Anti-Influenza A Virus Effect of Hypericum perforatum L. Extract

To study the antiviral effect of Hypericum perforatum L. extract (HPE) on influenza A virus (IAV) (H1N1) in vitro and in vivo. Cytopathic effect (CPE) and neutral red (NR) dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney (MDCK) cells which were infected with IAV in vitro. HPE was effective against influenza A virus (IAV) in vitro, with a 50% effective concentration (EC50) of 40 μg/mL. The mean 50% cytotoxic concentration (CC50) in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC50 values of 5.0 μg/mL; the mean CC50 for ribavirin was 520 μg/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin's minimum effective dose was 40 mg/kg/day with the LD50 determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD50 dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza.     

Antiviral activity of recombinant cyanovirin-N against HSV-1

In this study, a standard strain of HSV-1 (strain SM₄₄) was used to investigate the antiviral activity of the recombinant Cyanovirin-N (CV-N) against Herpes simplex virus type 1 (HSV-1) in vitro and in vivo. Cytopathic effect (CPE) and MTT assays were used to evaluate the effect of CV-N on HSV-1 in Vero cells. The number of copies of HSV-DNA was detected by real-time fluorescence quantitative PCR (FQ-PCR). The results showed that CV-N had a low cytotoxicity on Vero cells with a CC₅₀ of 359.03±0.56 μg/mL, and that it could not directly inactivate HSV-1 infectivity. CV-N not only reduced the CPE of HSV-1 when added before or after viral infection, with a 50% inhibitory concentration (IC₅₀) with 2.26 and 30.16μg/mL respectively, but it also decreased the copies of HSV-1 DNA in infected host cells. The encephalitis model for HSV-1 infection was conducted in Kunming mice, and treated with three dosages of CV-N (0.5, 5 &; 10 mg/kg) which was administered intraperitoneally at 2h, 3d, 5d, 7d post infection. The duration for the appearance of symptoms of encephalitis and the survival days were recorded and brain tissue samples were obtained for pathological examination (HE staining). Compared with the untreated control group, in the 5mg/kg CV-N and 10mg/kg CV-N treated groups, the mice suffered light symptoms and the number of survival days were more than 9d and 14d respectively. HE staining also showed that in 5mg/kg CV-N and 10mg/kg CV-N treated groups, the brain cells did not show visible changes, except for a slight inflammation. Our results demonstrated that CV-N has pronounced antiviral activity against HSV-1 both in vitro and in vivo, and it would be a promising new candidate for anti-HSV therapeutics.     

Antiviral Activity of Antimicrobial Lipopeptide from Bacillus subtilis fmbj Against Pseudorabies Virus, Porcine Parvovirus, Newcastle Disease Virus and Infectious Bursal Disease Virus in Vitro

Bacillus subtilis fmbj can produce lipopeptide antimicrobial substance, whose main components were surfactin and fengycin. In the study, the antiviral activity of antimicrobial lipopeptides (AMLs) from B. subtilis fmbj (CGMCC No. 0934) against Pseudorabies Virus (PRV), Porcine Parvovirus (PPV), Newcastle Disease Virus (NDV) and Infectious Bursal Disease Virus (IBDV) was evaluated in vitro. The AMLs represented a direct inactivation effect on cell-free virus stocks of PRV, PPV, NDV and IBDV, and it could effectively inhibit infection and replication of the NDV and IBDV, but failed to affect PRV and PPV. The AMLs were represented higher toxicity for the Porcine Kidney (PK-15) cells (50% cytotoxic concentration (CC₅₀) value was 32.87 μM) and lower for the Chicken Embryo Fibroblasts (CEF) cells (CC₅₀ value was 89.16 μM). The Selectivity index of AMLs on PRV, PPV, NDV and IBDV was 1.44, 2.23, 8.40 and 12.19, respectively.     

Revelation of Antiviral Activities by Artificial Sulfation of a Glycosaminoglycan from a Marine Pseudomonas

Sulfated derivatives of a glycosaminoglycan containing l-glutamic acid produced by a marine Pseudomonas species, No. 42 strain, were prepared by the method of dicyclohexyl-carbodiimide-mediated reaction. Both low and high degrees of sulfation of the polysaccharides (products A1 and A2, respectively) were investigated for their antiviral activities against influenza virus type A (FluV-A) and B (FluV-B) in MDCK cells. Both preparations showed antiviral activity against FluV-A at the 50% antiviral effective concentration of 17.3 and 5.2 μg/ml, respectively, whereas they had no antiviral activity against FluV-B. No cytotoxicity of either product was noted against MDCK cells at the 50% cytotoxic concentration of 100 μg/ml. Received April 4, 1998; accepted July 24, 1998.     

Total alkaloids from Alstonia scholaris inhibit influenza a virus replication and lung immunopathology by regulating the innate immune response

BackgroundAlstonia scholaris is a folk medicine used to treat cough, asthma and chronic obstructive pulmonary disease in China. Total alkaloids (TA) from A. scholaris exhibit anti-inflammatory properties in acute respiratory disease, which suggests their possible anti-inflammatory effect on influenza virus infection.PurposeTo assess the clinical use of TA by demonstrating their anti-influenza and anti-inflammatory effects and the possible mechanism underlying the effect of TA on influenza A virus (IAV) infection in vitro and to reveal the inhibitory effect of TA on lung immunopathology caused by IAV infection.MethodsAntiviral and anti-inflammatory activities were assessed in Madin-Darby canine kidney (MDCK) and A549 cells and U937-derived macrophages infected with influenza A/PR/8/34 (H1N1) virus. Proinflammatory cytokine levels were measured by real-time quantitative PCR and Bio-Plex assays. The activation of innate immune signaling induced by H1N1 virus in the absence or presence of TA was detected in A549 cells by Western blot. Furthermore, mice were infected intranasally with H1N1 virus and treated with TA (50, 25 and 12.5 mg/kg/d) or oseltamivir (60 mg/kg/d) for 5 days in vivo. The survival rates and body weight were recorded, and the viral titer, proinflammatory cytokine levels, innate immune cell populations and histopathological changes in the lungs were analyzed.ResultsTA significantly inhibited viral replication in A549 cells and U937-derived macrophages and markedly reduced cytokine and chemokine production at the mRNA and protein levels. Furthermore, TA blocked the activation of pattern recognition receptor (PRR)- and IFN-activated signal transduction in A549 cells. Critically, TA also increased the survival rate, reduced the viral titer, suppressed proinflammatory cytokine production and innate immune cell infiltration and improved lung histopathology in a lethal PR8 mouse model.ConclusionTA exhibits anti-viral and anti-inflammatory effects against IAV infection by interfering with PRR- and IFN-activated signal transduction.     

Tautomeric and non-tautomeric N-substituted 2-iminobenzimidazolines as new lead compounds for the design of anti-influenza drugs: An in vitro study

A series of 1,3-disubstituted 2-iminobenzimidazolines as well as a number of their tautomeric analogs were synthesized. The synthesized compounds were tested for their cytotoxicity against MDCK cells and for inhibiting activity against influenza virus A/California/07/09 (H1N1)pdm09. Based on the results obtained, 50% cytotoxic concentration (CC₅₀), 50% inhibiting concentration (IC₅₀) and selectivity index (SI) were calculated for each compound. It was found that some of synthesized benzimidazole derivatives (7 of 22, 32%) possess strong virus-inhibiting activity against pandemic influenza virus (IC₅₀’s in low micromolar range) with quite moderate cytotoxicity (CC₅₀ in the range of thousands micromoles). Due to their high selectivity (highest SI’s = 50–83) these compounds are of significant interest for further in vivo experiments as well as for further structural optimization and drug development.     

Synthesis and Biological Evaluation of Substituted Indole and Its Analogs as Influenza A Virus Inhibitors

Influenza A virus (IAV), a highly pathogenic virus to human beings, is most susceptible to mutation and thus causes rapid, severe global pandemics resulting in millions of fatalities worldwide. Since resistance to the existing anti‐influenza drugs is developing, innovative inhibitors with a different mode of action are urgently needed. The lead compound 6092B‐E5 has proven to be an effective antiviral reagent in our previous work. Using the principles of substitution and bioisosterism of the indole ring, six series of novel anti‐IAV target products were designed, synthesized and evaluated for their antiviral effect in this work. Compounds D₁ , D₃ , D₉ , G₁ , G₃ , G₁₂ and G₂₃ were identified as promising anti‐IAV candidates with excellent anti‐IAV efficacy (IC₅₀ values of 3.06–5.77 μm ) and low cytotoxicity (CC₅₀ values up to and beyond 100 μm ). This work represents a successful application of the substitution and bioisosteric replacement strategy for the discovery of novel antiviral molecules that can be used for further structural optimization.     

Synthesis of Camphecene and Cytisine Conjugates Using Click Chemistry Methodology and Study of Their Antiviral Activity

A series of camphecene and quinolizidine alkaloid (?)‐cytisine conjugates has been obtained for the first time using ‘click’ chemistry methodology. The cytotoxicity and virus‐inhibiting activity of compounds were determined against MDCK cells and influenza virus A/Puerto Rico/8/34 (H1N1), correspondingly, in in vitro tests. Based on the results obtained, values of 50 % cytotoxic dose (CC₅₀), 50 % inhibition dose (IC₅₀) and selectivity index (SI) were determined for each compound. It has been shown that the antiviral activity is affected by the length and nature of linkers between cytisine and camphor units. Conjugate 13 ((1R,5S)‐3‐(6‐{4‐[(2‐{(E)‐[(1R,4R)‐1,7,7‐trimethylbicyclo[2.2.1]heptan‐2‐ylidene]amino}ethoxy)methyl]‐1H‐1,2,3‐triazol‐1‐yl}hexyl)‐1,2,3,4,5,6‐hexahydro‐8H‐1,5‐methanopyrido[1,2‐a][1,5]diazocin‐8‐one), which contains cytisine fragment separated from triazole ring by –C₆H₁₂– aliphatic linker, showed the highest activity at relatively low toxicity (CC₅₀=168 μmol, IC₅₀=8 μmol, SI=20). Its selectivity index appeared higher than that of reference compound, rimantadine. According to theoretical calculations, the antiviral activity of the lead compound 13 can be explained by its influence on the functioning of neuraminidase.     

清热消炎复方制剂抗流感病毒作用的研究

为评价清热消炎复方制剂(简称AI)的抗流感病毒活性,我们以病毒唑为对照,通过在体外观察病毒致细胞病变效应(CPE)、MTT细胞染色检查病毒抑制率和检测病毒血凝滴度;在体内观察其对染毒小鼠的死亡保护作用,对小鼠流感病毒性肺炎的抑制作用,以及对小鼠肺内病毒增殖的影响,从而判定其抗流感病毒作用。结果发现AI在160ug/mL时能完全抑制流感病毒在MDCK细胞内的增殖复制作用。体内实验中0.1g/kg,0.5g/kg,1.2g/kg3个剂量均能明显降低染毒小鼠的致死率,延长平均存活时间:降低肺炎小鼠的肺指数和血凝滴度(P<0.01)。其作用与病毒唑相当。结论认为清热消炎复方制剂是一种有效的体内、体外抗流感病毒中药复方制剂。     

Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines

Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID₅₀) titers of the two cell lines reached 4.51×10⁶ cells/mL, 2.94Log₁₀(HAU/50 μL) and 8.49Log₁₀(virions/mL), and 5.97×10⁶ cells/mL, 3.88Log₁₀(HAU/50 μL), and 10.34Log₁₀(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.     

Zearalenone, deoxynivalenol and fumonisins in mixed-feed for laying hens

Fusarium toxins are secondary metabolites produced byfungi of these genera in many commodities under certain conditions. A study was carried out to investigate the co-occurrence of zearalenone (ZEN), deoxynivalenol (DON) and fumonisins (FB₁ and FB₂) in 52 samples of mixed-feed for poultry contaminated withFusarium verticillioides. The zearalenone and deoxynivalenol were checked using immunoaffinity column and the extraction of fumonisin was performed by strong anion exchange (SAX) solid phase column. Detection and quantification were determined by high performance liquid chromatography (HPLC). The limit of detection was 5 μg/kg for ZEN, 100 μg/kg for DON and 50 and 100 μg/kg for FB₁ and FB₂ respectively.Fusarium toxins were detected in 20 samples. Sixteen samples were positive for ZEN (30.7%) presenting levels that ranged from 7.4 μg/kg to 61.4 μg/kg (mean=27.0 μg/kg). 13.5% of the samples presented contaminations of DON, with levels ranging from 100.0 μg/kg to 253 μg/kg (mean=l18.07 μg/kg). FB₁ was detected in 19.2% of samples, with levels ranging from 50.0 μg/kg to 110.0 μg/kg (mean=73.6 μg/kg). FB2 was not detected in any sample. In positive samples simultaneously contamination with two or three mycotoxins were detected in 9 of them (17.3%).     

Antimalarial activity of endoperoxide compound 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol

Plasmodium falciparum, the major causative parasite for the disease, has acquired resistance to most of the antimalarial drugs used today, presenting an immediate need for new antimalarial drugs. Here, we report the in vitro and in vivo antimalarial activities of 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against P. falciparum and Plasmodium berghei parasites. The N-251 showed high antimalarial potencies both in the in vitro and the in vivo tests (EC₅₀ 2.3 × 10⁻⁸ M; ED₅₀ 15 mg/kg (per oral)). The potencies were similar to that of artemisinin in vitro and greater than artemisinin's activity in vivo (p.o.). In addition, N-251 has little toxicity: a single oral administration at 2000 mg/kg to a rat gave no health problems to it. Administration of N-251 to mice bearing 1% of parasitemia (per oral 68 mg/kg, 3 times a day for 3 consecutive days) resulted in a dramatic decrease in the parasitemia: all the 5 mice given N-251 were cured without any recurrence, with no diarrhea or weight loss occurring in the 60 days of experiment. N-251 deserves more extensive clinical evaluation, desirably including future trials in the human.     

The protective effect of arbutin against potassium bromate-induced oxidative damage in the rat brain

This study aimed to investigate the protective effects of arbutin (ARB) against brain injury induced in rats with potassium bromate (KBrO₃). The rats were divided into four groups as Group 1: Control (0.9% NaCl ml/kg/day p.), Group 2: KBrO₃ (100 mg/kg (gavage), Group 3: ARB (50 mg/kg/day p.), and Group 4: KBrO₃ + ARB (100 mg/kg (gavage) + 50 mg/kg/day p.). At the end of the fifth day of the study, the rats in all groups were killed, and their brain tissues were collected. In the collected brain tissues, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) levels were measured, and routine histopathological examinations were made. The MDA levels in the group that was exposed to KBrO₃ were significantly higher than those in the control group (p ˂ 0.001). In comparison to the KBrO₃ group, the MDA levels in the KBrO₃ + ARB group were significantly lower (p ˂ 0.001). It was observed that SOD and CAT enzyme activity levels were significantly lower in the KBrO₃ group compared to the control group (p ˂ 0.001), while these levels were significantly higher in the KBrO₃ + ARB group than in the KBrO₃ group (p ˂ 0.001). Additionally, the group that was subjected to KBrO₃ toxicity, as well as ARB administration, had much lower levels of histopathologic signs than the group that was subjected to KBrO₃ toxicity only. Consequently, it was found that KBrO₃ exposure led to injury in the brain tissues of the rats, and using ARB was effective in preventing this injury.     

Therapeutic efficacy of Hypericum perforatum L. extract for mice infected with an influenza A virus

Hypericum perforatum L., a plant used in Chinese herbal medicine, has been proven effective against many viral diseases. In the present study, the therapeutic efficacy of an extract of H.?perforatum (HPE) against influenza A virus (IAV) was investigated in mice. Whether HPE would be a promising agent for influenza treatment was evaluated by measuring the protection rate, mean survival days, lung index, and viral titer, as well as the secretion of IL-6, interleukin-10 (IL-10), tumour necrosis factor-α (TNF-α), and interferon-gamma (IFN-γ) in lung tissue and serum on days 3 and 5 post-infection. The results showed that HPE could reduce the lung index and viral titer of mice infected with IAV, decrease mortality, and prolong the mean survival time. HPE decreased the concentration of IL-6 and TNF-α in lung tissue and serum on day 5 post-infection. In contrast, HPE enhanced the lung and serum levels of IL-10 and IFN-γ on the days 3 and 5 post-infection. Our study indicates that HPE has significant therapeutic efficacy for mice infected with IAV. The possible reasons for these results were concluded to be pertaining to up-regulating the expression of IL-10 and IFN-γ, and down-regulating the secretion of IL-6 and TNF-α in lung and serum.     

Micropropagation of Chokecherry and Pincherry cultivars

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of chokecherry (Prunus virginiana L.), `Garrington', and pincherry (P. pensylvanica L.f), `Mary Liss' and `Jumping Pound', were examined using various combinations of growth regulators. Dormant winter buds were used as explants. MSMO medium supplemented with 0.49 μM IBA and either 4.44 or 8.87 μM BA was found to be optimal for culture initiation of both species and cultivars. GA₃ (28.89 μM) significantly reduced (p=0.0001) the number of successfully established cultures. BA concentrations 8.87–12.82 μM gave optimal shoot proliferation in chokecherry and 4.44 μM BA in both cultivars of pincherry. Auxin treatments were required for ex vitro rooting of approximately 10 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (84%) was obtained with IBA/NAA (9.80/2.69 μM). A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%) mixture, was also effective (75%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Human Dietary Exposure to Fumonisin B1 from Iranian Maize Harvested During 1998–2000

Fumonisin B₁ (FB₁) is the most abundant of the fumonisin mycotoxins, mainly produced in maize by F. verticillioides and F. proliferatum. A previous study on the FB1 contamination of maize harvested in Mazandaran and Isfahan Provinces of Iran in 1998 and 1999 demonstrated contamination in both provinces. This present study was undertaken to further investigate the variation in levels of contamination and to estimate possible levels of human exposure to fumonisins in Iran. The mean level of FB₁ in 49 visually healthy maize samples collected from Mazandaran Province during 2000 was 6.14 mg/kg, which is higher than that found during 1998 and 1999 (2.27 and 3.18 mg/kg, respectively). Although these levels are higher than the Iranian legislative limits for fumonisins in maize intended for humans, the relatively low estimated consumption of maize in Iran (3.3 g/person/day) implies that average exposures (0.011 and 0.215 μg/kg body weight/day in Isfahan and Mazandaran, respectively) are within the provisional maximum tolerable daily intake of 2 μg/kg body weight/day set by the Joint FAO/WHO Expert Committee on Food Additives. Nevertheless, certain sections of the population who may consume higher amounts of maize or who may replace all or some of their consumption of other cereals with maize, could well exceed this limit.     

Anti-HIV-1 efficacy of extracts from medicinal plants

The anti-HIV-1 activities of butanol, hexane, chloroform and water extracts from four widely used folk medicinal plants (Sophora flavescens, Tulipa edulis, Herba ephedra, and Pachyma hoelen Rumph) were evaluated in this study. The hexane extract of Pachyma hoelen Rumph, PH-4, showed effective inhibition against HIV-1. The 50% effective concentration (EC₅₀) of PH-4 was 37.3 μg/ml in the p24 antigen assay and 36.8% in the HIV-1 recombinant RT activity test (at 200 μg/ml). In addition, the PH-4 showed the protective effect on the infected MT-4 cells, with a 58.2% rate of protection. The 50% cytotoxic concentration (CC₅₀) of PH-4 was 100.6 μg/ml. These results suggest that PH-4 from Pachyma hoelen Rumph might be the candidate for the chemotherapy agent against HIV-1 infection with further study.     

Antiviral Activities of Marine Pseudomonas Polysaccharides and Their Oversulfated Derivatives

A marine Pseudomonas species WAK-1 strain simultaneously produces extracellular glycosaminoglycan and sulfated polysaccharide. Among the antiviral activities tested for these polysaccharides, the latter showed anti-HSV-1 activity in RPMI 8226 cells (50% effective concentration is 1.4 μg/ml). Oversulfated derivatives of these polysaccharides prepared by dicyclohexylcarbodiimide-mediated reaction for both polysaccharides showed antiviral activities against influenza virus type A (for glycosaminoglycan, 50% effective concentration is 11.0 μg/ml; for another, 2.9 μg/ml). Glycosaminoglycan, sulfated polysaccharide, and their chemically synthesized oversulfated derivatives did not show antiviral activities against influenza virus type B and human immunodeficiency virus type 1. No cytotoxicity of these products was noted against host cells at the 50% cytotoxic concentration of 100 μg/ml, except that naturally occurring sulfated polysaccharide had 50% cytotoxicity against MT-4 cells at 8–21 μg/ml. Received May 1, 1998; accepted July 24, 1998.     

The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn²⁺, CO₂, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn²⁺ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn²⁺ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO₂ concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO₂ concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO₂. The activity of extracellular CA at a CO₂ concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO₂ concentration was at 0.35 mL/L CO₂. Light intensity from 4.0 mW/cm² to 5.6 mW/cm² was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn²⁺, CO₂, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.