Application of Plant Proteases in Meat Tenderization: Recent Trends and Future Prospects.

Papain, bromelain, and ficin are commonly used plant proteases used for meat tenderization. Other plant proteases explored for meat tenderization are actinidin, zingibain, and cucumin. The application of plant crude extracts or powders containing higher levels of compounds exerting tenderizing effects is also gaining popularity due to lower cost, improved sensory attributes of meat, and the presence of bioactive compounds exerting additional benefits in addition to tenderization, such as antioxidants and antimicrobial effects. The uncontrolled plant protease action could cause excessive tenderization (mushy texture) and poor quality due to an indiscriminate breakdown of proteins. The higher cost of separation and the purification of enzymes, unstable structure, and poor stability of these enzymes due to autolysis are some major challenges faced by the food industry. The meat industry is targeting the recycling of enzymes and improving their stability and shelf-life by immobilization, encapsulation, protein engineering, medium engineering, and stabilization during tenderization. The present review critically analyzed recent trends and the prospects of the application of plant proteases in meat tenderization.

Synthesis and evaluation of the antiproliferative efficacy of BRM270 phytocomposite nanoparticles against human hepatoma cancer cell lines.

BRM270 is the most leading phytochemical extract that possesses potent anticancer properties. A major challenge associated with this drug is its low bioavailability and thus requires high dosages for cancer treatment. Here, we report the novel nano-synthesis of phyto-composite, BRM270 for the first time by mechanical milling method with specific modifications for enhanced cytotoxicity against HepG2 human hepatoma cancer cells. Unlike free BRM270 and other phytomedicines, BRM270 nanoparticles (BRM270 NPs) are well-dispersed and small sized (23 to 70 nm) which is believed to greatly enhanced cellular uptake. Furthermore, the acidic tumor microenvironment attracts BRM270 NPs enhancing targeted therapy while leaving normal cells less affected. The comparative cytotoxicity analysis using MTT assay among the three treatment groups, such as free BRM270, BRM270 NPs, and doxorubicin demonstrated that BRM270 NPs induced greater cytotoxicity against HepG2 cells with an effective drug concentration of 12 µg/ml. From FACS analysis, we observed an apoptotic cell death of 44.4% at BRM270 NPs treated cells while only 12.5% found in the free BRM270 treated cells. Further, the comparative relative expression profiling of the candidate genes were showed significant (p < 0.05) down-regulation of IL6, BCL2, p53, and MMP9 in the BRM270 NPs treated cells, compared to the free BRM270 and doxorubicin. Indeed, the genes, CASPASE 9 and BAX have shown significant (p < 0.05) upregulation in cells treated with BRM270 NPs as compared to counter treatment groups. The investigation of the signal pathways and protein-protein network associations were also carried out to elucidate the functional insights underlying anti-cancer potential of BRM270 NPs in HepG2 cells. Taken together, our findings demonstrated that these uniquely engineered BRM270 NPs effectively enter into the cancer cells due to its acidic microenvironment thereby inducing apoptosis and regulate the cell-proliferation in-vitro at extremely low dosages.

Insights into phytase-containing transgenic Lemna minor (L.) as a novel feed additive.

This study assessed the effect of supplementation of novel transgenic phytase on growth performance and bone mineralization in Korean native broiler chickens. The experiment was designed using four dietary groups: those with a diet supplemented with (A) recombinant phytase, (B) transgenic phytase from the plant Lemna minor, (C) or wild-type L. minor as well as (D) a control group that was supplemented with commercially available feed. Three hundred 1-day-old Korean native broiler chicks were used and divided into these four dietary treatment groups having three replicates of 25 birds each (n = 75). The results showed increases in growth performance and bone mineralization in Groups B and C; compared with Groups A and D. Hematological analyses revealed notable contrasts in erythrocyte sedimentation rate, red blood cell count, and hemoglobin levels among the experimental groups, whereas no impacts of dietary treatment were observed on total eosinophil, lymphocyte, heterophil, monocyte, and basophil levels. The relative expression profiling of candidate genes showed that the genes involved in growth response, meat quality, and P-Ca metabolism were significantly highly expressed in the phytase-supplemented groups. Hence, it is suggested that dietary supplementation with transgenic phytase plant L. minor for enhancing growth performance is a promising new approach in the broiler feed industry. To the best of our knowledge, we report here the most comprehensive analysis using a broiler model that provides a workable platform for further research on the cost-effective production of feed with different compositions that might be beneficial in the livestock feed industry.

Nanoformulations of curcumin: an emerging paradigm for improved remedial application.

Curcumin is a natural polyphenol and essential curcuminoid derived from the rhizome of the medicinal plant Curcuma longa (L.) is universally acknowledged as "Wonder drug of life". It is a vital consumable and restorative herb, commonly keened for several ailments such as cancer, arthritis, pain, bruises, gastrointestinal quandaries, swelling and much more. Despite its enormous curative potential, the poor aqueous solubility and consequently, minimal systemic bioavailability with rapid degradation are some of the major factors which restrict the utilization of curcumin at medical perspective. However, to improve its clinically relevant parameters, nanoformulation of curcumin is emerging as a novel substitute for their superior therapeutic modality. It enhances its aqueous solubility and targeted delivery to the tissue of interest that prompts to enhance the bioavailability, better drug conveyance, and more expeditious treatment. Subsequent investigations are endeavored to enhance the bio-distribution of native curcumin by modifying with felicitous nano-carriers for encapsulation. In this review, we specifically focus on the recent nanotechnology based implementations applied for overcoming the innate constraints of native curcumin and additionally the associated challenges which restrict its potential therapeutic applications both in vivo and in-vitro studies, as well as their detailed mechanism of action, have additionally been discussed.

Wogonin suppresses stem cell-like traits of CD133 positive osteosarcoma cell via inhibiting matrix metallopeptidase-9 expression.

BACKGROUND: Several efforts have been deployed to cure osteosarcoma, a high-grade malignant bone tumour in children and adolescents. However, some challenges such as drug resistance, relapse, and tumour metastasis remain owing to the existence of cancer stem cells (CSC). There is an urgent need to develop cost-effective and safe therapies. METHODS: Wogonin, an extract from the root of Scutellaria baicalensis, has long been considered as a promising natural and safe compound for anti-tumourigenesis, particularly to inhibit tumour invasion and metastasis. Hoechst 33,342 staining, wound healing assay, sphere formation assay, western blotting, and gelatin zymography assays were performed in CD133 positive osteosarcoma cell. RESULTS: In this study, we examined the effect of Wogonin on the mobility of human osteosarcoma CSC. Wogonin induces apoptosis of human osteosarcoma CSC, inhibits its mobility in vitro via downregulation of MMP-9 expression, and represses its renewal ability. CONCLUSIONS: We demonstrated that Wogonin decreases the renewal capacity of CSC. By inhibiting the formation of and reducing the size of spheres, Wogonin at a concentration of 40-80 µM effectively minimizes potential risk from CSC. Taken together, we have demonstrated a new approach for developing a potential therapy for osteosarcoma.

Anti-tumor activity of wogonin, an extract from Scutellaria baicalensis, through regulating different signaling pathways.

Wogonin is a plant flavonoid compound extracted from Scutellaria baicalensis (Huang-Qin or Chinese skullcap) and has been studied thoroughly by many researchers till date for its anti-viral, anti-oxidant, anti-cancerous and neuro-protective properties. Numerous experiments conducted in vitro and in vivo have demonstrated wogonin's excellent tumor inhibitory properties. The anti-cancer mechanism of wogonin has been ascribed to modulation of various cell signaling pathways, including serine-threonine kinase Akt (also known as protein kinase B) and AMP-activated protein kinase (AMPK) pathways, p53-dependent/independent apoptosis, and inhibition of telomerase activity. Furthermore, wogonin also decreases DNA adduct formation with a carcinogenic compound 2-Aminofluorene and inhibits growth of drug resistant malignant cells and their migration and metastasis, without any side effects. Recently, newly synthesized wogonin derivatives have been developed with impressive anti-tumor activity. This review is the succinct appraisal of the pertinent articles on the mechanisms of anti-tumor properties of wogonin. We also summarize the potential of wogonin and its derivatives used alone or as an adjunct therapy for cancer treatment. Furthermore, pharmacokinetics and side effects of wogonin and its analogues have also been discussed.

Novel phyto-derivative BRM270 inhibits hepatocellular carcinoma cells proliferation by inducing G2/M phase cell cycle arrest and apoptosis in xenograft mice model.

Hepatocellular carcinoma (HCC) is a major threat to human health worldwide and development of novel antineoplastic drug is demanding task. BRM270 is a proprietary combination of traditional medicinal herbs, has been shown to be effective against a wide range of stem-like cancer initiating cells (SLCICs). However, the underlying mechanism and antitumor efficacy of BRM270 in human hepatocellular carcinoma (HCC) cells have not been well elucidated till date. Here we studied the tumoricidal effect of BRM270 on human-CD133+ expressing stem-like HepG-2 and SNU-398 cells. Gene expression profiling by qPCR and specific cellular protein expressions was measured using immunocytochemistry/western blot analysis. In vivo efficacy of BRM270 has been elucidated in the SLCICs induced xenograft model. In addition, 2DG-(2-Deoxy-d-Glucose) optical-probe guided tumor monitoring was performed to delineate the size and extent of metastasized tumor. Significant (P<0.05) induction of Annexin-V positive cell population and dose-dependent upregulation of caspase-3 confirmed apoptotic cell death by pre/late apoptosis. In addition, bright field and fluorescence microscopy of treated cells revealed apoptotic morphology and DNA fragmentation in Hoechst33342 staining. Levels of c-Myc, Bcl-2 and c-Jun as invasive potential apoptotic marker were detected using qPCR/Western blot. Moreover, BRM270 significantly (P<0.05) increased survival rate that observed by Kaplan-Meier log rank test. In conclusion, these results indicate that BRM270 can effectively inhibit proliferation and induce apoptosis in hepatoma cells by down-regulating CyclinD1/Bcl2 mediated c-Jun apoptotic pathway.

Epigenetic induction of epithelial to mesenchymal transition by LCN2 mediates metastasis and tumorigenesis, which is abrogated by NF-κB inhibitor BRM270 in a xenograft model of lung adenocarcinoma.

Tumor initiating cancer stem-like cells (TICSCs) have recently become the object of intensive study. Human-Lipocalin-2 (hLCN2) acts as a biomarker for cancers. The aim of the present study was to explore new insights regarding the potential role of LCN2 in inducing epithelial to mesenchymal transition (EMT) by transfecting LCN2 into CD133+-A549-TICSCs and its cross-talk with the NF-κB signaling pathway in adenocarcinoma of the lung. Furthermore, EMT was confirmed by transcriptomic analysis, immunoblotting and immunocyto/histochemical analyses. Tumorigenesis and metastasis were confirmed by molecular therapeutics tracer 2DG infrared optical probe in BALB/cSIc-nude mice. It was observed that the CD133+-expressing-LCN2-A549 TICSCs population increased in adenocarcinoma of the lung compared to the normal lung tissue. The expressions of genes involved in stemness, adhesion, motility and drug efflux was higher in these cells than in their non-LCN2 expressing counterparts. The present study revealed that elevated expression of LCN2 significantly induced metastasis via EMT. Overexpression of LCN2 significantly increased stemness and tumor metastasis by modulating NF-κB cellular signaling. BRM270, a novel inhibitor of NF-κB plays a significant role in the EMT reversal. BRM270, a naturaceutical induces cell shrinkage, karyorrhexis and programmed cell death (PCD) which were observed by Hoechst 33342 staining while flow cytometry analysis showed significant (P<0.05) decrease in cell population from G0-G1 phases. Also, 2DG guided in vivo model revealed that BRRM270 significantly (P<0.0003) reduced tumor metastasis and increased percent survival in real-time with complete resection. An elaborate study on the novel concept with respect to linking of naturaceutics as selective and potential anticancer agent that eliminates the elevated LCN2 induced EMT and tumor dissemination through cooperation with the NF-κB signaling as the baseline data for the planning of new therapeutic strategies was conducted for the first time. Our results also illustrate a molecular mechanistic approach for 2DG-guided molecular imaging-based cancer therapy using BRM270 as a novel cancer therapeutic drug to enhance the effect of doxorubicin (Dox)-resistant LCN2 induced metastasis of solid tumors in nude mice.

The novel inhibitor BRM270 downregulates tumorigenesis by suppression of NF-κB signaling cascade in MDR-induced stem like cancer-initiating cells.

The nuclear factor κB (NF-κB) and interleukin-6 (IL-6) contribute to multidrug resistance (MDR) in tumor chemotherapy. The essential phenomenon of oncogenic activation of NF-κB in cancer-initiating cells showing MDR resulting from increased IL-6 expression is still unclear. Cancer stem cells (CSCs) have been the objective of intensive study. The aim of this study was to investigate the selective and potential efficacy of BRM270 against stem-like cancer-initiating cells (SLCICs) via the molecular mechanisms of its anticancer effects. Co-regulation of NF-κB and Cdk6 might be new arena to mitigate tumorigenesis. In the present study phyto-drug based approach provides a new avenue in understanding the amelioration and regulatory mechanisms in CSCs. In the present study, an in vivo tumor metastasis model of osteosarcoma was established by injecting Cal72 and SaOS-2 SLCICs into the right lower flank of nude mice. Later the development of tumor was analyzed by LICOR Biosciences (Pearl image analyzer). Significant suppression of activation of NF-κB and LPS-induced gene expression and apoptosis by BRM270 was confirmed by FACS, western blotting and qPCR. Further, both p65 and Cdk6 were significantly (P<0.05) overexpressed in BRM270 non-treated Cal72 SLCICs compared to treated group. BRM270 directly dephosphorylated RelA and selectively inhibited NF-κB transcriptional activity, resulting in decreased expression of interleukin-6, a cytokine implicated in cancer metastasis. BRM270-mediated cell shrinkage, pyknosis, karyorrhexis and programmed cell death (PCD) were observed by Hoechst 33342 staining while flow cytometry analysis showed significant (P<0.05) decrease in cell population from G0-G1 phases. These findings suggest that activation of the oncogenic Cdk6-NF-κB pathway, resulting from increased IL-6 expression, plays a central role in CD133 expressing SLCICs augmented MDR and neoplasia. This study proposes targeting of NF-κB, and Cdk6 with IL-6 as potential targets for PCD and treatment of chemotherapeutic resistance of CSCs to design novel therapies for their elimination.

Evaluation of the antioxidant, anti-inflammatory, and anticancer activities of Euphorbia hirta ethanolic extract.

This study evaluated the chemical composition, antioxidant, anti-inflammatory and anticancer activities of a Euphorbia hirta L. extract. The antioxidant activities of whole E. hirta ethanol extract were determined by electron spin resonance spectrophotometric analysis of 1,1-diphenyl-2-picryl-hydrazyl (DPPH), hydroxyl, and alkyl radical levels and by using an online high-performance liquid chromatography (HPLC)-2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay. The E. hirta ethanol extract (0.5 mg/mL) exhibited DPPH-scavenging activity of 61.19% ± 0.22%, while the positive control (0.5 mg/mL ascorbic acid) had 100% ± 0.22% activity. The concentration of the extract required to trap 50% of DPPH (IC50) was 0.205 mg/mL. Online HPLC analysis of the extract also showed strong antioxidant activity. The anti-inflammatory activity of the E. hirta extract was assessed in lipopolysaccharide-induced RAW 264.7 macrophages. The anti-inflammatory activity was highest in the presence of 200 µg/mL E. hirta extract, and nitric oxide production was decreased significantly (p < 0.05). The extract also showed selective anticancer activity at a concentration of 100 µg/mL (p < 0.05). These results indicated that E. hirta may warrant further investigation for the development of antioxidant, anti-inflammatory, and anticancer herbal medications.

Phase II trial of sorafenib in conjunction with chemotherapy and as maintenance therapy in extensive-stage small cell lung cancer.

OBJECTIVES: Sorafenib is a multi-tyrosine kinase inhibitor of Raf kinase, VEGFR, and PDGFR. Angiogenesis is important for growth and progression of SCLC. This trial was conducted to evaluate whether the combination of cisplatin and etoposide plus concurrent and sequential sorafenib could prolong survival in patients with previously untreated SCLC. METHODS: Previously untreated patients with extensive stage SCLC were treated with cisplatin and etoposide days 1, 2, 3 for four cycles, concurrent with sorafenib 200 mg orally bid starting day 1 cycle 1. Patients with no disease progression after four cycles continued sorafenib 400 mg orally bid as maintenance for maximum of 12 months. The primary endpoint was 1 year survival with response rate and safety as secondary endpoints. RESULTS: A total of 18 patients were enrolled with 17 evaluable patients. One patient had a complete response, seven patients had a partial response (overall response rate of 47 %) and one patient had stable disease. Overall median survival was 7.4 months and 1 year survival was 25 %. The most common treatment-related adverse events included fatigue, anorexia, rash, diarrhea, neutropenia and weight loss. Grade 5 GI bleeding, pulmonary hemorrhage and neutropenia occurred in one pt (6 %) each. Accrual was halted on the basis of safety profile as well as preliminary efficacy data. CONCLUSIONS: The combination of platinum based chemotherapy and sorafenib has significant toxicity at current dose levels and is associated with disappointing efficacy data.