Effective extraction of bioactive alkaloids from the roots of Stephania tetrandra by deep eutectic solvents-based ultrasound-assisted extraction.

The efficient and green extraction of bioactive ingredients from natural plants play a vital role in their corresponding drug effects and subsequent studies. Recently, deep eutectic solvents (DESs) have been considered promising new green solvents for efficiently and selectively extracting substances from varied plants. In this work, an environment-friendly DESs-based ultrasonic-assisted extraction (DESs-UAE) procedure was developed for highly efficient and non-polluting extraction of alkaloids from the roots of Stephania tetrandra (ST). A total of fifteen different combinations of DESs, compared with traditional organic solvents (methanol and 95% ethanol) and water, were evaluated for extraction of bioactive alkaloids (FAN and TET) from ST, and the results revealed that DESs system made up of choline chloride and ethylene glycol with mole ratio of 1:2 exhibited the optimal extraction efficiency for alkaloids. Additionally, a four-factor and three-level Box-Behnken design (BBD), a particular pattern of response surface methodology (RSM), was used to optimize extraction conditions. RSM results indicated that the maximum extraction yields of FAN, TET, and TA were attained 7.23, 13.36, 20.59 mg/g, respectively, within extraction temperature of 52 °C, extraction time of 82 min, DES water content of 23% (v/v), and liquid-solid ratio of 23 mL/g. The measured results were consistent with the predicted values. Notably, the optimized DES extraction efficiency of TA, according to the experimental data analysis, is 2.2, 3.3 and 4.1 times higher than methanol, 95% ethanol and water, respectively. Meanwhile, based on 3D response surface plots, interactive effects plots and contour maps, the effects of the aforementioned four essential factors on the extraction yield and their interactions on the response were visualized. The results revealed that the mutual interactions between extraction temperature and liquid-solid ratio exhibited positive effects on all responses, while extraction time and water content in DES posed a negative effect. Therefore, these results suggest that DESs, as a class of novel green solvents, with the potential to substitute organic solvent and water, can be widely and effectively applied to extract bioactive compounds from natural plants.

Metagenomic Analysis of in Vitro Ruminal Fermentation Reveals the Role of the Copresent Microbiome in Plant Biomass Degradation.

In vitro ruminal fermentation is considered an efficient way to degrade crop residue. To better understand the microbial communities and their functions during in vitro ruminal fermentation, the microbiome and short chain fatty acid (SCFA) production were investigated using the metagenomic sequencing and rumen simulation technique (RUSITEC) system. A total of 1677 metagenome-assembled genomes (MAGs) were reconstructed, and 298 MAGs were found copresenting in metagenomic data of the current work and 58 previously ruminal representative samples. Additionally, the domains related to pectin and xylan degradation were overrepresented in the copresent MAGs compared with total MAGs. Among the copresent MAGs, we obtained 14 MAGs with SCFA-synthesis-related genes positively correlated with SCFA concentrations. The MAGs obtained from this study enable a better understanding of dominant microbial communities across in vivo and in vitro ruminal fermentation and show promise for pointing out directions for further research on in vitro ruminal fermentation.

Systematic study on dynamic pyrolysis behaviors, products, and mechanisms of weathered petroleum-contaminated soil with Fe2O3.

Weathered petroleum-contaminated soil (WPCS) with a high proportion of heavy hydrocarbons is difficult to remediate. Our previous research demonstrated that Fe2O3-assisted pyrolysis was a cost-effective technology for the remediation of WPCS. However, the pyrolysis behaviors, products, and mechanisms of the WPCS with Fe2O3 are still unclear. In this study, a combination of Thermogravimetric-Fourier transform infrared spectroscopy (TG-FTIR) and pyrolysis gas chromatography/mass spectrometry (Py-GC/MS) techniques were used to explore these pyrolysis characteristics. The thermal desorption/degradation of light and heavy hydrocarbons in the WPCS mainly occurred at 200-400 °C and 400-550 °C, respectively. The activation energy of thermal reaction of heavy hydrocarbons was decreased in the presence of Fe2O3 during the WPCS pyrolysis processes. In the process, the released inorganic gaseous products were mainly H2O and CO2, while the released organic gaseous compounds were primarily cycloalkanes, alkanes, acids/esters, alcohols, and aldehydes. Compared with the WPCS pyrolysis without Fe2O3, the yields of gaseous products released during the WPCS pyrolysis with Fe2O3 were reduced significantly, and some gaseous products were even not detected. This phenomenon was contributed by the following two reasons: 1) heavy hydrocarbons in the WPCS were more easily transformed into coke in the presence of Fe2O3 during pyrolysis; 2) some released gaseous products were reacted with Fe2O3 and fixed on the soil particles. Therefore, the WPCS pyrolysis with Fe2O3 can effectively reduce the burden of tail gas treatment. Criado method analysis results suggested that the reaction mechanism of heavy hydrocarbons during the WPCS pyrolysis with Fe2O3 was rendered as the synergic effects of diffusion, order-based, and random nucleation and growth reactions.

Multifunctional MnO2/Ag3SbS3 Nanotheranostic Agent for Single-Laser-Triggered Tumor Synergistic Therapy in the NIR-II Biowindow.

Regulating the level of reactive oxygen species (ROS) in a tumor is an efficient and innovative anticancer strategy. However, the therapeutic efficacy of ROS-based therapies, such as chemodynamic therapy (CDT) and photodynamic therapy (PDT), offers finite outcomes due to the oxygen dependence and limited concentration of hydrogen peroxide (H2O2) and overexpression of glutathione (GSH) within the tumor microenvironment (TME), so a single therapeutic strategy is insufficient to completely eliminate tumors. Therefore, we demonstrated an omnipotent nanoplatform MnO2/Ag3SbS3 (abbreviated as MA) with strong optical absorbance in the NIR-II biowindow and oxygen self-sufficient ROS-mediated ability, which not only relieves tumor hypoxia significantly but also enhances the photothermal therapy (PTT)/PDT/CDT efficacy. By 1064 nm laser irradiation, MnO2/Ag3SbS3 nanoparticles (NPs) reveal a favorable photothermal conversion efficiency of 23.15% and achieve a single-laser-triggered NIR-II PTT/PDT effect, resulting in effective tumor elimination. Once internalized into the tumor, MnO2/Ag3SbS3 NPs will be degraded to Mn2+ and Ag3SbS3. The released Ag3SbS3 NPs as a NIR-II phototherapy agent could be utilized for photoacoustic imaging-guided NIR-II PDT/PTT. Mn2+ could be used as a Fenton-like catalyst to continuously catalyze endogenous H2O2 for generating highly virulent hydroxyl radicals (•OH) for CDT and O2 for PDT, enhancing the efficiency of PDT and CDT, respectively. Meanwhile, Mn2+ realizes magnetic resonance imaging-guided accurate tumor therapy. Moreover, the MnO2/Ag3SbS3 NPs could deplete intracellular GSH in TME to promote oxidative stress of the tumor, further strengthening ROS-mediated antitumor treatment efficacy. Overall, this work presents a distinctive paradigm of TME-responsive PDT/CDT/PTT in the second near-infrared biowindow by depleting GSH and decomposing H2O2 for efficient and precise cancer treatment.

Naofucong Ameliorates High Glucose Induced Hippocampal Neuron Injury Through Suppressing P2X7/NLRP1/Caspase-1 Pathway.

P2X7/NLRP1/caspase-1 mediated neuronal injury plays an important role in diabetic cognitive impairment and eventually inflammatory cascade reaction. Chinese herbal compound Naofucong has been mainly used to treat cognitive disorders in Traditional Chinese Medicine The present study aimed to investigate whether its neuroprotective effects might be related to the inhibition of P2X7R/NLRP1/caspase-1 mediated neuronal injury or not. In this study, high glucose-induced HT22 hippocampal neurons were used to determine Naofucong-containing serum neuronal protective effects. Lentiviruses knock out of TXNIP and P2X7R was used to determine that protective effects of Naofucong was related to inflammatory response and P2X7/NLRP1/caspase-1 mediated neuronal injury. NAC was also used to inhibit oxidative stress, so as to determine that oxidative stress is an important starting factor for neuronal injury of HT22 cells cultured with high glucose. Naofucong decreased apoptosis, IL-1ß and IL-18 levels in high glucose-induced HT22 hippocampal neuron cells. Naofucong suppressed NLRP1/caspase-1 mediated neuronal injury, and P2X7 was involved in process. HT22 cells cultured in high glucose had an internal environment with elevated oxidative stress, which could promote neuronal injury. The current study demonstrated that Naofucong could significantly improve high glucose-induced HT22 hippocampal neuron injury, which might be related to suppress P2X7R/NLRP1/caspase-1 pathway, which provides novel evidence to support the future clinical use of Naofucong.

Nao-Fu-Cong ameliorates diabetic cognitive dysfunction by inhibition of JNK/CHOP/Bcl2-mediated apoptosis in vivo and in vitro.

Chinese herbal compound Nao-Fu-Cong (NFC) has been mainly used to treat cognitive disorders in Traditional Chinese Medicine (TCM). The present study aimed to investigate whether its neuroprotective effects might be related to the inhibition of JNK/CHOP/Bcl2-mediated apoptosis pathway or not. We randomly assigned STZ (60 mg·kg-1)-induced diabetic rats into control group, diabetic model group and NFC groups (low-dose, medium-dose and high-dose). The primary culture of hippocampal neurons were transferred into different culture media on the third day. The cells were then divided into control group, high-glucose group, NFC (low-dose, medium-dose and high-dose) groups, CHOP si-RNA intervention group, JNK pathway inhibitor SP600125 group and oxidative stress inhibitor N-acetylcysteine (NAC) group. NFC significantly improved the cognitive function of diabetic rats, and had neuroprotective effect on hippocampal neurons cultured in high glucose. Further research results showed that NFC could reduce the apoptosis of hippocampal neurons in rats with diabetic cognitive dysfunction. NFC had inhibitory effects on CHOP/JNK apoptosis pathway induced by high glucose, and also decreased the levels of ROS and increased the mitochondrial membrane potential. These suggested that the neuroprotective effect of NFC might be related to the inhibition of CHOP and JNK apoptotic signaling pathways, and the cross pathway between oxidative stress and mitochondrial damage pathway.

Effect of icariin on apoptosis in hippocampal neurons cultured in high glucose.

OBJECTIVE: To investigate effect of icariin on apoptosis in hippocampal neurons cultured in high glucose, and the possible mechanism behind the action. METHODS: Hippocampus was obtained from newborn 24 h Sprague-Dawley rats and then primarily cultured. Then hippocampal neurons were divided into normal control group, high glucose group, icariin group, icariin + protein kinase B (Akt) inhibitor group and Akt agonist group. After each group was cultured in different conditioned medium for 72 h, we detected the apoptosis of neurons with flow cytometry, and the expression of Akt, p-Akt, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) with western blot. RESULTS: Compared with the normal control group, the apoptosis rate of hippocampal neurons increased significantly (P < 0.01), and p-Akt/Akt and Bcl-2/Bax decreased significantly (P < 0.01) in high glucose group. Compared with the high glucose group, the apoptosis rate of hippocampal neurons decreased significantly (P < 0.01), and p-Akt/Akt and Bcl-2/Bax increased significantly (P < 0.05)in icariin group and Akt agonist group. Compared with the icariin + Akt inhibitor group, the apoptosis rate of hippocampal neurons decreased significantly (P < 0.01), and p-Akt/Akt and Bcl-2/Bax increased significantly (P < 0.01) in icariin group. CONCLUSION: Icariin could reduce the apoptosis of neurons cultured in high glucose, which may be achieved by increasing the phosphorylation of Akt protein in Akt signal pathway, then increasing the expression of Bcl-2 and inhibiting the expression of Bax.

Effect of Chinese herbal compound Naofucong () on the inflammatory process induced by high glucose in BV-2 cells.

OBJECTIVE: To determine the effect of medicated serum of Chinese herbal compound Naofucong (, NFC) on the microglia BV-2 cells viability and the transcription and expression of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in microglia BV-2 cells to further explore the mechanisms underlying the protective effect of NFC on inflammatory process induced by high glucose. METHODS: The microglia BV-2 cells incubated in vitro were divided into different groups: the control group (25 mmol/L glucose), the model group (75 mmol/L glucose), high glucose media containing different dose medicated serum of NFC. After being cultured for 24 h, changes in IL-6 and TNF-α were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The expression of surface marker CD11b of activated microglia was measured by confocal laser scanning microscope and Western blot. Nuclear factor-κB (NF-κB) p-p65 expression was analyzed by Western blot. RESULTS: The model group obviously increased the expression of microglial surface marker CD11b and NF-κB p-p65 (all P<0.01), induced a signifificant up-regulation of release and the mRNA expression of IL-6 and TNF-α (P<0.01 or P<0.05). The medicated serum of NFC could obviously down-regulate the transcription and expression of surface marker CD11 b and NF-κB p-p65 (all P<0.01), and inhibit the mRNA and protein expression (P<0.01 or P<0.05) of inflflammatory cytokines, such as IL-6 and TNF-α, in microglia BV-2 cells cultured with high glucose for 24 h. CONCLUSIONS: The inhibition of microglial activation and IL-6 and TNF-α expression induced by high glucose may at least partly explain NFC therapeutic effects on diabetes-associated cognitive decline diseases. Its underlying mechanism could probably be related to the inhibition of NFC on NF-κB phosphorylation.

Puerarin reduces apoptosis in rat hippocampal neurons culturea in high glucose medium by modulating the p38 mitogen activated protein kinase and c-Jun N-terminal kinase signaling pathways.

OBJECTIVE: To investigate the neuroprotective etfect of puerarin on rat hippocampal neurons cultured in high glucose medium, and to examine the role of the p38 mitogen activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) signaling pathways in this effect. METHODS: Primary cultures of hippocampal neurons were prepared from newborn Sprague Dawley rats. Neuron-specific enolase immunocytochemistry was used to identify neurons. The neurons were cultured with normal medium (control group) or with high-glucose medium (high-glucose group), and puerarin (puerarin group), a p38 MAPK inhibitor (SB239063; p38 MAPK inhibitor group) or a JNK inhibitor (SP600125; JNK inhibitor group) were added. After 72 h of treatment, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was performed to detect apoptosis, and western blotting was used to assess protein levels of p-p38, p38, p-JNK and JNK. RESULTS: In the high-glucose group, the neuronal apoptosis rate and the p-p38/p38 and p-JNK/JNK ratios were higher than in the control group. The p38 MAPK and JNK inhibitors prevented this increase in the apoptosis rate. The apoptosis rates in the puerarin group, the p38 MAPK inhibitor group and the JNK inhibitor group were significantly decreased compared with the high-glucose group. Moreover, protein levels of p-p38 and p-JNK were significantly reduced, and the p-p38/p38 and p-JNK/JNK ratios were decreased in the puerarin group compared with the high-glucose group. In addition, compared with the high-glucose group, p-p38 levels and the p-p38/p38 ratio were reduced in the p38 MAPK inhibitor group, and p-JNK levels and the p-JNK/JNK ratio were decreased in the JNK inhibitor group. CONCLUSION: Puerarin attenuates neuronal apoptosis induced by high glucose by reducing the phosphorylation of p38 and JNK.

Separation of alkaloids from herbs using high-speed counter-current chromatography.

Alkaloids represent a most widespread group of bioactive natural products. Because of their alkalinity and structural diversity, the fractionation and purification of the alkaloids from herbs can often present a number of practical difficulties using the conventional chromatographic techniques. High-speed counter-current chromatography (HSCCC) is a liquid-liquid partition chromatography with a support-free liquid stationary phase, and is gaining more and more popularity as a viable separation technique for bioactive compounds from natural resources. In the present review, focus is placed on the separation of alkaloids by both conventional HSCCC and pH-zone-refining counter-current chromatography (CCC) techniques from herbs. The review presents the separation of over 120 different alkaloid compounds from more than 30 plant species by the conventional HSCCC and pH-zone-refining CCC. Based on the data from the literature, the proper solvent systems for the separation of alkaloids by the conventional HSCCC and pH-zone-refining CCC are also summarized.

Combined microwave-assisted extraction and high-speed counter-current chromatography for separation and purification of xanthones from Garcinia mangostana.

A microwave-assisted extraction (MAE) method is presented for the extraction of xanthones, α-mangostin and γ-mangostin from Garcinia mangostana. The MAE conditions including extraction temperature, liquid/solid ratio, extraction time and concentration of ethanol were optimized with an orthogonal test, and 5 g sample was extracted with the optimized conditions. The crude extraction of MAE was successfully isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (0.8:0.8:1:0.6, v/v) in one-step separation. The separation yielded 75 mg of α-mangostin at 98.5% purity, and 16 mg of γ-mangostin at 98.1% purity from 360 mg crude extract of G. mangostana in less than 7h. The purity of the two xanthones was determined by HPLC. Their structures were further identified by ESI-MS, (1)H NMR and (13)C NMR.

Application of high-speed counter-current chromatography for preparative separation of cyclic peptides from Vaccaria segetalis.

Following an initial clean-up step on silica gel, high-speed counter-current chromatography (HSCCC) was used to separate cyclic peptides from an extract of the seeds of Vaccaria segetalis. The two-phase solvent system used for HSCCC separation was composed of petroleum ether-ethyl acetate-methanol-water at an optimized volume ratio of 0.5:3.5:1:5. From 190 mg of crude extract, 38.0 mg of segetalin B and 28.5 mg of segetalin A were obtained with purities of 98.1% and 95.6% as determined by HPLC, respectively. The chemical structures of the target compounds were confirmed by high resolution electrospray ionization time of flight MS (HRESI-TOF-MS) and (1)H NMR analyses.

Minimally invasive plate internal fixation for calcaneal fractures.

OBJECTIVE: To assess the clinical efficacy of minimally invasive plate internal fixation for the treatment of calcaneal fractures. METHODS: Manual reduction, rectification of deformity, and cold compress with traditional Chinese medicine were used preoperatively to relieve swelling and pain. A small incision was made to expose the articular facet and to perform anatomic reduction and plate fixation. Self-made traditional Chinese pharmaceutics were applied postoperatively on the surface of the wound to accelerate bony union. RESULTS: All the 40 patients were followed up for at least 1 year postoperatively. According to the Maryland scoring system, the excellent and good rate was 87.5%. CONCLUSION: Minimally invasive plate internal fixation has the advantages of relatively mild injury, reliable fixation, good recovery, and rare complications in the treatment of intraarticular fractures.

[Correlation of Ca2+ current features of nasopharyngeal carcinoma cells with different metastatic potentiality to their moving abilities].

BACKGROUND & OBJECTIVE: The metastatic potentiality of malignancies is closely associated with their biological dynamic properties, which are affected by intracellular Ca2+ current activity. This study was to investigate the correlation of Ca2+ current features of sub-clonal nasopharyngeal carcinoma (NPC) cell lines 5-8F and 6-10B with different metastatic potentiality to their moving abilities. METHODS: 5-8F cells, with higher metastatic potentiality, and 6-10B cells, with lower metastatic potentiality, were cultured with herbal medicine-containing serum, which holds significant metastasis-inhibiting effect on tumor cells. Cell proliferation was assessed by MTT assay. The expression of nm23-H1 was detected by Western blot. Intracellular Ca2+ current features were detected with patch clamp technique in a whole cell recording way, while cell moving ability was determined by streak culturing assay. RESULTS: The expression of nm23-H1 was significantly lower in 5-8F cells than in 6-10B cells (2.3+/-0.2 vs. 2.9+/-0.4). The Ca2+ release-activated Ca2+ influx current (ICRAC) was significantly lower in 5-8F cells than in 6-10B cells [(-1.39+/-0.36) nA vs. (-0.66+/-0.40) nA, P < 0.05]. The number of cells moved across the streak was significantly higher in 5-8F cells than in 6-10B cells (350+/-3 vs. 246+/-1, P< 0.05). When cultured with herbal medicine-containing serum, no significant difference in proliferation was found between 5-8F cells and 6-10B cells; the expression of nm23-H1 was significantly higher in 5-8F cells than in 6-10B cells(3.9+/-0.1 vs.1.0+/-0.1,P<0.05)û the ICRAC was decreased to (-1.27+/-0.35) nA in 5-8F cells and decreased to (-0.37+/-0.23) nA in 6-10B cell, and the inhibition rate was significantly higher in 5-8F cells than in 6-10B cells [(1.90+/-0.47)% vs. (0.46+/-0.12)%, P < 0.05]û the number of cells moved across the streak was significantly lower in 5-8F cells than in 6-10B cells (94+/-6 vs. 229+/-6, P < 0.05). CONCLUSIONS: There are significant differences in nm23-H1 protein expression, ICRAC level and cell moving ability between 5-8F and 6-10B cells. Medicine intervention could inhibit Ca2+ current and moving ability of 5-8F cells, and meanwhile increase the nm23-H1 activity.

[Effects of Jianpi Yishen Recipe on recurrence, metastasis and life span of mice transplanted with proventriculus squamous carcinoma after tumorectomy].

OBJECTIVE: To observe the effects of Jianpi Yishen Recipe (JPYSR), a compound Chinese herbal medicine, on recurrence, metastasis and life span of mice transplanted with proventriculus squamous carcinoma cells after tumorectomy. METHODS: JPYSR was orally administered to 615 mice transplanted with proventriculus squamous carcinoma cells in the palma of left hind limb after tumorectomy. The local tumor recurrence, lung metastasis and life span of the mice were evaluated and compared with those of the 5-FU-treated mice and untreated mice. RESULTS: The pulmonary metastasis rate was 94.4% and the recurrence rate was 94.44% in untreated group. The pulmonary metastasis rate was 68.4%, the inhibition rate of pulmonary metastasis was 27.5%, the recurrence rate was 78.95%, and the inhibition rate of tumor recurrence was 65.35% in JPYSR-treated group. The average and median life spans were obviously prolonged in JPYSR-treated group, as compared with those in untreated group. The life-prolonging rate was 100%. CONCLUSION: JPYSR can effectively inhibit the local recurrence and pulmonary metastasis of the transplanted proventriculus squamous carcinoma in mice after tumorectomy, and prolong the life span.

[Effect of combination of glutamine and mitomycin C on human gastric carcinoma cell strain MGC-803].

OBJECTIVE: To evaluate the effect of combination of glutamine (GLN) and mitomycin C (MMC) on the human gastric carcinoma cell line MGC-803 in vitro. METHODS: The effects of GLN and MMC were measured by MTT assay, and the interaction between the two agents was evaluated by the median-effect principle. Flow cytometry was used for cell cycle analysis. RESULTS: GLN did not significantly stimulate the cell growth in vitro. High-concentration of GLN could inhibit the cell growth. MMC could effectively inhibit the cell growth in a time-dependent manner. The interaction of these two agents showed a weak antagonistic activity (1 < CI < 1.2703). MMC induced remarkable S-phase arrest. Low-dose GLN has limited effect on the S-phase arrest of MMC, while high-dose GLN significantly attenuated the S-phase arrest and lowered the proliferation index of MGC-803 cell. CONCLUSIONS: Combination of GLN and MMC has a a weak and dose-dependent antagonistic activity in the treatment of gastric carcinoma cell line MGC-803. The combination of high-dose MMC and low-dose GLN may achieve better efficacy.

Influence of mannan oligosaccharide, Ligustrum lucidum and Schisandra chinensis on parameters of antioxidative and immunological status of broilers.

The study was conducted to evaluate effects of dietary supplementation with Ligustrum lucidum (LL, 10 g/kg), Schisandra chinensis (SC, 10 g/kg), LL (10 g/kg) + mannan oligosaccharides (MOS, 50 mg/kg), or SC (10 g/kg) + MOS (50 mg/kg) on growth performance and parameters of antioxidative and immunological status of broilers. The results showed that feeding LL, SC, LL + MOS, or SC + MOS had no significant effect on growth performance of broilers relative to the control. However, compared to the control, LL, SC, LL + MOS, or SC + MOS significantly decreased malondialdehyde concentration in serum, thigh, and heart of broilers. In addition, glutathione reductase activity of heart and sera of the birds were significantly elevated by supplementation LL, SC, LK + MOS, or SC + MOS. Furthermore, LL, SC, LL + MOS, or SC + MOS significantly improved antibody titres against Newcastle disease virus and lymphocyte proliferation of broilers (p < 0.05). Whereas, no cooperating effect between LL (or SC) and MOS on antioxidant status and immunity of broilers were found.