Hydrolysable tannins as a potential therapeutic drug for the human fibrosis-associated disease.

Fibrosis is a pathological change with abnormal tissue regeneration due to a response to persistent injury, which is extensively related to organ damage and failure, leading to high morbidity and mortality worldwide. Although the pathogenesis of fibrosis has been comprehensively elucidated, there are few effective therapies for treating fibrotic diseases. Natural products are increasingly regarded as an effective strategy for fibrosis with numerous favorable functions. Hydrolysable tannins (HT) are a type of natural products that have the potential to treat the fibrotic disease. In this review, we describe some biological activities and the therapeutic prospects of HT in organ fibrosis. Furthermore, the underlying mechanisms of inhibition of HT on fibrotic organs in relation to inflammation, oxidative stress, epithelial-mesenchymal transition, fibroblast activation and proliferation, and extracellular matrix accumulation are discussed. Understanding the mechanism of HT against fibrotic diseases will provide a new strategy for the prevention and attenuation of fibrosis progression.

Tannic acid attenuates intestinal oxidative damage by improving antioxidant capacity and intestinal barrier in weaned piglets and IPEC-J2 cells.

Tannic acid (TA) has received widespread attention for its beneficial biological function with antioxidant capacity. This study investigated the protective role of TA on the intestinal antioxidant capacity and intestinal barrier in weaned piglets and porcine intestinal epithelial cells (IPEC-J2). A total of 18 weaned piglets were randomly allocated into two groups (n = 9) and fed with a basal diet (control, CON) and a basal diet containing 1,000 mg/kg TA for two weeks. The in vivo results showed that treatment with TA increased both glutathione peroxidase (GSH-PX) activity and the protein expression of ZO-1 in the jejunum of weaned piglets, and reduced the level of malondialdehyde (MDA) in the serum and the mRNA and protein expression of Keap1 in the jejunum of weaned piglets. Furthermore, in vitro results indicated that TA treatment effectively alleviated tert-butyl hydroperoxide (TBH)-induced oxidative stress in IPEC-J2 cells, improved the antioxidant capacity by elevating the cell redox state and activating the Nrf2 pathway, and improved the intestinal barrier by upregulating the mRNA and protein expression of intestinal tight junction proteins and increasing the transepithelial electrical resistance (TEER) value. In conclusion, these results confirmed that TA relieves oxidative injury and improves intestinal barrier function and intestinal antioxidant capacity by activating the Nrf2 signaling pathway. These findings suggest that TA has the potential application in alleviating oxidative stress in the intestine of weaned piglets.

Dietary high protein-induced diarrhea and intestinal inflammation by activation of NF-κB signaling in piglets.

The present study aimed to investigate whether inflammation-associated responses in piglets are induced by high protein (HP) through activating nuclear factor kappa B (NF-κB) signaling. Sixteen piglets (35 d of age, Duroc × [Landrace × Yorkshire], weaned at d 21, initial BW = 9.70 ± 0.11 kg) were allocated to 18% and 26% CP (HP group) at random, comprising 8 replicate pens per treatment. The piglets were slaughtered to collect intestinal tissues when apparent, persistent, and stable diarrhea syndromes happened (on d 12). No significant differences were observed in their growth performance (P > 0.05), but reduction by 19.11%, 25.31%, 23.64% of ADFI, ADG, and G:F, respectively was detected in the HP group. The HP group had greater (P = 0.002) diarrhea rates. Furthermore, dietary HP had lower ileal villus height (VH; P = 0.048), ratio of villus height to crypt depth (VH/CD ratio; P = 0.016), and colonic CD (P = 0.034), as well as had the trend (P = 0.075) to reduce the ileal villus absorptive area. Moreover, HP diets significantly elevated the goblet cell numbers in the ileal villi (P = 0.016) and colonic crypts (P < 0.001) and up-regulated (P = 0.012) the mRNA expression of mucin2 (Muc2) in the ileum. In addition, HP diets increased the myeloperoxidase concentration in the ileum (P = 0.002) and colon (P = 0.007) of piglets. Dietary HP significantly down-regulated the mRNA expression of tumor necrosis factor-α (TNF-α; P < 0.001) in the ileum, induced nitric oxide synthase (iNOS; P = 0.040) and interleukin-22 (IL-22; P = 0.008) in the colon, and inclined to down-regulate interleukin-1ß (IL-1ß; P = 0.076) expression in the colon. The relative protein abundance of Galectin-3 (P = 0.046) in the colon and the ratio of phosphorylation NF-κB to NF-κB (p-NF-κB/NF-κB ratio) in the ileum of HP piglets were also greater (P = 0.038). These results suggest that dietary HP may cause diarrhea in piglets by activating NF-κB signaling induced intestinal inflammation.

Herbal Extract Mixture Modulates Intestinal Antioxidative Capacity and Microbiota in Weaning Piglets.

Recently, herbal extracts have been applied in multiple aspects, such as medicine and animal feed. Different compositions of herbal extract mixture (HEM) have various components and diverse functions. This study aimed to evaluate the effects of HEM (Lonicera japonica, Astragalus membranaceus, Eucommia folium, and Codonopsis pilosula) on intestinal antioxidant capacity and colonic microbiota in weaned pigs. A total of 18 piglets [Duroc × (Landrace × Yorkshire)] with the initial body weight of 5.99 ± 0.13 kg (weaned at 21 days) were randomly divided into two groups (n = 9): the control group (CON, basal diet) and the HEM treatment group (HEM, 1,000 mg/kg HEM + basal diet). The experiment period lasted for 14 days. Our results showed that dietary supplementation with HEM modulated the antioxidant capacity through decreasing the activity of superoxide dismutase (SOD) in the ileum and glutathione peroxidase (GSH-PX) in the serum, and decreasing the mRNA expression of Kelch like-ECH-associated protein 1 (Keap1) in the jejunum and the protein level of Keap1 in the ileum. Moreover, the HEM group modified the composition of colonic microbiota with affecting relative abundances of the Firmicutes and Bacteroidetes at the phylum level. Taken together, supplementation of HEM can regulate the antioxidant capacity and modify the composition of colonic bacteria in weaning piglets. This study provides new insights into the combination effects of herbal extracts on weaning piglets.

Effects of dietary gamma-aminobutyric acid supplementation on amino acid profile, intestinal immunity, and microbiota in ETEC-challenged piglets.

Enterotoxigenic Escherichia coli (ETEC) infection is the most common cause of diarrhea in piglets, and ETEC could increase intestinal gamma-aminobutyric acid (GABA)-producing bacteria to affect intestinal immunity. However, the effect of GABA on ETEC-infected piglets is still unclear. This study aims at investigating the impact of dietary GABA supplementation on the growth performance, diarrhea, intestinal morphology, serum amino acid profile, intestinal immunity, and microbiota  in the ETEC-infected piglet model. Eighteen piglets were randomly divided into two groups, in which the piglets were fed with a basal diet with 20 mg kg-1 GABA supplementation or not. The experiment lasted for three weeks, and the piglets were challenged with ETEC K88 on the fifteenth day. The results showed that dietary GABA reduced the feed conversion ratio, promoted the kidney organ index but did not affect the diarrheal score and small intestinal morphology in ETEC-challenged piglets. Ileal mucosal amino acids (such as carnosine and anserine) and serum amino acids (including threonine and GABA) were increased upon GABA supplementation. GABA enhanced ileal gene expression of TNF-α, IFN-γ, pIgR, and MUC2, while inhibited the ileal expression of IL-18 in ETEC-challenged piglets. GABA supplementation also highly regulated the intestinal microbiota by promoting community richness and diversity and reducing the abundance of the dominant microbial population of the ileal microbiota. Collectively, GABA improves growth performance, regulates the serum amino acid profile, intestinal immunity, and gut microbiota in ETEC-challenged piglets. This study is a fine attempt to reveal the function of GABA in ETEC-infected piglets. It would contribute to the understanding of the roles of exogenous nutrition on the host response to ETEC infection.

Effects of dietary supplementation with herbal extract mixture on growth performance, organ weight and intestinal morphology in weaning piglets.

BACKGROUND: Many countries are increasingly prohibiting the addition of antibiotics in livestock diets. Therefore, herb extracts have gradually drawn attention to substitute antibiotics. Our present study aimed to determine the effects of herbal extract mixture (HEM) in dietary on growth performance, organ weight, intestinal morphology and intestinal nutrient transporters in weaned pigs. METHODS: 27 piglets (Duroc × [Landrace × Yorkshire]; Body Weight (BW) = 5.99 ± 0.13 kg) were weaned at day 21 and randomly divided into three groups (n = 9 piglets/group). All piglets received a basal diet containing similar amounts of nutrients for 14 days. The three groups were the control (no additive), the antibiotics (375 mg/kg chlortetracycline, 20%, 500 mg/kg enramycin, 4%, 1,500 mg/kg oxytetracycline calcium, 50%) and the HEM group (1000 mg/kg extract mixture of golden-and-silver honeysuckle, huangqi, duzhong leaves and dangshen). After 14 d of treatment, we collected tissue samples to measure organ weight, intestinal parameters, intestinal morphology, digestive enzyme activities and intestinal mRNA expression of nutrient transporters. RESULTS: The HEM group had no effects on growth performance and organ weight of weaned pigs. But compared with the control group, both HEM and antibiotics improved intestinal morphology, and HEM elevated the expression of nutrient transporters in ileum (SLC6A9, SLC15A1, and SLC5A1). HEM significantly decreased the activities of maltase in ileum and the ratio of small intestinal weight to BW than control group. CONCLUSIONS: These results indicate benefit effects of the supplementation of HEM in diet, including modulating intestinal morphology and increasing the mRNA expression of nutrients transporters. These findings suggest that HEM provides novel insights into a variety of herbal extract mixtures to replace antibiotics in animal production.

Effects of dietary microencapsulated tannic acid supplementation on the growth performance, intestinal morphology, and intestinal microbiota in weaning piglets.

Antibiotics are commonly overused to reduce weaning stress that leads to economic loss in swine production. As potential substitutes of antibiotics, plant extracts have attracted the attention of researchers. However, one of the plant extracts, tannic acid (TA), has an adverse effect on the growth performance, palatability, and intestinal absorption in weaning piglets when used at a large amount. Thus, this study aimed to investigate the effects of a proper dose of microencapsulated TA on the growth performance, organ and intestinal development, intestinal morphology, intestinal nutrient transporters, and colonic microbiota in weaning piglets. Forty-five Duroc × [Landrace × Yorkshire] (initial body weight = 5.99 ± 0.13 kg, weaned days = 21 d) piglets were randomly divided into five treatment groups (n = 9) and raised in 14 d. The piglets in the control group were raised on a basal diet; the piglets in the antibiotic test group were raised on a basal diet with three antibiotics (375 mg/kg Chlortetracycline 20%, 500 mg/kg Enramycin 4%, 1,500 mg/kg Oxytetracycline calcium 20%); and the other three groups were raised on a basal diet with three doses of microencapsulated TA (TA1, 500 mg/kg; TA2, 1,000 mg/kg; TA3, 1,500 mg/kg). All the piglets were raised in the same environment and given the same amount of nutrients for 2 wk. The results showed that both TA1 and TA2 groups had no adverse effect on the growth performance, organ weight and intestinal growth, and the pH value of gastrointestinal content. TA2 treatment improved the duodenal morphology (P < 0.05), increased the gene expression level of solute carrier family 6, member 19 and solute carrier family 15, member 1 (P < 0.05) in the ileum, and modulated the colonic bacteria composition (P < 0.05), but inhibited the activity of maltase in the ileum (P < 0.05) and the jejunal gene expression level of solute carrier family 5, member 1 (P < 0.05). In conclusion, our study suggests that a dosage between 500 and 1,000 mg/kg of microencapsulated TA is safe to be included in the swine diet and that 1,000 mg/kg of microencapsulated TA has beneficial effects on intestinal morphology, intestinal nutrient transporter, and intestinal microbiota in weaning piglets. These findings provide new insights into suitable alternatives to antibiotics for improving growth performance and colonic microbiota.

The effects of dietary supplementation with hyodeoxycholic acid on the differentiation and function of enteroendocrine cells and the serum biochemical indices in weaned piglets1.

Bile acid, a cholesterol metabolite, promotes gastrointestinal tract digestion and absorption of cholesterol, lipids, and fat-soluble vitamins. It is a signaling regulatory molecule that influences a variety of endocrinal and metabolic activities. This study investigated the effects of hyodeoxycholic acid (HDCA) as a dietary supplement on endocrine cell differentiation and function and weaned piglet serum biochemical indices. Sixteen piglets [Duroc × (Landrace × Yorkshire)] were individually housed and weaned at 21 d of age (BW of 6.14 ± 0.22 kg). Uniform weight animals were randomly assigned to 1 of 2 treatments (8 replicate pens per treatment and 1 piglet per pen). The treatments were 1) base diet (control) and 2) base diet supplemented with 2 g/kg of HDCA. Control and HDCA piglet numbers of chromogranin A (CgA)-positive cells per crypt did not differ. HDCA CgA-positive cells numbers decreased (P < 0.05) in the jejunal villi showed a tendency to decrease (P < 0.10) in the ileal villi and showed tendency toward an increase (P < 0.10) in the duodenal villi compared with the controls. The HDCA diet led to a decline in glucagon-like peptide 2 (P < 0.01) concentrations, but did not affect plasma glucagon-like peptide 1. HDCA supplementation increased (P < 0.05) the mRNA expression of jejunal Insm1, Sst, PG, and Gast, but decreased (P < 0.05) duodenal expression of Insm1, jejunal Pdx1, and ileal NeuroD1. HDCA elevated globulin and immunoglobulin A (P < 0.05) serum concentrations and decreased the albumin/globulin ratio (P < 0.05). Total protein and immunoglobulin G serum levels tended to increase compared with the control group. These results indicate that dietary HDCA at 2 g/kg may regulate enteroendocrine cell differentiation and play a role in increasing weaned piglet humoral immunity.

The effects of dietary supplementation with hyodeoxycholic acid on the differentiation and function of enteroendocrine cells and the serum biochemical indices in weaned piglets.

Bile acid, a cholesterol metabolite, promotes gastrointestinal tract digestion and absorption of cholesterol, lipids, and fat-soluble vitamins. It is a signaling regulatory molecule that influences a variety of endocrinal and metabolic activities. This study investigated the effects hyodeoxycholic acid (HDCA) as a dietary supplement on endocrine cell differentiation and function and weaned piglet serum biochemical indices. Sixteen piglets (Duroc × [Landrace × Yorkshire]) were individually housed and weaned at 21 days of age (body weight of 6.14 ± 0.22 kg). Uniform weight animals were randomly assigned to one of two treatments (eight replicate pens per treatment and one piglet per pen). The treatments were 1) base diet (control); and 2) base diet supplemented with 2 g/kg of HDCA. Control and HDCA piglet numbers of CgA-positive cells per crypt did not differ. HDCA CgA-positive cells numbers decreased (P < 0.05) in the jejunal villi, showed a tendency to decrease (P < 0.10) in the ileal villi, and showed tendency toward an increase (P < 0.10) in the duodenal villi compared to the controls. The HDCA diet led to a decline in GLP-2 (P < 0.01) concentrations, but did not affect plasma GLP-1. HDCA supplementation increased (P < 0.05) the mRNA expression of jejunal Insm1, Sst, PG, and Gast, but decreased (P < 0.05) duodenal expression of Insm1, jejunal Pdx1, and ileal NeuroD1. HDCA elevated GLO and IgA (P < 0.05) serum concentrations and decreased the A/G ratio (P < 0.05). TP and IgG serum levels tended to increase compared to the control group. These results indicate that dietary HDCA at 2 g/kg may regulate enteroendocrine cell differentiation and play a role in increasing weaned piglet humoral immunity.

Effects of dietary gamma-aminobutyric acid supplementation on the intestinal functions in weaning piglets.

This study aims to investigate the effects of dietary gamma-aminobutyric acid (GABA) supplementation on the growth performance, intestinal immunity, intestinal GABAergic system, amino acid profiles and gut microflora of the weaned piglets. Totally sixteen healthy piglets were randomly assigned into two groups to be fed with the basal diet (Con group) or the basal diet with GABA (20 mg kg-1) supplementation. Body weights and feed intakes were monitored weekly. Piglets were sacrificed after 3 weeks of GABA supplementation to collect the blood, ileum, ileal mucosa and luminal content. Immune-associated factors, GABAergic system, amino acid profiles, and microbiota in the ileum and serum amino acid profiles were explored. The results showed that GABA supplementation improved the growth performance and modulated the intestinal immunity with inhibiting the gene expressions of IL-22, proinflammatory cytokines (IL-1 and IL-18), and Muc1, but promoted the expressions of anti-inflammatory cytokines (IFN-γ, IL-4, and IL-10), TLR6 and MyD88. GABA regulated a few components of the intestinal GABAergic system, increased the levels of most amino acids in the ileal mucosa but reduced the serum amino acid profiles. GABA regulated the population and diversity of intestinal microbiota, such as the abundances of the dominant microbial populations, the community richness, and diversity of the ileal microbiota. In conclusion, GABA supplementation modulated the intestinal functions, including intestinal immunity, intestinal amino acid profiles and gut microbiota, and the results can be helpful for understanding the functions of GABA in the intestine.

Tannic acid modulates intestinal barrier functions associated with intestinal morphology, antioxidative activity, and intestinal tight junction in a diquat-induced mouse model.

Oxidative stress is more likely to occur in the intestine compared to other organs because it is located at the interface between an organism and its luminal environment. Tannic acid (TA) is reported to serve as an antioxidant, antimicrobial, anticarcinogenic and antimutagenic agent in various models. In the present study, we evaluated the effects of TA on body weight, intestinal morphology, antioxidative activity, and intestinal barrier in diquat-induced oxidative stress mouse model. The results showed that TA had failed to affect antioxidative enzymes in diquat-challenged mice, while the concentration of 2.5 mg kg-1 to 10 mg kg-1 TA had no negative effect on body weight and enhanced the colon length in mice. The dose of 2.5 mg kg-1 TA ameliorated the morphological damage in the jejunum by increasing the villus height and crypt depth, activated the antioxidative pathway by decreasing jejunal protein expression of Kelch like-ECH-associated protein 1 (KEAP1) and increasing protein expression of Nuclear factor erythroid 2-related factor 2 (NRF2), and affected the intestinal barrier by inhibiting the jejunal mRNA expression of claudin and promoting mRNA expression of zonula occludens (zo-1). In conclusion, the pretreatment of TA in a mouse model of oxidative stress failed to change the antioxidative enzymes but modulated the jejunal morphology, colon length, antioxidative pathway and intestinal barrier in the diquat oxidative model.

Effects of dietary tryptophan supplementation in the acetic acid-induced colitis mouse model.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract and is strongly associated with intestinal immunity and the microbiome. Tryptophan (Trp) is an inflammatory inhibitor and modulator of the intestinal microflora. We explored the serum profile of amino acids and the effects of diet supplementation with Trp (1.0 g kg-1) on intestinal immunity and microbiota in the acetic acid-induced colitis mouse model. We analyzed the survival rate, colonic morphological parameters, profiles of serum amino acids, microbiota in colonic contents and the relative gene abundance of intestinal proinflammatory cytokines. Although the dietary Trp supplementation failed to improve the survival rate and ameliorate the morphological parameters of colon in mice with colitis, Trp modulated the general serum amino acid profile by reducing the amino acid profiles of threonine, methionine and proline, affected intestinal immunity by inhibiting the colonic expression of interleukin-22 and changed the microbiota by reducing the abundance of Candidatus, Clostridium and Coprococcus at the genus level. In conclusion, dietary Trp supplementation in a mouse model of colitis did not ameliorate the survival rate and morphological parameters of colon but did modulate the serum amino acid profiles, intestinal immunity and microbiota. These findings enhance our understanding of the roles of Trp in the metabolism of serum amino acids, intestinal immunity and microbiota.

Effect of dietary soy oil, glucose, and glutamine on growth performance, amino acid profile, blood profile, immunity, and antioxidant capacity in weaned piglets.

Weaning stress results in gastrointestinal dysfunction and depressed performance in pigs. This study aimed to investigate the effect of soy oil, glucose, and glutamine on the growth and health of weaned piglets. Compared with those in the glutamine group, piglets in the glucose and soy oil groups had greater average daily gain, average daily feed intake, and gain: feed ratio from day 0 to 14, and gain: feed ratio for the overall period. There were no differences with regard to serum amino acids among the three groups on day 14, except glycine and threonine. The serum concentration of histidine, serine, threonine, proline, and cysteine was the highest in the glutamine group, while the content of glycine and lysine in the soy oil group on day 28 was the highest among all groups. Piglets fed with glutamine had greater serum glucose and creatinine on day 14, high-density lipoprotein on day 28, and serum IgG and IgM on day 28. Piglets in the glutamine group demonstrated lower serum total superoxide dismutase on day 14 and 28; however, they demonstrated higher total superoxide dismutase and total antioxidant capacity in the duodenum and ileum on day 14. Weaned pigs supplemented with glucose or soy oil demonstrate better growth performance possibly due to their enhanced feed intake, whereas those supplemented with glutamine may have improved immunity and intestinal oxidative capacity.

Transfection of neurotrophin-3 into neural stem cells using ultrasound with microbubbles to treat denervated muscle atrophy.

Neurotrophin-3 (NT-3) has potential as a therapeutic agent for the treatment of patients with denervated muscle atrophy. However, the endogenous secretion of NT-3 is low and exogenous NT-3 lacks sufficient time to accumulate due to its short half-life. The transfection of NT-3 has been demonstrated to have a beneficial effect on denervated muscle and motor endplates. Neural stem cells (NSCs) differentiate into neurons and form motor endplate nerve-muscle connections. It has been previously demonstrated that local and noninvasive transfection can be performed using ultrasound with microbubbles (MBs). In the current study, hematoxylin and eosin, acetylcholinesterase and gold chloride staining, as well as transmission electron microscopy, were performed to verify the effects of this treatment strategy. The results demonstrated that using ultrasound with MBs for the transfection of NT-3 into NSCs, and their subsequent transplantation in vivo, attenuated the atrophy of denervated muscle and reduced motor endplate degeneration. This noninvasive, efficient and targeted treatment strategy may therefore be a potential treatment for patients with denervated muscle atrophy.

Ethanolic extract of fructus Alpinia oxyphylla protects against 6-hydroxydopamine-induced damage of PC12 cells in vitro and dopaminergic neurons in zebrafish.

In an attempt to understand the neuroprotective effect of Fructus Alpinia oxyphylla (AOE) and to elucidate its underlying mechanism of action, the ethanolic extract of AOE was investigated using zebrafish and PC12 cell models. AOE prevented and restored 6-hydroxydopamine (6-OHDA)-induced dopaminergic (DA) neuron degeneration and attenuated a deficit of locomotor activity in a zebrafish (Danio rerio) model of Parkinson's disease (PD). Treatment with AOE increased the viability of 6-OHDA-treated PC12 cells in vitro in a dose-dependent manner by attenuating cellular apoptosis. However, protocatechuic acid (PCA) and chrysin, two known polyphenol components of AOE, could not reproduce the neuroprotective activity of AOE in the PD zebrafish or PC12 cell models. A mechanistic study found that the protective effect of AOE against 6-OHDA-induced neuronal injury involved anti-inflammatory action (down-regulation of gene expression of IL-1ß and TNF-α) and anti-oxidative action (inhibition of NO production and iNOS expression in PC12 cells). Moreover, the PI3K-AKT pathway might be part of the mechanism of neuroprotection of AOE. The results of this research are expected to provide a scientific rationale for the use of AOE in the treatment of PD. However, it is important that the active components that contribute to the neuroprotective action of AOE are identified and characterized.

Eriocaulon buergerianum extract protects PC12 cells and neurons in zebrafish against 6-hydroxydopamine-induced damage.

BACKGROUND: Ericaulon buergerianum (Gujingcao) is an ophthalmic, anti-inflammatory and antimicrobial Chinese medicinal herb. This study aims to investigate the neuroprotective effects of Ericaulon buergerianum ethanol extract (EBE) and to elucidate its underlying action mechanism. METHODS: The viability of dopaminergic (DA) neuron in zebrafish was examined by anti-tyrosine hydroxylase (TH) immunostaining. The locomotor activity of zebrafish was assessed with a digital video tracking system. The viability and cellular damage of the PC12 cells were determined by MTT and LDH assays respectively. The nuclear morphological changes in apoptotic cells were evaluated with DNA staining by Hoechst 33342 dye. Intracellular nitric oxide (NO) was quantified by DAF-FM diacetate staining. The expression of inducible nitric oxide synthase (iNOS) was determined by Western blot. RESULTS: EBE inhibited the 6-OHDA-induced decrease in total distance of movement in zebrafish. Pretreatments of EBE (25, 50, 100 and 200 µg/ml) increased the viability of 6-OHDA-damaged PC12 cells in a dose dependent manner. Protection against 6-OHDA-induced nuclear fragmentation and accumulation of apoptotic bodies was also observed in EBE pretreated cells. Anti-oxidative (inhibition of NO production and iNOS expression in PC12 cells in vitro) activities of EBE are related to its neuroprotective effects in 6-OHDA-induced DA neuron damage. CONCLUSION: EBE exhibited significant neuroprotective activities in zebrafish, including recovery of dopaminergic neuron loss caused by 6-OHDA in a dose-dependent manner in vivo, inhibition of 6-OHDA-induced decrease of total distance in movement in zebrafish. The iNOS-NO pathway may be involved.