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) [33], imperatorin (2) [34,35], isopimpinellin (3) [36], auraptenol (4) [37], 8-epoxypentylcoumarin (5) [38], meranzin hydrate (6) [36], 2′-deoxymetranzin hydrate (7) [36], 8-methylbutenalcoumarin (8) [39], and micromarin-F (9) [39,40].

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 [9]. 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 [41]. 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 [19,21]. The recent opinion states that the degradable compounds with a low LC₅₀/EC₅₀ ratio may still be considered when selecting candidate compounds [19]. 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[20]. 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) 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.

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.

Compounds 1, 8 and 9 were the first subgroup to be compared. They have the same coumarin structures, except that the C-5′ groups were 5′-CH₃ (1), 5′-CHO (8), 5′-CH₂OH (9), respectively (as shown in Figure 3). The EC₅₀ values against B. albicostatus were 5′-CH₃ (19.01 μmol mL⁻¹) < 5′-CHO (26.24 μmol mL⁻¹) < 5′-CH₂OH (42.04 μmol mL⁻¹), and the EC₅₀ values against B. neritina were 5′-CHO (15.00 μmol mL⁻¹) < 5′-CH₃ (30.98 μmol mL⁻¹) < 5′-CH₂OH (47.62 μmol mL⁻¹). It was suggested that C-5′ groups could affect the activities.

Compounds 1, 5, 6 and 7 have the same structures, except that the groups between C-2′ and C-3′ have a double bond (1), expoy (5), dihydroxy (6), hydroxy (7), respectively (as shown in Figure 2). The EC₅₀ values against B. albicostatus were hydroxy (17.17 μmol mL⁻¹) < double bond (19.01 μmol mL⁻¹) < expoy (28.69 μmol mL⁻¹) < dihydroxy (127.19 μmol mL⁻¹), and The EC₅₀ values against B. neritina were double bond (30.98 μmol mL⁻¹) < hydroxy (33.31 μmol mL⁻¹) < dihydroxy (65.58 μmol mL⁻¹) < expoy (143.42 μmol mL⁻¹). It was suggested that groups between C-2′ and C-3′ could affect the activities too.

The linear furanocoumarin 2 has the same isoamylene chain as simple Coumarin 1 (as shown in Figure 4). They have close EC₅₀ values against B. albicostatus (Compound 1, 19.01 μmol mL⁻¹ and Compound 2, 12.56 μmol mL⁻¹) but different EC₅₀ values against B. neritina (Compound 1, 19.01 μmol mL⁻¹ and Compound 2, >185.19 μmol mL⁻¹). It seems that the existence of the furan ring could affect the activity against B. neritina.

The fruits of C. monnieri, which were collected from Zhejiang Province, China in October 2006, were purchased from a local medicine store in Xiamen, Fujian Province, China. The plant material was identified by Dr. Yang Qiu, Department of Pharmacy, Xiamen University. A voucher specimen (SCZ-2006-10) is now deposited at the College of Oceanography and Environmental Science, Xiamen University. The fruits of C. monnieri (2.5 kg) were extracted with methanol (3 L) by maceration for 2 weeks at room temperature. The extract was desiccated by a vacuum rotary evaporator. The extract was dissolved in water and the solution was partitioned with EtOAc to obtain the EtOAc extract in yield of 35.6 g.

The EtOAc extract (35.6 g) from C. monnieri was chromatographed over a silica gel column using a gradient solvent system (petroleum ether-EtOAc = 20:1→1:1) to give five subfractions (F1–F5). The F2 fraction was separated over silica gel (petroleum ether-EtOAc = 12:1) to yield Compounds 1 (5 g) and 2 (750 mg). The F3 fraction was separated over silica gel (petroleum ether-EtOAc = 8:1) to yield Compound 3 (32 mg). The F4 fraction was separated over silica gel (petroleum ether-EtOAc = 5:1) and Sephadex LH-20 filtration (MeOH) to yield Compound 4 (12 mg).

The nuclear magnetic resonance (NMR) spectra were recorded with a Bruker Avance-600 FT NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) in CDCl₃ with Tetramethylsilan (TMS) as a reference. electrospray ionization mass spectrometry (ESI-MS) data were recorded on an AB 3200Q TRAP spectrometer (AB SCIEX, Boston, MA, USA). Structural elucidation of the pure compounds was based on interpretation of their spectral data (NMR, MS) and comparison with published values.

Compounds 5–9 were prepared by standard organic synthesis procedures described in references [39,42,43], and the synthesis of these osthole derivatives is illustrated in Figure 2. Epoxide 5 was prepared by the epoxidation of 1 with m-chloroperbenzoic acid (m-CPBA). Diol 6 was synthesized from 5 by acid hydrolysis. Tertiary alcohol 7 was prepared by the oxymercuration of 1. Aldehyde 8 was synthesized from 2 with the oxidant SeO₂. Secondary alcohol 9 was prepared by the reduction of 8. The synthetic procedures are described in the supplementary materials.

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 [9,20], 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 [44]. Test samples were dissolved in EtOAc and the methods for measuring activities were based on references [9,20,44]. 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 [45]. 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.