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Myriophyllum spicatum-released allelopathic polyphenols inhibiting growth of blue-green algae Microcystis aeruginosa
PII: S0043-1354(00)00039-7
Wat. Res. Vol. 34, No. 11, pp. 3026±3032, 2000 7 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
www.elsevier.com/locate/watres
MYRIOPHYLLUM SPICATUM-RELEASED ALLELOPATHIC POLYPHENOLS INHIBITING GROWTH OF BLUE-GREEN ALGAE MICROCYSTIS AERUGINOSA SATOSHI NAKAI*M, YUTAKA INOUE, MASAAKI HOSOMI and AKIHIKO MURAKAMIM Department of Chemical Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka, Koganei, Tokyo, 184-8588, Japan (First received 1 April 1999; accepted 1 August 1999) AbstractÐA culture solution of macrophyte Myriophyllum spicatum was subjected to algal assaydirected fractionation on the basis of polarity and molecular weight. As the water-soluble fraction below molecular weight 1000 was the only fraction to inhibit the growth of blue-green algae Microcystis aeruginosa, it was analyzed by analytical high-performance liquid chromatography (HPLC) and atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in order to identify M. spicatum-released growth-inhibiting allelochemicals. Both HPLC and APCI-MS revealed the release of four polyphenols exhibiting growth inhibition eects, i.e., ellagic, gallic and pyrogallic acids and (+)catechin. A quantitative investigation of their respective inhibitory eects showed that gallic and pyrogallic acids are more inhibitory than (+)-catechin and ellagic acid, and that the autoxidized products of each polyphenol demonstrated growth inhibition. Finally, when the collective activity of a mixture of the polyphenols was examined, synergistic growth inhibition of M. aeruginosa occurred. 7 2000 Elsevier Science Ltd. All rights reserved Key wordsÐMyriophyllum spicatum, allelochemical, polyphenol, synergistic growth inhibition, autoxidation, blue-green algae
INTRODUCTION
With regard to the antagonistic relationship occurring between algae and macrophytes in natural and experimental aquatic ecosystems (Hasler and Jones., 1949), the competition for available nutrients and light is generally known to inhibit algal growth. Hogetsu et al. (1960) proposed another mechanism, in which macrophytes release allelochemicals that inhibit algal growth. Such a growth inhibition mechanism strongly suggests that macrophytes could be used to control undesirable algae. We recently investigated the allelopathic eects produced by nine species of macrophytes (Nakai et al., 1999) and found that Myriophyllum spicatum produced the most inhibitory eects on two species of problem-causing blue-green algae (Microcystis aeruginosa and Phormidium tenue ). Moreover, the inhibitory eects of M. spicatum were demonstrated to be due to its release of allelochemicals, thereby con®rming the feasibility of using macrophytes as a control tool for algal growth. *Author to whom all correspondence should be addressed. Tel.: +81-42-388-7070; fax: +81-42-381-4201; e-mail: [email protected]
Planas et al. (1981) found that an extract of M. spicatumÐwhich included 12 kinds of phenols and polyphenols, e.g., gallic acid and ellagic acidÐcould inhibit algal growth. They did not, however, investigate the inhibitory eects produced by each individual compound. Regarding other species of the genus Myriophyllum, Saito et al. (1989) showed that the growth of blue-green algae Anabaena ¯os-aquae and M. aeruginosa could be inhibited by the hydrolyzable tannins eugeniin and 1-desgalloyl eugeniin extracted from M. brasiliense and as well by gallic and ellagic acids, which are components of these hydrolyzable tannins. Later, Aliotta et al. (1992) extracted three polyphenols from M. verticillatum and con®rmed the associated inhibitory eects on algal growth. Now, in view of the fact that phenols, and especially polyphenols, have high water solubility due to their hydroxyl groups, and that M. spicatum contains phenols and polyphenols (Planas et al., 1981), it is reasonable to surmise that M. spicatum releases phenols and/or polyphenols which cause the resultant growth inhibition of algae. In fact, Gross et al. (1996) showed that M. spicatum released tellimagrandin II and ellagic acid, and that each compound produced an inhibitory eect. Questions
3026
Myriophyllum spicatum-released polyphenols
remain, though, because the amounts released by M. spicatum have not been evaluated, and some phenolic compounds released by it have not been identi®ed. Although the feasibility of controlling algal growth by the addition of allelopathic macrophyte M. spicatum and/or its allelochemicals has been demonstrated, further research must be carried out to establish this as a safe, eective method for aquatic ecosystem management. Essential tasks are (1) to reveal the allelopathic compounds released from M. spicatum, (2) to determine a quantitative relationship that clari®es how the inhibition of algal growth is aected by the concentration of each allelochemical, and (3) to study collective activity of the allelochemicals on algal growth inhibition. Toward this end, here we identify the allelochemicals released by M. spicatum and report on a quantitative investigation of the growth inhibitory eects of the identi®ed allelochemicals. In addition, collective activity of the identi®ed allelochemicals and their inhibitory eects were investigated to determine whether or not the identi®ed allelochemicals demonstrate synergistic growth inhibition of algae. MATERIALS AND METHODS
Algae and M. spicatum As one of the most undesirable blue-green algae in Japan, Microcystis aeruginosa (NIES-87) obtained from the microbial collection of the National Institute for Environmental Studies (NIES), Japan was used for algal assays. M. spicatum was collected from the Asakawa River, Tokyo, Japan and then cultivated in a 20-fold dilution Gorham's medium (Zehnder and Gorham, 1960) using a light intensity of 3000 lux at 258C for 3 days. Algal assays were used to: (1) accomplish assay-directed fractionation of the culture solution (Fig. 1); (2) analyze concentration-dependent inhibitory eects of each identi®ed allelochemical; and (3) determine collective activity of the identi®ed allelochemicals as a result of their inhibitory eects on M. aeruginosa's growth. Under a light intensity
3027
of 5000 lux, M. aeruginosa was cultivated in triplicate in a modi®ed C (CB) medium (Watanabe and Satake, 1991) at 258C for about 10±15 days, during which time its growth was monitored by determining cell numbers with a hemocytometer (Thoma JHS, Hishikaki). Maximum growth was determined from data obtained at the stationary phase. Identi®cation of allelochemicals Figure 1 graphically shows the procedure employed for identifying the allelochemicals released by M. spicatum. The M. spicatum-cultured solution was prepared by culturing the macrophyte (100 g wet lÿ1) in a 20-fold dilution of Gorham's medium for 3 days without rotation, after which the solution was subjected to the algal assay-directed fractionation on the basis of polarity and molecular weight of the allelochemicals. Because the results showed that the ultra®ltered water-soluble fraction (UWF) with molecular weight <1000 was the only one to inhibit growth (data not shown), this fraction was analyzed using high-performance liquid chromatography (HPLC) followed by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). HPLC and APCI-MS The UWF (100 ml) was subjected to HPLC on an ODS column (TSK-gel ODS 80TS, 250 4.6 mm, TOSOH) at 358C using two eluents (Zhu et al., 1992): that is, eluent A (0.025% H3PO4 in water) and eluent B (0.025% H3PO4 in methanol) using an elution pro®le 0±55 min 0±19.8% B, 55±90 min 19.8±47.8% at a constant ¯ow rate of 1 ml minÿ1. As the detection device, we used an electrochemical detector (HP1049A, Hewlett Packard) set as follows: mode, amperometry; working electrode, glassy carbon; potential, 0.9 V (vs Ag/AgCl). Qualitative con®rmation of the allelochemicals detected in the UWF was performed by HPLC using a spike test in which the UWF was spiked with commercially available standard compounds. HPLC results were veri®ed by subjecting concentrated UWF to APCI-MS. Brie¯y, the residue obtained by freeze-drying 2 l of the UWF was dissolved in 10 ml of methanol and concentrated to a 2 ml solution using gaseous N2. The concentrated sample (20 ml) was then subjected to APCI-MS analysis in which a HPLC system separated the concentrated UWF and an APCI-MS detector (HP1100 MSD, Hewlett Packard) operated at the conditions listed in Table 1. The concentrated UWF was separated by an ODS column (Develosil ODS UG, 250 2.1 mm, Yokogawa Analytical Systems) operated at 408C using eluent C (0.05% CF3COOH in pure water) and eluent D (0.05% CF3COOH in 80% methanol) with an elution pro®le: 0± 55 min 0±33% D, 55±90 min 33±75% at a constant ¯ow rate of 0.2 ml minÿ1.
Table 1. Operating conditions of the APCI-MS detector
Fig. 1. Analytical process used in identi®cation of allelochemicals released by M. spicatum.
Instrument Fragmentor
HP1100MSD 80 V (0±30 min) 120 V (30±90 min)
Nebulizer
Nitrogen gas (50 psi)
Drying gas
Nitrogen gas (10 l minÿ1, 3508C)
Mode
Positive selected ion monitoring (positive SIM)
Mass fragment
m/z m/z m/z m/z
127 (PA) 1771 (GA) 291 (CATECH) 303 (EA)
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Satoshi Nakai et al.
Assaying allelochemicals and their autoxidized products To obtain respective dose±response relationships between added amounts of each identi®ed allelochemical and their inhibitory eects on the growth of M. aeruginosa, algal assays were performed in which the amount of added allelochemical was varied. That is, commercially obtained allelochemicals were respectively dissolved in methanol and ®ltered through an autoclaved membrane ®lter (0.22 mm, Millipore), after which a small volume of the ®ltrate (<1.5 ml) was added to the algal medium (100 ml) and M. aeruginosa was immediately inoculated (0104±105 cells mlÿ1). Control experiments were performed by substituting methanol for the ®ltrate. The inhibitory eects of allelochemical-autoxidized products were con®rmed by ensuring full autoxidation of the allelochemical in the algal medium, i.e., sucient time was allowed to pass between the addition of the ®ltrate and the inoculation of M. aeruginosa. Full autoxidation was HPLC-veri®ed by con®rming that the allelochemical was no longer present in the algal medium. Estimation of allelochemical collective activity The collective activity of allelochemicals on growth inhibition of M. aeruginosa was estimated by comparing experimentally obtained inhibitory eects produced by a mixture of the identi®ed allelochemicals with those predicted using a calculation based on the inhibitory eects of an individual allelochemical at the concentrations used to obtain the dose±response relationships. The predicted inhibitory eect (PIE) of a mixture of the allelochemicals was calculated by Colby's equation (Colby, 1967), i.e.,
PIE % A B C N= 100nÿ1 ,
1
where A, B, C, . . . N are the normalized maximum growth with allelochemical A, B, C, . . . N at the added amount a, b, c, . . . n mg lÿ1, respectively, and n is the total number of allelochemicals considered. The PIE indicates the predicted normalized maximum growth of M. aeruginosa when inhibited by a mixture of allelochemicals. An experimental normalized maximum growth of M. aeruginosa that is above or below the PIE respectively indicates an antagonistic or synergistic response. RESULTS AND DISCUSSION
Identi®cation of allelochemicals Figure 2(a) shows the resultant HPLC chromatogram of the UWF. Comparing retention time of the occurred peaks with that of authentic standards, peak 1 at 11.89 min, 2 at 16.44 min, 3 at 48.25 min, 4 at 87.10 min are considered to indicate polyphenols, i.e., pyrogallic acid (PA), gallic acid (GA), (+)-catechin (CATECH), and ellagic acid (EA) whose structures are shown in Fig. 3, however many unknown peaks are presented. Tellimagrandin II (Gross et al., 1996) is thought to elute between GA (peak 2) and EA (peak 4), although this cannot be con®rmed because we could not obtain an analytical grade standard sample of this compound.
Fig. 2. Chromatograms of the water-soluble fraction of the culture solution of M. spicatum: (a) not spiked, (b) spiked with standard samples (PA, GA, CATECH, EA and six phenolic compounds).
Myriophyllum spicatum-released polyphenols
Fig. 3. Structures of polyphenols PA, GA, CATECH and EA detected in the UWF.
Note that Planas et al. (1981) reported that PA, GA and EA are produced by M. spicatum, but not CATECH. Results of the spike test showed no change in the retention time of these four polyphenols (Fig. 2(a) and (b)), thereby con®rming their presence in the M. spicatum-cultured solution. Using their peak areas, the concentration of each polyphenol was calculated. GA (62.8 mg lÿ1) and EA (76.6 mg lÿ1) were presented in much higher concentrations than PA (5.2 mg lÿ1) and CATECH (16.6 mg lÿ1). Although another six phenolic compounds, such as syringic acid, were also examined in the spike test, they were not presented in the M. spicatum-cultured solution. When standard samples of the four polyphenols were subjected to APCI-MS, PA, GA, CATECH and EA were detected as proton-added ions (MH+): m/z = 127, 171, 291 and 303 at a retention time of 10.58, 12.46, 37.18 and 73.83 min, respect-
3029
ively (data not shown). Figure 4 shows the total ion chromatogram (TIC) obtained from APCI-MS of the concentrated UWF using the positive SIM mode (m/z = 127, 171, 291 and 303), where the peaks at 10.59, 12.46 and 73.86 min have mass fragment ions m/z = 127, 171 and 303 corresponding to those of PA, GA and EA. The peaks at 20.68 and 26.19 min, however, indicate the presence of other unknown compounds in the concentrated UWF. These results con®rm HPLC which indicated that the UWF contained PA, GA and EA released from M. spicatum; however the presence of CATECH is not con®rmed. This result might be due to the fact that it occurs at concentrations below detector thresholds. Since CATECH was not found by Planas et al. (1981), we decided to con®rm whether or not M. spicatum contains CATECH. Brie¯y, a methanol extract of M. spicatum, obtained using our previously described method (Nakai et al., 1996), was subjected to APCI-MS using the positive SIM mode (m/z = 291), with results showing a peak at 37.16 min (Fig. 5), which corresponds to that of CATECH standard compound, thereby indicating that CATECH is also released by M. spicatum. Inhibitory eects of polyphenols Figure 6 shows the eect of PA on the growth curve of M. aeruginosa, where the inhibitory eects are apparent as the concentration increases. While the addition of PA concentrations over 1.26 mg lÿ1 (10 mM) signi®cantly inhibited the growth of M. aeruginosa, it still survived at this concentration. Figure 7 shows dose±response relationships for the four polyphenols, where each polyphenol demonstrates some degrees of concentration-depen-
Fig. 4. TIC chromatogram obtained from APCI-MS analysis of concentrated UWF using the positive SIM mode (m/z = 127, 171, 291 and 303).
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Satoshi Nakai et al.
Fig. 5. Mass chromatogram (m/z = 291) obtained from APCI-MS analysis of the methanol extract of M. spicatum.
dent inhibitory eect. The concentrations at which each compound inhibited normal growth of M. aeruginosa by 50% were determined, i.e., the EC50 concentration of PA, GA, CATECH and EA were 0.65, 1.0, 5.5 and 5.1 mg lÿ1, respectively. Thus, among these polyphenols, PA and GA demonstrated strong growth inhibition of M. aeruginosa, whereas growth inhibition by CATECH and EA was weak. In comparison with results by Saito et al. (1989), who used a dierent strain of M. aeruginosa (NIES-44), the EC50 concentrations of GA and EA (1.0 and 5.1 mg lÿ1) are comparable (3.2 and 7.6 mg lÿ1). Polyphenols such as PA and GA are known to be easily autoxidized by dissolved oxygen in a basic solution (Inescu et al., 1978; Doona and Kustin, 1993). Due to this characteristic, whether or not autoxidation occurred during the algal assay was
Fig. 6. Growth curve of M. aeruginosa as aected by indicated concentrations of PA. Symbols correspond to (w) control, (R) 0.63 [mg lÿ1], (q) 1.26 [mg lÿ1], (*) 2.52 [mg lÿ1], (r) 5.04 [mg lÿ1].
con®rmed. After adding to the algal medium an amount of individual compound which was con®rmed to inhibit the growth of M. aeruginosa (PA, 1.2; GA, 1.5; CATECH, 7; and EA, 7.5 mg lÿ1), the presence of each compound was found to vanish over time (PA and GA, 1 h; CATECH, 8 days; and EA, 12 days). In the experiments to determine whether or not the autoxidized products of these polyphenols inhibit growth of M. aeruginosa, i.e., after inoculating the algal medium with M. aeruginosa following complete autoxidation of the above amounts, results showed that growth inhibition occurred. Figure 8 shows the eects of autoxidized products of each polyphenol on the maximum growth of M. aeruginosa, where the maximum growth is inhibited to 0% relative to that of controls, thereby indicat-
Fig. 7. Respective inhibitory eects of the polyphenols PA, GA, CATECH and EA on the maximum growth of M. aeruginosa. Symbols correspond to (w) PA, (R) GA, (q) CATECH, (*) EA. Bars indicate standard deviation (n = 3).
Myriophyllum spicatum-released polyphenols
Fig. 8. Inhibitory eects of autoxidized products of each polyphenol (PA, GA, CATECH and EA) on the maximum growth of M. aeruginosa.
ing that autoxidized products of the four polyphenols also demonstrate growth inhibition eects. Collective activity of polyphenols In the experiments to determine how algal growth is inhibited by the collective action of a mixture of the polyphenols, the used concentrations of the polyphenols were determined on the basis of both their detected concentration in the M. spicatum-cultured solution and their autoxidation rate, i.e., PA, 297; GA, 596; CATECH, 44; and EA, 570 mg lÿ1. The PIE of this mixture was determined using equation 1 in which n = 4 and the values of normalized maximum growth of M. aeruginosa after adding PA, GA, CATECH and EA were obtained from Fig. 7. That is, we used the normalized maximum growth with PA at 297 mg lÿ1, A 100%; with GA at 596 mg lÿ1, B 65±100%; with CATECH at 44 mg lÿ1, C 100%; and with EA at 570 mg lÿ1, D 94±96%. Substituting values into equation 1 gives PIE=61±96%, which indicates that the mixture of the four polyphenols is pre-
Fig. 9. Growth inhibition of M. aeruginosa by a mixture of PA, GA, CATECH and EA. Symbols correspond to (w) control, (R) polyphenol mixture.
3031
dicted to inhibit the normalized maximum growth of M. aeruginosa by 61±96% relative to that of controls. As shown in Fig. 9, at the obtained PIE, the mixture of the four polyphenols completely inhibits growth of M. aeruginosa to 0% relative to controls. Where the actual inhibitory eects of the mixture are stronger than the PIE, this indicates that the mixture induces synergistic actions as part of their inhibitory eects on M. aeruginosa. Accordingly, if further research can determine the appropriate combination to be added for optimal growth inhibition, then utilization of such additives may prove to be a safe and eective method for practically controlling algal growth in aquatic ecosystems. Contribution of the four polyphenols to the allelopathic eects of M. spicatum During the three-day cultivation period of M. spicatum, it probably continuously releases various amounts of PA, GA, CATECH and EA which in turn are autoxidized into products that accumulate in the culture solution. Regarding PA and GA, due to their rapid autoxidation (1 h), comparatively large amounts of their autoxidized products will be present. In addition, because the autoxidized products of each polyphenol inhibited the growth of M. aeruginosa (Fig. 8), it is reasonable to assume that the polyphenols together with their autoxidized products contribute to allelopathic eects produced by M. spicatum. It is for this reason that research directed at further elucidating the allelopathic eects of M. spicatum must consider the inhibitory eects produced by the collective activity of allelochemicals together with those produced by the autoxidation of allelochemicals such as polyphenols. CONCLUSIONS
Using HPLC and APCI-MS analysis, we found that (1) macrophyte M. spicatum releases four polyphenols, i.e., PA, GA, CATECH and EA, each of which inhibits the growth of blue-green algae M. aeruginosa, and (2) the autoxidized products of these polyphenols also produce growth inhibition eects. A quantitative investigation of their respective inhibitory eects on the maximum growth of M. aeruginosa showed that PA and GA produce strong inhibitory eects compared to those of CATECH and EA which are much weaker. When the calculated PIE of a mixture of these polyphenols was compared to experimentally obtained inhibitory eects, it becomes apparent that synergistic actions are collectively induced by the polyphenols as part of their inhibitory eects on M. aeruginosa. AcknowledgementsÐWe are grateful to the National Institute for Environmental Studies, Japan, for supplying M. aeruginosa.
3032
Satoshi Nakai et al. REFERENCES
Aliotta G., Molinaro A., Monaco P., Pinto G. and Previtera L. (1992) Three biologically active phenylpropanoid glucosides from Myriophyllum verticillatum. Phytochem. 31(1), 109±111. Colby S. R. (1967) Calculating synergistic and antagonistic responses of herbicide combinations. Weeds 15, 20±22. Doona C. J. and Kustin K. (1993) Kinetics and mechanism of pyrogallol autoxidation: calibration of the dynamic response of an oxygen electrode. Int. J. Chem. Kinet. 25, 239±247. Gross E. M., Meyer H. and Schilling G. (1996) Release and ecological impact of algicidal hydrolysable polyphenols in Myriophyllum spicatum. Phytochem. 41(1), 133± 138. Hasler A. D. and Jones E. (1949) Demonstration of the antagonistic action of large aquatic plants on algae and rotifers. Ecol. 30(3), 359±364. Hogetsu K., Okanishi R. and Sugawara H. (1960) Studies on the antagonistic relationship between phytoplankton and rooted aquatic plants. Jap. J. Limnol. 21, 124±130, (in Japanese). Inescu G. H., Matei F. and Duca A. L. (1978) Kinetic aspects of the oxidation reactions of gallic acid using an oxygen-selective electrode. Revue Roumaine de Chimie 23(1112), 1611±1617. Nakai S., Hosomi M., Okada M. and Murakami A.
(1996) Control of algal growth by macrophytes and macrophyte-extracted bioactive compounds. Wat. Sci. and Technol. 34(78), 227±235. Nakai S., Inoue Y., Hosomi M. and Murakami A. (1999) Growth inhibition of blue-green algae by the allelopathic eects of macrophytes. Wat. Sci. and Technol. 39(8), 47±53. Planas D., Sarhan F., Dube L., Godmaire H. and Cadieux C. (1981) Ecological signi®cance of phenolic compounds of Myriophyllum spicatum. Verh. Internat. Verein. Limnol. 21, 1492±1496. Saito K., Matsumoto M., Sekine T. and Murakoshi I. (1989) Inhibitory substances from Myriophyllum brasiliense on growth of blue-green algae. J. Nat. Prod. 52(6), 1221±1226. Watanabe M. M. and Satake N. (1991) NIES-collection list of strains. In Microalgae and Protozoa, 3rd ed. National Institute for Environmental Studies Environmental Agency, Japan, pp. 31±32. Zehnder A. and Gorham P. R. (1960) Factors in¯uencing the growth of Microcystis aeruginosa KUTZ. EMEND. Can. J. Microbiol. 6, 645±660. Zhu J., Ng J. and Filippich L. J. (1992) Determination of tannic acid and its phenolic metabolites in biological ¯uids by high-performance liquid chromatography. J. Chromatgr. 577, 77±85.
Wat. Res. Vol. 34, No. 11, pp. 3026±3032, 2000 7 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
www.elsevier.com/locate/watres
MYRIOPHYLLUM SPICATUM-RELEASED ALLELOPATHIC POLYPHENOLS INHIBITING GROWTH OF BLUE-GREEN ALGAE MICROCYSTIS AERUGINOSA SATOSHI NAKAI*M, YUTAKA INOUE, MASAAKI HOSOMI and AKIHIKO MURAKAMIM Department of Chemical Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka, Koganei, Tokyo, 184-8588, Japan (First received 1 April 1999; accepted 1 August 1999) AbstractÐA culture solution of macrophyte Myriophyllum spicatum was subjected to algal assaydirected fractionation on the basis of polarity and molecular weight. As the water-soluble fraction below molecular weight 1000 was the only fraction to inhibit the growth of blue-green algae Microcystis aeruginosa, it was analyzed by analytical high-performance liquid chromatography (HPLC) and atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in order to identify M. spicatum-released growth-inhibiting allelochemicals. Both HPLC and APCI-MS revealed the release of four polyphenols exhibiting growth inhibition eects, i.e., ellagic, gallic and pyrogallic acids and (+)catechin. A quantitative investigation of their respective inhibitory eects showed that gallic and pyrogallic acids are more inhibitory than (+)-catechin and ellagic acid, and that the autoxidized products of each polyphenol demonstrated growth inhibition. Finally, when the collective activity of a mixture of the polyphenols was examined, synergistic growth inhibition of M. aeruginosa occurred. 7 2000 Elsevier Science Ltd. All rights reserved Key wordsÐMyriophyllum spicatum, allelochemical, polyphenol, synergistic growth inhibition, autoxidation, blue-green algae
INTRODUCTION
With regard to the antagonistic relationship occurring between algae and macrophytes in natural and experimental aquatic ecosystems (Hasler and Jones., 1949), the competition for available nutrients and light is generally known to inhibit algal growth. Hogetsu et al. (1960) proposed another mechanism, in which macrophytes release allelochemicals that inhibit algal growth. Such a growth inhibition mechanism strongly suggests that macrophytes could be used to control undesirable algae. We recently investigated the allelopathic eects produced by nine species of macrophytes (Nakai et al., 1999) and found that Myriophyllum spicatum produced the most inhibitory eects on two species of problem-causing blue-green algae (Microcystis aeruginosa and Phormidium tenue ). Moreover, the inhibitory eects of M. spicatum were demonstrated to be due to its release of allelochemicals, thereby con®rming the feasibility of using macrophytes as a control tool for algal growth. *Author to whom all correspondence should be addressed. Tel.: +81-42-388-7070; fax: +81-42-381-4201; e-mail: [email protected]
Planas et al. (1981) found that an extract of M. spicatumÐwhich included 12 kinds of phenols and polyphenols, e.g., gallic acid and ellagic acidÐcould inhibit algal growth. They did not, however, investigate the inhibitory eects produced by each individual compound. Regarding other species of the genus Myriophyllum, Saito et al. (1989) showed that the growth of blue-green algae Anabaena ¯os-aquae and M. aeruginosa could be inhibited by the hydrolyzable tannins eugeniin and 1-desgalloyl eugeniin extracted from M. brasiliense and as well by gallic and ellagic acids, which are components of these hydrolyzable tannins. Later, Aliotta et al. (1992) extracted three polyphenols from M. verticillatum and con®rmed the associated inhibitory eects on algal growth. Now, in view of the fact that phenols, and especially polyphenols, have high water solubility due to their hydroxyl groups, and that M. spicatum contains phenols and polyphenols (Planas et al., 1981), it is reasonable to surmise that M. spicatum releases phenols and/or polyphenols which cause the resultant growth inhibition of algae. In fact, Gross et al. (1996) showed that M. spicatum released tellimagrandin II and ellagic acid, and that each compound produced an inhibitory eect. Questions
3026
Myriophyllum spicatum-released polyphenols
remain, though, because the amounts released by M. spicatum have not been evaluated, and some phenolic compounds released by it have not been identi®ed. Although the feasibility of controlling algal growth by the addition of allelopathic macrophyte M. spicatum and/or its allelochemicals has been demonstrated, further research must be carried out to establish this as a safe, eective method for aquatic ecosystem management. Essential tasks are (1) to reveal the allelopathic compounds released from M. spicatum, (2) to determine a quantitative relationship that clari®es how the inhibition of algal growth is aected by the concentration of each allelochemical, and (3) to study collective activity of the allelochemicals on algal growth inhibition. Toward this end, here we identify the allelochemicals released by M. spicatum and report on a quantitative investigation of the growth inhibitory eects of the identi®ed allelochemicals. In addition, collective activity of the identi®ed allelochemicals and their inhibitory eects were investigated to determine whether or not the identi®ed allelochemicals demonstrate synergistic growth inhibition of algae. MATERIALS AND METHODS
Algae and M. spicatum As one of the most undesirable blue-green algae in Japan, Microcystis aeruginosa (NIES-87) obtained from the microbial collection of the National Institute for Environmental Studies (NIES), Japan was used for algal assays. M. spicatum was collected from the Asakawa River, Tokyo, Japan and then cultivated in a 20-fold dilution Gorham's medium (Zehnder and Gorham, 1960) using a light intensity of 3000 lux at 258C for 3 days. Algal assays were used to: (1) accomplish assay-directed fractionation of the culture solution (Fig. 1); (2) analyze concentration-dependent inhibitory eects of each identi®ed allelochemical; and (3) determine collective activity of the identi®ed allelochemicals as a result of their inhibitory eects on M. aeruginosa's growth. Under a light intensity
3027
of 5000 lux, M. aeruginosa was cultivated in triplicate in a modi®ed C (CB) medium (Watanabe and Satake, 1991) at 258C for about 10±15 days, during which time its growth was monitored by determining cell numbers with a hemocytometer (Thoma JHS, Hishikaki). Maximum growth was determined from data obtained at the stationary phase. Identi®cation of allelochemicals Figure 1 graphically shows the procedure employed for identifying the allelochemicals released by M. spicatum. The M. spicatum-cultured solution was prepared by culturing the macrophyte (100 g wet lÿ1) in a 20-fold dilution of Gorham's medium for 3 days without rotation, after which the solution was subjected to the algal assay-directed fractionation on the basis of polarity and molecular weight of the allelochemicals. Because the results showed that the ultra®ltered water-soluble fraction (UWF) with molecular weight <1000 was the only one to inhibit growth (data not shown), this fraction was analyzed using high-performance liquid chromatography (HPLC) followed by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). HPLC and APCI-MS The UWF (100 ml) was subjected to HPLC on an ODS column (TSK-gel ODS 80TS, 250 4.6 mm, TOSOH) at 358C using two eluents (Zhu et al., 1992): that is, eluent A (0.025% H3PO4 in water) and eluent B (0.025% H3PO4 in methanol) using an elution pro®le 0±55 min 0±19.8% B, 55±90 min 19.8±47.8% at a constant ¯ow rate of 1 ml minÿ1. As the detection device, we used an electrochemical detector (HP1049A, Hewlett Packard) set as follows: mode, amperometry; working electrode, glassy carbon; potential, 0.9 V (vs Ag/AgCl). Qualitative con®rmation of the allelochemicals detected in the UWF was performed by HPLC using a spike test in which the UWF was spiked with commercially available standard compounds. HPLC results were veri®ed by subjecting concentrated UWF to APCI-MS. Brie¯y, the residue obtained by freeze-drying 2 l of the UWF was dissolved in 10 ml of methanol and concentrated to a 2 ml solution using gaseous N2. The concentrated sample (20 ml) was then subjected to APCI-MS analysis in which a HPLC system separated the concentrated UWF and an APCI-MS detector (HP1100 MSD, Hewlett Packard) operated at the conditions listed in Table 1. The concentrated UWF was separated by an ODS column (Develosil ODS UG, 250 2.1 mm, Yokogawa Analytical Systems) operated at 408C using eluent C (0.05% CF3COOH in pure water) and eluent D (0.05% CF3COOH in 80% methanol) with an elution pro®le: 0± 55 min 0±33% D, 55±90 min 33±75% at a constant ¯ow rate of 0.2 ml minÿ1.
Table 1. Operating conditions of the APCI-MS detector
Fig. 1. Analytical process used in identi®cation of allelochemicals released by M. spicatum.
Instrument Fragmentor
HP1100MSD 80 V (0±30 min) 120 V (30±90 min)
Nebulizer
Nitrogen gas (50 psi)
Drying gas
Nitrogen gas (10 l minÿ1, 3508C)
Mode
Positive selected ion monitoring (positive SIM)
Mass fragment
m/z m/z m/z m/z
127 (PA) 1771 (GA) 291 (CATECH) 303 (EA)
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Satoshi Nakai et al.
Assaying allelochemicals and their autoxidized products To obtain respective dose±response relationships between added amounts of each identi®ed allelochemical and their inhibitory eects on the growth of M. aeruginosa, algal assays were performed in which the amount of added allelochemical was varied. That is, commercially obtained allelochemicals were respectively dissolved in methanol and ®ltered through an autoclaved membrane ®lter (0.22 mm, Millipore), after which a small volume of the ®ltrate (<1.5 ml) was added to the algal medium (100 ml) and M. aeruginosa was immediately inoculated (0104±105 cells mlÿ1). Control experiments were performed by substituting methanol for the ®ltrate. The inhibitory eects of allelochemical-autoxidized products were con®rmed by ensuring full autoxidation of the allelochemical in the algal medium, i.e., sucient time was allowed to pass between the addition of the ®ltrate and the inoculation of M. aeruginosa. Full autoxidation was HPLC-veri®ed by con®rming that the allelochemical was no longer present in the algal medium. Estimation of allelochemical collective activity The collective activity of allelochemicals on growth inhibition of M. aeruginosa was estimated by comparing experimentally obtained inhibitory eects produced by a mixture of the identi®ed allelochemicals with those predicted using a calculation based on the inhibitory eects of an individual allelochemical at the concentrations used to obtain the dose±response relationships. The predicted inhibitory eect (PIE) of a mixture of the allelochemicals was calculated by Colby's equation (Colby, 1967), i.e.,
PIE % A B C N= 100nÿ1 ,
1
where A, B, C, . . . N are the normalized maximum growth with allelochemical A, B, C, . . . N at the added amount a, b, c, . . . n mg lÿ1, respectively, and n is the total number of allelochemicals considered. The PIE indicates the predicted normalized maximum growth of M. aeruginosa when inhibited by a mixture of allelochemicals. An experimental normalized maximum growth of M. aeruginosa that is above or below the PIE respectively indicates an antagonistic or synergistic response. RESULTS AND DISCUSSION
Identi®cation of allelochemicals Figure 2(a) shows the resultant HPLC chromatogram of the UWF. Comparing retention time of the occurred peaks with that of authentic standards, peak 1 at 11.89 min, 2 at 16.44 min, 3 at 48.25 min, 4 at 87.10 min are considered to indicate polyphenols, i.e., pyrogallic acid (PA), gallic acid (GA), (+)-catechin (CATECH), and ellagic acid (EA) whose structures are shown in Fig. 3, however many unknown peaks are presented. Tellimagrandin II (Gross et al., 1996) is thought to elute between GA (peak 2) and EA (peak 4), although this cannot be con®rmed because we could not obtain an analytical grade standard sample of this compound.
Fig. 2. Chromatograms of the water-soluble fraction of the culture solution of M. spicatum: (a) not spiked, (b) spiked with standard samples (PA, GA, CATECH, EA and six phenolic compounds).
Myriophyllum spicatum-released polyphenols
Fig. 3. Structures of polyphenols PA, GA, CATECH and EA detected in the UWF.
Note that Planas et al. (1981) reported that PA, GA and EA are produced by M. spicatum, but not CATECH. Results of the spike test showed no change in the retention time of these four polyphenols (Fig. 2(a) and (b)), thereby con®rming their presence in the M. spicatum-cultured solution. Using their peak areas, the concentration of each polyphenol was calculated. GA (62.8 mg lÿ1) and EA (76.6 mg lÿ1) were presented in much higher concentrations than PA (5.2 mg lÿ1) and CATECH (16.6 mg lÿ1). Although another six phenolic compounds, such as syringic acid, were also examined in the spike test, they were not presented in the M. spicatum-cultured solution. When standard samples of the four polyphenols were subjected to APCI-MS, PA, GA, CATECH and EA were detected as proton-added ions (MH+): m/z = 127, 171, 291 and 303 at a retention time of 10.58, 12.46, 37.18 and 73.83 min, respect-
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ively (data not shown). Figure 4 shows the total ion chromatogram (TIC) obtained from APCI-MS of the concentrated UWF using the positive SIM mode (m/z = 127, 171, 291 and 303), where the peaks at 10.59, 12.46 and 73.86 min have mass fragment ions m/z = 127, 171 and 303 corresponding to those of PA, GA and EA. The peaks at 20.68 and 26.19 min, however, indicate the presence of other unknown compounds in the concentrated UWF. These results con®rm HPLC which indicated that the UWF contained PA, GA and EA released from M. spicatum; however the presence of CATECH is not con®rmed. This result might be due to the fact that it occurs at concentrations below detector thresholds. Since CATECH was not found by Planas et al. (1981), we decided to con®rm whether or not M. spicatum contains CATECH. Brie¯y, a methanol extract of M. spicatum, obtained using our previously described method (Nakai et al., 1996), was subjected to APCI-MS using the positive SIM mode (m/z = 291), with results showing a peak at 37.16 min (Fig. 5), which corresponds to that of CATECH standard compound, thereby indicating that CATECH is also released by M. spicatum. Inhibitory eects of polyphenols Figure 6 shows the eect of PA on the growth curve of M. aeruginosa, where the inhibitory eects are apparent as the concentration increases. While the addition of PA concentrations over 1.26 mg lÿ1 (10 mM) signi®cantly inhibited the growth of M. aeruginosa, it still survived at this concentration. Figure 7 shows dose±response relationships for the four polyphenols, where each polyphenol demonstrates some degrees of concentration-depen-
Fig. 4. TIC chromatogram obtained from APCI-MS analysis of concentrated UWF using the positive SIM mode (m/z = 127, 171, 291 and 303).
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Satoshi Nakai et al.
Fig. 5. Mass chromatogram (m/z = 291) obtained from APCI-MS analysis of the methanol extract of M. spicatum.
dent inhibitory eect. The concentrations at which each compound inhibited normal growth of M. aeruginosa by 50% were determined, i.e., the EC50 concentration of PA, GA, CATECH and EA were 0.65, 1.0, 5.5 and 5.1 mg lÿ1, respectively. Thus, among these polyphenols, PA and GA demonstrated strong growth inhibition of M. aeruginosa, whereas growth inhibition by CATECH and EA was weak. In comparison with results by Saito et al. (1989), who used a dierent strain of M. aeruginosa (NIES-44), the EC50 concentrations of GA and EA (1.0 and 5.1 mg lÿ1) are comparable (3.2 and 7.6 mg lÿ1). Polyphenols such as PA and GA are known to be easily autoxidized by dissolved oxygen in a basic solution (Inescu et al., 1978; Doona and Kustin, 1993). Due to this characteristic, whether or not autoxidation occurred during the algal assay was
Fig. 6. Growth curve of M. aeruginosa as aected by indicated concentrations of PA. Symbols correspond to (w) control, (R) 0.63 [mg lÿ1], (q) 1.26 [mg lÿ1], (*) 2.52 [mg lÿ1], (r) 5.04 [mg lÿ1].
con®rmed. After adding to the algal medium an amount of individual compound which was con®rmed to inhibit the growth of M. aeruginosa (PA, 1.2; GA, 1.5; CATECH, 7; and EA, 7.5 mg lÿ1), the presence of each compound was found to vanish over time (PA and GA, 1 h; CATECH, 8 days; and EA, 12 days). In the experiments to determine whether or not the autoxidized products of these polyphenols inhibit growth of M. aeruginosa, i.e., after inoculating the algal medium with M. aeruginosa following complete autoxidation of the above amounts, results showed that growth inhibition occurred. Figure 8 shows the eects of autoxidized products of each polyphenol on the maximum growth of M. aeruginosa, where the maximum growth is inhibited to 0% relative to that of controls, thereby indicat-
Fig. 7. Respective inhibitory eects of the polyphenols PA, GA, CATECH and EA on the maximum growth of M. aeruginosa. Symbols correspond to (w) PA, (R) GA, (q) CATECH, (*) EA. Bars indicate standard deviation (n = 3).
Myriophyllum spicatum-released polyphenols
Fig. 8. Inhibitory eects of autoxidized products of each polyphenol (PA, GA, CATECH and EA) on the maximum growth of M. aeruginosa.
ing that autoxidized products of the four polyphenols also demonstrate growth inhibition eects. Collective activity of polyphenols In the experiments to determine how algal growth is inhibited by the collective action of a mixture of the polyphenols, the used concentrations of the polyphenols were determined on the basis of both their detected concentration in the M. spicatum-cultured solution and their autoxidation rate, i.e., PA, 297; GA, 596; CATECH, 44; and EA, 570 mg lÿ1. The PIE of this mixture was determined using equation 1 in which n = 4 and the values of normalized maximum growth of M. aeruginosa after adding PA, GA, CATECH and EA were obtained from Fig. 7. That is, we used the normalized maximum growth with PA at 297 mg lÿ1, A 100%; with GA at 596 mg lÿ1, B 65±100%; with CATECH at 44 mg lÿ1, C 100%; and with EA at 570 mg lÿ1, D 94±96%. Substituting values into equation 1 gives PIE=61±96%, which indicates that the mixture of the four polyphenols is pre-
Fig. 9. Growth inhibition of M. aeruginosa by a mixture of PA, GA, CATECH and EA. Symbols correspond to (w) control, (R) polyphenol mixture.
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dicted to inhibit the normalized maximum growth of M. aeruginosa by 61±96% relative to that of controls. As shown in Fig. 9, at the obtained PIE, the mixture of the four polyphenols completely inhibits growth of M. aeruginosa to 0% relative to controls. Where the actual inhibitory eects of the mixture are stronger than the PIE, this indicates that the mixture induces synergistic actions as part of their inhibitory eects on M. aeruginosa. Accordingly, if further research can determine the appropriate combination to be added for optimal growth inhibition, then utilization of such additives may prove to be a safe and eective method for practically controlling algal growth in aquatic ecosystems. Contribution of the four polyphenols to the allelopathic eects of M. spicatum During the three-day cultivation period of M. spicatum, it probably continuously releases various amounts of PA, GA, CATECH and EA which in turn are autoxidized into products that accumulate in the culture solution. Regarding PA and GA, due to their rapid autoxidation (1 h), comparatively large amounts of their autoxidized products will be present. In addition, because the autoxidized products of each polyphenol inhibited the growth of M. aeruginosa (Fig. 8), it is reasonable to assume that the polyphenols together with their autoxidized products contribute to allelopathic eects produced by M. spicatum. It is for this reason that research directed at further elucidating the allelopathic eects of M. spicatum must consider the inhibitory eects produced by the collective activity of allelochemicals together with those produced by the autoxidation of allelochemicals such as polyphenols. CONCLUSIONS
Using HPLC and APCI-MS analysis, we found that (1) macrophyte M. spicatum releases four polyphenols, i.e., PA, GA, CATECH and EA, each of which inhibits the growth of blue-green algae M. aeruginosa, and (2) the autoxidized products of these polyphenols also produce growth inhibition eects. A quantitative investigation of their respective inhibitory eects on the maximum growth of M. aeruginosa showed that PA and GA produce strong inhibitory eects compared to those of CATECH and EA which are much weaker. When the calculated PIE of a mixture of these polyphenols was compared to experimentally obtained inhibitory eects, it becomes apparent that synergistic actions are collectively induced by the polyphenols as part of their inhibitory eects on M. aeruginosa. AcknowledgementsÐWe are grateful to the National Institute for Environmental Studies, Japan, for supplying M. aeruginosa.
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