CN100439353C - Extraction and separation method and application of chamaejama chamaejasma chromone in Daphne chamaejasmin - Google Patents

Abstract

The present invention relates to a method for extracting and separating chamaejasma chromone from chamaejasma chamaejasme and its application, which isolates and identifies a broad-spectrum bactericidal activity that can inhibit and kill various plant pathogenic bacteria from the root of chamaejasma chamaejas Composition, i.e. chamaejasma chromogenone, the present invention comprises the following steps: using Daphne chamaejasme root as raw material, using 4 to 10 times the weight of ethyl acetate or acetone or a mixture of ethyl acetate and acetone as solvent, using 30°C ~55°C ultrasonic normal pressure continuous extraction 3 times, each extraction time 30-50min. The extract was filtered, concentrated and column chromatographic to obtain 60%-99% chamaejasmin chromone.

Description

Extraction and separation method and application of chamaejama chamaejasma chromone in Daphne chamaejasmin

1. Technical field:

The present invention relates to a comprehensive utilization of chamaejasma chamaejasme in the field of medicine, veterinary medicine and pesticide intermediates or original medicines, in particular to a method for extracting and separating chamaejasma chamaejasme from chamaejasma chamaejasme and its application as a plant fungicide .

2. Background technology:

Stellera Chamaejasme (Stellera Chamaejasme L.) is a plant of the genus Stellera Chamaejasme. Its aliases include Stellera Chamaejasme and Broken Heart Grass. Its root is the poisonous matrix of traditional Chinese medicine. As early as 1959, in the "China Soil Pesticides", there is a record of the application of chamaejasma chamaejasme as a botanical pesticide. The water extract is sprayed on the leaves of plants to prevent and control leaf bean aphids, cabbage aphids, leaf rust, etc. In recent years, some researchers have successively developed plant pesticides containing chamaejasma crude extract, and some have applied for patents. However, these botanical pesticides of chamaejasma are all insecticides, and a few of them have clear active ingredients and contents, while most of the patents do not know their insecticidal active ingredients. Zhang Jie et al. (patent application No. 00116146) invented a method for preparing a broad-spectrum bioinsecticide from Eupatorium adenophorum, Daphne chamaejasme and Deervine. The insecticide prepared by this method belongs to a compound preparation, and its insecticidal active ingredient Unclear, the object of action does not contain plant pathogenic bacteria. Zhang Kejie et al. (patent application No. 02133769) described the process of preparing biopesticide wolfsin aqueous emulsion from the roots and stems of chamaejasma. The product prepared by this method belongs to a single preparation, and the effective insecticidal component and its content are clear, ranging from 1.1% to 5%. . However, its target of action is only plant pests, and does not contain plant pathogenic bacteria. Liu Shigui etc. (patent application No. 02133971) disclose the method for preparing plant insecticide by chamaejasma chamaejasma extract, azadirachtin and motherwort alkaloid etc., the product that this method obtains is compound preparation, wherein the The active ingredients are not clear, and the target of action is also only plant pests. Han Mingli et al. (patent application No. 02100771.3) described the preparation method of 0.3% chamaejamajasme phytowatercide from Daphne chamaejasme. The product is a unilateral preparation, the active ingredient is chamaejasmin, and the object of action is still only plant pests. The production process only involves the crude extract of chamaejasma root, not the separation of chamaejasmin. To sum up, it can be seen that chamaejasma chamaejasme is mainly used as a botanical pesticide as an insecticide, and the research and development of its fungicide is still blank.

Niwa et al. (Tetrahydron Letters, 1984, 25(34): 3735-3738) first isolated and identified chamaejasma chromone from the root of Daphne chamaejasma. Subsequently, studies by Feng Baomin (Chinese herbal medicine, 2001, 32(1): 14-15) and others confirmed the research results of Niwa et al. These studies have a common feature, that is, they all belong to the basic theoretical research of phytochemistry or natural product chemistry. The research methods are all conventional methods in the laboratory, with low extraction efficiency and complicated separation methods, which are not suitable for industrial scale production at all. Secondly, all the research reports on chamaejasmin chromone seldom involve the study of its biological activity, and there is no research on chamaejasma chromone on plant pathogenic bacteria, that is, as a plant fungicide.

3. Contents of the invention:

In order to solve the deficiencies in the above-mentioned background technology, the present invention provides a method for extracting and separating chamaejasma chromone from Daphne chamaejasma and its application. The specific content is to isolate and identify a broad-spectrum bactericidal active ingredient that can inhibit and kill a variety of plant pathogenic bacteria from the root of Daphne chamaejasma, that is, chamaejasma chromone, and develop a method suitable for large-scale extraction from chamaejasma root A production method for isolating chamaejasma chromone, the production method and process are simple, economical and reasonable, and at the same time, provides the application of chamaejasma chromone as a broad-spectrum plant bactericidal active ingredient.

To achieve the above object, the technical solution adopted in the present invention is:

A method for extracting and separating chamaejasma chromogenone from Daphne chamaejasma, which is special in that it includes the following steps: adding chamaejasma root powder into a solvent for extraction, and then conducting the first silica gel column chromatography for rough separation to obtain a The crude fraction was subjected to silica gel column chromatography for the second time to obtain the secondary crude fraction, and then the secondary crude fraction was purified.

The above-mentioned operation steps include: taking Daphne chamaejasma root as raw material, using 4-10 times the weight of ethyl acetate or acetone or a mixture of ethyl acetate and acetone as solvent, and using 30°C-55°C ultrasonic pressure to extract continuously for 3 times, Each extraction time is 30-50 minutes. The extract is filtered, concentrated and column chromatographed to obtain 60%-99% chamaejasme chromone.

The above operation steps include:

(1) Grinding the root of chamaejasma to a certain extent, according to the ratio of 1:4 to 1:10 of the weight (kg) of chamaejasma root powder and the volume of solvent (L), use intermittent ultrasonic extraction, and the extraction temperature is 30°C to 55°C , each batch of raw materials is continuously extracted 3 times with the same solvent, each extraction time is 30-50min, the ultrasonic power is 1.8W, each ultrasonic time is 6s, and the time interval between two ultrasonic waves is 4s;

(2) After each extraction, use negative pressure or centrifugal filtration to collect the filtrate, combine the three extracts, and evaporate the solvent under negative pressure to obtain a crude extract. The recovered solvent can be used repeatedly. The total extraction rate depends on the solvent. The types of different types are different, generally 7% to 21%, the content of chamaejasma chromone in the crude extract is 5% to 25%, the crude extract does not need to be separated and purified, and can be directly used to prepare plant fungicide emulsion in water. Other organic solvents can also be used for secondary ultrasonic extraction of the crude extract, and the primary or secondary extract of the obtained crude extract is used for the crude separation of chamaejasma chromone;

量的大约200目的柱层析硅胶，将拌样后的硅胶在常温下晾干，加到硅胶柱的顶端，进行干柱层析，依次采用氯仿、乙酸乙酯进行洗脱，收集乙酸乙酯流份，负压蒸除溶剂后，得一次粗分物，该粗分物中的狼毒色原酮含量为45％～55％，可直接用于配制医用或农用杀菌剂；(3) Dissolve the crude extract with acetone or methanol or a mixture of the two, make a solution with a mass concentration of about 20%, and add 3 to 7

Amount of about 200 mesh column chromatography silica gel, dry the mixed silica gel at room temperature, add to the top of the silica gel column, perform dry column chromatography, use chloroform and ethyl acetate for elution in sequence, and collect ethyl acetate Fractions, after the solvent is evaporated under negative pressure, a crude fraction is obtained, and the content of chamaejasma chromanone in the crude fraction is 45% to 55%, which can be directly used to prepare medical or agricultural fungicides;

(4) The primary crude fraction is subjected to column chromatography in a method similar to (3), except that a mixed solvent of chloroform-acetone 7:3 (V/V) is used as an eluent, and chamaejasma chromogen The ketone standard product was used as a control, and the fractions were detected by TLC or HPLC, and the fractions containing chamaejasma chromone were combined, and after the solvent was evaporated under negative pressure, the secondary crude fraction was obtained. The content of chamaejasma chromone is 80%-90%, which can be directly used to prepare medical or agricultural fungicides;

(5) Dissolve the secondary crude fraction with methanol, put it on a sephadex LH-20 column for purification, collect the fractions containing chamaejasma chromone, evaporate the solvent under negative pressure, and obtain the fine-quality chamaejasma chromone with a content of 95% %～99%, it can be used as standard sample or analytical pure chemical reagent.

In the above-mentioned steps (1), the extraction solvent is selected from one of chloroform, ethyl acetate, acetone, ethanol or methyl alcohol, and the extraction scheme includes: A. directly carry out ultrasonic extraction with ethyl acetate or acetone, material (kg)/liquid ( L) ratio is controlled at 1:4 to 1:10, and the extraction temperature is 30°C to 55°C, the obtained crude extract is a light yellow amorphous powder containing 5% to 10% chamaejamae chromone, which is directly used for Column chromatography separation. The extraction rates of ethyl acetate and acetone are 5% to 6% and 7% to 8% respectively; B. First, ultrasonic extraction is carried out with methanol, the extraction conditions are the same as the ethyl acetate method, and the obtained crude extract can be directly used to prepare plant sterilization The extraction rate is 18% to 25%. Acetone is used for secondary ultrasonic extraction of the methanol crude extract. The extraction conditions are the same as the ethyl acetate method. The obtained acetone crude extract is equivalent to 10% of the plant material used. % to 12%, it is a yellow amorphous powder, containing 5% to 10% chamaejamae chromone, which is directly used for column chromatography separation.

In the above step (2), the crude extract is made into a solution with a mass concentration of about 20% with acetone, and 3 to 7 times the mass of silica gel for column chromatography is added, and the silica gel after mixing the sample is dried at room temperature or heated with 40°C hot air. Dry it under pressure, then add it to the top of the silica gel column for the first dry column chromatography rough separation, the specification of the silica gel column is: height/diameter 10:1, the particle size of the silica gel filler is 100-300 mesh, and the amount used is the sample amount 1 to 3 times that of chloroform, first eluted with chloroform until the effluent was almost colorless, and then eluted with ethyl acetate until TLC or HPLC detected that no chamaejasma chromone flowed out, combined the ethyl acetate fractions, After evaporating the solvent, the primary crude fraction was obtained, and its mass was about 40% to 50% of the sample amount. The chamaejamae chromanone content of the crude fraction is 45%-55%.

In the above step (3), the primary crude fraction is made into a saturated solution with acetone, and added to the top of the silica gel column for wet column silica gel chromatography. The specification of the silica gel column is: height/diameter 20:1. The particle size of the silica gel filler is 100-300 mesh, and its dosage is 50-100 times of the sample amount. The mixed solvent of chloroform-acetone 7:3 (V/V) is used as the eluent to collect the After distilling off the solvent to obtain the main fraction of the main fraction, a yellow amorphous powder, its mass is about 40% to 50% of the sample amount, and the content of chamaejasma chromone is 80% to 90%.

In the above step (4), the secondary crude fraction is made into a saturated solution with methanol, and purified with methanol-balanced sephadex LH-20 in advance. The specifications of the column are: height/diameter 10:1, sephadex LH-20 The particle size is 200-400 mesh, and the loading amount is 50-100 times of the amount of sephadex LH-20 in the column. With methanol as the eluent, the fraction containing chamaejasmin chromone is concentrated under reduced pressure to obtain chamaejasma chromogen The quality of ketone quality is about 85%-90% of the sample amount, which is equivalent to 0.5%-1.5% of the raw material of chamaejasma root, and the purity is 95%-99%.

The physical parameter of above-mentioned wolfbane chromone is as follows:

Chromone chamaejama, white powder, mp 245°C (decomposition), [α] _D =+127.434° (c 0.076, MeOH), UV (MeOH, nm): λ _max 296, 260, 212. IR(KBr): υ _max cm ^-1 3401 (OH), 1654 (C=O), 1630, 1511 (C=C), 1238, 1174, 1070 (CO). FAB MS e/z: 543 ([M+1] ⁺ , 100), 344 ([M-HOC ₆ H ₄ CH ₂ C ₆ H ₄ OH+1] ⁺ , 38), 199 ([HOC ₆ H ₄ CH _2C6H4OH ] ₊ ^, 65) _. ¹ H NMRδ(CD ₃ OD): 4.64 (1H, d, J=12Hz, H-12), 5.80 (2H, s, H-16, H-18), 6.13 (1H, d, J=2.0Hz, H-6), 6.20 (1H, d, J=2.0Hz, H-8), 6.36 (1H, d, J=12.0Hz, H-11), 7.94 (1H, s, H-2). The following four Group peaks are H-21, H-22, H-24, H-25, H-27, H-28, H-30, H-31: 6.75 (1H, d, J=7.0Hz), 6.64 (1H , d, J=7.0Hz), 7.07 (1H, d, J=7.0Hz), 7.20 (1H, d, J=7.0Hz). ¹³ C NMR δ(CD ₃ OD): 48.32 (C-11), 54.09(C-12), 94.36(C-8), 96.04(C-16), 96.04(C-18), 99.78(C-6), 105.24(C-10), 106.17(C-14), 115.53 (C-28), 115.53(C-30), 115.69(C-22), 115.69(C-24), 121.75(C-3), 129.61(C-25), 129.61(C-21), 130.51( C-27), 130.51(C-31), 134.68(C-26), 135.61(C-20), 156.36(C-29), 156.39(C-23), 156.94(C-2), 158.48(C -9), 163.22(C-5), 165.03(C-15), 165.03(C-19), 165.22(C-7), 165.73(C-17), 181.04(C-4), 204.22(C- 13);

HPLC spectrum: eluent CH ₃ OH/H ₂ O (V/V) 47:5, Waters ALC/JPC-201 high pressure liquid chromatography, the column is Nova.Pak C18, 150mm×4mm (id), particle size 4.5μm, flow rate 0.7ml·min ^-1 , ultraviolet detector, detection wavelength 296nm.

An application of chamaejasma chamaejasme chromone in Daphne chamaejasme as a plant fungicide, which is special in that: the fungicide uses the root extract of Daphne chamaejasme as a raw material, and chamaejasba chromone as the main bactericidal active ingredient, For the prevention and treatment of diseases caused by apple dry rot (Bakeri Rehm), wheat scab (Fusarium graminearum), tomato early blight (Alternariasolani), pumpkin blight (Fusarium bulbigenum), corn leaf spot (Exserohilum turcicum), tobacco Plant diseases caused by Alternaria alternata and Phytophthora capsici.

The concentration of above-mentioned chamaejasma chromanone is 2200 mg·L ^-1 .

Compared with prior art, the advantages and effects that the present invention has are as follows:

The present invention adopts the biological activity tracking method and the chemical separation method for the first time to isolate and identify a broad-spectrum bactericidal active ingredient that has inhibitory and killing effects on various plant pathogenic bacteria, i.e. chamaechromone (chamaechromone). ). At the same time, a production process for extracting and separating chamaejasma chromone from the root of chamaejasma has been developed. The extraction efficiency is high, the separation method is simple, and it is suitable for industrial scale production.

4. Description of drawings:

Fig. 1 is a process flow diagram of the present invention;

Fig. 2 is the chemical structural formula of chamaejasma chromone;

Figure 3 is the HPLC spectrum of chamaejamae chromone.

5. Specific implementation methods:

1. Referring to Fig. 1, the present invention adopts 4-step method to obtain different contents of chamaejamae chromone, namely extraction, the first silica gel column chromatography crude fractionation, the second silica gel column chromatography crude fractionation, sephadexLH-20 purification , the content of chamaejamae chromone in the target substance obtained by separation at each step is 5%-25%, 45%-55%, 80%-90%, and 95%-99%, respectively. Its technological process is as follows:

1.1. Crush the root of chamaejasma to a certain extent, and its fineness is not strictly required, generally 1mm in diameter. According to the ratio of the weight (kg) of the wolfba root powder and the solvent volume (L) of 1:8, intermittent ultrasonic extraction is adopted, and the extraction temperature is 40°C. Each batch of raw materials was continuously extracted three times with the same solvent, and the extraction time was 40 min each time. The ultrasonic power is 1.8W, each ultrasonic time is 6s, and the time interval between two ultrasonic waves is 4s. If the ultimate goal is to separate the fine product of chamaejasma chromanone, ethyl acetate or acetone or ethyl acetate/acetone 1:1 (V/V) is selected as the solvent. If the ultimate goal is to extract crude extracts for the preparation of Daphne chamaejasme fungicide, methanol is selected as the solvent.

1.2. For solid-liquid separation during extraction, one of the three methods of negative pressure filtration, centrifugal filtration or press filtration can be used, and centrifugal filtration is best. The filtrate was depressurized by a water pump, and the solvent was distilled off at 38°C to obtain a crude extract. The recovered solvent can be used repeatedly. The total extraction rate varies according to the type of solvent, and the extraction rates of ethyl acetate, acetone and methanol are 5.2%, 6.4% and 21%, respectively. The contents of chamaejamae chromone in the three kinds of crude extracts are about 20%, 6% and 5% respectively. The three kinds of crude extracts can be directly used to prepare water emulsion of plant fungicides without separation and purification. If the ultimate goal is to separate the chamaejasma chromogenone, choose ethyl acetate or acetone extract.

1.3. When carrying out the first silica gel column chromatography on the crude extract of ethyl acetate or acetone, choose acetone to dissolve the crude matter and prepare a solution with a mass concentration of about 20%. Then, add about 200 mesh column chromatography silica gel equivalent to 3-7 mass of the crude extract, mix quickly, and dry at room temperature. Add it to the top of a silica gel column equivalent to 1/8 of the mass of the mixed sample silica gel for dry column chromatography. Elution was carried out sequentially with chloroform and ethyl acetate. Chromone of chamaema chamaeme was present in the fraction of ethyl acetate, and the solvent was evaporated under reduced pressure with a water pump to obtain a crude fraction. The chamaejamae chromanone content in the crude fraction is 45%-55%, which can be directly used to prepare medical or agricultural fungicides, and can also be sold as the technical medicine of agricultural fungicides.

1.4. When performing silica gel column chromatography on the primary crude fraction, conventional dry column chromatography is used. A mixed solvent of chloroform-acetone 7:3 (V/V) was used to pack the column, and at the same time, it was used as the eluent. With the standard substance of chamaejasma chromone as the control, the fractions were detected by TLC method or HPLC method. All the fractions containing chamaejasma chromone were combined, and the solvent was distilled off under negative pressure to obtain the secondary crude fraction. The chamaejamae chromanone content in the crude fraction is 80%-90%, which can be directly used to prepare medical or agricultural fungicides.

1.5. Purification of the secondary crude fraction was carried out using sephadex LH-20 gel chromatography. The specification of the column is: height/diameter 10:1. Sephadex LH-20 has a particle size of 300 mesh. The loading amount is 1/50 times of the amount of sephadex LH-20 in the column. Using methanol as the eluent, collect and combine the fractions containing only chamaejasmin chromone, concentrate under reduced pressure to obtain the fine chamaejasma chromone, and the yield is about 85% to 90% of the sample amount. The whole process The total yield is equivalent to 0.5% to 1.5% of the raw material of chamaejasma root, and the purity is 95% to 99%.

2. The application of chamaejasma chromone as a plant fungicide

The invention discovers for the first time the new function and new use of chamaejasma chromone in plant sterilization, and fills in the research blank of chamaejasma and chamaejasma chromone in plant sterilization. The results of the study confirmed that chamaejasba chromogenone at a concentration of 2200 mg·L ^-1 is effective against apple dry rot (Bakeri Rehm), wheat scab (Fusarium graminearum), tomato early blight (Alternariasolani), pumpkin blight (Fusarium bulbigenum) , the inhibition rates of plant pathogens such as Exserohilum turcicum, Alternaria alternata and Phytophthora capsici. were 81.1%, 80.0%, 77.3%, 87.8%, 98.5%, respectively, 96.8%, 81.0%. Therefore, chamaema chromone is expected to become a new plant source agricultural fungicide.

3. Referring to Fig. 2, Fig. 2 is the chemical structural formula of chamaejasma chromone, and the physical parameters are as follows:

Chromone chamaejama, white powder, mp 245°C (decomposition), [α] _D =+127.434° (c 0.076, MeOH), UV (MeOH, nm): λ _max 296, 260, 212. IR(KBr): υ _max cm ^-1 3401 (OH), 1654 (C=O), 1630, 1511 (C=C), 1238, 1174, 1070 (CO). FAB MS e/z: 543 ([M+1] ⁺ , 100), 344 ([M-HOC ₆ H ₄ CH ₂ C ₆ H ₄ OH+1] ⁺ , 38), 199 ([HOC ₆ H ₄ CH _2C6H4OH ] ₊ ^, 65) _. ¹ H NMRδ(CD ₃ OD): 4.64 (1H, d, J=12Hz, H-12), 5.80 (2H, s, H-16, H-18), 6.13 (1H, d, J=2.0Hz, H-6), 6.20 (1H, d, J=2.0Hz, H-8), 6.36 (1H, d, J=12.0Hz, H-11), 7.94 (1H, s, H-2). The following four Group peaks are H-21, H-22, H-24, H-25, H-27, H-28, H-30, H-31: 6.75 (1H, d, J=7.0Hz), 6.64 (1H , d, J=7.0Hz), 7.07 (1H, d, J=7.0Hz), 7.20 (1H, d, J=7.0Hz). ¹³ C NMRδ(CD ₃ OD): 48.32 (C-11), 54.09 (C-12), 94.36(C-8), 96.04(C-16), 96.04(C-18), 99.78(C-6), 105.24(C-10), 106.17(C-14), 115.53( C-28), 115.53(C-30), 115.69(C-22), 115.69(C-24), 121.75(C-3), 129.61(C-25), 129.61(C-21), 130.51(C -27), 130.51(C-31), 134.68(C-26), 135.61(C-20), 156.36(C-29), 156.39(C-23), 156.94(C-2), 158.48(C- 9), 163.22(C-5), 165.03(C-15), 165.03(C-19), 165.22(C-7), 165.73(C-17), 181.04(C-4), 204.22(C-13 );

See Figure 3, HPLC spectrum: eluent CH ₃ OH/H ₂ O (V/V) 47:5, WatersALC/JPC-201 high pressure liquid chromatograph, column is Nova.Pak C18, 150mm×4mm (id) , particle size 4.5μm, flow rate 0.7ml·min ^-1 , ultraviolet detector, detection wavelength 296nm.

Claims (1)

1. A method for extracting and separating chamaejamae chromone in Daphne chamaejasma, characterized in that it comprises the following steps: (1) Pulverize the wolfba root, according to the ratio of the weight kg of the wolfba root powder and the solvent volume L 1:4 to 1:10, use intermittent ultrasonic extraction, the extraction temperature is 30°C to 55°C, and each batch of raw materials uses ethyl acetate Esters or acetone or a mixture of ethyl acetate and acetone were extracted continuously for 3 times, each extraction time was 30-50min, the ultrasonic power was 1.8W, each ultrasonic time was 6s, and the time interval between two ultrasonic waves was 4s; (2) After each extraction, use centrifugal filtration to collect the filtrate, combine the 3 extracts, and evaporate the solvent under negative pressure to obtain a crude extract. The recovered solvent can be used repeatedly. The total extraction rate varies according to the type of solvent And different, generally 7% to 21%, the content of chamaejasma chromone in the crude extract is 5% to 25%; (3) Dissolve the crude extract with acetone or methanol or a mixture of the two, make a solution with a mass concentration of about 20%, and add 200 mesh column chromatography silica gel equivalent to 3 to 7 times the mass of the crude extract under constant stirring , dry the silica gel after mixing the sample at normal temperature, add it to the top of the silica gel column, carry out dry column chromatography, use chloroform and ethyl acetate to elute in turn, collect the ethyl acetate fraction, and evaporate the solvent under negative pressure , to obtain a crude fraction, the chamaejamae chromone content in the crude fraction is 45% to 55%; (4) The primary crude fraction is subjected to column chromatography in a method similar to (3), except that the mixed solvent of chloroform-acetone 7:3 volume ratio is used as the eluent, and the standard substance of chamaejasma chromone As a control, the TLC method or HPLC method was used to detect each fraction, and the fractions containing chamaejasma chromone were combined, and after the solvent was evaporated under negative pressure, the secondary crude fraction was obtained. Protoketone content is 80% to 90%; (5) Dissolve the secondary crude fraction with methanol, put it on a dextran LH-20 column for purification, collect the fractions containing chamaejasma chromone, evaporate the solvent under negative pressure, and obtain the chamaejasma chromone boutique, the content 95% to 99%.

Images