (75:25), 100 mg/kg ES and 75 mg/kg SA (50:50), 50 mg/kg ES and 112.5 mg/kg SA (25:75), and only 150 mg/kg SA. Pregnant mice were orally treated with combined ES and SA on day 1-4th of pregnancy. On the 4th day, mice were infected with 107 CFU/mL of E. coli and continuously treated with ES and SA until the 16th day of pregnancy. After treatment, the kidney and liver were prepared for histological examination using H&E staining. The blood serum was collected in each stage of pregnancy and measured by ELISA assays. RESULTS: Combined ES and SA gave an impact on altering the prolactin level. Combined ES and SA at ratio dose 75:25 was able to restore progesterone to normal levels (P < 0.05). The level of estradiol (E2) was relatively stable in the presence of E. coli and treatment. Treatment with 200 mg/kg ES, combined 50 mg/kg ES and 112.5 mg/kg SA (25:75) and 100 mg/kg ES and 75 mg/kg SA (50:50) demonstrated an immunomodulatory effect on the Gr1+ cell of E. coli treated-pregnant mice. E. coli infection significantly increased renal tubules and hepatic necrosis in pregnant mice compared to control (P < 0.05). Combined SA and ES at ratio dose 75:25 significantly demonstrated remarkable renal and hepatic protection activity in infected pregnant mice. CONCLUSION: The present study provided the establishment of combined ES and SA could be used to invent potent hormonal balancing agent and hepato-renal protective agent in infected pregnant mice.

Single garlic oil modulates T cells activation and proinflammatory cytokine in mice with high fat diet.

BACKGROUND: Hyperlipidemia triggers atherosclerosis by involving immune cells, such as T-cells. T-cells plays a role in worsening conditions during a high-fat diet (HFD). OBJECTIVE: The research aimed to analyze the role of single garlic oil (SGO) on T-cells activation and its proinflammatory cytokine expression in HFD mice. METHODS: Mice were divided into six groups: ND (normal diet); HFD (high-fat diet without treatment); HFD + Simv (HFD + simvastatin 2.6 mg/kg body weight); and HFD + SGO 1-3 (high-fat diet + single garlic oil in a dose of 12.5, 25, and 50 mg/kg body weight), respectively. Treatments were orally given every day for 45 days. At the end of treatments, lymphocytes were isolated from mice spleen. The relative number of T-cells and proinflammatory cytokines were measured using flow-cytometry and analyzed using one-way ANOVA (p < 0.05). RESULT: Our result indicated that HFD mice had lower naive T cells (CD4+CD62L+) than normal mice (p < 0.05). SGO treatment in HFD mice increased the relative number of naïve T cells. HFD treatment increased the expression of TNF-α and IFN-γ through NF-κB expression. Furthermore, SGO treatment improved the expression of TNF-α and IFN-γ. CONCLUSION: Our study suggests that SGO could act as a promising prospect for therapy to improve chronic inflammation in a HFD.