Natural product; Balanus albicostatus and Bugula neritina

Four natural products were purified from the fruits of C. monnieri, and five other compounds, which occur in trace amounts in nature, were obtained by chemical synthesis. General chemical structure characteristics of these compounds are described in the supplementary materials. The spectrum data were consistent with those of references, and nine compounds were identified as osthole (1), imperatorin (2), isopimpinellin (3), auraptenol (4), 8-epoxypentylcoumarin (5), meranzin hydrate (6), 2′-deoxymetranzin hydrate (7) [36], 8-methylbutenalcoumarin (8), and Micromarin-F (9). [1]

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AF Activity of Compounds [1]
The EC50 and LC50 values of Compounds 1–9 against B. albicostatus and B. neritina are summarized in Table 1. The detailed rates of settlement and mortality against B. albicostatus and B. neritina for Compounds 1–9 are shown in the supplementary materials. The standard requirement established by the US Navy program as a potency criterion for natural antifoulants was that of being active at less than 25 μg mL−1 in static bioassays. All compounds except meranzinhydrate (6) had EC50 values lower than 25 μg mL−1 and showed inhibitory activities against barnacle settlement. Among these, Compounds 1, 2, 4, and 7 showed high inhibitory activities against barnacle settlement with EC50 values <5 μg mL−1. The calculated therapeutic ratio (LC50/EC50) of greater than one was considered for potential use in environmentally compatible AF coatings. The AF compounds with LC50/EC50 ratio higher than 15.0 were considered as non-toxic AF agents, and the compounds with a LC50/EC50 ratio lower than 5.0 were considered as toxic AF agents. The recent opinion states that the degradable compounds with a low LC50/EC50 ratio may still be considered when selecting candidate compounds. The LC50/EC50 ratio of Compounds 1, 2, 4, 5, 7 and 8 was higher than 5.0, indicating that these compounds are low-toxicity AF agents against the settlement of B. albicostatus larvae. All compounds except for 2, 3 and 5 showed inhibitory activities against bryozoan B. neritina settlement with EC50 values <25 μg mL−1, and the EC50 value of Compound 8 was lower than 5 μg mL−1. All compounds showed no significant mortality effect on B. neritina at a concentration of 50 μg mL−1. In previous research, we reported that the crude extracts of six common Chinese herbs showed AF activities against the cyprids of B. albicostatus, and we also identified two AF compounds from Sophora flavescens. In this study, we have identified four AF compounds (osthole, imperatorin, isopimpinellin, auraptenol) from the fruits of another Chinese herbal plant Cnidium monnieri. The results further demonstrate the value of herbal plants as a source of AF agents. Osthole (1) showed significant inhibitory activities against both B. albicostatus and B. neritina. Furthermore, some osthole derivatives (5, 7, 8 and 9/Micromarin-F) also showed established AF activity. It was suggested that the compound osthole should be considered as a potential lead compound for the design of new AF agents.

Impact of the Functional Groups on Antilarval Settlement Activities [1]
Because the nine compounds have a basic coumarin skeleton (benzo-α-pyrone ring) with different functional groups, the impact of functional groups on anti-larval settlement activities could be estimated to obtain preliminary information about the structure-activity relationship (SAR). In order to discuss the impact of functional groups, the concentration unit of bioassay results was converted to micromole per milliliter as shown in Figures 1 and 2.

AF Assay [1]
The barnacle B. albicostatus and the bryozoan B. neritina were used to test the AF activities of the natural products and synthetic derivatives. Adults of B. albicostatus were collected from the intertidal zone in Xiamen, Fujian Province, China. Based on the methods of references, after being released from the adults, the I–II stage nauplii were collected and reared to metamorphosis with Chaetoceros muelleri as food source. The larvae, which were metamorphosed to the cyprid stage, were stored in the dark at 5 °C until use for bioassays. Adult colonies of B. neritina were collected from a fish farm near Pozhao Island, Zhangzhou, Fujian Province, China. After exposure to the overhead room light, the adults released the larvae, which were harvested and immediately used. Test samples were dissolved in EtOAc and the methods for measuring activities were based on references. Percentages of larval settlement, swimming and death were calculated. The EC50 value (the concentration that reduced the settlement rate by 50% relative to the control) and LC50 value (the concentration that resulted in 50% mortality) of the compounds were calculated using the Spearman–Karber method. The differences between the experimental treatments and controls were analyzed with one-way ANOVA followed by a Dunnet post hoc test. The significance level was defined as p < 0.05.

[1]. Coumarins from the Herb Cnidium monnieri and Chemically Modified Derivatives as Antifoulants against Balanus albicostatus and Bugula neritina Larvae. Int. J. Mol. Sci. 2013, 14.

Micromarin F has been reported in Micromelum minutum with data available.

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In the search for new environmental friendly antifouling (AF) agents, four coumarins were isolated from the herbal plant Cnidium monnieri, known as osthole (1), imperatorin (2), isopimpinellin (3) and auraptenol (4). Furthermore, five coumarin derivatives, namely 8-epoxypentylcoumarin (5), meranzin hydrate (6), 2′-deoxymetranzin hydrate (7), 8-methylbutenalcoumarin (8), and micromarin-F (9) were synthesized from osthole. Compounds 1, 2, 4, 7 showed high inhibitory activities against larval settlement of Balanus albicostatus with EC50 values of 4.64, 3.39, 3.38, 4.67 μg mL−1. Compound 8 could significantly inhibit larval settlement of Bugula neritina with an EC50 value of 3.87 μg mL−1. The impact of functional groups on anti-larval settlement activities suggested that the groups on C-5′ and C-2′/C-3′ of isoamylene chian could affect the AF activities.
In conclusion, four coumarins were isolated from the herb C. monnieri, five other coumarins were prepared by chemical synthesis from osthol. All compounds were identified and tested for AF activities; most of them showed inhibitory activities against barnacle or bryozoan settlement. Among these compounds, osthole could be considered as a good lead compound in AF agent discovery, since it was present in a high quantity, was of simple structure and had substantial AF activities against both B. albicostatus and B. neritina. Furthermore, some preliminary information about the structure-activity relationship of these coumarins was given and the results showed that the groups on C-5′ and C-2′/C-3′ of the isoamylene chain could affect the AF activities.[1]

C15H16O4

260.29

413.184

7336-40-5

23758

Typically exists as solid at room temperature

1.24g/cm3

683.2ºC at 760 mmHg

367ºC

1.583

2.6

1

4

4

19

386

0

C/C(=C\CC1=C(C=CC2=C1OC(=O)C=C2)OC)/CO

NYBDJZVNEBTWCZ-XCVCLJGOSA-N

InChI=1S/C15H16O4/c1-10(9-16)3-6-12-13(18-2)7-4-11-5-8-14(17)19-15(11)12/h3-5,7-8,16H,6,9H2,1-2H3/b10-3+

8-[(E)-4-hydroxy-3-methylbut-2-enyl]-7-methoxychromen-2-one

Micromarin F; 73292-93-0; 8-(4-Hydroxy-3-methylbut-2-en-1-yl)-7-methoxy-2H-chromen-2-one; 1443627-08-4; 8-((E)-4-hydroxy-3-methylbut-2-enyl)-7-methoxychromen-2-one; 8-[(E)-4-hydroxy-3-methylbut-2-enyl]-7-methoxychromen-2-one;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)