J Appl Phycol (2012) 24:847–856 DOI 10.1007/s10811-011-9703-2
The antialgal activity of 40 medicinal plants against Microcystis aeruginosa Yang-Lei Yi & Yi Lei & Yue-Bang Yin & Hong-Yu Zhang & Gao-Xue Wang
Received: 22 January 2011 / Revised and accepted: 18 July 2011 / Published online: 17 August 2011 # Springer Science+Business Media B.V. 2011
Abstract In search of a botanical algicide, 40 traditional medicinal plants were screened for antialgal activity against the bloom-forming cyanobacterium Microcystis aeruginosa using coexistence culture system assay. The results of the coexistence assay showed that significant inhibition of the algae at 800 mg L−1 were observed for methanolic extracts of the root of Salvia miltiorrhiza (Radix Salviae Miltiorrhizae), rhizome of Acorus tatarinowii (Rhizoma Acori), rhizome of Polygonum cuspidatum (Rhizoma Polygoni), cortex of Phellodendron amurense (Cortex Phellodendri), and fruits of Crataegus pinnatifida (Fructus Crataegi). Methanol extract of these plants were further partitioned with petroleum ether, chloroform, ethyl acetate, and acetone to obtain allelopathically active fractions with various polarity. Among these fractions tested, the ethyl acetate extract of S. miltiorrhiza was observed to be more efficient than the other plant extracts with the inhibitory rate (IR) of 91.3% at 800 mg L−1 and 50% effective concentrations (EC50) values 98.9 mg L−1 after 7 days, followed by chloroform extracts of A. tatarinowii, S. miltiorrhiza, and P. cuspidatum, and the petroleum ether extracts of A. Y.-L. Yi : Y.-B. Yin : G.-X. Wang (*) Northwest A & F University, Xinong Road 22nd, Yangling, Shaanxi 712100, China e-mail:
[email protected] G.-X. Wang e-mail:
[email protected] Y. Lei College of life science, Northwest University, Xi’an, Shaanxi 710069, China H.-Y. Zhang Chinese Academy of Fishery Sciences, Beijing 100141, China
tatarinowii with EC 5 0 102.5, 111.5, 122.9, and 130.0 mg L−1, respectively. Keywords Antialgal activity . Crude extract . Herbal medicine plants . Microcystis aeruginosa
Introduction In recent years, there has been a growing awareness worldwide of the problems associated water blooms in eutrophic water bodies, which causes deterioration of water quality, mass mortalities of fish and shellfish (Nagayama et al. 2003). Moreover, human illness and even death can result from eating seafood contaminated by some bloom-forming algae (Galvao et al. 2009). Microcystis aeruginosa is the dominant species of freshwater bloom-causing cyanobacteria and is known to produce cyanobacterial hepatotoxins, the microcystins (Oh et al. 2000). These soluble peptides, which are lethal or harmful to many kinds of aquatic organisms (Chen et al. 2009), damage the livers of higher animals (Park et al. 2009; Herranz et al. 2010). Therefore, the control of microcystinproducing Microcystis has become an important environmental and public health concern. In an effort to manage and mitigate the potentially devastating effects of harmful algal blooms (HABs), several control techniques have been applied including yellow loess (Lee et al. 2008) and clay minerals (Pan et al. 2006). Although these methods have been found to be locally effective, yellow loess and clay cause secondary effects on bottom-dwelling organisms. Chemical agents such as copper sulfate (Han et al. 2001) and hydrogen peroxide (Drabkova et al. 2007) are effective in controlling blooms within a short period after application, but their usage in
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aquatic ecosystems is potentially dangerous (Jeong et al. 2000). Biological agents, such as bacteria (Kang et al. 2008), viruses (Yoshida et al. 2006), planktonic ciliates (Mayali and Doucette 2002), and fungi (Sigee et al. 1999), have disadvantages such as difficulty of application, and in some cases, they have the potential of disastrous ecological consequences. None of these methods can specifically control harmful algal blooms without causing problems towards other organisms. Therefore, there is an urgent need to develop highly effective, biodegradable, and environmentally safe algicides for controlling harmful algal blooms. Currently, investigators have diverted their approach towards the development of botanical algicides. Natural allelochemicals from different organisms have been considered as the source for potential algicides (Jüttner et al. 2001; Chung et al. 2007; Park et al. 2009). Several varieties of macrophytes and higher plants have been found to inhibit algae growth effectively. Barley straw has been shown to inhibit algal growth (Ball et al. 2001; Ó hUallacháin and Fenton 2010) and remains the only treatment in widespread use. According to Nakai et al. (2000), Myriophyllum spicatum inhibited M. aeruginosa coexisting in a culture. The methanol extracts of oak tree decreased the cell density of M. aeruginosa by over 90% for 7 days (Park et al. 2006). Gross et al. (2003) have also reported that the methanol extract of Najas marina and acetone extract of Ceratophyllum demersum showed strong inhibitory effects on various filamentous or chroococcal cyanobacteria. Other plants have also been screened for antialgal activities, including brown-rotted wood (Pillinger et al. 1995), Flourensia cernua leaves (Tellez et al. 2001), Ruta graveolens (Meepagala et al. 2005), and Arundo donax (giant reed) shoots (Hong et al. 2010). Therefore, a good possibility exists that the growth of noxious algae can be controlled by using antialgal allelopathic extract of plants. Medicinal plants have long been used in traditional medicine in Asia, and there is increasing research into the utilization of traditional plant-based medicines to control algae growth (Zhou et al. 2007, 2008; Liu et al. 1998), but little information is available on the use of medicinal plants for the treatment of the bloom-forming cyanobacterium M. aeruginosa. Considering the popularity of medicinal plants and herbs and their low toxicities, the present work seeks to exploit the antialgal activity of these plants, by screening 40 traditional herbal medicine plants for antialgal activity against M. aeruginosa and to separate the allelopathically active fractions of selected plants.
Materials and methods Microcystis aeruginosa (toxic strain FACHB-905, isolated from Dianchi Lake in Yunnan, China) was provided by the
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FACHB collection (Freshwater Algae Culture of Hydrobiology Collection, Chinese Academy of Sciences). Before the experiment, the algae were cultivated at 25°C under a 14:10-h light–dark cycle at 90 μmol of photons m−2 s−1 in sterile BG11 medium (Li and Hu 2005). Fresh plant material from each of the selected species (see Table 1) was collected in 2010. These were identified by Prof. X.L. He (Northwest A&F University, Shaanxi, China), and voucher references have been deposited in the College of Life Science, Northwest A&F University, China. The plants were washed thoroughly, air-dried under sunlight for a week and finally oven-dried at 45°C for 48 h. The dried plant materials were crushed manually and then with a mortar and pestle, finally reduced to fine powder using a strainer (30–40 mesh). The powdered samples were freezedried at −45°C to ensure a complete removal of water. Each plant was authenticated by comparing their TLC and HPLC profiles (Fig. 1) with those given in the Pharmacpoeia of the People’s Republic of China (2010 edition). Activity guided screening of plants The dry powder (100 g) of each species was extracted with methanol (1,000 mL × 3 times) for 48 h. The methanol filtrates were separately filtered and evaporated under reduced pressure in a vacuum rotary evaporator. The methanolic extracts were then screened for their antialgal activity. Only S. miltiorrhiza, A. tatarinowii, P. cuspidatum, P. amurense, and C. pinnatifida showed strong antialgal activity when compared with others, and crude methanolic extracts of these selected plants were further fractionated by solid-phase extraction (SPE) to isolate allelopathically active fractions. The crude extracts of selected plants were fractionated with petroleum ether, chloroform, ethyl acetate, acetone, and methanol for 12 h for complete extraction, and the process was repeated three times. The ratio of sample to solvent was 1:10 (m/v). Each extract was subsequently filtered and concentrated under reduced pressure in a vacuum rotary evaporator until the solvents were completely evaporated to get more or less solidified crude extracts. These crude extracts were dissolved in 40 mL of DMSO to get 200 g L-1(sample/ solvent) of stock solutions which were used for preparation of the desired concentrations for bioassay. Antialgal bioassay Most of the extracts produced by plants have low water solubility, and therefore cannot be directly bioassayed. Usually, a small amount of organic solvents such as DMSO and methanol are used to increase the water solubility of extracts, which are then bioassayed. In this study, 0.3% (v/v) of DMSO was used because the growth of algae was not
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Table 1 Plants used in this study, their part used, voucher references, yield of extracts, and inhibitory rate against M. aeruginosa (n=3) Species
Medical names
Family
Yield (%) Plant part used
Voucher IR % references (means ± SD)
Rhus chinensis Mill.
Galla Chinensis
Anacardiaceae
8.2
Stems
SZ-1,221
23.8±4.4
Acorus tatarinowii Schott
Rhizoma acori graminei
Araceae
8.3
Rhizome
SZ-1,031
91.5±4.2
Pinellia ternata (Thunb.) Breit. Acanthopanax senticosus
Rhizoma Pinelliae Radix Acanthopanacis
Araceae Araliaceae
2.3 7.9
Rhizome Roots
SZ-1,225 SZ-1,037
14.6±2.8 12.4±2.1
Acanthopanax gracilistylus W. W. Smith Cortex Acanthopanacis
Araliaceae
9.1
Root bark
SZ-1,140
11.5±3.3
Epimedium brevicornum Maxim. Tripterygium wilfordii Hook. F.
Herba Epimedii Radix Tripterygii
Berberidaceae Celastraceae
4.1 7.9
Leaves Roots
SZ-1,127 SZ-1,239
23.6±4.6 20.8±3.9
Quisqualis indica L
Fructus Quisqualis
Combretaceae
12.3
Fruits
SZ-1,032
25.4±5.4
Isatis indigotica Fort.
Radix Isatidis
Cruciferae
6.9
Roots
SZ-1,058
34.4±1.5
Euphorbia humifusa Willd. Ricinus communis L
Herba Euphorbiae Humifrsae Euphorbiaceae Semen Ricini Euphorbiaceae
5.4 9.3
Leaves Seeds
SZ-1,226 SZ-1,027
44.5±5.5 36.4±3.9
Croton tiglium L
Semen Crotonis Tiglii
Euphorbiaceae
5.6
Fruits
SZ-1,063
2.6±2.1
Hydnocarpus hainanensis (Merr.) Sleum. Semen Hydnocarpi
Flacourtiaceae
8.5
Seeds
SZ-1,041
48.3±4.9
Aesculus chinensis Bge.
Caulis Spatholobi
Hippocastanacease
4.1
Seeds
SZ-1,039
72.3±2.1
Curculigo orchioides Gaertn. Salvia miltiorrhiza Bge. Psoralea corylifolia L
Rhizoma Curculiginis Radix salviae Fructus Psoraleae
Hypoxidaceae Labiatae Leguminosae
5.4 7.6 8.3
Rhizome Roots Fruits
SZ-1,024 SZ-1,015 SZ-1,022
45.1±3.5 95.3±4.6 9.6±1.2
Glycyrrhiza uralensis Fisch. Ophiopogon japonicus (Thunb.) Ker-Gawl. Lilium pumilum DC. Strychnos nux-vomica L Paeonia lactiflora Pall.
Radix Glycyrrhizae Raidix Ophiopogonis
Leguminosae Liliaceae
6.7 6.4
Root Roots
SZ-1,323 SZ-1,237
−11.3±2.4 −2.7±3.4
Bulbus Lilii Semen Strychni Raidix Paeoniae Rubra
Liliaceae Loganiaceae Paeoniaceae
10.7 5.2 5.7
Bulbs Seeds Roots
SZ-1,059 SZ-1,020 SZ-1,219
−13.5±2.6 53.2±3.1 55.7±2.7
Areca catechu L Phytolacca americana L
Semen Arecae Radix Phytolaccae
Palmae Phytolaccaceae
11.8 4.3
Seeds Roots
SZ-1,113 SZ-1,214
24.7±3.2 17.8±3.5
Polygonum cuspidatum Sieb. et Zucc.
Rhizoma Polygoni
Polygonaceae
3.8
Stem
SZ-1,117
89.8±4.5
Rumex nepalensis Spreng. Aconitum carmichaeli Debx. Crataegus pinnatifida Bge.
Radix Rumicis Radix Aconiti Fructus Crataegi
Polygonaceae Ranunculaceae Rosaceae
11.6 6.1 9.2
Roots Roots Fruits
SZ-1,335 SZ-1,055 SZ-1,187
24.4±5.8 27.2±4.5 92.9±3.8
Prunus mume Sieb. et Zucc. Phellodendron amurense Rupr. Dictamnus dasycarpus Turcz. Houttuynia cordata Thunb. Brucea javanica (L) Merr. Solanum nigrum L Lycium barbarum L Bupleurum chinense DC. Cnidium monnieri (L) Cuss. Foeniculum vulgare Mill. Alpinia officinarum Hance. Zingiber officinale Rosc.
Fructus Mume Cortex phellodendri Cortex Dictamni Herba Houttyniae Fructus Bruceae Herba Solani Nigri Fructus Lycii Radix Bupleuri Fructus Cnidii Fructus Foeniculi Rhizoma Alpiniae Officinari
Rosaceae Rutaceae Rutaceae Saururaceae Simaroubaceae Solanaceae Solanaceae Umbelliferae Umbelliferae Umbelliferae Zingiberaceae
6.5 10.7 7.2 3.6 5.9 8.3 8.7 4.6 4.1 5.6 3.6
Fruits Stem Roots bark Leaves Fruits Leaves Fruits Roots Fruits Fruits Rhizome
SZ-1,156 SZ-1,045 SZ-1,050 SZ-1,419 SZ-1,028 SZ-1,048 SZ-1,033 SZ-1,124 SZ-1,043 SZ-1,072 SZ-1,230
45.8±3.7 92.5±5.3 28.6±1.7 69.3±6.4 60.7±5.1 24.8±2.4 −6.9±2.5 45.2±2.3 33.7±3.5 6.5±2.8 53.2±4.6
Rhizoma Zingiberis
Zingiberaceae
12.6
Rhizome
SZ-1,026
23.1±4.6
influenced at this concentration, as confirmed by a previous study (Yasushi et al. 2003). Inhibition assay of algal growth was performed using the US Environmental Protection Agency standard method (1989) with some modifications. The crude methanolic extracts and the allelopathically active fractions were tested
against M. aeruginosa in 100-mL conical flasks containing 20 mL culture media, to which 106 cells mL−1 of M. aeruginosa were inoculated. The designed concentration gradients of extract were added, the final concentrations in the test solution were 0, 50, 100, 200, 400, and 800 mg L−1, negative control groups containing no plant extract were set
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Fig. 1 The HPLC profiles of P. amurense, P. cuspidatum, and S. miltiorrhiza. Chromatographic condition: Hyper ODS-2 C18 (250× 10 mm) column, flow rate of 1.0 mL min−1 at 25°C, and a capacity of 10 μL. a, b Mobile phase: acetonitrile–1% phosphoric acid (50:50, v/v);
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detection wavelength 265 nm. c, d mobile phase: acetonitrile–water (23:77, v/v); detection wavelength 306 nm. e, f mobile phase: methanol– water (75:25, v/v); detection wavelength 270 nm
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up under the same conditions as the test groups. To eliminate the possible effects of DMSO on the algae, another control containing the corresponding percentage of DMSO was also included. Cultures were shaken by hand thrice daily, and algal growth was monitored microscopically by cell counting with a hemocytometer everyday. The extracts effects on algae were tested for 7 days. Each experiment included triplicate treatments, and the experiments were repeated twice. The percentage growth inhibition at specific test substance concentration was calculated compared to the control group. The inhibitory effect of the extracts on the algal growth was estimated by inhibition over control which is defined by the following formula: IRð%Þ ¼ ½1 ðN =N0 Þ 100 Where N0 and N are the numbers of cells in the control and extract-added cultures, respectively. Concentration analysis of microcystin A study of the effects of allelopathically active fractions on the microcystin (microcystin-LR) concentration of Microcystis strain FACHB-905 also was carried out. Active fractions were added into algal medium at the concentrations of 800 mg mL−1; the algae were then inoculated to give the cell density of approximately 106 cells mL−1. The algae were incubated under the same conditions described above for 1 week, and the Microcystin-LR was analyzed by HPLC-UV methods and quantified using an external gravimetric standard of microcystin-LR (Axxora) on the 3rd and 7th day. Each experiment included triplicate treatments and was conducted in duplicate. For the microcystin extraction, 50-mL culture were filtered through glass microfiber filters (Whatman GF/C; 25 mm diameter), immediately freeze-dried, and stored at −20°C. The extracellular (dissolved) microcystin concentration was also analyzed from the filtrate. According to Lawton et al. (1994), microcystins were extracted with 75% (v/v) aqueous methanol. Each sample was extracted three times with 10 mL of 75% methanol for 30 min. Cell disruption and filter dispersal were carried out with ultrasonication for 15 min. Afterwards, samples were shaken for 30 min and centrifuged at 12,000×g for 10 min, then the supernatant was applied directly to preconditioned ODS SepPak 6 mL 500 mg C-18 cartridge (Waters) for solidphase extraction. After sample application, the cartridge was washed with water and 20% (v/v) aqueous methanol (10 mL each). The microcystins were finally eluted from the C-18cartridge with 90% (v/v) aqueous methanol, evaporated to dryness, reconstituted in 1 mL methanol, and applied to high-performance liquid chromatography system (L-2,000, HITACHI) analysis with an UV-detector set at 238 nm. The column was a 4.6×250 mm Hyper
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ODS-2 C18. The mobile phase was phosphate buffer and methanol (58:42) (pH 2) at room temperature, and a flow rate of 1 mL min−1. Data analysis When algal growth was significantly inhibited, the effective concentration causing a 50% inhibitory response at 7 days (EC50) was estimated with logistic fitting. The data were analyzed by one-way ANOVA and expressed as the arithmetic mean ± standard deviation (SD). Differences was determined by Tukey’s test in SPSS statistical software (SPSS Inc., USA) with P values less than 0.05 being accepted as significant.
Results The inhibitory activities on M. aeruginosa for the plant species examined were evaluated and the percentage inhibitions are shown in Table 1. Of the plant extracts tested, S. miltiorrhiza, A. tatarinowii, P. cuspidatum, P. amurense, and C. pinnatifida were found to have significant antialgal activity at 800 mg L−1. The algal cell stopped growing and remained long in an inhibited state. The IR values of the extracts varied from 89.8% to 95.3%. DMSO as the negative controls did not show any inhibition of M. aeruginosa. Interestingly, roots of O. japonicus, roots of G. uralensis, fruits of L. barbarum, and bulbs of L. pumilum stimulated algae growth with IR values of −2.7%, −11.3%, −6.9%, and −13.5%, respectively. The antialgal efficacies of different extracts of S. miltiorrhiza, A. tatarinowii, P. cuspidatum, P. amurense, and C. pinnatifida are shown in Table 2 and Fig. 2, which indicated that the ethyl acetate extract of S. miltiorrhiz was the most active fraction with IR of 91.3% and EC50 of 98.9 mg L−1. High antialgal activity against M. aeruginosa was also observed in the chloroform and methanol extracts with IR of 74.4 and 70.3%, and EC50 of 111.5 and 369.6 mg L−1, respectively. Followed by the petroleum ether and acetone extracts and the maximum IR were 48.8 and 37.5%, respectively. In the case of A. tatarinowii and P. cuspidatum, the chloroform extracts were observed to be the most effective with IR of 85.4 and 72.5%, and EC50 of 102.5 and 122.9 mg L−1 after 7 d of post-treatment, respectively. The petroleum ether extracts were the next most effective, with IR of 70.6% and 66.9%, and EC50 of 130.0 and 203.2 mg L−1. The ethyl acetate P. cuspidatum extract showed optimal antialgal activity with IR of 70.0% and EC50 of 238.7 mg L−1. The acetone and methanol extracts of P. cuspidatum showed little activity with antialgal
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Table 2 Algicidal efficacy of extracts from S. miltiorrhiza, A. tatarinowii, P. cuspidatum, P. amurense, and C. pinnatifida against Microcystis aeruginosa after 7 days of exposure (n=3) Plants
Extraction solvent
IR % (means ± SD) EC50 (mg L-1)
S. miltiorrhiza Chloroform
Discussion 74.4±1.2
111.5
91.3±2.5
98.9
70.3±1.9 70.6±3.1
369.6 130.0
85.4±2.5
102.5
66.9±1.7 72.5±2.4
203.2 122.9
Ethyl acetate
70.0±1.4
238.7
Petroleum ether Chloroform
70.0±1.8 66.9±2.3
157.8 173.3
64.3±2.6
327.7
Ethyl acetate Methanol A. tatarinowii Petroleum ether Chloroform P. cuspidatum Petroleum ether Chloroform P. amurense
significantly higher than control (P
