- freely available
Int. J. Mol. Sci. 2013, 14(1), 1197-1206; doi:10.3390/ijms14011197
Published: 9 January 2013
Abstract: 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.
Marine biofouling on man-made structures causes serious technical and economic problems by threatening mariculture facilities, shipping facilities, vessels, and seawater pipelines [1–6]. Although the metal-based biocides such as tributyltin and cuprous oxide can effectively control biofouling, they also affect non-target organisms and are difficult to degrade [7,8]. Thus, the International Maritime Organization (IMO Resolution A., 895 21, 25/11/1999) established the ban of antifouling coatings containing tributyltin (TBT). Furthermore, the discharge of copper from antifouling paints is currently under scrutiny in many countries, because the cuprous oxide is associated with heavy metal pollution and high environmental risk. Obviously, there is a high demand for new antifouling (AF) agents that are not only effective but also environmentally friendly. Since the natural products already exist in the environment and are commonly biodegradable, it is suggested that these natural products with AF activity are promising and are environmentally friendly alternatives for classical AF agents [3,9–11].
So far, most of the AF natural products have been obtained from marine organisms [9,11–19], but some natural products, which were found in herbal plants, have also proved to be AF agents [20,21]. Compared with marine organisms, many species of herbal plants are easily obtained on an industrial scale because of the rapid development of the herbal industry . Cnidium monnieri, one of the most popular traditional herbs, has a wide distribution in China. Its fruits have been used for treatment of impotence, renal disease, dermatosis, and colpitis . The lipophilic extract of C. monnieri fruits can significantly inhibit the settlement of cyprids .
The barnacle Balanus albicostatus and bryozoan Bugula neritina were chosen for screening AF agents as model organisms. The barnacle B. albicostatus is one of the dominant fouling species in East Asian waters and is a great threat to mariculture facilities, shipping facilities, vessels, and seawater pipelines [24–26]. The marine bryozoan B. neritina is an important fouling organism in tropical and temperate waters and attached to seawater cages, buoys and dock pilings at certain times of the year [27–29]. Both B. albicostatus and B. neritina are excellent species for anti-fouling bioassays because their adults can be easily collected and the larvae can be conveniently maintained in controlled laboratory conditions [20,30].
In this study, four coumarins including osthole have been isolated from the fruits of C. monnieri, and have been tested for AF activities against B. albicostatus and B. neritina. Furthermore, five other coumarins were synthesized from osthole and tested for AF properties. These five synthesic coumarins occuring only in trace amounts in nature were difficult to purify from the plant, but were easy to obtain through a simple synthesis processes. Among these compounds, osthole was the main chemical composition of C. monnieri fruits . In China, osthole has been isolated from the widely cultivated herbal plant C. monnieri and commercialized as medicine or pesticide . Consequently, osthole is in abundant supply at low price on the market, which makes it possible for large-scale antifouling testing. Also, because these coumarins share a similar molecular skeleton, the relationship between the functional groups and AF activity are discussed.
2. Results and Discussion
2.1. Isolation, Synthesis and Identification of These Compounds
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) [34,35], isopimpinellin (3) , auraptenol (4) , 8-epoxypentylcoumarin (5) , meranzin hydrate (6) , 2′-deoxymetranzin hydrate (7) , 8-methylbutenalcoumarin (8) , and micromarin-F (9) [39,40].
2.2. AF Activity of Compounds
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 [19,21]. 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) 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.
2.3. Impact of the Functional Groups on Antilarval Settlement Activities
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.
3. Experimental Section
3.1. Plant Material and Extraction
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.
3.2. Isolation of Bioactive Compounds and Structural Identification
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.
3.3. Chemical Synthesis of Osthole Derivatives
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.
3.4. AF Assay
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 . 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 . 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.
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.
The project was supported by the earmarked fund for Modern Agro-industry Technology Research System (nycytx-47), the National Natural Science Foundation of China under contract Nos. 41106121 and 40906078, and the Fundamental Research Funds for the Central Universities of China under contract No. 2010111025. We are also grateful to Junda Lin for his help with English.
- Conflict of InterestThe authors declare no conflict of interest.
- Lewis, J.A. Marine biofouling and its prevention on underwater surfaces. Mater. Forum 1998, 22, 41–61. [Google Scholar]
- Rittschof, D. Natural product antifoulants: One perspective on the challenges related to coatings developments. Biofouling 2000, 15, 119–127. [Google Scholar]
- Yebra, D.M.; Kiil, S.; Dam-Johansen, K. Antifouling technology-past, present and future steps towards efficient and environmentally friendly antifouling coatings. Prog. Org. Coat 2004, 50, 75–104. [Google Scholar]
- Krug, P.J. Defense of Benthic Invertebrates against Surface Colonization by Larvae: A Chemical Arms Race. In Antifouling Compounds; Fusetani, N., Clare, A.S., Eds.; Springer: Berlin, Germany, 2006; pp. 1–53. [Google Scholar]
- Ralston, E.; Swain, G. Bioinspiration-the solution for biofouling control? Bioinspir. Biomim 2009, 4, 1–9. [Google Scholar]
- Schultz, M.P.; Bendick, J.A.; Holm, E.R.; Hertel, W.M. Economic impact of biofouling on a naval surface ship. Biofouling 2011, 27, 87–98. [Google Scholar]
- Abbott, A.; Abel, P.D.; Arnold, D.W.; Milne, A. Cost-benefit analysis of the use of TBT: The case for a treatment approach. Sci. Total Environ 2000, 258, 5–19. [Google Scholar]
- Champ, M.A. A review of organotin regulatory strategies pending actions, related costs and benefits. Sci. Total Environ 2000, 258, 21–71. [Google Scholar]
- Rittschof, D. Fouling and Natural Products as Antifoulants. In Recent Advances in Marine Biotechnology; Fingerman, M., Nagabhushanam, R., Thompson, M.F., Eds.; Oxford & IBH Publishing Company: New Delhi, India, 1999; pp. 245–257. [Google Scholar]
- Rittschof, D. Natural Product Antifoulants and Coatings Developments. In Marine Chemical Ecology; McClintock, J.B., Baker, B.J., Eds.; CRC Press: Boca Raton, FL, USA, 2001; pp. 543–566. [Google Scholar]
- Fusetani, N. Biofouling and antifouling. Nat. Prod. Rep 2004, 21, 94–104. [Google Scholar]
- Sears, M.A.; Gerhart, D.J.; Rittschof, D. Antifouling agents from marine sponge Lissodendoryx isodictyalis Carter. J. Chem. Ecol 1990, 16, 791–799. [Google Scholar]
- Fusetani, N.; Hiroto, H.; Okino, T.; Tomono, Y.; Yoshimura, E. Antifouling activity of isocyanoterpenoids and related compounds isolated from a marine sponge and nudibranchs. J. Nat. Toxins 1996, 5, 249–259. [Google Scholar]
- Clare, A.S.; Rittschof, D.; Gerhart, D.J.; Hooper, I.R.; Bonaventura, J. Antisettlement and narcotic action of analogues of diterpene marine natural product antifoulants from octocorals. Mar. Biotechnol 1999, 1, 427–436. [Google Scholar]
- Cho, J.Y.; Choi, J.S.; Kang, S.E.; Kim, J.K.; Shin, H.W.; Hong, Y.K. Isolation of antifouling active pyroglutamic acid, triethyl citrate and di-n-octylphthalate from the brown seaweed Ishige okamurae. J. Appl. Electrochem 2005, 17, 431–435. [Google Scholar]
- Qi, S.H.; Zhang, S.; Qian, P.Y.; Xiao, Z.H.; Li, M.Y. Ten new antifouling briarane diterpenoids from the South China Sea gorgonian Junceella juncea. Tetrahedron 2006, 62, 9123–9130. [Google Scholar]
- Qi, S.H.; Zhang, S.; Yang, L.H.; Qian, P.Y. Antifouling and antibacterial compounds from the gorgonians Subergorgia suberosa and Scripearia gracillis. Nat. Prod. Res 2008, 22, 154–166. [Google Scholar]
- Raveendran, T.V.; Limna Mol, V.P.; Parameswaran, P.S. Natural Product Antifoulants from the octocorals of Indian waters. Int. Biodeter. Biodegr 2011, 65, 265–268. [Google Scholar]
- Qian, P.Y.; Xu, Y.; Fusetani, N. Natural products as antifouling compounds: Recent progress and future perspectives. Biofouling 2010, 26, 223–234. [Google Scholar]
- Feng, D.Q.; Ke, C.H.; Lu, C.Y.; Li, S.J. Herbal plants as a promising source of natural antifoulants: Evidence from barnacle settlement inhibition. Biofouling 2009, 25, 181–190. [Google Scholar]
- Zhou, X.J.; Zhang, Z.; Xu, Y.; Jin, C.L.; He, H.P.; Hao, X.J.; Qian, P.Y. Flavone and isoflavone derivatives of terrestrial plants as larval settlement inhibitors of the barnacle Balanus amphitrite. Biofouling 2009, 25, 69–76. [Google Scholar]
- Yang, S.L.; Zhang, B.G.; Chen, J.; Li, X.E.; Wei, J.H. Strategies for the cultivation of Chinese medicinal plants for the everlasting development and utilization of this great treasure house. Chin. Trad. Herb. Drugs 1999, 30, 870–873. [Google Scholar]
- Chen, Y.; Zhang, G.; Yu, Z. The advancement in the chemical and pharmacological study of the fruits of Cnidium monnieri. J. Shenyang Pharm. Univ 2006, 23, 256–260. [Google Scholar]
- Utinomi, H. Comments on some new and already known cirripedes with emended taxa, with special reference to the parietal structure. Publ. Seto. Mar. Biol. Lab 1967, 15, 199–237. [Google Scholar]
- Newman, W.A.; Ross, A. Revision of balanomorph barnacles, including catalog of the species. Mem. San Diego Soc. Nat. Hist 1976, 9, 101–108. [Google Scholar]
- Lee, C.; Kim, C.H. Larval development of Balanus albicostatus Pilsbry (Cirripedia, Thoracica) reared in the laboratory. J. Exp. Mar. Biol. Ecol 1991, 147, 231–244. [Google Scholar]
- Woollacott, R.M.; Zimmer, R.L. Attachment and metamorphosis of the cheilo-ctenostome bryozoan Bugula neritina (Linne’). J. Morphol 1971, 134, 351–382. [Google Scholar]
- Walters, L.J. Post-settlement success of the arborescent bryozoan Bugula neritina (L.): The importance of structural complexity. J. Exp. Mar. Biol. Ecol 1992, 164, 55–71. [Google Scholar]
- Yu, X.J.; Yan, Y.; Gu, J.D. Attachment of the biofouling bryozoan Bugula neritina larvae affected by inorganic and organic chemical cues. Int. Biodeter. Biodegr 2007, 60, 81–89. [Google Scholar]
- Dahms, H.U.; Dobretsov, S.; Qian, P.Y. The effect of bacterial and diatom biofilms on the settlement of the bryozoan Bugula neritina. J. Exp. Mar. Biol. Ecol 2004, 313, 191–209. [Google Scholar]
- Feng, Y.; Zhang, L.; Cai, J.N.; Wang, Z.T.; Xu, L.S.; Zhang, Z.X. Analysis of Cnidium monnieri fruits in different regions of China. Talanta 2001, 53, 1155–1162. [Google Scholar]
- Yang, X.H.; Liu, S.J.; Yie, H.Z. The inspection and extract of active constituents from the fruits of Cnidium Monnieri (L.) Cusson. J. Guangzhou Med 2003, 34, 67–68. [Google Scholar]
- Fujioka, T.; Furumi, K.; Fujii, H.; Okabe, H.; Mihashi, K.; Nakano, Y.; Matsunaga, H.; Katano, M.; Mori, M. Antiproliferative constituents from umbelliferae plants. V. A new furanocoumarin and falcarindiol euranocoumarin ethers from the root of Angelica japonica. Chem. Pharm. Bull 1999, 47, 96–100. [Google Scholar]
- Elgamal, M.H.A.; Elewa, N.H.; Elkhrisy, E.A.M.; Duddeck, H. 13C Chemical shift and carbon-proton coupling constants of some furocoumarins and furochromones. Phytochemistry 1979, 18, 139–143. [Google Scholar]
- Harkar, S.; Razdan, T.K.; Waight, E.S. Steroids, chromone and coumarins from Angelica officinalis. Phytochemistry 1984, 23, 419–426. [Google Scholar]
- Chang, C.T.; Doong, S.L.; Tsai, I.L.; Chen, I.S. Coumarins and anti-HBV constituents from Zanthoxylum schionifolium. Phytochemistry 1997, 45, 1419–1422. [Google Scholar]
- Cai, J.N.; Basnet, P.; Wang, Z.T.; Komatsu, K.; Xu, L.S.; Tani, T. Coumarins from the fruits of Cnidium monnieri. J. Nat. Prod 2000, 63, 485–488. [Google Scholar]
- Concannon, S.; Ramachandran, V.N.; Smyth, W.F. A study of the electrospray ionisation and ion trap fragmentation of hemiterpenoid and dimeric coumarin derivatives. Rapid Commun. Mass Spectrom 2000, 14, 2260–2270. [Google Scholar]
- Koul, S.K.; Dhar, K.L.; Thakur, R.S. Regiospecific oxidation of osthol [7-methoxy-8-(isopentenyl)coumarin] with selenium dioxide. Indian J. Chem 1978, 17, 285–287. [Google Scholar]
- Ito, C.; Otsuka, T.; Ruangrungsi, N.; Furukawa, H. Chemical constituents of Micromelum minutum. Isolation and structural elucidation of new coumarins. Chem. Pharm. Bull 2000, 48, 334–338. [Google Scholar]
- Rittschof, D.; Lai, C.H.; Kok, L.M.; Teo, S.L.M. Pharmaceuticals as antifoulants: Concept and principles. Biofouling 2003, 19, 207–212. [Google Scholar]
- Hitotsuyanagi, Y.; Kojima, H.; lkuta, H.; Takeya, K.; ltokawa, H. Identification and structure—activity relationship studies of osthol, a cytotoxic principle from Cnidium Monnieri. Bioorg. Med. Chem. Lett 1996, 6, 1791–1794. [Google Scholar]
- Lin, J.R.; Wang, Y.C.; Xu, Q.H.; Jin, R.H.; Tian, W.S. Formal synthesis of 1α,25-dihydroxyvitamin D3 by utilizing the intact skeleton of diosgenin. Acta Chem. Sinica 2007, 65, 1685–1692. [Google Scholar]
- Bryan, J.P.; Kreider, L.; Qian, P.Y. Settlement of the serpulid polychaete Hydroides elegans (Haswell) on the arborescent bryozoan Bugula neritina (L.): Evidence of a chemically mediated relationship. J. Exp. Mar. Biol. Ecol 1998, 220, 171–190. [Google Scholar]
- Hamilton, M.A.; Russo, R.C.; Thurston, R.V. Trimmed Spearman–Karber method for estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol. 1978, 12, 417–417. (Correction to: Hamilton M.A., Russo R.C., Thurston R.V. (1977)). [Google Scholar]
|Table 1. Antilarval settlement activities of Compounds (1–9) against Balanus albicostatus and Bugula neritina.|
|Tested sample||B. albicostatus||B. neritina|
|EC50(μg mL−1)||LC50(μg mL−1)||LC50/EC50||EC50(μg mL−1)|
|Meranzin hydrate (6)||35.36||>100||UD||18.23|
|2′-deoxymetranzin hydrate (7)||4.67||>50||>10.7||9.06|
© 2013 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).