Genomic analysis revealed adaptive mechanism to plant-related fermentation of Lactobacillus plantarum NCU116 and Lactobacillus spp.

Lactobacillus plantarum NCU116 is the first sequenced strain derived from traditional Chinese sauerkraut (TCS). Since NCU116 manifested outstanding probiotic effects in vitro and in vivo, it is crucial to comprehend a clear genetic background for NCU116. Functional re-annotation and comparative analysis were performed to excavate the unique and representative genes in NCU116, in order to investigate its metabolic preference and adaptive mechanism. Horizontal gene transfer (HGT) seemed to occur frequently, which endows NCU116 with a strong ability to transport carbohydrates, as a strain-specific fructose/mannose-PTS was identified, and opu and osmC coding genes were retrieved as NCU116-specific. In addition, a strain-specific type I R/M system and several prophage loci were found in NCU116, which could play vital roles in self-defense mechanism. Pathways of bacterial metabolism on plant-related substrates fermentation were then generated by reconstruction of associated pathways. Moreover, a unique potential plantaricin-producing locus with high homology to that of JDM1 was defined in the genome of NCU116, which could be very important for the preservation of fermented-food. Our results would provide critical basis for the application of NCU116 in food and pharmaceuticals industries.

Interactions between Lactobacillus plantarum NCU116 and its environments based on extracellular proteins and polysaccharides prediction by comparative analysis.

Lactic acid bacteria (LAB) play a significant role in food industry and artisan fermented-food. Most of the applicable LABs were commonly obtained from natural fermented food or human gut. And Lactobacillus plantarum NCU116 was screened from a LAB-dominated traditional Chinese sauerkraut (TCS). In order to comprehend the interaction between NCU116 and its environments, comparative genomics were performed to identify genes involved in extracellular protein biosynthesis and secretion. Four secretory pathways were identified, including Sec and FPE pathways, holins and efflux ABC transporter system. Then 348 potential secretory proteins were identified, including 11 alpha-amylases responsible for degradation of macromolecules, and 8 mucus binding proteins which attribute to adherence to intestine epithelium. Besides, EPS clusters of NCU116 (EPS116) were identified and analyzed by comparing to other strains, which suggested a novel genotype of EPS clusters. These findings could be critical to extend the application of NCU116 in food and pharmaceuticals industries.

Recent developments in Hericium erinaceus polysaccharides: extraction, purification, structural characteristics and biological activities.

Hericium erinaceus (H. erinaceus), an edible mushroom with medicinal value, has a long history of usage in China and other oriental countries. Polysaccharide is supposed to be one of the major bioactive compounds in H. erinaceus, which possesses immunomodulating, anti-cancer, antioxidant, gastroprotection and intestinal health promotion, neuroprotective, hepatoprotective, antihpyerglycemic and hypolipidemic activities. In this review, the current advancements on extraction, purification, structural characteristics and biological activities of polysaccharide from different sources (fruiting body, mycelium and culture broth) of H. erinaceus were summarized. Among these aspects, summaries of the structural characteristics focused on the purified polysaccharides. Meanwhile, comparisons on the structural characteristics among the purified polysaccharides obtained from above three sources were made. Moreover, their biological activities were introduced on the basis of in vivo and in vitro experiments, and some possible action mechanisms were listed. Furthermore, the structure-activity relationship of the polysaccharide was discussed. New perspectives for the future work of Hericium erinaceus polysaccharide were also proposed. HIGHLIGHTS Extraction, purification, structural characteristics and biological activities of Hericium erinaceus polysaccharide (HEP) were summarized. Structural characteristics of the purified polysaccharides from different sources (fruiting body, mycelium and culture broth) of Hericium erinaceus were summarized and compared. Structure-activity relationship of HEP was discussed, and new perspectives for the future work of this polysaccharide were proposed.

Tea Polysaccharide Prevents Colitis-Associated Carcinogenesis in Mice by Inhibiting the Proliferation and Invasion of Tumor Cells.

The imbalance between cell proliferation and apoptosis can lead to tumor progression, causing oncogenic transformation, abnormal cell proliferation and cell apoptosis suppression. Tea polysaccharide (TPS) is the major bioactive component in green tea, it has showed antioxidant, antitumor and anti-inflammatory bioactivities. In this study, the chemoprophylaxis effects of TPS on colitis-associated colon carcinogenesis, especially the cell apoptosis activation and inhibition effects on cell proliferation and invasion were analyzed. The azoxymethane/dextran sulfate sodium (AOM/DSS) was used to induce the colorectal carcinogenesis in mice. Results showed that the tumor incidence was reduced in TPS-treated AOM/DSS mice compared to AOM/DSS mice. TUNEL staining and Ki-67 immunohistochemistry staining showed that the TPS treatment increased significantly the cell apoptosis and decreased cell proliferation among AOM/DSS mice. Furthermore, TPS reduced the expression levels of the cell cycle protein cyclin D1, matrix metalloproteinase (MMP)-2, and MMP-9. In addition, in vitro studies showed that TPS, suppressed the proliferation and invasion of the mouse colon cancer cells. Overall, our findings demonstrated that TPS could be a potential agent in the treatment and/or prevention of colon tumor, which promoted the apoptosis and suppressed the proliferation and invasion of the mouse colon cancer cells via arresting cell cycle progression.

The Agr-Like Quorum Sensing System Is Required for Pathogenesis of Necrotic Enteritis Caused by Clostridium perfringens in Poultry.

Clostridium perfringens encodes at least two different quorum sensing (QS) systems, the Agr-like and LuxS, and recent studies have highlighted their importance in the regulation of toxin production and virulence. The role of QS in the pathogenesis of necrotic enteritis (NE) in poultry and the regulation of NetB, the key toxin involved, has not yet been investigated. We have generated isogenic agrB-null and complemented strains from parent strain CP1 and demonstrated that the virulence of the agrB-null mutant was strongly attenuated in a chicken NE model system and restored by complementation. The production of NetB, a key NE-associated toxin, was dramatically reduced in the agrB mutant at both the transcriptional and protein levels, though not in a luxS mutant. Transwell assays confirmed that the Agr-like QS system controls NetB production through a diffusible signal. Global gene expression analysis of the agrB mutant identified additional genes modulated by Agr-like QS, including operons related to phospholipid metabolism and adherence, which may also play a role in NE pathogenesis. This study provides the first evidence that the Agr-like QS system is critical for NE pathogenesis and identifies a number of Agr-regulated genes, most notably netB, that are potentially involved in mediating its effects. The Agr-like QS system thus may serve as a target for developing novel interventions to prevent NE in chickens.

Gamma-Aminobutyric Acid Increases the Production of Short-Chain Fatty Acids and Decreases pH Values in Mouse Colon.

Gamma-Aminobutyric acid (GABA) could regulate physiological functions in the gastrointestinal tract. The present study aimed to investigate the effect of GABA on colon health in mice. The female Kunming mice were given GABA at doses of 5, 10, 20 and 40 mg/kg/d for 14 days. Afterwards, the short-chain fatty acids (SCFAs) concentrations, pH values, colon index, colon length and weight of colonic and cecal contents were determined to evaluate the effects of GABA on colon health. The results showed that intake of GABA could increase the concentrations of acetate, propionate, butyrate and total SCFAs in colonic and cecal contents, as well as the weight of colonic and cecal contents. The colon index and length of the 40 mg/kg/d GABA-treated group were significantly higher than those of the control group (p < 0.05). In addition, decrease of pH values in colonic and cecal contents was also observed. These results suggest that GABA may improve colon health.

Advances on Bioactive Polysaccharides from Medicinal Plants.

In recent decades, the polysaccharides from the medicinal plants have attracted a lot of attention due to their significant bioactivities, such as anti-tumor activity, antioxidant activity, anticoagulant activity, antidiabetic activity, radioprotection effect, anti-viral activity, hypolipidemic and immunomodulatory activities, which make them suitable for medicinal applications. Previous studies have also shown that medicinal plant polysaccharides are non-toxic and show no side effects. Based on these encouraging observations, most researches have been focusing on the isolation and identification of polysaccharides, as well as their bioactivities. A large number of bioactive polysaccharides with different structural features and biological effects from medicinal plants have been purified and characterized. This review provides a comprehensive summary of the most recent developments in physiochemical, structural features and biological activities of bioactive polysaccharides from a number of important medicinal plants, such as polysaccharides from Astragalus membranaceus, Dendrobium plants, Bupleurum, Cactus fruits, Acanthopanax senticosus, Angelica sinensis (Oliv.) Diels, Aloe barbadensis Miller, and Dimocarpus longan Lour. Moreover, the paper has also been focused on the applications of bioactive polysaccharides for medicinal applications. Recent studies have provided evidence that polysaccharides from medicinal plants can play a vital role in bioactivities. The contents and data will serve as a useful reference material for further investigation, production, and application of these polysaccharides in functional foods and therapeutic agents.

Effect of encapsulated carvacrol on the incidence of necrotic enteritis in broiler chickens.

There is an urgent need to control necrotic enteritis (NE) caused by Clostridium perfringens in chickens when antibiotics are withdrawn from feed. Carvacrol has strong antimicrobial activity and its delivery to the animal intestine can be significantly enhanced after encapsulation. The present study has investigated the potential of encapsulated carvacrol in controlling NE. In general, micro-encapsulation of carvacrol in an alginate-whey protein matrix showed no adverse effect on its antimicrobial activity towards C. perfringens in either Brain Heart Infusion (BHI) broth or a simulated gastrointestinal model. The minimum inhibitory concentrations of both encapsulated and un-encapsulated carvacrol were approximately 200 µl/l against C. perfringens in BHI. In a broiler infection model with C. perfringens, the diets supplemented with encapsulated carvacrol at the dose of either 250 or 650 µg/g significantly reduced NE in the chicken intestine, which was close to the degree of lesions observed in bacitracin/salinomycin treated birds. Supplementation with either bacitracin/salinomycin or encapsulated carvacrol showed no significant impact on intestinal burden of Lactobacillus. However, the treatment with bacitracin/salinomycin or the low dose of encapsulated carvacrol reduced the level of C. perfringens in the ileum of birds at 35 days of age. These results suggest that our encapsulated carvacrol can be used to combat NE disease in chickens.

Structural Features of Alkaline Extracted Polysaccharide from the Seeds of Plantago asiatica L. and Its Rheological Properties.

Polysaccharide from the seeds of Plantago asiatica L. has many bioactivities, but few papers report on the structural and rheological characteristics of the alkaline extract. The alkaline extracted polysaccharide was prepared from seeds of P. asiatica L. and named herein as alkaline extracted polysaccharide from seeds of P. asiatica L. (PLAP). Its structural and rheological properties were characterized by monosaccharide composition, methylation, GC-MS and rheometry. PLAP, as an acidic arabinoxylan, was mainly composed of 1,2,4-linked Xylp and 1,3,4-linked Xylp residues. PLAP solution showed pseudoplastic behavior, and weak gelling properties at high concentration. Sodium and especially calcium ions played a significant role in increasing the apparent viscosity and gel strength.

Modulation of cytokine gene expression by selected Lactobacillus isolates in the ileum, caecal tonsils and spleen of Salmonella-challenged broilers.

Probiotics have been used to control Salmonella colonization in the chicken intestine. Recently, we demonstrated that certain selected Lactobacillus isolates were able to reduce Salmonella infection in the chicken spleen and liver as well as down-regulated Salmonella pathogenicity island 1 virulence gene expression in the chicken caecum. To further understand the mechanisms through which Lactobacillus protected chickens from Salmonella infection, the present study has investigated the Lactobacillus isolate(s)-induced host immune response of chickens to Salmonella enterica serovar Typhimurium infection. A thorough examination of cytokine gene expression in the ileum, caecal tonsils, and spleen on days 1 and 3 post-Salmonella infection showed a dynamic spatial and temporal response to Salmonella infection and Lactobacillus treatments. In most instances, it was evident that treatment of chickens with Lactobacillus isolates could significantly attenuate Salmonella-induced changes in the gene expression profile. These included the genes encoding pro-inflammatory cytokines [lipopolysaccharide-induced TNF factor, interleukin (IL)-6, and IL-8], T helper 1 cytokines [IL-12 and interferon (IFN)-γ], and T helper 2 cytokines (IL-4 and IL-10). Another important observation from the present investigation was that the response induced by a combination of Lactobacillus isolates was generally more effective than that induced by a single Lactobacillus isolate. Our results show that administration of certain selected Lactobacillus isolates can effectively modulate Salmonella-induced cytokine gene expression, and thus help reduce Salmonella infection in chickens.

Effect of Lactobacillus plantarum NCU116 on loperamide-induced constipation in mice.

Lactobacillus plantarum, as a probiotic, has many functional properties in human intestinal tract. This study examined the effects of L. plantarum NCU116 on loperamide-induced constipation in a mouse model. Loperamide (5 mg kg(-1)) was injected subcutaneously to induce constipation. Animals were divided to five groups: normal group, constipation group, constipation plus three doses of L. plantarum NCU116 groups (NCU116-L, 10(7) CFU/mL; NCU116-M, 10(8) CFU/mL; NCU116-H, 10(9) CFU/mL; respectively). Mice were treated with the probiotic for 15 d to assess the anti-constipation effects. Fecal parameters, intestinal transit ratio and the production of fecal short chain fatty acids, histological of colon and immunohistochemical in colonic interstitial cells of Cajal (ICC) by c-kit were all improved in L. plantarum NCU116-treated mice as compared to the constipation group. These results demonstrate that L. plantarum NCU116 enhanced gastrointestinal transit and alleviated in mice with loperamide-induced constipation.

Genistein modulates the anti-tumor activity of cisplatin in MCF-7 breast and HT-29 colon cancer cells.

The function of genistein (GEN) on tumor prevention and tumor promotion is discussed controversially. A possible interference of GEN with chemotherapy has been only rarely addressed so far. In this study, effects of GEN on the anti-tumor activity of cisplatin (CIS) were investigated in the presence and absence of estradiol (10(-10) M) in MCF-7 breast and HT-29 colon cancer cells. Cells were treated with graded concentrations of GEN (10(-4)-10(-6) M), E2, CIS and combinations. Cell growth, proliferation and apoptosis were determined as well as the expression level of PCNA, Ki67 and BCL-2 family members. CIS and GEN 10(-4) M inhibited cell growth and induced apoptosis in MCF-7 and HT-29 cells in the presence and absence of E2. Co-treatment with CIS and 10(-4)M GEN resulted in additive effects. In concentrations of 10(-5) and 10(-6) M, GEN stimulated cell growth in MCF-7 cells. It promoted proliferation, inhibited apoptosis and counteracted the anti-tumor activity of CIS in MCF-7 and HT-29 cells. Particularly the ability of CIS to induce apoptosis was antagonized. In ER alpha-positive MCF-7 cells, but not in ER alpha-negative HT-29 cells, E2 was able to neutralize the anti-CIS effects of GEN. Our data provide evidence that GEN in the absence of E2, a situation which occurs in postmenopausal women, directly affects the anti-tumor activity of cytostatic drugs like CIS. The exact molecular mechanism has to be investigated in future studies.

Highland Barley ß-Glucan Relieves Symptoms of Colitis via PPARα-Mediated Intestinal Stem Cell Proliferation.

Intestinal stem cells (ISCs) are necessary to maintain intestinal renewal. Here, we found that the highland barley ß-glucan (HBG) alleviated pathological symptoms and promoted the proliferation of intestinal stem cells in colitis mice. Notably, metabolomics studies showed that docosahexaenoic acid (DHA) was significantly increased by the HBG treatment. DHA is a ligand for peroxisome proliferator-activated receptor α (PPARα), which can promote ISC proliferation. Expectedly, HBG facilitated the expression of intestinal PPARα and the proliferation of ISCs in colitis mice. Further experiments verified that DHA significantly facilitated the expression of PPARα and the proliferation of ISCs in intestinal organoids. Intriguingly, the effect of DHA on ISC proliferation was reversed by the PPARα inhibitor. Together, our data indicate that HBG might accelerate PPARα-mediated ISC proliferation through DHA. This provides new insights into the effective application of polysaccharides in maintaining intestinal homeostasis.

Macrophage immunomodulatory activity of a purified polysaccharide isolated from Ganoderma atrum.

The objective of this study was to evaluate the immunomodulatory effects of the purified Ganoderma atrum polysaccharide (PSG-1) on murine macrophage cell line RAW264.7. Phagocytotic assay by fluorescein isothiocyanate-dextran internalization showed that PSG-1 stimulated the phagocytosis of macrophages. G. atrum polysaccharide increased the production of NO, and the level of mRNA expression of inducible nitric oxide synthase in a dose-response manner. G. atrum polysaccharide also dose-dependently induced the release of TNF-α and interleukin-1ß. Generation of reactive oxygen species was promoted by PSG-1, as determined by flow cytometry. Moreover, PSG-1 induced nuclear factor-κB activation by elevation of p65 nuclear translocation, suggesting that PSG-1 probably stimulated macrophage activities by activating the nuclear factor-κB pathway.

Isolation and characterization of potential Lactobacillus acidophilus strains isolated from pig feces.

The aim of this study was to isolate and characterize Lactic Acid Bacteria (LAB) from 16 feces samples of pig farm, and to evaluate the probiotic potential of these isolates as potential oral probiotic candidates. The selection process was based on the isolation, identification, and a series of experiments for the selection of appropriate candidates with beneficial properties. The results demonstrated that most of LAB showed relatively strong resistance to pH 2.5 and high bile salts (1%), and had good survival in simulated gastric and intestinal juice. 9 isolates displayed antimicrobial activities against Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa and Enterobacter sakazakii. Almost all isolates were sensitive to ampicillin, chloramphenicol, vancomycin and amoxicillin, and most of isolates exhibited resistance against tetracycline and vancomycin. The adhesion rates of LAB varied greatly. The results of the study suggested that the Lactobacillus acidophilus NCUA065001 have the important functional property of probiotic candidates to enhance gut integrity and could considered to be the potential antibiotic alternatives in the pig feed industry.

White hyacinth bean polysaccharide ameliorates diabetes via microbiota-gut-brain axis in type 2 diabetes mellitus rats.

Gut played a potent role in onset and progression of metabolic disorders, presenting an exciting direction for diabetes prevention. Here, the anti-diabetic effects of White hyacinth bean polysaccharides (WHBP) were observed, including the reduction of blood glucose levels and improvement of intestinal impairment in type 2 diabetes mellitus (T2DM) rats. Further data concerning intestinal protection suggested that WHBP restored intestinal barrier, as evidenced by inhibition of intestinal pathological damage, up-regulation of Zonula occluden-1 expression and manipulation of the redox system in T2DM rats. Moreover, WHBP-mediated anti-diabetic effects were in parallel with the adjustment of changes in gut microbiota composition of T2DM rats. Meanwhile, hypersecretion of corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone levels, which were critical coordinators of the hypothalamic-pituitary-adrenal (HPA) axis, were suppressed in T2DM rats exposed to WHBP, indicating that WHBP-mediated health benefits were referring to regulate brain feedback in reduction of HPA axis. Concomitantly, further suggested and expanded on gut-brain communication by data of microbial metabolites short-chain fatty acids, mediators of gut-brain interactions, were remarkably raised in cecum contents of T2DM rats subjected to WHBP. Collectively, WHBP performed anti-diabetic effects were associated with control of microbiota-gut-brain axis implicated in intestinal barrier, HPA axis, gut microbiota and their metabolites.

Antimicrobial activity of saponin-rich fraction from Camellia oleifera cake and its effect on cell viability of mouse macrophage RAW 264.7.

BACKGROUND: As a by-product of oil production, several million tons of Camellia oleifera cake is discarded every year in China. The aim of this study was to evaluate the antimicrobial activity of a saponin-rich fraction isolated from C. oleifera cake and investigate its effect on the cell viability of RAW 264.7, a macrophage-like cell line present in almost all mouse tissues. RESULTS: The saponin-rich fraction was isolated from C. oleifera cake in several steps and had a saponin content of 95.42 ± 0.10% (w/w). It showed significant inhibitory activity against the bacteria Staphylococcus aureus, Escherichia coli and Bacillus subtilis and the fungi Mucor racemosus, Aspergillus oryzae, Rhizopus stolonifer, Saccharomyces cerevisiae and Penicillium glaucum, with minimum inhibitory concentrations of 31.3, 31.3, 62.5, 250, 250, 250, 31.3 and 125 µg ml(-1) respectively. In addition, mouse macrophage RAW 264.7 pretreated with the saponin-rich fraction (80-200 µg mL(-1) ) exhibited a significant loss of cell viability in a dose-dependent manner. CONCLUSION: These results may be useful for the future application of saponins from C. oleifera cake. However, the potential use of the saponin-rich fraction as an antimicrobial agent should be decided according to the target micro-organisms in order to avoid causing harm in humans.

Structural characteristic of pectin-glucuronoxylan complex from Dolichos lablab L. hull.

Polysaccharides are important constituents in Dolichos lablab hull. Herein, pectin-glucuronoxylan complex from D. lablab hull designated as DLHP-3 (D. lablab hull polysaccharide,) was prepared by ion exchange and gel permeation chromatography, and further characterized by acid degradation and enzymatic hydrolysis, methylation combined with GC-MS, NMR and MALDI-TOF-MS analysis. Both of pectin and glucuronoxylan regions were found in DLHP-3. The glucuronoxylan region consisted of a →4)-ß-Xylp-(1→ backbone with branches of α-GlcpA-(1→ substituted at O-2 site, and the ratio of xylose to glucuronic acid was about 5:1. Acetyl groups were mainly attached to O-3 site of →2,4)-ß-Xylp-(1→ residues. The main chain of pectin region could be represented by →4)-α-GalpA-(1→4)-α-GalpA-(1→ and →2)-α-Rhap-(1→4)-α-GalpA-(1→ with partial methyl-esterification. The side chains were deduced to embrace arabinan and arabinogalactan linked to rhamnogalacturonan-I region. Pectin was probably covalently bound to glucuronoxylan. Our findings uncovered the molecular structure of pectin-glucuronoxylan complex from D. lablab hull.

Gastroprotective activity of polysaccharide from the fruiting body of Hericium erinaceus against acetic acid-induced gastric ulcer in rats and structure of one bioactive fraction.

This study aimed at investigating gastroprotective activity of Hericium erinaceus polysaccharide (HEP) and characterizing one of its bioactive fractions. Acetic acid-induced gastric ulcer (GU) rat model was used to evaluate the gastroprotective activity of HEP, while H2O2-induced injury GES-1 cell model was conducted to screen the bioactive fractions from HEP. Moreover, one of the bioactive fractions was characterized using methylation and 1D/2D NMR analysis. Results indicated HEP treatment could ameliorate acetic acid-induced GU in rats. HEP supplement decreased levels of interleukin-6, tumor necrosis factor-α and malondialdehyde and myeloperoxidase activity, and increased releases of nitric oxide, prostaglandin E2, epidermal growth factor, vascular endothelial growth factor and basic fibroblast growth factor and superoxide dismutase activity in gastric tissues of ulcerated rats. Five purified polysaccharides from HEP were screened to be bioactive fractions with cytoprotection on H2O2-induced injury in GES-1 cells. Among them, RP-S was characterized to be a (1 â†’ 6)-ß-D-glucan, whose backbone was composed of →6)-ß-D-Glcp-(1 â†’ residue and branched with T-ß-D-Glcp-(1 â†’ residue at O-3 position. In conclusion, HEP possessed gastroprotection against acetic acid-induced GU in rats and one of its bioactive fractions was a ß-D-glucan. This study supports the utilization of HEP in anti-GU and provides evidences for the structure of gastroprotective HEP.

Ganoderma atrum polysaccharide induces anti-tumor activity via the mitochondrial apoptotic pathway related to activation of host immune response.

Ganoderma atrum polysaccharide (PSG-1), the major active ingredient isolated from Ganoderma atrum, has been suggested as a candidate for cancer therapy. The aim of this study was to investigate the anti-tumor effect of PSG-1 using sarcoma 180 (S-180) transplanted mice and further to examine the molecular mechanisms of PSG-1-induced anti-tumor effect. Results showed that PSG-1 significantly inhibited tumor growth in S-180-bearing mice. PSG-1-induced tumor apoptosis was associated with the alteration of Bcl-2 family proteins, increase of reactive oxygen species generation, loss of mitochondrial membrane potential (Δψ(m) ), release of cytochrome c from the mitochondria into cytosol, and activation of caspase-3 and -9. Elevation of immune function was also shown during PSG-1-induced tumor apoptosis, as evidenced by increase of spleen and thymus indexes, lymphocyte proliferation, concentrations of tumor necrosis factor (TNF)-α, and interleukin-2 in serum. Furthermore, the combined treatment of PSG-1 and cyclophosphamide (CTX) results in an enhancement of the anti-tumor effect of CTX alone via increased host immune response. These results suggested that PSG-1 had a potent anti-tumor activity by induction of tumor apoptosis through mitochondrial pathways, and immunoenhancement effect of PSG-1 was related to its anti-tumor effect. In addition, PSG-1 enhanced CTX-induced anti-tumor activity in S-180-bearing mice.