KR101103345B1 - Composition for control of microalgae comprising sesquiterpene compounds - Google Patents

Abstract

The present invention is a plant extract prepared by extracting a plant selected from the group consisting of a plant of the genus Atractylodes , a plant of the genus Piper , a plant of the genus Curcuma , and a mixture thereof with a solvent, or is isolated therefrom. The present invention relates to a composition for controlling microalgae containing the identified sesquiterpene-based compound and a method for controlling microalgae using the same.

According to the present invention, the composition can selectively control the microalgae to be controlled to show different reactivity in the control. In addition, since the activity is increased during the mixing process rather than the individual treatment according to the mixed combination, it is possible to reduce the side effects to the environment by adding a small amount, and the economic efficiency will also be good because the excellent algae activity is maintained.

Microalgae, cyanobacteria, green algae, sesquiterpene compounds, plant extracts

Description

Composition for controlling microalgae containing sesquiterpene compound {COMPOSITION FOR CONTROL OF MICROALGAE COMPRISING SESQUITERPENE COMPOUNDS}

The present invention relates to a composition for controlling microalgae containing a sesquiterpene compound or a plant extract containing at least one of these compounds as an active ingredient, and a method for controlling harmful microalgae using the same.

 Harmful algal blooming (HAB) has caused various industrial problems, depending on their habitat. Algae outbreaks in lakes, streams, reservoirs, fish farms, etc. are associated with the generation of noxious toxins (Haider et al., Chemosphere 52: 1-21, 2003) and off-flavor substances such as soil odors (Duke et al., Weed Sci). 50: 138-151, 2002), water pollution causes discomfort and impedes leisure and industrial activities, causing a lot of losses, both directly and indirectly. On the other hand, in the golf course fairway, the grass dies due to the occurrence of soil algae, and in the freshwater reclamation, the occurrence of algae affects crop productivity by decreasing the rate of raising or lowering the soil temperature.

Recently, the number of useful microalgae has also increased in closed or non-closed conditions (Schenk et al. Bioenerg. Res. 1: 20-43, 2008). At this time, there is a need for the development of a new technology to selectively control the microalgae of the microalgae that is contaminated or cultivated by other harmful algae that interfere with the growth of useful microalgae.

Therefore, physical, biological, or chemical methods have been studied in various ways in order to effectively cope with the problem of microalgae generation. Among them, chemical methods are ones of high practicality, which mainly treat inorganic or organic compounds in water to treat algae. Many compounds have been used, including copper sulfate, as a method of inhibiting or eliminating development. Many of the compounds used in the past have been relatively inexpensive but have problems such as human toxicity, environmental safety for non-target organisms, and persistence. In the future, as the importance of human conservation and environmental protection is more important, it is necessary to discover new compounds that can replace these compounds. Recently, research on microalgae using natural or low-toxic biochemicals has been actively conducted (Schrader). KK, ACS Symposium Series 848: 195-208, 2003). However, natural algicides have a problem of weak activity or narrow working spectrum and high production price depending on the type.

Algal-active biochemicals or natural products reported so far include falcarindaiol (Min et al., Hanjacho 27: 275-284, 2007), L-2-azetidinecarboxylic acid (Kim et al., Aquatic Botany) 85: 1-6, 2006), Rutacridone epoxide (Meepagala et al., Phytochemistry 66: 2689-2695, 2005), Ethyl 2-methylacetoacetate (Li & Hu, Appl. Environ.Microbiol. 71: 6545-6553, 2005) and other compounds (Duke et al., Weed Sci. 50: 138-151, 2002; Schrader et al., Aquaculture 163: 85-99, 1998).

In addition, as a natural product for the algae patented or published in Korea, the oleander plant extract (Korean Patent No. 456467), the plant of the genus or its extract (Korean Patent No. 621917), azetidine derivative (Korean Patent No. 592798), Organic acid (Korean Patent No. 231912), Prodigiosin (Korean Patent No. 661175), Cypress (Korean Patent Publication No. 2006-0103310), Naturally Aging Enzyme Solution of Various Natural Materials (Korean Patent Publication No. 2005-0122919 No.), Bacillus culture filtrate (Korean Patent No. 610527), natural minerals and various plants (Korean Patent No. 623993), Myeongja tree fruit (Korean Patent No. 543747).

On the other hand, sesquiterpene-based compounds such as furanodiene, cariophyllene, alpha curcumin, bisabolene and the like have been extracted as essential oil components in many plants until now, and many reports have been made mainly on medicinal activity. That is, in the case of furanodiene, anticancer activity (Xiao et al., Cancer Biology & Therapy 6 (7): 1044-1050, 2007), anti-inflammatory and antiviral activity (Chinese Patent No. 1884271, 2006), antibacterial activity (Ogunwande Et al., International Journal of Aromatherapy, 15 (3): 147-152, 2005; Dolara et al., Planta Medica, 66 (4): 356-358, 2000), and in the case of cariophyllene, anticancer activity (Marongiu et al. , Journal of Essential Oil Research 16 (3): 256-261, 2004), antimicrobial activity (Costa et al., Phytochemistry, 69 (9): 1895-1899, 2008; Santos et al., Quimica Nova, 31 (7), 1648- 1652, 2008; Zhang et al., Journal of Integrative Plant Biology, 50 (1): 84-91, 2008; Sabulal et al., Phytochemistry 67 (22): 2469-2473, 2006; Pires et al., Natural Product Communications 1 (5), 395-398, 2006), mite-preventing activity (Furuno et al., Mokuzai Gakkaishi, 40 (1), 78-87, 1994), and the like, and anti-curcumin or bisabolene (Ibrahim et al., Food Chemistry, 113 (2): 575-577, 2009; Setzer et al., Natural Product Communicat ions 3 (8), 1367-1374, 2008; Stoyanova et al., Journal of Essential Oil-Bearing Plants 9 (1): 93-98, 2006; Schwob et al., Fitoterapia 73 (6): 511-513, 2002); Bacterial activity (McEnroe et al., Tetrahedron (1978), 34 (11): 1661-1664, 1978), anticancer activity (Setzer et al., Natural Product Communications 3 (8), 1367-1374, 2008;), antioxidant activity (Saroglou et al. , Flavor and Fragrance Journal 22 (2): 154-157, 2007), lipid metabolism control (Yasni et al., Food and Chemical Toxicology 32 (3): 273-278, 1994), and use as a fragrance. However, there have been no reports of cases of using these sesquiterpene compounds and plant extracts containing them as selective algicides of microalgae.

It is an object of the present invention to provide a microalgae control composition and a method of controlling the same, which contain a natural-friendly natural product as an active ingredient that selectively acts between microalgae species.

 The present inventors confirmed that the plant extract exhibits an excellent control effect against microalgae, the active ingredient in these plant extracts is a sesquiterpene-based compound, and the composition containing the same can be effectively used for selective control of microalgae. Completed. Accordingly, the present invention provides a composition for controlling microalgae containing a sesquiterpene-based compound or a plant extract containing at least one of these compounds as an active ingredient, and water or soil in which the microalgae inhabits the microalgal control composition. The present invention relates to a method for controlling microalgae, including treating the same. Hereinafter, the present invention will be described in more detail.

The present invention relates to a composition for controlling microalgae comprising a compound comprising at least one sesquiterpene-based compound of Formulas 1 to 5 below.

[Formula 1] Furanodiene (furanodiene)

[Chemical Formula 2] Caryophyllene

Caryophyllene oxide (caryophyllene oxide)

[4] alpha curcurene (alpha-curcumene)

[Formula 5] Beta-bisabolene (Beta-bisabolene)

The sesquiterpene compounds may be extracted from plants selected from the group consisting of plants of the genus Atractylodes , piper of the genus, plants of the genus Curcuma , and mixtures thereof. The art to raktil Roy des (Atractylodes) in plants, it is preferred to use a root of the generating (Atractylodes chinensis), sapju (Atractylodes japonica), baekchul (Atractylodes macrocephale). The rotifers (Piper) in plants hocho (Piper nigrum), pilbal (Piper longum), pepper etc. (Piper kadzura) the fruit or sentinel, the greater kyuma (Curcuma) in plants of the plant is Turmeric (turmeric) (Curcuma aromatica) It is recommended to use the roots of the plant, Curcuma zedoaria or Curcuma aeruginosa , Curcuma caesia , Curcuma angustifolia plants, more preferably Atractylodes chinensis roots, Curcuma aromatica roots, Piper nigrum berries and Selection from the group consisting of these mixtures is excellent in microalgae control effect.

The present invention also includes a plant extract prepared by extracting a plant selected from the group consisting of Atractylodes genus plants, Piper genus plants, Curcuma genus plants, and mixtures thereof with a solvent. It relates to a composition for controlling microalgae.

The plants used in the plant extracts are mentioned above. The plant may be dried and extracted, and the solvent may be selected from an organic solvent, and more specifically, selected from (C ₁ ~ C ₅ ) alcohol, hexane, ethyl acetate, acetone, methylene chloride, ethyl ether or a mixture thereof. Can be.

The plant extract in the present invention, creation ( Atractylodes chinensis ) Roots and shovels ( Atractylodes) japonica ) Roots, Eruption ( Atractylodes) macrocephale roots and piper nigrum ) Fruit, Piper longum fruit, Curcuma aromatica ) extracting a plant selected from the group consisting of roots and mixtures thereof (C ₁ ~ C ₅ ) alcohol to prepare an alcohol extract; And

Concentration of the hexane extract obtained by dissolving the alcohol extract in water, (C ₁ ~ C ₅ ) alcohol, or a mixture thereof and extracting with hexane;

It is characterized in that the hexane fraction prepared through.

In more detail, the extraction may use a conventional method, for example, by adding about 5 to 20 L of methanol to 1 kg of dried plant root or fruit and leaching at 20 to 60 ℃ for 6 hours to 20 days After the extract to obtain an extract, it can be concentrated under reduced pressure after filtration to obtain an extract. In another example, about 5 to 20 L of (C ₁ to C ₅ ) alcohol is added to 1 kg of dried plant roots or berries, and the organic solvent is leached at 20 to 60 ° C. for 6 hours to 20 days to obtain an alcohol extract. Extraction with one of the organic solvent extract can be obtained. And using the column chromatography to the organic solvent extract can be carried out a fraction process commonly used. The column that can be used for column chromatography includes a silica gel column, a reverse phase silica gel column or a Sephadex column, and the developing solvent is hexane, ethyl acetate, chloroform, methylene chloride, acetone, such as (C ₁ ~ C ₆ ) alcohol, etc. An organic solvent or a mixture thereof can be used. The plant extract according to the present invention, the sesquiterpene-based compound of Formula 1 to Formula 5 separated and purified therefrom exhibits a selective control effect on microalgae. In particular, yen Pew pyrano die of formula 1, 2, 4, 5, karioh propylene, alpha-curcumin, beta-Ibiza Vollenhoven is in (. Microcystis spp) Micro when seutiseu, Ana vena in (. Anabaena spp), come la thoria It shows very high control against harmful cyanobacteria such as genus Oscillatoria spp., And green algae such as Chlorella spp., Chlamydomonas spp and Senedesmus spp. It shows a relatively low control effect. However, the caryophyllene oxide of formula 3 shows a relatively higher control effect against the green algae than the southern algae.

 The plant extract fractions or isolates according to the present invention exhibit synergism in microalgae control depending on the combination. More specifically, the produced hexane fraction and the turmeric hexane fraction have a synergistic effect on microcystis control, and the generated methanol extract and the reef hexane fraction have a high synergism on microcistis control. In this case, the weight ratio of the resulting hexane fraction and the turmeric hexane fraction is 0.5 to 99.5: 99.5 to 0.5, and the weight ratio of the generated methanol extract and the reef hexane fraction is 0.5 to 99.5: 99.5 to 0.5, it is preferable to include, the mixing If one of the weight ratios is less than 0.5% or more than 99.5%, the synergy effect due to mixing may be insignificant, and therefore, mixing in the above range is preferable.

The control composition of the present invention is a pharmaceutically acceptable solid carrier and liquid carrier for stable expression of the effect, enhanced adhesion to the organism to be applied, and simplicity of transport and treatment within the range that does not inhibit the effect of the sesquiterpene-based compound. , Surfactants such as liquid diluents, liquefied gas diluents, solid diluents, or other suitable auxiliaries such as emulsifiers, dispersants or foaming agents. In addition, it is possible to use a mixture of other commonly used algae. The control composition of the present invention may preferably be formulated in emulsion, hydrated, granulated, powdered, capsule and gelled formulations, and is preferably provided as a contact through a formulation such as a donut for buoyancy of the formulation. .

The micro-algae in the present invention, more specifically, micro during seutiseu (Microcystis), Ana vena (Anabaena), come la thoria (Oscillatoria), Spirulina (Spirulina), Aphanizomenon Menon (Aphanizomenon), no dyulra Liao (Nodularia), nose keulrodi titanium (Cochlodinium), Stefano diseukeoseu (Stephanodiscus), chlorella (chlorella), Chlamydomonas (Chlamydomonas), means is selected from the three or four des mousse (Scenedesmus) and boats Rio nose kusu the group consisting of algae in the (Botryococcus).

The present invention is a composition for controlling the microalgae microcystis ( Microcystis ) , Anabaena ( Anabaena ) , Oscilltoria ( Oscillatoria ) , Spirulina ( Spirulina ), Aphanizomenon ( Aphanizomenon ) , Nodularia ( nodularia ), nose keulrodi titanium (Cochlodinium), Stefano diseukeoseu (Stephanodiscus), chlorella (chlorella), Chlamydomonas (Chlamydomonas), three or four des mousse (Scenedesmus) and boats Rio nose kusu (Botryococcus) the birds over any one genus selected from the genus birds format It provides a method of controlling microalgae, characterized in that the treatment in water or in the soil. In this case, the microalgae control composition may be treated in an amount of 0.01 μg / ml to 200 μg / ml in water or soil based on the active ingredient, more specifically, a sesquiter of Chemical Formulas 1 to 5 based on the final concentration of the treatment. It is preferable to treat the algae in water or soil so that the pen-based compound has a concentration of 0.5 µg / ml to 80 µg / ml, and the plant extract has a concentration of 3 µg / ml to 200 µg / ml. It can be appropriately adjusted in consideration of the density of growth, the degree of growth, the water environment.

According to the present invention, the composition can selectively control the microalgae to be controlled to show different reactivity in the control. In addition, since the activity is increased during the mixing process rather than the individual treatment according to the mixed combination, it is possible to reduce the side effects to the environment by adding a small amount, and the economic efficiency will also be good because the excellent algae activity is maintained. It is also possible to use a mixture of extract fractions can increase the utilization efficiency of the extract sample. Therefore, the mixture of the present invention can be usefully used as a harmful algae control agent which is eco-friendly and excellent in efficacy. In particular, the molecular weight is relatively low, it is easy to volatilize and can be well applied to the growth inhibition of floating harmful algae without remaining in the water environment for a long time. Meanwhile, when cultivating useful algae indoors or outdoors, it can be used to prevent damage caused by harmful algae contamination, and thus can be used to produce high quality microalgal biomass.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the present invention is not limited to the following examples.

Example 1 Control Effects of Plant Methanol Extracts on Cyanobacteria

(1) Preparation of Plant Methanol Extract

In order to investigate the control effect on the harmful algae of the plant extracts, methanol extracts of creation, weed and turmeric were prepared as follows.

① Creation ( Atractylodes chinensis )

600 g of commercially produced dry roots were crushed to 0.2 cm or less using a grinder, and then immersed in 6 L of methanol and left at room temperature for 7 days. After filtration and concentration under reduced pressure, a methanol extract was obtained. Refrigerated until.

② Hoar ( Piper nigrum )

1.5 kg of commercially available dried reed fruit was ground slightly using a grinder and then immersed in 2 L of methanol and left at room temperature for 7 days. After filtration and concentration under reduced pressure, a methanol extract was obtained. It was.

③ Curcuma aromatica )

To 600 g of commercially available turmeric root dried samples, 6 L of methanol was added and the extract obtained by leaching at room temperature for 7 days was filtered using a filter paper. The resulting methanol extract was concentrated completely under reduced pressure and then refrigerated until used in the experiment.

(2) Confirmation of control effect on seaweed

Growth inhibitory activity of the cyanobacteria of the plant methanol extract prepared in (1) was confirmed as follows. In the present example it was used rugi Labor (Microcystis aeruginosa UTEX 2388) when the blue-green algae micro seutiseu Oh received pre-sale at the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center as a test target birds.

First, microcistis aeruginosa subcultured was inoculated into the culture medium (Allen medium), and the initial concentration of algae at this time was about 0.08 at 670 nm using a microplate reader. Was made. 10 ml of the solution was injected into a 50 ml culture bottle, and 100 µl of the test solution and the control solution prepared as follows were administered to the strain culture. For test solution preparation, each methanol extract was dissolved in dimethylsulfoxide containing 100 μg / ml of Tween 20 ™ and then prepared at various concentrations using the same solvent. As a result, the final concentrations of dimethylsulfoxide and Tween 20 in the culture were about 1% and 2 μg / ml, respectively. On the other hand, a solution in which the solvent was added to distilled water under the same conditions was used as a control. The algae culture is that these test agent is administered after 25 ℃, 14 sigan photoperiod, light intensity 40-60 μmolm ^-2 s ^- and cultured under the condition of ^Fig. Six days after the test solution treatment, the absorbance at 670 nm was measured by using a microplate reader, and the following absorbance-preparation equation (1) (Kim et al., Aquatic Botany 85: 1-6, After investigating the growth of each algae through 2006), the algal control effect of the plant methanol extract was calculated as the percentage of dry weight inhibition (%) to the control group, and is shown in Table 1 below.

[Equation 1]

Y = -0.00837X ² + 0.2524X + 0.00383

In the above formula, Y represents absorbance and X represents dry weight.

As can be seen from the results in Table 1, all of the plant extracts showed high algal growth inhibitory activity at a treatment concentration of 50 μg / ml or less, and especially the extracts produced were 90% or more algae even at a low concentration of about 10 μg / ml. Growth inhibitory activity was shown.

Example 2 Isolation and Purification of Furanodiene Compound from Production

Atractylodes prepared in Example 1 chinensis ), methanol extract of the root was dissolved in distilled water, and then divided into two portions of equal amount of normal hexane, ethyl acetate, butanol and water in sequence and concentrated under reduced pressure. Inhibitory activity of microcistis aeruginosa, a harmful cyanobacteria, was investigated for each fraction in the same manner as in Example 1. As a result, as shown in Table 2, the normal hexane layer showed the highest algal growth inhibitory activity.

Eight normal hexane layers with high activity were prepared using normal hexane: ethyl acetate (13: 1, v / v) solvent with silica gel column chromatography (silica gel 60F, 70-230 mesh, 250 g, 4.2 × 60 cm). Fraction until. The fractions were named CH1 to CH8 according to the fractionation order. As a result of investigating the growth inhibitory activity of microcistis aeruginosa by the method of Example 1 for each fraction, as shown in Table 3 below, the CH-1 layer showed the highest algal growth inhibitory activity.

The fractions CH-1 were separated into two again using Sephadex LH-20 column chromatography using only methanol as a developing solvent, and were named CH-1-1 and CH-1-2. Growth inhibition activity of the fractionated CH-1-1 and CH-1-2 microcystis aeruginosa by the method of Example 1, 0.16, 0.32, 0.63, 1.25μg mL-1 treatment level In the case of CH-1-1, strong algae activity of 29.2, 56.0, 74.3 and 100%, respectively. The CH-1-1 was identified as a single substance through thin layer chromatography (TLC) analysis and analyzed by GC / MS. Furanodiene {(5Z, 9Z) -3,6 having a molecular weight of 216.32 , 10-trimethyl-4,7,8,11-tetrahydrocyclodeca [b] furan}. The algal activity of furanodiene was first identified in this study. On the other hand, in order to find a simpler extraction method, when the dry sample was directly extracted with normal hexane, ethyl acetate, acetone, and methanol, the extraction with normal hexane showed the highest inhibitory effect of about 85% even at 5μg mL-1. .

 Through the above experiments, furanodiene with strong algae activity has been isolated from the traditional medicines or diuresis, antipyretic, hypertension, dizziness, sweating, etc. It could be used as an algae control composition.

Example 3 Isolation and Purification of Cariophyllene Compound from Algae

Dried poncho prepared in Example 1 ( Piper nigrum ), methanol extract of the fruit was dissolved in distilled water, and then sequentially partitioned twice into equal amounts of normal hexane, ethyl acetate, butanol and water, and concentrated under reduced pressure. Inhibitory activity of microcistis aeruginosa, a harmful cyanobacteria, was investigated for each fraction in the same manner as in Example 1. As a result, as shown in Table 4, the normal hexane layer showed the highest algal growth inhibitory activity.

The normal hexane layer with high activity was converted to silica gel column chromatography (silica gel 60F, 70-230 mesh, 350 g, 7.0 × 55 cm) using normal hexane: ethyl acetate (3: 1, v / v) solvent. Again, fractions of up to four 100 ml. Four fractions were named PH-1 to PH-4 according to the fraction order of the fractions. In addition, the growth inhibition activity of microcistis aeruginosa was investigated for each fraction in the same manner as in Example 1. As shown in Table 5 below, the PH-2 layer showed the highest algal growth inhibitory activity.

The fraction PH-2 was separated by silica gel column chromatography (silica gel 60F, 70-230 mesh, 200 g, 3.0 × 60 cm) using a normal hexane: ethyl acetate (3: 1, v / v) developing solvent. Fractions were obtained and named PH2-1, PH-2-2, and PH2-3. The algal activity test showed that PH-2-1 and PH-2-2 showed excellent activity. PH-2-1 was again separated by silica gel column chromatography (silica gel 60F, 70-230 mesh, 200 g, 3.0 × 60 cm) using a normal hexane: ethyl acetate (4: 1, v / v) developing solvent. Two fractions (PH-2-1-A and PH-2-1-B) were obtained. At this time, PH-2-1-B was identified as a single substance by TLC analysis, and caryophyllene-oxide having a molecular weight of 220.35 through GC / MS analysis {(1R, 4R, 6R, 10S) -9-methylene-4 , 12,12-trimethyl-5-oxatricyclo [8.2.0.0] 4,6)] dodecane}.

On the other hand, PH-2-1-A was again subjected to silica gel column chromatography (silica gel 60F, 70-230 mesh, 200 g, 3.0 × 60 cm) using a normal hexane: ethyl acetate (95: 5, v / v) developing solvent. ) To obtain PH-2-1-A-1, and again through preparative TLC (preparative TLC (normal hexane: ethyl acetate = 99: 1, v / v)) pure material (PH-2-1-A -1-1), the material was analyzed by GC / MS analysis of trans-caryophyllene having a molecular weight of 204.35 trans-caryophyllene {trans- (1R, 9S) -8-methylene-4,11,11-trimethylbicyclo [7.2 .0] undec-4-ene}.

 The growth inhibition activity of microcistis aeruginosa was examined by the method of Example 1 with the two trans-caryophyllene and cariophyllene oxides identified above, and the concentrations of 0.63, 1.25, 2.5, 5, and 10 μg mL-1 were determined. PH-2-1-B (cariophyllene oxide) was 0, 0, 1.2, 40.9, 78.7%, and PH-2-1-A-1-1 (trans-cariophilene) was 21.5, respectively. The inhibitory activity of 81.9, 89.1, 96.5, and 97.1% showed relatively strong activity of trans-caryophyllene.

On the other hand, in order to find a simpler extraction method, 90% or more of the ethanol or methylene chloride extract was extracted at 5μg mL ^{- 1} when the reed fruit was directly extracted with normal hexane, ethyl acetate, methylene chloride, acetone, and ethanol. Eruginosa showed growth inhibition effect showed high activity.

 Through the above experiments, the algae active substance cariophyllene was isolated from the vinegar, which has been traditionally used as an herbal medicine such as abdominal pain, indigestion, vomiting, diarrhea, or a spicy or fragrant flavor. It has been shown that can be used as a bird control composition.

Example 4 Isolation and Purification of Alpha Curcumin and Beta Bisabolene Compounds from Turmeric

Dried Curcuma ( Curcuma) Prepared in Example 1 aromatica ) Methanol extract of the roots were dissolved in distilled water, and then concentrated in vacuo by dividing two times into the same amount of normal hexane, ethyl acetate, butanol and water layers. Inhibitory activity of microcistis aeruginosa, a harmful cyanobacteria, was investigated for each fraction in the same manner as in Example 1. As a result, as shown in Table 6, the normal hexane layer showed the highest algal growth inhibitory activity.

The relatively high activity of normal hexane layer was prepared using normal hexane: ethyl acetate (95: 5, v / v) developing solvent and silica gel column chromatography (silica gel 60F, 70-230 mesh, 220 g, 3.0 × 60 cm). Up to 7 again. Sequentially divided fractions were named CAH-1 to CAH-7. Growth inhibition activity of microcistis aeruginosa for each fraction was investigated by the method of Example 1, and as shown in Table 7, CAH-1 layer showed the highest algal growth inhibitory activity.

The fraction CAH-1 was separated into silica gel column chromatography (silica gel 60F, 70-230 mesh, 220 g, 3.0 × 60 cm) using only a developing solvent of normal hexane to obtain two fractions, which were CAH-1-. Named A and CAH-1-B. TLC analysis of the two fractions confirmed that the CAH-1-B is a single substance, and analyzed by GC / MS, the molecular weight is 202.34 alpha-curcumene (alpha-curcumene) [1-methyl-4-((S ) -6-methylhept-5-en-2-yl) -benzene

] Was identified. On the other hand, CAH-1-A was identified by the TLC analysis of five substances and subjected to preparative TLC (preparative TLC (developing only with normal hexane)) to obtain three fractions, CAH-1-A-1 to CAH- Named 1-A-3, of which CAH-1-A-3 was excellent in activity. The CAH-1-A-3 was subjected to preparative thin layer chromatography (TLC) again, and the preliminary TLC was developed twice only with normal hexane. As a result, two fractions of CAH-1-A-3-1 and CAH-1-A-3-2 were obtained, and the two fractions were treated at 1.25, 2.5, and 5 μg mL ^-1 concentrations. As a result, CAH-1-A-3-1 was 62.9, 89.9, and 100%, respectively, as a result of the growth inhibition activity assay of Microcistis aeruginosa. CAH-1-A-3-2 was found to be 10.1, 49.9 and 99.2%, respectively, and showed relatively good activity in CAH-1-A-3-1. This CAH-1-A-3-1 was identified as a single substance by TLC analysis and analyzed by GC / MS. As a result, beta bisabolene [(S) -1-methyl- having a molecular weight of 204 was obtained. 4- (5-methyl-1-methylene-4-hexenyl) cyclohexene

] Was identified. Through the above experiments, we have isolated the algae active substance cariophyllene from turmeric, which has been used as herbal medicine or medicine for traditional medicine such as jaundice, biliary stones, hepatitis, hyperlipidemia, hypertension, arteriosclerosis, gastric ulcer, and anticancer effect. Or it was shown that the extract containing it can be used as a microalgae control composition.

Example 5 Interaction with Freshwater Algae Inhibitory Activity by Mixture Treatment of Plant Extract Fractions

Methanol extract was obtained in the same manner as in Example 1 on the resulting roots, the reed fruit and the turmeric root. Meanwhile, in order to obtain a normal hexane extract, the methanol extract concentrated under reduced pressure was dissolved in distilled water, extracted twice with the same amount of hexane, and the hexane fraction layer was concentrated under reduced pressure. The methanol extract and hexane fraction thus obtained were used to investigate the algal activity interactions according to the mixing treatment.

Methanol extract or normal hexane fractions prepared above were prepared at the final concentrations of 0.0-5 ㎍ / ml and 0.0-5 ㎍ / ml, respectively, or in combination of various mixtures. It confirmed by the same method as Example 1. The mixed mixtures tested were produced methanol extract + reef hexane fraction, produced methanol extract + turmeric hexane fraction, generated hexane fraction + turmeric hexane fraction, generated hexane fraction + reef hexane fraction, reef hexane fraction + turmeric hexane fraction. The interaction between the two mixtures was evaluated by Colby S.R .. Weeds 15: 20-22, 1967. If the actual control value (observed value) shown by the admixture treatment is greater than the expected value (expected by Equation 2, expected value), then synergism, equality, or antagonism To indicate. Equation 2 is represented as in the following equation, and the results of the experiment are shown in FIGS. 1 and 2.

[Equation 2]

E = (X + Y)-XY / 100

Where X is% inhibition at p concentration of Compound A and Y is% inhibition at q concentration of Compound B.

The experimental results are shown in FIGS. 1 and 2 below, and when the effect of mixing treatment with hexane fractions was investigated, the resulting hexane fraction + turmeric hexane fraction had synergistic effect, whereas the resulting hexane fraction + reef hexane fraction, reef Hexane fraction + turmeric hexane fraction showed a slight synergistic or additive action. On the other hand, when mixed with the produced methanol extract, there was a high synergy in mixing with the hexane fraction, and showed only an additive effect with the turmeric hexane fraction.

As a result of the experiment, the combination showing excellent synergistic effect on the microcyst control among the combined concentrations of the resulting hexane fraction + turmeric hexane fraction was treated with 1.3 to 2.5 µg / ml of the produced hexane fraction + 1.3 to 2.5 µg / ml of the turmeric hexane fraction. It was. In addition, the combination showing excellent synergistic effect on microcistic control among the combined concentrations of the produced methanol extract + reef hexane fraction was treated with 1.3 μg / ml of the produced methanol extract + 1.3 μg / ml of the reef hexane fraction, and 0.6 to 2.5 μg of the produced methanol extract. / Ml + hexane fraction 2.5 μg / ml.

[Test Example] Selective Growth Inhibitory Activity Test of Microalgae of Sesquiterpene Compounds and Extract Fractions Containing the Same

Hexane fraction of Methanol extract of Produce Root, Rhododendron, Turmeric Root, extracted as in Example 5, and Caranophylene oxide separated from Furanodiene, Rhodiola, isolated from Produce as in Examples 1-4. Inhibition of growth inhibition of several freshwater algae with alpha curcumin and beta bisabolene isolated from caryophyllene and turmeric root was as follows.

First, the hexane fraction and the separated compound are dissolved in dimethyl sulfoxide containing 200 μg / ml of Tween 20, respectively, and then diluted in Allen's medium as in Example 1 Test solutions were prepared to reach the final concentration of the eggplant. At this time, the final concentrations of dimethylsulfoxide and tween 20 were about 1% and 2 μg / ml, respectively. As a control, a solution in which the solvent was added to distilled water under the same conditions was used. Freshwater algae in this study were classified as three harmful algae [ Microcystis] from the Korea Institute of Bioscience and Biotechnology. aeruginosa , UTEX 2388), Anabaena affinis , Oscillatoria tenuis UTEX1566)] and four kinds of green alga [Chlorella vulgaris (Chlorella vulgaris), three or four in mousse des (Scenedesmus spp.), in Chlamydomonas (Chlamydomonas spp.), boat Rio nose kusu brownie (Botryococcus braunii UTEX 572). The initial concentration of algae was such that the final concentration in the culture medium was absorbed at around 0.06 to 0.08 at 670 nm using a microplate reader. 10 ml of algal culture with test agent was dispensed into 100 ml culture bottles, which were 5 days for green algae and 6 for cyanobacteria at 25 ° C, 14 hours photoperiod, and 40-60 μmolm ^-2 s ^-1 of brightness. One day was incubated. After the incubation was terminated by measuring the absorbance of the culture or by analyzing the chlorophyll content.

In the case of absorbance measurement, the absorbance at 670 nm was measured using a microplate reader, and a previously set absorbance-building correlation was expressed by the following equations (1) and (3) to (5). Light, Aquatic Botany 85: 1-6, 2006) And after examining the degree of growth of each bird, the control effect on freshwater algae of the test solution was calculated as the percentage of growth inhibition on the control group.

Equation 1 is a correlation of absorbance-building in the microcistis aeruginosa, and Equation 3 is an absorbance-building correlation of Chlorella vulgaris or Climidomonas, and Equation 4 is an absorbance in Senedmus. It is a correlation between buildings, and Equation 5 below is the absorbance-analysis of buildings of Anavena affinis.

[Equation 1]

Y = -0.00837X ² + 0.2524X + 0.00383

Where Y is absorbance and X is dry weight

&Quot; (3) "

Y = 0.4304X + 0.0384

&Quot; (4) "

Y = 0.4212X + 0.0649

[Equation 5]

Y = 0.1734X + 0.0306

In Equations 1 and 3 to 5, Y represents absorbance and X represents building weight.

In addition, instead of the absorbance of the cultures, the oscillatoria tenis and Boturiocus brownies extracted chlorophyll from the cultures and analyzed their growth rates based on their contents. In other words, the oscillatoria tenuis filter the culture medium on the filter paper (GF / F filter, pore size Ф 0.45um, 47mm in diameter) and then put it in ethanol solvent to extract chlorophyll and absorbance at the wavelength of 648.5, 664.6, 750nm spectrophotometer Measure After that, the chlorophyll a content was determined by quantitative formula (Nusch EA, Arch . Hydrobiol . Beih . (Ergebn.Limnol.), 14 : 14-36, 1980). The growth inhibition of the corresponding microalgae was shown.

First, the inhibition of growth of microcistis aeruginosa and chlorella vulgaris with respect to the hexane fraction of the production root, the reed fruit, turmeric root, furanodiene, alpha curcumin, and beta bisabolene prepared in Example 5 above The experiment was conducted in the same manner, and the results are shown in Table 8 below.

When treated with hexane fractions, microcistis aeruginosa was well controlled at relatively low concentrations and chlorella vulgaris inhibited growth at higher concentrations. This property also showed the same trend when tested with furanodiene isolated from the production and alpha-curcumin and beta-bisabolene isolated from turmeric.

On the other hand, trans-caryophyllene and cariophyllene oxide isolated from the vinegar was tested for growth inhibition activity by the method described above with three species of algae and four species of green algae, as shown in Table 9 below. There was a clear difference between cyanobacteria and algae.

That is, according to the following Table 9, microcistis, anavena, oscillatoria, etc. belonging to the harmful cyanobacteria are easily inhibited by trans-cariophyllene, resulting in a growth inhibitory effect of 90% or more in the treatment of 5 µg / ml or more. However, the reaction was relatively insensitive to cariophyllene oxide. On the other hand, chlorella and chlamidomonas belonging to the algae showed a more sensitive reaction to cariophyllene oxide than to trans-cariophyllene. On the other hand, Boturiococcus, a kind of green algae, was slightly more sensitive to cariophyllene oxide than trans-cariophyllene, although the difference in reactivity between the two drugs was not greater than that of chlorella and chlamidomonas.

Table 1

[Table 2]

[Table 3]

Table 4

[Table 5]

Table 6

Table 7

Table 8

Table 9

1 is a graph showing that the mixture of the resulting root hexane fractionation layer and the turmeric root hexane fractionation layer has a synergistic effect on microcystis aeruginosa control.

2 is a graph showing that the mixture of the resulting root methanol extract and the lychee fruit hexane fractionation layer has a synergistic effect on microcystis aeruginosa control.

Claims (11)

A composition for controlling microalgae comprising one or two or more sesquiterpene compounds of Formulas 1 to 5 below. [Formula 1]

(2)

[Formula 3]

[Formula 4]

[Formula 5]

The method of claim 1, The sesquiterpene compound is a microalgae control composition, characterized in that derived from the extract of Atractylodes chinensis root, Curcuma aromatica root or Piper nigrum fruit. For the control of microalgae containing plant extract prepared by extracting a plant selected from the group consisting of plants of the genus Aactylodes, plants of the genus Piper, plants of the genus Curcuma, and mixtures thereof with a solvent Composition. The method of claim 3, The plant extracts are created by Atractylodes chinensis ) Root, Atractylodes japonica Root, Atractylodes macrocephale roots and piper nigrum ) Fruit, Piper longum fruit, Curcuma aromatica ) Microalgae control composition, characterized in that the extract or a concentrate prepared by extracting a plant selected from the group consisting of roots and mixtures thereof with a solvent. The method of claim 3, The solvent is a composition for controlling microalgae, characterized in that selected from (C ₁ ~ C ₅ ) alcohol, hexane, ethyl acetate, acetone, methylene chloride, ethyl ether or a mixture thereof. The method of claim 3, The plant extracts are created by Atractylodes chinensis ) Roots, Intract japonica ) Roots, Eruption ( Atractylodes) macrocephale roots and piper nigrum ) Fruit, Piper longum fruit, Curcuma aromatica ) extracting a plant selected from the group consisting of roots and mixtures thereof (C ₁ ~ C ₅ ) alcohol to prepare an alcohol extract; And Concentration of the hexane extract obtained by dissolving the alcohol extract in water, (C ₁ ~ C ₅ ) alcohol, or a mixture thereof and extracting with hexane; Composition for controlling microalgae, characterized in that the hexane fraction prepared through. The method of claim 6, The composition for controlling microalgae is a composition for controlling microalgae, characterized in that the weight ratio of the resulting hexane fraction and the turmeric hexane fraction is 0.5 to 99.5: 99.5 to 0.5. The method of claim 6, The composition for controlling microalgae is a composition for controlling microalgae, characterized in that the weight ratio of the resulting methanol extract and the reef hexane fraction is 0.5 to 99.5: 99.5 to 0.5. The method of claim 3, Wherein the microalgae is a micro when seutiseu (Microcystis), Ana vena (Anabaena), come la thoria (Oscillatoria), Spirulina (Spirulina), Aphanizomenon Menon (Aphanizomenon), no dyulra Liao (Nodularia), nose keulrodi titanium (Cochlodinium), Stephanodiscus , Chlorella ( Chlorella ), Chlamydomonas ( Chlamydomonas ), Scenedesmus and Scenerymus ( Botryococcus ) A composition for controlling microalgae, characterized in that selected from the group consisting of algae. Claim when the composition for any one of the micro-algae control is selected from 1 to 9, wherein the micro seutiseu (Microcystis), Ana vena (Anabaena), come la thoria (Oscillatoria), Spirulina (Spirulina), Aphanizomenon Menon (Aphanizomenon), no dyulra Liao (Nodularia), nose keulrodi titanium (Cochlodinium), Stefano diseukeoseu (Stephanodiscus), chlorella (chlorella), Chlamydomonas (Chlamydomonas), three or four des mousse (Scenedesmus) and boats selected from algae in the scenarios Imperial's (Botryococcus) Control method of microalgae, characterized in that the treatment in the water or soil in which one or more algae inhabit. The method of claim 10, The method of controlling microalgae, characterized in that the microalgae control composition is treated to 0.01 to 200 ㎍ / ㎖ in water or soil based on the active ingredient.

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