KR100688907B1 - Composition comprising an extract of turmeric or curcuminoids for controlling rice blast - Google Patents

Abstract

The present invention relates to a composition for controlling plant anthrax and rice blast disease, which contains a curcuminoids compound or turmeric extract comprising the same as an active ingredient, wherein the composition for controlling the present invention is harmless to humans and is environmentally friendly. As an environmentally friendly natural product disinfectant that does not cause pollution, it can be effectively used to produce high value-added organic products by effectively controlling plant anthrax and rice blast.

Description

COMPOSITION COMPRISING AN EXTRACT OF TURMERIC OR CURCUMINOIDS FOR CONTROLLING RICE BLAST Using Turmeric Extract or Curcuminoid Compound as an Active Ingredient

1 shows an electron impact mass spectrum of demethoxycurcumin,

Figure 2 shows the electron collision mass spectrum of curcumin,

3 shows the electron collision mass spectrum of bisdemethoxycurcumin,

Figure 4 shows the control activity of demethoxy curcumin pepper anthrax.

The present invention relates to a composition for controlling plant anthrax and rice blasts, which contains a curcuminoids compound or turmeric extract containing the same as an active ingredient.

Colletotrichum is a representative multi-pathogen that causes anthrax in economically important crops such as cucumbers, watermelons, peppers, and strawberries (Bailey et al., Biology, Pathology and Control 88-120, 1992; Elmer et al., Plant Dis ., 85: 216, 2001), anthrax is a big problem because its control occupies about 20 billion won in the domestic vegetable disease control market. Rice blasts, on the other hand, are one of the major pests of rice caused by rice blasts, such as Magnaporthe grisea (incomplete generation: Pyricularia oryzae ), and occur in almost all regions where rice is grown, especially rice. In Korea, Japan, and Southeast Asia, the occurrence and damage of this disease is a major factor in the decrease in yield.

Currently, the most effective control method for the plant diseases is the use of chemical disinfectant spraying, but this method has problems such as pollution of the natural ecosystem due to the chemical pesticide residues and causing induced toxicity. Resistant varieties, seedlings, biological control and biopesticides such as natural product fungicides or microbial fungicides are being developed as a means of controlling these problems and harmonizing with the environment. Since the skeleton can be used to prepare new synthetic fungicides, studies on this have been actively conducted (Baker et al., Phytopathology 73: 1148, 1983; Katz and Demain, Bacteriol. Rev. , 41: 449, 1977; Kim; Pest Manag. Sci ., 60: 803, 2004; Lange et al. , Pestic. Sci ., 39: 155, 1993; Porter and Fox, Pestic. Sci ., 39, 161, 1993; Powel and Jutsum, Pestic. Sci. ., 37: 315, 1993). However, there is almost no development of biopesticides that can effectively control anthrax or rice blasts, both domestically and globally.

On the other hand, turmeric is a perennial herb belonging to the Zingiberaceae family and is native to tropical Asia, and there are 140 kinds of ginger plants in the world, and about 50 kinds of plants of the genus Curcuma , such as turmeric, have. Among them, spring turf, autumn turf, and purple turf are known to have excellent medicinal effects. Spring turmeric is herbaceous, with turmeric, pink flowers in spring, and scientific name is Curcuma aromatica . Autumn turmeric is herbaceous grass or turmeric with white flowers in fall, scientific name is Curcuma longa . In addition, purple turmeric, also called acupuncture or bonsai , blooms in spring and its scientific name is Curcuma zedoaria . Turmeric is the main ingredient of curry and has a unique taste and aroma. In ancient times, turmeric has been shown to promote the circulation of qi, loosen the lumps, loosen the blood, and eliminate the blood, so that the bloating of the abdominal muscles, hemorrhage, nasal bleeding, hematuria, hemolim, and women It has been used to treat menstruation and jaundice. Recently, the clinical effects of turmeric on the prevention and treatment of liver cirrhosis, colon cancer, gastrointestinal diseases and dementia as well as hepatitis B, anticancer effects, mental illness, esophageal cancer, and biliary secretion have been reported. In addition, turmeric extract and curcumin isolated from turmeric have been reported to exhibit control activity against rice leaf blight, tomato blight and wheat rust (Kim et al ., J. Agric. Food Chem . 51: 1578, 2003).

Accordingly, the present inventors have conducted research to develop an environmentally friendly natural product disinfectant that is harmless to humans, and found that curcuminoid compounds or turmeric extracts containing the same exhibit very high control against anthrax and rice blasts of plants. The present invention was completed by confirming that it can be used as a natural fungicide for controlling anthrax and rice blasting plants.

It is an object of the present invention to provide an environmentally friendly control composition derived from natural products, which is harmless to the human body and exhibits excellent control against plant anthrax and rice blasts without causing environmental pollution.

In addition, an object of the present invention is to provide a method for controlling plant anthrax and rice blast using the composition for the control.

In order to achieve the above object, the present invention provides a composition for controlling plant anthrax and rice blast bottle containing curcuminoid compound of Formula 1 or turmeric extract comprising the same:

Wherein R ¹ and R ² are each independently H or OCH ₃ .

In addition, the present invention provides a method for controlling plant anthrax and rice blast by treating the control composition to crops or soil.

Hereinafter, the present invention will be described in detail.

The present invention is derived from natural products, harmless to the human body and does not cause environmental pollution, and has a curcuminoid compound of the formula (1 ) or a turmeric extract containing the same as the active ingredient showing a high control against plant anthrax and rice blast Provided are compositions for controlling plant anthrax and rice blast.

Preferred curcuminoid compounds exhibiting control activity against plant anthrax and rice blast in the present invention can be used demethoxy curcumin of formula (2) , curcumin of formula (3) and bisdemethoxycurcumin of formula (4 ):

In the present invention, turmeric is preferable to use spring turmeric ( Curcuma aromatica ; herbal name: turmeric), autumn turmeric ( Curcuma longa ; native name: turmeric, turmeric) and purple turmeric ( Curcuma zedoaria ; herbal : bonsai, bonsul ) Do.

Turmeric extract according to the present invention can be prepared by extracting the root of turmeric or its dried matter with an organic solvent. The organic solvents usable in the present invention are methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, ethyl acetate, acetone and the like, and preferred are ethanol and 50-90% ethanol aqueous solution.

In a preferred embodiment of the present invention, about 5 to 20 L, preferably 8 to 12 L of lower alcohol is added to 1 kg of dried turmeric root and leached at 20 to 60 ° C, preferably at room temperature for 6 hours to 3 days. Extract. The extract obtained therefrom is filtered to remove impurities and then concentrated under reduced pressure to obtain an organic solvent extract. In the present invention, it may further be carried out a fractionation process commonly used in the art.

Curcuminoid compound represented by the formula (1 ), preferably demethoxy curcumin, curcumin or bisdemethoxy curcumin can be separated, purified or chemically synthesized from turmeric extract.

Specifically, demethoxycurcumin, curcumin and bisdemethoxycurcumin can be prepared by a method comprising separating and purifying the organic solvent extract of turmeric root prepared as described above by column chromatography. In this case, the column may be a silica gel column, a reverse phase silica gel column or a Sephadex column, and the eluent may be an organic solvent such as hexane, ethyl acetate, chloroform, methylene chloride, lower alcohol, and a mixture thereof.

In a preferred embodiment of the present invention, the dried turmeric root is extracted with ethanol and concentrated under reduced pressure, and the ethanol extract obtained by dissolving with distilled water, and then extracted with an organic solvent such as ethyl acetate and concentrated under reduced pressure, and then concentrated Purification of demethoxycurcumin, curcumin and bisdemethoxycurcumin is purified by purification with one silica gel column chromatography and one Sephadex LH-20 column chromatography.

Methanol extract of turmeric according to the present invention, the control activity of the demethoxycurcumin, curcumin and bisdemethoxycurcumin isolated and purified from red pepper anthracnose, rice blast, rice leaf blight, tomato ash, tomato blight, wheat red 7 plant diseases of rust and barley powdery mildew showed that methanol extracts of turmeric against pepper anthrax, rice blast and wheat red rust, and demethoxy curcumin against pepper anthrax, rice blast and tomato blight, curcumin and bis Demethoxycurcumin showed high control activity against rice blast (see Table 1 , Table 3 and Figure 4 ).

Demethoxy curcumin, curcumin, bisdemethoxy curcumin or turmeric extract containing the same of the present invention exhibiting the above characteristics is harmless to the human body and does not cause environmental pollution because it exhibits high control against plant anthrax and rice blast It can be usefully used as a natural fungicide of plant anthrax and rice blast.

Curcuminoid compounds in the compositions of the invention, preferably demethoxycurcumin, curcumin or bisdemethoxycurcumin, may be in the form of acceptable salts for agricultural compositions.

The control composition of the present invention may further include a substance contained in a conventionally used insecticide or fungicide, and as an active ingredient, pharmaceutically acceptable as an excipient in addition to the turmeric extract, demethoxycurcumin, curcumin or bisdemethoxycurcumin It may further comprise surfactants such as solid carriers, liquid carriers, liquid diluents, liquefied gas diluents, solid diluents, or other suitable auxiliaries such as emulsifiers, dispersants or foaming agents. The control composition of the present invention may be used by mixing the active ingredient and the excipients to be formulated into an agricultural composition, and the method of formulating into an agricultural composition may be applied to all methods commonly used in the art.

The control composition of the present invention is preferably a formulation of a hydrating agent, granule, powder, emulsion, spray, smokescreen, capsule and gel, and is preferably provided as a contact through a formulation such as donut for buoyancy of the composition. Do.

In order to effectively control plant anthrax and / or rice blast, the control composition of the present invention has a concentration of 333.3 μg / ml to 3000 μg / ml of turmeric extract, and 250 μg / of demethoxycurcumin, curcumin and bisdemethoxycurcumin, respectively. It is preferable to include in a concentration of ㎖ to 1000 ㎍ / ㎖, this can be appropriately adjusted in consideration of the type of crop, growth degree, cropland environment, the degree of development of plant diseases.

The control composition of the present invention formulated as described above can control plant anthrax or rice blast by treating to plants or areas requiring control of plant anthrax and / or rice blast. The preferred method is to apply the control composition so as to be in direct contact with the plant pathogens to control, the most preferred is a method of mixing the soil or spraying directly to the crop.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Control Activity against Plant Disease of Turmeric Extract

To the roots of commercially available Curcuma longa , 1 L of methanol or 1 L of distilled water was added per 100 g of dry sample, and the extract obtained by leaching at room temperature for 24 hours was filtered using a filter paper. The methanol extract thus obtained was concentrated under reduced pressure and refrigerated until use in experiments. The water extract was filtered and immediately refrigerated.

In order to investigate the control activities of plant extracts of methanol and water extracts of turmeric, the most common plant diseases are pepper anthrax, rice blast, rice leaf blight, tomato ash fungus, tomato blight, wheat rust and wheat powdery mildew. Activity experiments were performed as follows. First, the methanol extract was dissolved in methanol and diluted with 250 μg / ml Tween 20 solution to prepare final concentrations of 3000 μg / ml, 1000 μg / ml and 333.3 μg / ml. At this time, the final concentration of methanol was 5% level. The water extract was diluted with 10 ml of 250 μg / ml Tween 20 solution, taking the amounts corresponding to 1 g of dry sample and 0.1 g, respectively. At this time, a solution in which 0.5 ml of methanol (final concentration 5%) and 2.5 mg of Tween 20 (final concentration 250 µg / ml) was added to 10 ml of distilled water was used as a negative control. Methanol extracts, water extracts and control samples thus prepared were sprayed onto the foliar of plant seedlings 1 day before inoculation of the pathogen, and then dried at room temperature for 24 hours.

Rice, tomatoes, barley and wheat used in the experiments were filled with water or soil for horticulture in 70 cm diameter plastic pots, and then seeded, and seeded and grown in a greenhouse at 25 ± 5 ° C. for 1 to 4 weeks. Rice blast is sprayed with spore suspension (5 × 10 ⁵ spores / ml) of Magnaporthe grisea ( Korea Research Institute of Chemical Technology) , which is a causative agent of blast disease, in seedlings of 3 to 4 leaves. After the wet treatment, the incubation was induced by incubation for 5 days in a constant temperature room of 25 ℃. Rice leaf blight is a cultured product obtained by incubating for 7 days in a medium (90 g of wheat bran, 15 g of chaff, 100 ml of distilled water) of Thanatephorus cucumeris, a causative agent of leaf blight, The seedlings were inoculated to 5-leaf seedlings and wet-treated for 4 days on 25 ° C., and then incubated in a constant temperature room at 25 ° C. for 4 days to induce the onset. The tomato late blight is caused by Phytophthora infestans ( Gangneung University) After inoculation of the drift suspension from the drift sac (10 ⁵ scavenger sac / ml), it was wet-treated at 25 ° C. for 2 days and incubated at 25 ° C. for 2 days to induce the onset. Tomato gray mold disease was treated with spore suspension (10 ⁶ spores / ml) of Botrytis cinerea ( Korea Research Institute of Chemical Technology) , which is a causative agent of gray mold disease, in tomato seedlings of 3-4 leaves. In fact, it was incubated for 3 days to induce onset. Wheat red rust sprays spores of Puccinia recondita ( Inchon National University) , the causative agent of rust, known as biolactic bacteria, in a single-leaf seedling, suspended in 250 µg / ml Tween 20 solution with 0.67 g spores / ℓ. Treatment was carried out for one day in a 20 ℃ humid room and then transferred to a 20 ℃ constant temperature room and incubated for 6 days to induce the onset. Barley powdery mildew is inoculated with spores of Erysiphe graminis f. Sp. Hordei ( Korea Research Institute of Chemical Technology) , which is the causative agent of powdery mildew, passaged from host plants, in barley seedlings. Incubation was induced by incubation for 7 days in a constant temperature room. The area ratios of rice blast, wheat rust, and wheat flour were 7 days after inoculation, 8 days after inoculation for rice leaf blight, and 3 and 4 days after inoculation for tomato ash and tomato late blight, respectively.

On the other hand, for the control activity test for pepper anthrax, filled with about 70% of horticultural soil in a plastic pot of diameter 7.0 cm, sowing the seeds of the most pepper, grow them up to 5-6 leaf period in the greenhouse and then prepared turmeric Methanol extract and water extract samples were foliar sprayed. At this time, dithianon (Sungbo Chemical), which is used as a fungicide for pepper anthrax, was used as a positive control. After spraying the sample was dried at room temperature for 24 hours, and then spray inoculated with a spore suspension (1 × 10 ⁶ spores / ml) of the pepper anthrax pathogen Colletotrichum coccodes (Chungbuk National University). After 2 days onset in a wet room and left in a constant temperature room at 25 ℃ 2 days, the disease area ratio was investigated 4 days after inoculation.

The control value was calculated according to the following Equation 1 using the lesion area ratio obtained therefrom, and the control activities of methanol extract and water extract of turmeric against the seven plant diseases are shown in Table 1 below.

As shown in Table 1 , methanol extract of turmeric showed very high control against pepper anthrax, rice blast and wheat red rust. However, the water extract showed only weak control activity against red pepper anthrax.

When 100 g of autumn turmeric dry sample was extracted with methanol, the weight of the extract was 7.92 g. Compared with the concentration of the water extract of turmeric, the water extract corresponds to 79.2 mg of methanol extract in 1 g dry sample. Therefore, the control activity for pepper anthrax is much better than the water extract of methanol extract, it can be seen that the material exhibiting control activity for pepper anthrax is not water-soluble but fat-soluble.

Example 2 Determination of Extraction Solvent Conditions for Separation of Active Ingredients from Turmeric

To determine the optimal extraction solvent conditions for the separation of active ingredients from turmeric, 10 g of dry urine sample was placed in a beaker, followed by hexane, ethyl acetate, methanol, 50% methanol, 50% ethanol and 50% ethanol solution. Each of the six solvents was added 100 ml each and leached at room temperature for 12 hours. Each solvent extract obtained therefrom was filtered and concentrated under reduced pressure. 0.5 g and 0.25 g of each solvent extract sample was dissolved in acetone or methanol during drying. The hexane and ethyl acetate extracts were dissolved in 1 ml of acetone, methanol, 50% aqueous methanol solution, ethanol and 50% aqueous ethanol extract. It was dissolved in 0.5 ml of methanol. The concentration of each sample was adjusted to 10 ml with 250 μg / ml Tween 20 solution, and the concentration was diluted to 0.05 g dry sample / ml and 0.025 g dry sample / ml. It investigated in the same manner as in Example 1.

As a result, as shown in Table 2 , the 50% ethanol aqueous solution extract and the ethanol extract showed the best control activity against pepper anthrax, and using 50 to 100% ethanol as the extraction solvent was most effective in extracting the active ingredient from turmeric. It was confirmed to be efficient. In addition to 50 to 100% ethanol, ethyl acetate and 50 to 100% methanol may also be used as useful extractants.

Example 3 Isolation and Purification of the Controlled Active Ingredient

8 L of ethanol was added to 800 g of the dried sample of autumn urine, and the ethanol extract obtained by leaching at room temperature for 1 day was filtered using filter paper, and the filtrate was concentrated under reduced pressure. The ethanol extract of turmeric was dissolved in 3 L of distilled water, and then extracted three times with 3 L of hexane, ethylacetate, and butanol. As a result of investigating the control activity against the pepper anthracnose of each of these extracts, it was confirmed that the ethyl acetate extract showed the best control activity.

Thus, in order to separate the active ingredient from the ethyl acetate extract (33.37 g), silica gel column chromatography (6.6 ×) was used as the developing solvent in the hexane: ethyl acetate mixed solution (33.3% → 100% ethyl acetate concentration gradient). 60 cm, Kaigel gel 60, 70-230 mesh, 500 g: Merck). The eluate obtained therefrom was separated and analyzed by thin layer chromatography (Kaigel gel 60 F254, 0.25 mm; Merck) using hexane: ethyl acetate mixed solution (1: 1, v / v) as a developing solvent. Seven fractions of F1 to F7 according to the pattern of TLC (Rf value range: F1 = 0.47 to 0.74, F2 = 0.37 to 0.59, F3 = 0.37 to 0.47, F4 = 0.31 to 0.47, F5 = 0.2 to 0.29, F5 = 0.1) -0.19, F6 = 0.05-0.1, F7 = 0.01-0.05), each fraction was dissolved in methanol, and then diluted to 250 µg / ml solution with 250 µg / ml solution. The control activities for pepper anthrax were investigated.

As a result, the F2, F3 and F4 fractions showed high control activity, and these three fractions were mixed (named C1) and analyzed again by silica gel column chromatography. Specifically, 4.5 g of the C1 fraction was dissolved in chloroform and then loaded into a column (3.6 × 60 cm, Kaigel gel 60, 70-230 mesh, 200 g), followed by chloroform: methanol mixture (1% → 10% methanol farming tool). E) was eluted using the developing solvent. Six fractions of C11 to C16 (Rf value range: C11 = 0.5 to 0.76, C12 = 0.41 to 0.5, C13 = 0.18, C14 = 0.11 to 0.18, C15 depending on TLC pattern in chloroform: methanol (95: 5) solvent) = 0.04-0.11, C16 = 0.01-0.04), and each fraction was prepared at a concentration of 2,000 µg / ml, and the control activity was investigated for pepper anthrax in the same manner as in Example 1.

As a result, it showed high control activity in the C13 and C14 fractions. Thus, two samples were mixed again (509 mg) and dissolved in a methylene chloride: hexane: methanol mixture (5: 5: 1, v / v / v), followed by a Sephadex LH-20 column (3.6 × 45 cm; 100 g And Sigma) chromatography. The column was eluted using methylene chloride: hexane: methanol mixture (5: 5: 1, v / v / v) as a developing solvent, from which yellowish 150 mg of sterile active ingredient A was purely separated.

On the other hand, the C12 (2.28 g) and C15 fractions (600 mg) did not show control activity in the pepper anthrax, but were found to contain a yellowish substance which was estimated to be a derivative of the active ingredient A isolated from the C13 and C14 fractions. Both samples were analyzed using Sephadex LH-20 column chromatography in the same manner as above. Through this process, the active ingredient B (256.6 mg), which is slightly more polar than the active ingredient A, was separated from the C12 fraction, and the active ingredient C (443 mg), which was slightly more polar than the active ingredient A, was purified from the C15 fraction.

Example 4 Structure Determination of Demethoxycurcumin, Curcumin and Bisdemethoxycurcumin

In Example 3, three active ingredients purely separated from methanol extract of turmeric were analyzed using a mass spectrometer (JEOL JMS-DX303; JEOL Ltd., Tokyo, Japan). Electron impact mass spectrum showed that active ingredient A showed molecular ion [M + 1] ⁺ at m / z 338, with major segment ions at m / z 320, 242, 217 , 177, 147 (base peak), 119, 91 and the like was detected by the structure of the demethoxycurcumin of the formula ( 1 ). As a result of mass spectrometry, the active ingredient B showed the molecular ion [M + 1] ⁺ at m / z 368, and the major segment ions were m / z 350, 272, 244, 217, 190, 177 (base peak), 137 , 117 and the like were identified as curcumin of the formula ( 2 ) ( FIG. 2 ). On the other hand, the active ingredient C showed that the molecular ion [M + 1] ⁺ was found at m / z 308, and the main segment ions were m / z 290, 280, 212, 202, 187, 173, 160 and 147 (basic peak). , 133, 119, 107, 91 and the like was identified as bisdemethoxycurcumin of the formula ( 3 ) ( Fig. 3 ). As described above, all of the active ingredients A, B, and C separated from the methanol extract of turmeric as a material showing control activity were identified as curcuminoid-based materials.

Example 5 Control Activity of Turmeric Extract-Derived Active Ingredients against Seven Plant Diseases

In order to investigate the in vivo control activity of the demethoxy curcumin, curcumin and bisdemethoxy curcumin identified in Example 4, including rice anthracnose disease, rice blast, rice leaf blight, tomato gray mold, tomato blight A total of seven plant diseases, including wheat rust and wheat flour, were tested. After dissolving each of the three active ingredients in acetone, 250 μg / ml Tween 20 solution was added to adjust the final concentration to 250, 500 and 1000 μg / ml, and the final acetone concentration of all samples was adjusted to 10%. At this time, a solution containing 10% acetone and 250 µg / ml Tween 20 was used as a control. Two pots were used for each plant disease, and the active ingredient samples were sprayed with foliar spray for 24 hours and then inoculated with each plant pathogen. The control activity against these seven plant diseases was investigated according to the method described in Example 1, and the results are shown in Table 3 below.

As shown in Table 3 , demethoxycurcumin showed high control activity against pepper anthrax, rice blast and tomato late blight among seven plant diseases. In particular, demethoxy curcumin showed high control activity against the other two curcuminoid active ingredients against pepper anthrax, indicating that the turmeric extract was mainly caused by demethoxycurcumin. Can be ( FIG. 4 ). Curcumin, on the other hand, showed control against rice blast and tomato late blight. In the case of bisdemethoxy curcumin, curcumin-like level of control activity was shown only in rice blast among seven plant diseases.

As described above, the control composition of the present invention using the curcuminoid-based compound or turmeric extract containing the same as an active ingredient is harmless to the human body from natural products and does not cause environmental pollution to plant anthrax and rice blast bottles. Because it shows high control activity, it can be useful for developing eco-friendly natural product disinfectant and producing high value-added organic products.

Claims (8)

Curcuminoid compound of Formula 1; Or a composition for controlling a rice blast bottle containing turmeric extract containing the curcuminoid compound as an active ingredient extracted with an organic solvent selected from the group consisting of methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, ethyl acetate, and mixtures thereof : <Formula 1>

Wherein R ¹ and R ² are each independently H or OCH ₃ . The method of claim 1, Curcuma turmeric ( Curcuma aromatica ), autumn turmeric ( Curcuma longa ) or purple turmeric ( Curcuma zedoaria ), characterized in that the composition. The method of claim 1, The composition of the turmeric extract is prepared by adding 5 to 20 L of an organic solvent to 1 kg of dried turmeric root and leaching at 20 to 60 ° C. for 3 hours to 3 days. delete The method of claim 1, A composition characterized in that the curcuminoid compound is demethoxycurcumin of formula 2 , curcumin of formula 3 or bisdemethoxycurcumin of formula 4 : <Formula 2>

<Formula 3>

<Formula 4>

The method of claim 1, A curcuminoid compound is separated and purified by fractionating turmeric extract by column chromatography. The method of claim 1, Turmeric extract is characterized in that it comprises a concentration of 333.3 μg / ㎖ to 3000 μg / ㎖. The method of claim 1, Composition comprising a curcuminoid compound at a concentration of 250 μg / ml to 1000 μg / ml.

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