KR20180060430A - Antibacterial active composition of strawberry leaf extract and method for controlling anthrax using the same - Google Patents

Abstract

The present invention relates to a strawberry leaf extract having antibacterial activities in Colletotrichum gloeosporioides and an antibacterial composition including the same. The strawberry leaf extract of the present invention is prepared by mixing 1 L of methanol with 0.5 to 1.5 kg of strawberry leaves to obtain a mixed solution, leaving the mixed solution alone at 28 to 32°C for 10 to 15 hours, extracting the mixed solution to obtain an extracted material, filtering the extracted material to obtain a filtrate, and concentrating the filtrate, thereby removing methanol from the filtrate to prepare the extract. The strawberry leaf extract according to the present invention has inhibited activities of bacteria including Colletotrichum coccodes (A), Fusarium oxyspourum (B), Glomerella cingulate (C), Rhizoctonia solani (D), Phytophthora cactorum (E), and P. capsici (F). Further, the strawberry leaf extract according to the present invention has an effect that the degree of inhibiting activities of anthracnose mycelia of the strawberry leaf extract has increased as the content of the extract has been increased.

Description

[0001] The present invention relates to an antimicrobial active composition of a strawberry leaf extract and an antifungal active composition for controlling anthrax,

The present invention relates to a strawberry leaf extract having antibacterial activity against anthrax bacteria and an antimicrobial active composition containing the same. More specifically, the present invention relates to a strawberry leaf extract having antibacterial activity against anthrax bacteria and an antimicrobial composition having an antifungal effect against anthrax bacteria containing the extract.

The present invention relates to an antimicrobial composition having a controlling effect against anthrax bacteria by a methanol extract composition from strawberry leaves.

The strawberry is a strawberry belonging to Rosaceae of the dicotyledonous plant ( Fragaria ananassa Duch) are extensively planted in the northern hemisphere in the country of origin or in the southern hemisphere. Most cultivars are varieties of Virginia strawberries and Chilean berries.

Strawberries have beard roots. Leaves come from roots. There are three leaves with many hairs. There are sawtooths on the edges. The flower is almost white, but the red flower also blooms, and several are gathered on the thin peduncle. The peduncle is like a stem that carries the earth.

In calyx, the fruit that is often called seed is buried. It is rich in vitamin C, and contains iron and other minerals.

Most of the antimicrobial agents currently in use are manufactured through chemical synthesis, which is costly and has many limitations such as causing side effects. Therefore, recently, researches for separating new antimicrobial substances from natural products are actively conducted. In order to isolate new antimicrobial substances from natural products, it should be considered that the antimicrobial spectrum is wide and safe even without long-term administration without adverse effects.

Anthrax anthracnose is an anthrax disease caused by infection with anthrax. It is a kind of plant disease such as red pepper, tomato, etc., and crops such as rice, bean, cucumber and asteraceae, persimmon, plum tree, peach tree, It can be caused by the parasitism of different kinds of anthrax bacteria in fruit trees such as trees and amber, banana, cabbage and flower.

The greatest damage to pepper is the damage caused by anthrax. However, since the anthracnose of pepper is difficult to control and the damage is large, a lot of pesticides and the like for controlling anthrax are developed, but it is difficult to effectively control.

The most effective control of pepper anthracnose is to prevent rainwater from splashing directly into the fruit with a rainfed facility. Several studies have reported that the control of anthracnose by rain forests is more than 95%, and the yield of nonpolished red pepper plants is about twice as high as that of non - planted ones. In addition, it is most important to remove the sick fruids by discovery and incinerate as more than tens of millions of anthrax infectious agents are formed in one diseased fruit and they are blown away by several kilometers by wind and rain.

Korean Registered Patent No. 10-0699333 B1 (Mar. 26, 2007)

It is an object of the present invention to provide a strawberry leaf methanol extract having antibacterial activity against anthrax. The present invention also provides an antimicrobial composition comprising the methanol extract of Strawberry Leaf.

The present invention provides a methanol extract of strawberry leaf having an anthrax control effect of a crop, which is prepared by pulverizing strawberry leaves with a pulverizer, extracting with an extraction solvent, and filtering. Water or C1-C5 alcohol is used as an extraction solvent, and methanol (CH3OH) is preferable as an extraction solvent for preparing an extract.

The strawberry leaf extract of the present invention is prepared by mixing 0.5-1.5 kg of strawberry leaves with 1 L of methanol and then allowing it to stand at 28-32 ° C for 10-15 hours to extract, then filter and concentrate to remove methanol to produce an extract. In addition, the extract can be prepared by mixing 0.5 to 5 kg of strawberry leaves in 10 L of distilled water, extracting the mixture at 25 to 30 ° C. for 12 to 48 hours, filtering, and concentrating.

In vitro tests on the activity and growth of anthrax mycelia of the strawberry leaf methanol extract showed that when the control, methanol, 0.31 mg / ml methanol extract, 0.64 mg / ml methanol extract and 2.50 mg / ml methanol extract were added, Growth is inhibited, and anthrax mycelial activity is significantly inhibited by the amount of methanol extract.

Strawberry leaf methanol extract is colletotrichum coccodes (A) , Fusarium oxysporum (B) , Glomerella cingulata (C) , Rhizoctonia solani (D), Phytophthora cactorum (E), P. capsici (F) has an effect on the inhibition of bacterial activity, the strawberry leaves (L), the tap (P), the green unripe strawberry (G) and ripe strawberry ( R) was extracted with methanol and two kinds of antimicrobial substances were found by bioautogram.

The methanol extract of the strawberry leaf was analyzed by GC-MS chromatogram method, and it was found that the antibacterial substance was tyrosol and sitosterol.

(A) and β-sitosterol (B) on the mycelial growth of Colletotrichum coccodes . In FIG. 4, when the control, methanol, 0.01 mg / ml tyrosol, 0.10 mg / ml tyrosol and 1.00 mg / ml tyrosol were added respectively, the degree of inhibition of anthracnose activity was inhibited. . In order to investigate the antimicrobial activity of sitosterol, β-sitosterol, 0.01mg / ml of β-sitosterol, 0.10mg / ml of β-sitosterol and 1.00mg / ml of β-sitosterol were added to the control. Antimicrobial activity was inhibited and there was no significant difference in inoculation amount.

The present invention is to provide a strawberry leaf methanol extract having an anthrax control effect. The strawberry leaf methanol extracts were colletotrichum coccodes (A) , Fusarium oxysporum (B) , Glomerella cingulata (C) , Rhizoctonia inhibited solani (D), Phytophthora cactorum ( E), P. capsici (F) bacteria activity.

The inhibitory effect of methanol extract of strawberry leaves on the activity of anthrax mycelia was more effective as the content of extract increased, and the effect of methanol extract of strawberry leaves on the control was enhanced. The inhibitory effect of methanol extract, 0.64 mg / ml methanol extract and 2.50 mg / ml methanol extract on inhibition of mycelial growth of anthrax was investigated in terms of methanol content As the number of patients increased, the number of patients increased.

The methanol extract of strawberry leaf of the present invention has an antifungal effect on not only branches and crops such as pepper, tomatoes but also anthracnose of flowers, soybean and crops, cabbage, banana, and firewood. Analysis of methanol extracts of strawberry leaves revealed that the antimicrobial agents effective against pepper anthracnose were silver tyrosol and sitosterol.

Fig. 1 shows the results of in vitro tests on the activity and growth of anthrax mycelia of methanol extract of strawberry leaves.
2 is a graph showing the degree of inhibition of bacterial activity such as anthrax bacteria.
FIG. 3 is a graph showing that methanol extract of strawberry leaves is analyzed to find that the antimicrobial substance is tyrosol and sitosterol.
Figure 4 shows inhibition by tyrosol (A) and beta-sitosterol (B) on mycelial growth of anthrax.

In the present invention, the leaves of the strawberry ( Fragaria ananassa Duch) are used, and strawberry leaves have three leaves with a long leaf in the crown and have sawtooth on the edge. Other than Rubus crataegifolius), bokbunja berries (R. coreanus MIQ), native raspberry (R. parvifolius Linne var. tiphyllus) , Zheng bokbunja strawberries (R. coreanus for. concolor T.Lee) , bear strawberry (R. phoenicolasius Maximowicz), jangttalgi ( R. hirsutus Thunberg, R. corchorifoius L Fil, R. ribesioideus Maisumira, R. buergeri Miquel and R. oldhamil Maximowicz can be used.

The strawberry leaf used in the production of the strawberry leaf extract of the present invention is preferably used as the dried strawberry, and may be pulverized by a pulverizer to increase the extraction efficiency. As the drying method of the strawberry leaf, it is possible to use any of bion, shade, hot air drying, freeze drying and natural drying methods. Preferably, a freeze-drying method can be used.

In addition, the strawberry leaf extract of the present invention can be produced using a method known in the art. That is, the extract of the present invention can be prepared by cutting the raw material to a predetermined size, extracting it with an extraction solvent, and filtering it. As the extraction solvent, water or a C1 to C5 alcohol may be used. Methanol (CH3OH) is preferable as an extraction solvent for preparing the extract of the present invention.

The strawberry leaf extract of the present invention is prepared by mixing 0.5-1.5 kg of strawberry leaves with 1 L of methanol and then allowing to stand at 28 to 32 ° C for 10 to 15 hours for extraction, then filtering and concentrating to remove methanol to prepare an extract. When the filtrate is concentrated by the above method, the methanol extract of strawberry leaves is reduced to 1/100 of the original methanol capacity (1 L). The obtained methanol extract of strawberry leaf is diluted with water and used to prevent anthrax of pepper.

The methanol extract from the strawberry leaf extract thus extracted and concentrated is diluted with water and used. Preferably, the diluted methanol extract of strawberry leaves is diluted to 100 to 300 times when the methanol extract of filtered and purified strawberry leaves is diluted with water. 1 part by weight of the methanol extract of strawberry leaf is mixed and diluted with 100 parts by weight to 300 parts by weight of water use.

In the example of the present invention, 1 kg of strawberry leaves was added to 1 L of methanol of 80% to 90%, and the mixture was allowed to stand at 30 ° C for 12 hours, filtered, and concentrated to produce a strawberry leaf extract

The strawberry leaf extract according to the present invention prepared as described above was tested for its effect on red pepper anthracnose by the different test sites as in Examples 1 and 2 described below.

However, the strawberry leaf methanol extract of the present invention has an antifungal effect on not only branches and crops such as pepper, tomatoes but also anthracnose of flowers, soybean, crops, cabbage, banana, and firewood.

In FIG. 1, when the control, methanol, 0.31 mg / ml methanol extract, 0.64 mg / ml methanol extract and 2.50 mg / ml methanol extract were injected into the anthrax mycelium of the strawberry leaf methanol extract, It shows the growth size of mycelium. FIG. 1 shows that anthrax mycelial activity is significantly inhibited by the amount of the methanol extract.

Figure 2 is a graph coccodes (A) , Fusarium oxyspourum (B) , Glomerella cingulata (C) , Rhizoctonia solani (D) , Phytophthora shows an extent cactorum (E), P. capsici ( F) Inhibition of bacterial activity, extracts the leaf (L) of strawberry, tap (P), the green unripe strawberry (G) and ripe strawberries (R) methanol We found two antimicrobials in the bioautogram. The white part inhibits bacterial activity, and the methanol extract of strawberry leaf (L) shows antibacterial activity in all bacteria.

FIG. 3 shows the results of GC-MS chromatogram analysis of the methanol extract of the strawberry leaf, and found that the antibacterial substance was tyrosol and sitosterol.

Figure 4 shows inhibition by tyrosol (A) and beta-sitosterol (B) on mycelial growth of anthracnose ( Colletotrichum coccodes ). In FIG. 4, when the control, methanol, 0.01 mg / ml tyrosol, 0.10 mg / ml tyrosol and 1.00 mg / ml tyrosol were added respectively, the degree of inhibition of anthracnose activity was inhibited. . In order to investigate the antimicrobial activity of sitosterol, β-sitosterol, 0.01mg / ml of β-sitosterol, 0.10mg / ml of β-sitosterol and 1.00mg / ml of β-sitosterol were added to the control. Antimicrobial activity was inhibited and there was no significant difference in inoculation amount.

≪ Example 1 >

1. Test Overview

In Example 1, in order to select an anti-anthracnose agent for red pepper (including red pepper), the above-mentioned methanol-extracted strawberry leaf extract was tested in the pepper cultivar 'Super Maniata' and 'Papillot' varieties. The test site was 'Namyang-eup Siri' in Hwaseong City, Gyeonggi Province, and the test period was from August 2, 2016 to August 30, 2016.

2. Test method

The strawberry leaf extract of Colletotrichum spp. Was subjected to the following treatments in the pepper cultivars (Super Manithe and Papi Red) as follows. The drug efficacy test was conducted on 'Super Manithe', and the weakness test was conducted on 'Super Manithe' and 'Pappy Red'.

Test agent chief ingredient
content
(weight%) Drug efficacy test Weak test Commissioned company Dilution factor
And usage
When and how to process Standard amount Quantity Seoul National University
Strawberry leaf methanol
extract

Propyne
Wetting agent (contrast)

No treatment
0.5
(01 + 0.4)


70

-
300 times



500

-
Every 7 days
4 foliage processing
(8/2, 8/9, 8/16, 8/23)

"

-
300 times
(8/2)


-

-
150 times
(8/2)


-

-
Seoul National University


-

-

The diluted solution containing the strawberry leaf extract was sprayed with 150 L / 10a using an engine power disperser. In the above table, the content of the main component in the strawberry leaf methanol extract is 0.5% by weight, and 0.1% by weight of tyrosol and 0.4% by weight of β-sitosterol are contained in 0.5% by weight of the extract. That is, in the methanol extract of strawberry leaves, tyrosol and? -Sitosterol are the main components for inhibiting the activity of the anthracnose bacterium, and the methanol extract of strawberry leaf containing the main ingredient is diluted 300 times, Respectively.

The cultivation of pepper in the cultivated area was carried out by vinyl mulching and cultivation. The official date was 5/5/2010, the planting distance was 150cm X 35cm, and no other medicines were treated or mixed during the test period.

The placement of the test area and the cultivation area were carried out by the repeated three rounds as follows.

division
Treated water Number of repeats Muzzle count Area per unit Required area Total area required About 3 3 9 20㎡ 180㎡ 225㎡ about
year pepper 3 3 9 5㎡ 45㎡ Sweet pepper 3 3 9 5pot 45pot 45pot

The meteorological situation at the spraying day was as follows.

Month / day Rainfall (mm) High / Low Temperature (℃) Average temperature (℃) 8/1 - 32.5 / 25.7 28.3 8/2 ^※ 0.0 30.7 / 25.4 27.7 8/3 - 34.7 / 24.4 29.2 8/8 - 35.0 / 24.5 29.3 8/9 ^※ - 34.3 / 26.4 29.6 8/10 - 34.1 / 25.3 29.0 8/15 0.0 34.1 / 25.4 28.8 8/16 ^※ 0.0 34.4 / 24.2 29.0 8/17 0.3 34.6 / 24.7 29.0 8/22 0.0 35.1 / 25.1 29.3 8/23 ^※ 0.3 33.6 / 26.3 28.9 8/24 0.0 33.8 / 25.9 29.2

3. Survey method and criteria

The irradiation method and judgment criteria in Example 1 were carried out as follows.

division Research Item Number of investigations Date of investigation Investigation method Drug efficacy test Disease rate 1 time 8/30 After 7 days of the final drug treatment, Weak test Presence or absence of weakness 3rd time 8/5, 8/7, 8/9 3, 5, 7 days after medication treatment

4. Test scores

end. In the efficacy test, the effect of controlling the anthracnose of pepper on the 7th day after the final treatment was measured and the following results were obtained.

Test agent
Disease rate (%) Significant difference
(DMRT) The control
(%) Ⅰ repeat Ⅱ repeat Ⅲ repeat Average Strawberry leaf methanol extract
Propinab Wetting Agent (Contrast)
No treatment 9.4
5.9
30.6 11.8
6.4
33.0 6.0
5.4
28.9 9.1
5.9
30.8 b
c
a 70.5
80.8
-

The CV% (coefficient of variation%) in the above table was 8.1%.

I. The results of the weakness test on the 3rd, 5th and 7th day after the chemical treatment were as follows. Test agent Test crop Weak degree (0 ~ 5) Remarks
Standard amount Quantity
Strawberry leaf methanol extract pepper
(Super Manithe, Papi Red)
0
0
No Weak

5. Test results of Example 1

In order to carry out this test, the experiment was performed by the repeated three rounds of the nodule method. In the test of drug efficacy test, the mortality rate of 200 or more per 7 days after the final drug treatment was examined. The amount of the standard dose and the weakness after the dose treatment were investigated. Significant differences between the drugs were tested by Duncan's multiple range test (DMRT) at a 95% significance level.

In Example 1, the average rate of non-treatment of pepper anthracnose was 30.8, which was a sufficient condition to evaluate the drug. The control effect of the disclosed material (strawberry leaf methanol extract) was 70.5% after 7 days of the final treatment And statistical significance was confirmed by statistical significance between drugs. In addition, there was no weakness as a result of investigating whether or not the standard amount and the amount of the disclosure material were foliarized.

That is, in Example 1, the methanol extract of Strawberry Leaf showed an excellent effect as a control effect of 70.5%, which was statistically significant as compared with control. As a result of the weak test, there was no weakness in both the standard amount and the dose, and the strawberry leaf methanol extract is considered to be practical for anthrax of pepper.

The methanol extract of strawberry leaf of the present invention has an antifungal effect on not only branches and crops such as pepper, tomatoes but also anthracnose of flowers, soybean and crops, cabbage, banana, and firewood.

≪ Example 2 >

1. Test Overview

In Example 2, in order to select an antifungal agent for anthrax of pepper (including red pepper), the above-mentioned methanol-extracted strawberry leaf extract was tested in the pepper cultivar 'Super Manitta' and 'Pappy Red' Cheongju-si Cheongwon-gu, Seoul, and the test period was from August 3, 2016 to August 31, 2016.

2. Test method

Anthracnose of pepper varieties (Super Manithe and Papi Red) ( Colletotrichum spp.) were treated as follows. The drug efficacy test was conducted on 'Super Manithe', and the weakness test was conducted on 'Super Manithe' and 'Papill Red'.

Test agent chief ingredient
content
(%) Drug efficacy test Weak test Commissioned company Dilution factor
And usage
When and how to process Standard amount Quantity Seoul National University
Strawberry leaf methanol
extract


Propyne
Wetting agent (contrast)

No treatment
0.5
(0.1 + 0.4)


70


-
300 times



500


-
Every 7 days
4 foliage processing
(8/3, 8/10, 8/17, 8/24)

"

-
300 times
(8/3)


-


-
150 times
(8/3)


-


-
Seoul National University


-


-

The diluted solution containing the strawberry leaf extract was sprayed with 150 L / 10a using an engine power disperser.

In the above table, the content of the main component in the strawberry leaf methanol extract is 0.5% by weight, and 0.1% by weight of tyrosol and 0.4% by weight of β-sitosterol are contained in the 0.5% by weight main component. That is, in the methanol extract of strawberry leaves, tyrosol and? -Sitosterol are the main components for inhibiting the activity of the anthracnose bacterium, and the methanol extract of strawberry leaf containing the main ingredient is diluted 300 times, Respectively.

The cultivation of pepper in the cultivated area was carried out by vinyl mulching and cultivation. The planting date was set on May 17, 2016, and the planting distance was 80 cm × 35 cm.

The placement of the test area and the cultivation area were carried out by the repeated three rounds as follows.

division
Treated water Number of repeats Muzzle count Area per unit Required area Total area required About 3 3 9 20㎡ 180㎡ 225㎡ about
year pepper 3 3 9 5㎡ 45㎡ Sweet pepper 3 3 9 5pot 45pot 45pot

The meteorological situation of the spraying day was as follows.

Month / day Rainfall (mm) High / Low Temperature (℃) Average temperature (℃) 8/2 4.9 30.1 / 22.8 27.2 8/3 ^※ 0.1 32.1 / 23.0 27.1 8/4 - 33.4 / 24.3 28.9 8/9 22.4 34.5 / 25.3 28.9 8/10 ^※ - 34.4 / 26.0 29.8 8/11 - 35.0 / 24.5 29.7 8/16 0.0 34.1 / 25.4 29.4 8/17 ^※ - 34.4 / 25.8 29.7 8/18 - 35.2 / 25.9 30.2 8/23 0.9 33.8 / 25.2 28.5 8/24 ^※ 0.0 34.0 / 25.1 29.4 8/25 0.0 33.8 / 24.3 28.6

3. Survey method and criteria

The irradiation method and judgment criteria in Example 1 were carried out as follows.

division Research Item Number of investigations Date of investigation Investigation method Drug efficacy test Disease rate 1 time 8/31 After 7 days of the final drug treatment, Weak test Presence or absence of weakness 3rd time 8/6, 8/8, 8/10 3, 5, 7 days after medication treatment

4. Test scores

end. In the efficacy test, the efficacy of anti - anthracnose anti - anthracnose was measured on the 7th day after the final treatment, and the following results were obtained.

Test agent
Disease rate (%) Significant difference
(DMRT) The control
(%) Ⅰ repeat Ⅱ repeat Ⅲ repeat Average Strawberry leaf methanol extract
Propinab Wetting Agent (Contrast)
No treatment 4.8
4.2
22.0 5.8
2.1
13.1 6.6
3.0
28.9 5.7
3.1
17.7 b
b
a 67.8
82.5
-

In the table above, CV% (coefficient of variation%) was 29.0%.

I. The results of the weakness test on the 3rd, 5th and 7th day after the drug treatment were as follows.

Test agent Test crop Weak degree (0 ~ 5) Remarks
Standard amount Quantity
Strawberry leaf methanol extract pepper
(Super Manithe, Papi Red)
0
0
No Weak

5. Test results of Example 2

In order to carry out this test, the experiment was performed by the repeated three rounds of the nodule method. In the test of drug efficacy test, the mortality rate of 200 or more per 7 days after the final drug treatment was examined. The amount of the standard dose and the weakness after the dose treatment were investigated. Significant differences between the drugs were tested by Duncan's multiple range test (DMRT) at a 95% significance level.

In Example 2, the untreated average mortality of pepper anthracnose was 17.7%, which was sufficient to evaluate the drug. The control effect of the publicly available material (strawberry leaf methanol extract) was 67.8% after 7 days of the final treatment And showed a comparable control effect. In addition, there was no weakness as a result of investigating whether or not the standard amount and the amount of the disclosure material were foliarized.

That is, in Example 1, the methanol extract of Strawberry Leaf showed an excellent effect as a control effect of 67.8%, which was statistically significant as compared with control. As a result of the weak test, there was no weakness in both the standard amount and the dose, and the strawberry leaf methanol extract is considered to be practical for anthrax of pepper.

The methanol extract of strawberry leaf of the present invention has an antifungal effect on not only branches and crops such as pepper, tomatoes but also anthracnose of flowers, soybean and crops, cabbage, banana, and firewood.

Claims (6)

In a strawberry leaf extract having an antitumor effect against anthrax,
An antifungal active composition having an anthrax control effect characterized in that the substance having antibacterial activity against anthrax disease contained in the strawberry leaf extract is tyrosol and sitosterol
The method according to claim 1,
Wherein the strawberry leaf extract is prepared by mixing 0.5-1.5 kg of strawberry leaves with 1 L of methanol and then allowing to stand for 10-15 hours at 28-32 ° C to extract an antifungal active composition.
The method according to claim 1,
Wherein the strawberry leaf extract is prepared by mixing 0.5 to 5 kg of strawberry leaves with 10 L of distilled water and then allowing to stand for 12 to 48 hours at 25 to 30 캜 to extract an antifungal active composition.
4. The method according to any one of claims 1 to 3,
The strawberry leaves used for the strawberry leaf extract include Fragaria ananassa Duch and Rubus crataegifolius , R. coreanus MIQ, R. parvifolius Linne var. Tiphyllus, R. berry strawberry ( R. coreanus for concolor T.Lee, R. phoenicolasius Maximowicz, R. hirsutus Thunberg, R. corchorifoius L Fil, R. ribesioideus Maisumira, R. strawberry ( R. The present invention relates to an antimicrobial active composition having an anthrax control effect, which is characterized in that the leaves of buergeri Miquel and R. oldhamil Maximowicz are methanol extracts of strawberry leaves extracted with C1 to C5 alcohol.
4. The method according to any one of claims 1 to 3,
The strawberry leaf extract has an effect on anthrax control when it is sprayed on a crop comprising leaves, crops such as pepper and tomato, as well as crops such as flowers, soybean, crops, cabbage, banana and pumpkin Lt; / RTI >
A method for producing a strawberry leaf extract having antibacterial activity,
The method of preparing the strawberry leaf extract comprises mixing 0.5-1.5 kg of strawberry leaves with 1 L of methanol and then allowing to stand for 10-15 hours at 28-32 ° C.,
Wherein the methanol mixed in the strawberry leaves is 80% to 90% methanol.

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