Screening and evaluating honokiol from Magnolia officinalis against Nocardia seriolae infection in largemouth bass (Micropterus Salmoides).

Nocardiosis caused by Nocardia seriolae is a major threat to the aquaculture industry. Given that prolonged therapy administration can lead to a growth of antibiotic resistant strains, new antibacterial agents and alternative strategies are urgently needed. In this study, 80 medicinal plants were selected for antibacterial screening to obtain potent bioactive compounds against N. seriolae infection. The methanolic extracts of Magnolia officinalis exhibited the strongest antibacterial activity against N. seriolae with the minimal inhibitory concentration (MIC) of 12.5 µg/ml. Honokiol and magnolol as the main bioactive components of M. officinalis showed higher activity with the MIC value of 3.12 and 6.25 µg/ml, respectively. Sequentially, the evaluation of antibacterial activity of honokiol in vivo showed that honokiol had good biosafety, and could significantly reduce the bacterial load of nocardia-infected largemouth bass (p < .001). Furthermore, the survival rate of nocardia-infected fish fed with 100 mg/kg honokiol was obviously improved (p < .05). Collectively, these results suggest that medicinal plants represent a promising reservoir for discovering active components against Nocardia, and honokiol has great potential to be developed as therapeutic agents to control nocardiosis in aquaculture.

Ophiopogon japonicus inhibits white spot syndrome virus proliferation in vivo and enhances immune response in Chinese mitten crab Eriocheir sinensis.

White spot syndrome virus (WSSV) is a fatal pathogen threatening global crustacean industry with no commercially available drugs to control. Herbal medicines have been widely used to treat a number of viral infections, which could offer a rich reserve for antiviral drug discovery. Here, we evaluated the inhibition activities of 30 herbal medicines against WSSV in Chinese mitten crab Eriocheir sinensis. A WSSV infection model in E. sinensis was firstly established in order to determine the antiviral effects of the plant extracts and to explore the potential action mechanisms. Results showed that the highest anti-WSSV activity was obtained by the treatment of Ophiopogon japonicus extract (93.03%, 100 mg/kg). O. japonicus treatment decreased viral loads in a dose-dependent manner and significantly improved the survival of WSSV-challenged crabs. O. japonicus reduced the expression of vital genes in viral life cycle in vivo, particularly for the immediate-early stage gene ie1. Further results indicated that O. japonicus could repress the JAK-STAT signaling pathway to block ie1 transcription. Moreover, O. japonicus could modulate certain immune genes such as the myosin, toll-like receptor, crustin, and prophenoloxidase in the interactions between WSSV and crabs. The up-regulated expression of pro-autophagic factors (Gabarap and Atg7) and elevated levels of antioxidant enzymes (SOD, CAT and GSH) suggested that O. japonicus may induce autophagy and attenuate WSSV-induced oxidative stress. Taken together, O. japonicus could inhibit WSSV proliferation and improve the survival of WSSV-challenged crabs. Thus, O. japonicus may have the potential to be developed as a preventive or therapeutic agent against WSSV, and its effective compounds merit further isolation and identification.

Ursolic acid from Prunella vulgaris L. efficiently inhibits IHNV infection in vitro and in vivo.

Infectious hematopoietic necrosis virus (IHNV) is a fish viral pathogen that causes severe disease and huge economic losses in the salmonid aquaculture industry. However, anti-IHNV drugs currently are scarce. For the purpose of seeking out anti-IHNV drugs, the anti-IHNV activities of 32 medicinal plants were investigated by using epithelioma papulosum cyprini (EPC) cells. Among these plants, Prunella vulgaris L. (PVL) showed the strongest inhibition on IHNV replication with an inhibitory percentage of 99.3% at the concentration 100 mg/L. Further studies demonstrated that ursolic acid (UA), a major constituent of PVL, also showed a highly effective anti-IHNV activity. The half-maximal inhibitory concentration (IC50) at 72 h of UA on IHNV was 8.0 µM. Besides, UA could significantly decrease cytopathic effect (CPE) and the viral titer induced by IHNV in EPC cells. More importantly, UA also showed a strong anti-IHNV activity in vivo, as indicated by increasing the survival rate of rainbow trout and inhibiting viral gene expression. Intraperitoneal injection of UA increased the relative percentage of survival of rainbow trout by 18.9% and inhibited IHNV glycoprotein mRNA expression by > 90.0% in the spleen at the 1st-day post-infection. Altogether, UA was expected to be a therapeutic agent against IHNV infection in aquaculture.

Synthesis and antiviral activity of a new arctigenin derivative against IHNV in vitro and in vivo.

Viral diseases in aquaculture were challenging because there are few preventative measures and/or treatments. Our previous study indicated that imidazole arctigenin derivatives possessed antiviral activities against infectious hematopoietic necrosis virus (IHNV). Based on the structure-activity relationship in that study, a new imidazole arctigenin derivative, 4-(8-(2-ethylimidazole)octyloxy)-arctigenin (EOA), was designed, synthesized and its anti-IHNV activity was evaluated. By comparing inhibitory concentration at half-maximal activity (IC50), we found that EOA (IC50 = 0.56 mg/L) possessed a higher antiviral activity than those imidazole arctigenin derivatives in our previous study. Besides, EOA could significantly decrease cytopathic effect (CPE) and viral titer induced by IHNV in epithelioma papulosum cyprinid (EPC) cells. In addition, EOA significantly inhibited apoptosis induced by IHNV in EPC cells. Further data verified that EOA inhibited IHNV replication in rainbow trout, with reducing 32.0% mortality of IHNV-infected fish. The results suggested that EOA was more stable with a prolonged inhibitory half-life in the early stage of virus infection (1-4 days). Consistent with above results, EOA repressed IHNV glycoprotein gene expression in virus sensitive tissues (kidney and spleen) in the early stage of virus infection. Moreover, histopathological evaluation showed that tissues from the spleen and kidney of fish infected with IHNV exhibited pathological changes. But there were no lesions in any of the tissues from the control group and EOA-treaten group. In accordance with the histopathological assay, EOA could elicited anti-inflammation response in non-viral infected rainbow trout by down-regulating the expression of cytokine genes (IL-8, IL-12p40, and TNF-α). Altogether, EOA was expected to be a therapeutic agent against IHNV infection in the field of aquaculture.

Isolation of anti-Saprolegnia lignans from Magnolia officinalis and SAR evaluation of honokiol/magnolol analogs.

To control the fish fungal pathogen Saprolegnia, the effects of the petroleum ether extracts of Magnolia officinalis were evaluated by a rapeseed (Brassicanapus) microplate method in vitro. By loading on an open silica gel column and eluting with petroleum ether-ethyl acetate-methanol, honokiol (C18H18O2) and magnolol (C18H18O2) were isolated from Magnolia officinalis. Saprolegnia parasitica growth was inhibited significantly when honokiol concentration was >8.0 mg/L, and magnolol concentration was >9.0 mg/L, with EC50 values of 4.38 and 4.92 mg/L, respectively. Six honokiol and magnolol derivatives were designed, synthesized and evaluated for their anti-Saprolegnia activity. According to the results, double bond and hydroxyl played an important role in inhibiting Saprolegnia. Mechanistically, through the scanning electron microscope observation, honokiol and magnolol could cause the Saprolegnia parasitica mycelium tegumental damage including intensive wrinkles and nodular structures. Moreover, compared to traditional drugs kresoxim-methyl (LC50 = 0.66 mg/L) and azoxystrobin (LC50 = 2.71 mg/L), honokiol and magnolol showed a lower detrimental effect on zebrafish, with the LC50 values of 6.00 and 7.28 mg/L at 48 h, respectively. Overall, honokiol and magnolol were promising lead compounds for the development of commercial drugs anti-Saprolegnia.

Synthesis and in vitro activities evaluation of arctigenin derivatives against spring viraemia of carp virus.

Spring viraemia of carp virus (SVCV) is a viral fish pathogen causing high mortality in several carp species and other cultivated fish. However, robust anti-SVCV drugs currently are extremely scarce. For the purpose of seeking out anti-SVCV drugs, here a total of 35 arctigenin derivatives were designed, synthesized and evaluated for their anti-viral activities. By comparing the inhibitory concentration at half-maximal activity (IC50) of the 15 screened candidate drugs (max inhibitory response surpassing 90%) in epithelioma papulosum cyprini (EPC) cells infected with SVCV, 2Q and 6 A were chosen for additional validation studies, with an IC50 of 0.077 µg/mL and 0.095 µg/mL, respectively. Further experiments revealed that 2Q and 6 A could significantly decrease SVCV-induced apoptosis and have a protective effect on cell morphology at 48 and 72 h post-infection. Moreover, the reactive oxygen species (ROS) induced upon SVCV infection could be obviously inhibited by 2Q and 6 A, while SVCV-infected cells were clearly observed. On account of these findings, 2Q and 6 A could have a promising application for the treatment of infection of SVCV and provide a considerable reference for novel antivirals in aquaculture.

Magnolol protects Ctenopharyngodon idella kidney cells from apoptosis induced by grass carp reovirus.

Many natural products from medicinal plants are small molecular weight compounds with enormous structural diversity and show various biological activities. Magnolol is a biphenol compound rich in the stem bark of Magnolia officinalis Rehd et Wils., and is able to suppress viral replication in GCRV-infected grass carp (Ctenopharyngodon idella) kidney (CIK) cells in the previous study. In this study, in vivo studies demonstrated that magnolol was efficient to restrain the replication of GCRV and repair the low level of superoxide dismutase and total antioxidant capacity in serum at the non-toxic concentration in vivo. Furthermore, magnolol inhibited CIK cell apoptosis induced by GCRV and kept the normal cellular morphological structure, reflecting in the protection of CIK cells from cell swelling, the formation of apoptotic bodies, the disappearance of cellular morphology and nuclear fragmentation. Reverse transcript quantitative polymerase chain reaction (RT-qPCR) showed that magnolol facilitated the expression of apoptosis-inhibiting gene bcl-2, while suppressed the expression of apoptosis-promoting gene bax in GCRV-infected cells. Besides, RT-qPCR and enzyme activity assays proved that magnolol suppressed the expression of caspase 3, caspase 8 and caspase 9. Moreover, interactions between magnolol and proteins were predicted by using the STITCH program, which revealed that ten proteins including caspase 3, were involved in the apoptosis pathway, p53 signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway and toll-like receptor signaling pathway. Further assays were performed to test the effect of magnolol on apoptosis pathway, which showed that magnolol dramatically inhibited the activity of caspase 3 rather than those of caspase 8 and caspase 9. Collectively, the present study revealed that magnolol heightened the resistance of grass carp against GCRV infection and refrained GCRV-induced apoptosis, which may be attributed to the direct interaction of magnolol with caspase 3. The present results make a contribution to understanding the mechanisms by which small-molecule drugs possess antiviral activities, and lay a foundation for the development of broad-spectrum antiviral compounds in aquaculture industry.

Magnolol and honokiol from Magnolia officinalis enhanced antiviral immune responses against grass carp reovirus in Ctenopharyngodon idella kidney cells.

Medicinal plants have been widely used for a long history. Exploration of pharmacologically active compounds from medicinal plants present a broad prevalent of application. By examining viral mRNA expression in GCRV-infected Ctenopharyngodon idella kidney (CIK) cells treated with thirty kinds of plant extracts, we identified Magnolia officinalis Rehd et Wils. was able to preferably suppress viral replication. Further studies demonstrated that the main ingredients of magnolia bark, namely, magnolol and honokiol presented protective pharmacological function when treated GCRV-infected CIK cells with a concentration of 2.00 µg/ml and 1.25 µg/ml, respectively. Furthermore, reverse transcript quantitative polymerase chain reaction (RT-qPCR) and western blot showed that both magnolol and honokiol were efficient to restrain the replication of GCRV in CIK cells at non-toxic concentration (2.51 ± 0.51 µg/ml for magnolol, and 3.18 ± 0.61 µg/ml for honokiol). Moreover, it was found that magnolol and honokiol promoted the expression of immune-related genes. Magnolol obviously significantly increased the expression of interferon (IFN) regulatory factor (IRF)7 rather than that of IRF3 in the GCRV-infected cells, leading to the activation of type I IFN (IFN-I). Simultaneously, magnolol drastically facilitated the expression of interleukin (IL)-1ß, but failed to induce the molecules in nuclear factor (NF)-κB pathways. Differently, honokiol strikingly motivated not only the expression of IL-1ß, but also those of tumor necrosis factor α (TNFα) and NF-κB. Interestingly, though honokiol motivated the expression of IFN-ß promoter stimulator 1 (IPS-1), IRF3 and IRF7, it failed to up-regulate the expression of IFN-I, indicating that honokiol enhanced the host innate antiviral response to GCRV infection via NF-κB pathways. Collectively, the present study revealed that magnolol and honokiol facilitated the expression of innate immune-related genes to strengthen the innate immune signaling responses to resist GCRV infection, which contributed to understanding the mechanisms by which small-molecule drugs possessed antiviral activities. In addition, these results lay a foundation for the development of broad-spectrum antiviral compounds in aquaculture industry.

In vitro immunocompetence of two compounds isolated from Polygala tenuifolia and development of resistance against grass carp reovirus (GCRV) and Dactylogyrus intermedius in respective host.

The present study was undertaken to isolate some compounds from methanol extract of Polygala tenuifolia and evaluate their immunostimulatory properties and antiviral activity using grass carp Ctenopharyngodon idella kidney (CIK) cells and GCRV. By applying insecticidal bioassay-guided, chromatography techniques and successive recrystallization, two purified compounds were obtained. The changes of expression of selected immune genes (Mx1, IL-1ß, TNFα, MyD88 and IgM) in C. idella kidney cell lines were evaluated after exposure to these isolated compounds. The results showed that compound 1 and 2 up-regulated to varying degrees of Mx1, IL-1ß, TNFα, and MyD88 in C. idella kidney cells. WST-8 kit assay verified the two compounds has no toxic effects on CIK cell, and furthermore, have in vitro antivirus activity. Especially, that there is keeping 79% cell viability when exposure to compound 2 (100 mg L(-1)). According to in vivo insecticidal assays against Dactylogyrus intermedius, compound 2 exhibited higher efficacy than compound 1, which was found to be 87.2% effective at the concentrations of 5 mg L(-1) and safe to goldfish (Carassius auratus). Besides, the purified compounds were identified by spectral data as: (1) 1,5-Anhydro-D-glucitol and (2) 3,4,5-trimethoxy cinnamic acid. Overall, the results indicate that bath administration of these compounds modulates the immune related genes in C. idella kidney cells and to some extent, eliminate the virus and parasitic infections.

Aquatic environmental safety assessment and inhibition mechanism of chemicals for targeting Microcystis aeruginosa.

Cyanobacteria are a diverse group of Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against vertebrates, invertebrates, fungi, bacteria and cell lines. Recently the main methods of controlling cyanobacteria are using chemicals, medicinal plants and microorganism but fewer involved the safety research in hydrophytic ecosystems. In search of an environmentally safe compound, 53 chemicals were screened against the developed heavy cyanobacteria bloom Microcystis aeruginosa using coexistence culture system assay. The results of the coexistence assay showed that 9 chemicals inhibited M. aeruginosa effectively at 20 mg L(-1) after 7 days of exposure. Among them dimethomorph, propineb, and paraquat were identified that they are safe for Chlorella vulgaris, Scenedesmus obliquus, Carassius auratus (Goldfish) and Bacillus subtilis within half maximal effective concentration (EC50) values 5.2, 4.2 and 0.06 mg L(-1) after 7 days, respectively. Paraquat as the positive control observed to be more efficient than the other compounds with the inhibitory rate (IR) of 92% at 0.5 mg L(-1). For the potential inhibition mechanism, the chemicals could destroy the cell ultrastructure in different speed. The safety assay proved dimethomorph, propineb and paraquat as harmless formulations or products having potential value in M. aeruginosa controlling, with the advantage of its cell morphology degrading ability.

Developmental toxicity in rare minnow (Gobiocypris rarus) embryos exposed to Cu, Zn and Cd.

Using rare minnow (Gobiocypris rarus) embryos as experimental model, the developmental toxicity of Cu, Zn and Cd were investigated following exposure to 0.001-1.000mg/L for 72h, and the toxicity effects were evaluated by larval malformation rate, heart rate, pericardial area, spontaneous movements, tail length, enzyme activities and biomarker genes. Our results revealed that increased malformation rate provide a gradual dose-response relationship, and the most pronounced morphological alteration was heart and body malformations. Values of 72h EC50 with their 95 percent confidence intervals on G. rarus embryos were 0.103 (0.072-0.149)mg/L for Cu, 0.531 (0.330-1.060)mg/L for Zn, 0.219 (0.147-0.351)mg/L for Cd. Enzyme activities can be regard as a type of low-dose stimulation and high-dose inhibition. Stress and metabolism-related genes (hsp70, cyp1a and mt) were significantly up-regulated, development-related genes (wnt8a, vezf1 and mstn) were significantly down-regulated after the treatment by Cu, Zn and Cd. Overall, the present study points out Cu, Zn and Cd are highly toxic to G. rarus embryos. The information presented in this study will be helpful in fully understanding the toxicity induced by Cu, Zn and Cd in fish embryos.

Effect of intestinal autochthonous probiotics isolated from the gut of sea cucumber (Apostichopus japonicus) on immune response and growth of A. japonicus.

The study isolated 224 bacteria from the intestine of Apostichopus japonicus, then selected and identified three of the bacteria (HS1, HS7, and HS10) which demonstrated amylase, lipase, and protease production capacity as candidate probiotics for sea cucumbers. The three potential probiotics showed no pathogenicity both in hemolytic assays on sheep blood agar plates and after immersing sea cucumbers in a suspension of the bacteria. To reveal the effects of these three potential probiotics on the innate immunity of sea cucumbers, total coelomocyte counts, respiratory burst activity, superoxide dismutase activity, lysozyme activity, acid phosphatase activity, and phagocytic activity by coelomocytes were examined after feeding with four different diets for up to 28 days. Also the specific growth rate and survival rate were investigated after a 60-day feeding trial. Sea cucumbers were fed with 4 diets: one control, three diets supplemented with 1 × 10(9) cell g(-1) of HS1, HS7, and HS10 for 28-60 days. Results showed that sea cucumbers fed diets containing HS1, HS7, and HS10 had led to an enhanced cellular and humoral immune response, notably higher total coelomocytes counts, respiratory burst activity, lysozyme activity, acid phosphatase activity, and phagocytic activity, as recorded during the four weeks of probiotics administration. On the other hand, the survival rate among dietary treatments ranged from 90.71 to 97.97% with significant improvement (P < 0.05) compared to that of the control; and the growth rate observed in the sea cucumbers fed HS1 and HS7 showed sharp increases after 60 days feeding. The present study confirmed the potential beneficial effects of Pseudoalteromonas elyakovii HS1, Shewanella japonica HS7, and Vibrio tasmaniensis HS10 as dietary probiotics in A. japonicus.

Screening of plant extracts for anthelmintic activity against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus).

With the aim of finding natural anthelmintic agents against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus), 26 plants were screened for antiparasitic properties using in vivo anthelmintic efficacy assay. The results showed that Caesalpinia sappan, Lysima chiachristinae, Cuscuta chinensis, Artemisia argyi, and Eupatorium fortunei were found to have 100% anthelmintic efficacy at 125, 150, 225, 300, and 500 mg L(-1) after 48 h of exposure. Crude extract of the five plants were further partitioned with petroleum ether, chloroform, ethyl acetate, methanol, and water to obtain anthelmintically active fractions with various polarity. Among these fractions tested, the ethyl acetate extract of L. chiachristinae was found to be the most effective with a 50% effective concentration (EC50) value of 5.1 mg/L after 48 h of exposure. This was followed by ethyl acetate extract of C. chinensis (48 h-EC50 = 8.5 mg L(-1)), chloroform extracts of C. sappan (48 h-EC50 = 15.6 mg L(-1)), methanol extract of C. chinensis (48 h-EC50 = 15.9 mg L(-1)), and chloroform and petroleum ether extract of L. chiachristinae (EC50 values of 17.2 and 21.1 mg/L, respectively), suggesting that these plants, as well as the active fractions, provide potential sources of botanic drugs for the control of D. intermedius in aquaculture.

In vivo anthelmintic effect of flavonol rhamnosides from Dryopteris crassirhizoma against Dactylogyrus intermedius in goldfish (Carassius auratus).

The purpose of the present study was to assess the anthelmintic property of plant-derived polyphenolic compounds extracted and isolated from Dryopteris crassirhizoma against Dactylogyrus intermedius in goldfish. The active ethyl acetate extract was loaded on an open silica gel column and eluted with chloroform-methanol. According to (1)H-nuclear magnetic resonance (NMR), (13)C-NMR, and mass spectral data, the structures of three purified compounds were identified as protocatechuic acid, sutchuenoside A, and kaempferitrin. Among these compounds, sutchuenoside A and kaempferitrin were observed to be effective with median effective concentration (EC50) of 3.01 and 2.71 mg L(-1), respectively. The alterations in the tegument of the parasites treated with isolated compound were examined using scanning electron microscopes. Ultrastructural micrographs revealed shrinkage of body surface, dense tegumental folds, and disheveled protuberances. The structural deformities in the treated parasites were indicative of an efficient anthelmintic activity of the isolated compound kaempferitrin. In addition, the 48-h median lethal concentration for sutchuenoside A and kaempferitrin against goldfish were 12.03- and 11.98-fold higher than corresponding EC50. The present results showed that ethyl acetate extract of D. crassirhizoma may be considered as a potent source, and sutchuenoside A and kaempferitrin as new natural parasitic agents against D. intermedius.

Antiprotozoal activity of medicinal plants against Ichthyophthirius multifiliis in goldfish (Carassius auratus).

Ichthyophthiriasis is a widespread disease in aquaculture and causes mass mortalities of fish. The development of new antiprotozoal agents for the treatment of Ichthyophthirius multifiliis infections is of increasing interest. The aim of the present study was to investigate the efficacy of 30 medicinal plants against I. multifiliis. The results showed that the methanol extracts of Magnolia officinalis and Sophora alopecuroides displayed the highest antiprotozoal activity against theronts, with 4-h LC(50) values estimated to be 2.45 and 3.43 mg L(-1), respectively. Concentrations of 2.5, 5.0, 10.0, and 20.0 mg L(-1) of M. officinalis extracts resulted in tomont mortality of 9.7, 43.7, 91.3, and 100% at 20 h, respectively. From 40 to 320 mg L(-1) of S. alopecuroides extracts, tomont mortality increased from 29.7 to 100%. Antiprotozoal efficacy against settled tomonts (2 and 10 h) was also applied; the results indicated that encysted I. multifiliis tomonts were less susceptible to these plant extract treatments. In vivo experiments demonstrated that high concentrations of M. officinalis and S. alopecuroides extracts could kill tomonts, and M. officinalis significantly reduced its reproduction (P < 0.05). These results suggested that the methanol extracts of M. officinalis and S. alopecuroides have the potential to be used as an eco-friendly approach for the control of I. multifiliis.

Effects of aqueous extract of Capsicum frutescens (Solanaceae) against the fish ectoparasite Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is an important fish ectoparasite that often results in significant economic losses to freshwater aquaculture. The search of alternative substances to control infections of I. multifiliis became stringent after malachite green, an effective and widely used chemotherapeutant, is banned on fish farms because of its carcinogenicity and teratogenicity. In this study, the effects of the aqueous extract of Capsicum frutescens, which is readily available and affordable, were evaluated under in vitro and in vivo conditions. The results in the in vitro conditions showed that the aqueous extracts of C. frutescens with the ratios (V (SS)/V (T)-V (SS), the volume of stock solution; V (T), the volume of total solution) of 1:32 and 1:64 led to more than 70 % mortality of I. multifiliis theronts during 4 h of exposure and significantly reduced the survival of the tomonts and the total number of theronts released by the tomonts within 22 h (P < 0.05). A 96-h bioassay was carried out to determine the acute toxicity of the aqueous extract of C. frutescens to goldfish. No visible effect was observed in the treatments with the aqueous extracts of C. frutescens with the ratios (V (SS)/V (T)) of 1:32, 1:64 and 1:128, while in the other treatments, the erratic behaviour of fish was noted. In addition, in vitro tests demonstrated that the aqueous extract of C. frutescens had an adverse effect on I. multifiliis trophonts in situ. Fish treated with the aqueous extracts of C. frutescens in ratios V (SS)/V (T) of 1:32 and 1:64 carried significantly fewer parasites than the control and the other treatments (P < 0.05). These results suggest, therefore, that aqueous extracts of C. frutescens have potential for the control of ichthyophthiriasis in the aquaculture industry, though further phytochemical studies will need to be performed for isolation and identification of the active compounds.

Bioassay-guided isolation and identification of active compounds from Semen pharbitidis against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus).

The present study was undertaken to isolate the active compounds responsible for the anthelmintic activity of methanol extract of Semen pharbitidis against Dactylogyrus intermedius in goldfish (Carassius auratus). The active methanol extract was fractionated on silica gel column chromatography in a bioassay-guided fractionation, eventually yielding two bioactive compounds: palmitic acid and pharnilatin A by comparing spectral data (NMR and ESI-MS) with literature values. According to in vivo anthelmintic assays, they were found to be 50% effective at the concentrations (EC(50)) of 5.3 and 1.4 mg L(-1), respectively. The promising palmitic acid and pharnilatin A from S. pharbitidis were also subjected to acute toxicity tests for the evaluation of their safety to the host (goldfish). After 48h exposure, the mortalities of goldfish were recorded, and the established LC(50) values were 2.45- and 5.29-fold higher than the corresponding EC(50), demonstrating that pharnilatins A may have better application potential than palmitic acid. The present results provide evidence that pharnilatins A might be potential source of new anti-parasitic drug for the control of Dactylogyrus.

Screening of 42 medicinal plants for in vivo anthelmintic activity against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus).

In the present study, methanol extracts of 42 traditional medicinal plants with potent anthelmintic activity against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus) were investigated. Cinnamomum cassia, Lindera aggregata, and Pseudolarix kaempferi exhibited 100% activity and were selected for further evaluation by applying five solvents (petroleum ether, chloroform, ethyl acetate, methanol, and water) for the extraction of the samples, followed by the in vivo bioassay. Among the extracts tested, water and methanol extracts of C. cassia showed the highest efficacies with EC(50) values of 13.2 and 12.3 mg L(-1), showing 100% efficacy against D. intermedius at 30.0 and 40.0 mg L(-1), followed by methanol extract of L. aggregata which demonstrated 100% efficacy at 60.0 mg L(-1) with EC(50) value of 17.1 mg L(-1) after 48 h of exposure. Methanol and ethyl acetate extract of P. kaempferi, which exhibited a 100% efficacy against D. intermedius at 60.0 and 50.0 mg L(-1), revealed similar activity with EC(50) values of 23.5 and 23.3 mg L(-1), respectively. Acute toxicity of these active extracts was investigated on goldfish for 48 h and the corresponding median lethal concentrations (LC(50)) of 56.9, 31.3, 88.7, 168.2, and 165.7 mg L(-1), respectively. These findings indicated that these extracts of the three plants can be developed as preferred natural antiparasitic agents for the treatment of D. intermedius.

In vivo anthelmintic activity of Dryopteris crassirhizoma, Kochia scoparia, and Polygala tenuifolia against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus).

In order to find natural agents against Dactylogyrus intermedius in goldfish, petroleum ether, chloroform, ethyl acetate, acetone, and methanol extracts of three medicinal plants (Dryopteris crassirhizoma, Kochia scoparia, and Polygala tenuifolia) were screened for antiparasitic properties using in vivo anthelmintic efficacy assay. Among these extracts investigated, methanolic extract of D. crassirhizoma was observed the most effective with EC50 value of 22.97 mg L⁻¹ after 48 h of exposure, which exhibited a 100% efficacy against D. intermedius at 60.00 mg L⁻¹, followed by the methanolic extracts of K. scoparia and P. tenuifolia with EC50 values of 31.28 and 154.79 mg L⁻¹, showing 100% efficacy against D. intermedius at 60.00 and 500.00 mg L⁻¹, respectively. In addition, acute toxicity assay indicated that 48-h LC50 values of methanolic extracts of D. crassirhizoma, K. scoparia, and P. tenuifolia were 4.10-, 2.27-, and 5.00-fold higher than the corresponding EC50. The obtained results demonstrated that methanolic extracts of D. crassirhizoma, K. scoparia, and P. tenuifolia have the potential for the development of novel therapy for the control of D. intermedius in aquaculture.

In vivo evaluation of anthelmintic potential of medicinal plant extracts against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus).

In the present study, an attempt has been made to petroleum ether, chloroform, ethyl acetate, methanol, and water extracts of Prunus amygdalus Batsch seeds (Semen amygdali), Cimicifuga foetida L. rhizomes (Rhizoma Cimicifugae), Peucedanum decursivum (Miq.) Maxim roots (Radix Peucedani), Momordica cochinchinensis Spreng. seeds (Semen Momordicae), and Bupleurum chinense DC. roots (Radix Bupleuri chinensis) for their in vivo anthelmintic activity against monogenean Dactylogyrus intermedius in goldfish (Carassius auratus). The results showed that the efficacies of methanol, chloroform, and ethyl acetate extracts from R. Bupleuri chinensis were found to be, in this order, more effective than others with the 48 h-EC(50) and EC(90) values of 3.5 and 6.9, 6.0 and 8.4, 7.4 and 11.2 mg/L, respectively, followed by ethyl acetate extract of R. cimicifugae and chloroform extract of R. peucedani with EC(50) 189.2 and 240.4 mg/L. The promising methanol, chloroform, and ethyl acetate extracts from R. Bupleuri chinensis were subjected to acute toxicity tests for the evaluation of their safety to the host. After 48-h exposure, the mortalities of goldfish were recorded, and the established LC(50) values were 10.1-, 4.2-, and 8.4-fold higher than the corresponding EC(50). These results indicated that the three extracts from R. Bupleuri chinensis exhibit potential to be used as preferred natural antiparasitics for the control of the D. intermedius, especially for the methanolic one.