CN111869671B

CN111869671B - Application of trihydroxybenzoic acid substituted monoterpene glycoside compounds in resisting phytopathogen

Info

Publication number: CN111869671B
Application number: CN202010896976.7A
Authority: CN
Inventors: 周学明; 林芳霞; 杨静雨; 陈光英; 宋鑫明; 李小宝
Original assignee: Hainan Normal University
Current assignee: Hainan Normal University
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2021-09-14
Anticipated expiration: 2040-08-31
Also published as: CN111869671A

Abstract

The invention discloses application of trihydroxybenzoic acid substituted monoterpene glycoside compounds in resisting phytopathogen, wherein the structural formula of the trihydroxybenzoic acid substituted monoterpene glycoside compounds is shown as follows, wherein R is₁、R₂、R₃、R₄Are respectively and independently selected from-H or 3,4, 5-trihydroxybenzoic acid. The invention discovers for the first time that the trihydroxybenzoic acid substituted monoterpene glycoside compound has obvious and broad-spectrum activity against phytopathogen and can be used for preparing the medicines against the phytopathogen.

Description

Application of trihydroxybenzoic acid substituted monoterpene glycoside compounds in resisting phytopathogen

Technical Field

The invention relates to the technical field of phytochemistry, in particular to application of trihydroxybenzoic acid substituted monoterpene glycoside compounds extracted and separated from melaleuca alternifolia leaves in resisting phytopathogens.

Background

Melaleuca alternifolia (scientific name: Melaleuca alternifolia (Maiden & belche) Cheel) is a plant of the genus Melaleuca of the family Myrtaceae. Native to the 23.5 ° coastal region of australia and the northern part of the northern field. At present, China is cultivated in Hainan, Guangdong, Guangxi, Chongqing and other places. Melaleuca alternifolia is a commercial crop with high economic value, and essential oil can be extracted from fresh branches and leaves of the melaleuca alternifolia, which is called tea tree oil. It can kill fungus and bacteria on skin surface effectively, without toxicity and irritation, and has inhibiting effect on some viruses, so that it is widely used in medicine, food preservative, cosmetics, skin care and health product. At present, reports on the activity of trihydroxybenzoic acid substituted monoterpene glycosides extracted and separated from melaleuca alternifolia leaves on resisting phytopathogen are not found.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide the application of trihydroxybenzoic acid substituted monoterpene glycoside compounds extracted and separated from melaleuca alternifolia in resisting phytopathogens.

The technical scheme adopted by the invention is as follows:

the application of the trihydroxybenzoic acid substituted monoterpene glycoside compounds in resisting phytopathogens is as follows:

wherein R is₁、R₂、R₃、R₄Are respectively and independently selected from-H or 3,4, 5-trihydroxybenzoic acid.

One of the preferred structures is that R₁Is 3,4, 5-trihydroxybenzoic acid, R₂Is 3,4, 5-trihydroxybenzoic acid, R₃is-H, R₄is-H.

Second of the preferred structures, R₁Is 3,4, 5-trihydroxybenzoic acid, R₂is-H, R₃is-H, R₄is-H.

The plant pathogenic bacteria are bacteria, and more preferably cabbage black spot pathogenic bacteria, tobacco black shank pathogenic bacteria, pepper anthracnose pathogenic bacteria, rice flax leaf disease pathogenic bacteria, kumquat sand skin disease pathogenic bacteria, sugarcane pineapple disease pathogenic bacteria, corn big spot pathogenic bacteria, tea round spot pathogenic bacteria or Gonggan chain lattice.

The invention has the following beneficial effects:

the invention discovers for the first time that the trihydroxybenzoic acid substituted monoterpene glycoside compound has the activity of resisting plant pathogenic fungi and can be used for preparing the medicine for resisting the plant pathogenic fungi.

Drawings

FIG. 1: compound 1 structural formula;

FIG. 2: the compound 2 has a structural formula.

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

Example 1:

preparation of monoterpene glycosides:

(1) taking 3Kg of melaleuca alternifolia branch and leaf powder, leaching for 3 times and 7 days each time by using 70% v/v ethanol to obtain an extracting solution, and concentrating the extracting solution under reduced pressure to obtain 320g of melaleuca alternifolia extract;

(2) diluting the melaleuca alternifolia extract with water to obtain a suspension, sequentially extracting with petroleum ether and ethyl acetate, concentrating the ethyl acetate extract to obtain an extract, performing silica gel column chromatography (200-300 meshes) with a petroleum ether-ethyl acetate mixed solvent (100: 0-0: 100, V/V) and an ethyl acetate-methanol mixed solvent (100: 0-0: 100, V/V) in an increasing polarity manner, and collecting fractions about 500mL each time. Similar fractions were pooled and split into 8 fractions, Fr.1-8, by TLC (10% by volume chloroform in chloroform-methanol as developing solvent). Fr.5 performing Sephadex LH-20 column chromatography, eluting with chloroform-methanol (50:50, V/V); then prepared by high performance liquid phase, eluting with 30% acetonitrile water by volume to obtain compound 1(8mg) and compound 2(10 mg).

The structural identification of the 2 compounds is as follows:

compound 1: is white powder with optical rotation of

HR-ESI-MS m/z 635.1988[M-H]^-Is combined with¹H-NMR and¹³c NMR indicates that the molecular formula is C₃₀H₃₆O₁₅The unsaturation was calculated to be 13. According to¹H,¹³C-NMR and DEPT showed that the compound consisted of one glucose, two 3,4, 5-trihydroxybenzoic acids and one monoterpene. According to HMBC, the following results are obtained: the hydrogen at the 4-and 6-positions of glucose, respectively, is associated with the carboxyl carbon of two 3,4, 5-trihydroxybenzoic acids, indicating that both 3,4, 5-trihydroxybenzoic acids are via estersThe bond is respectively connected with the 4 th position and the 6 th position of the glucose; furthermore, the correlation between the hydrogen at the 1-position of glucose and the carbon at the 2-position of monoterpene indicates that monoterpene is attached to the 1-position of glucose via an ether linkage. The structure of the compound is shown in FIG. 1. Process for preparation of Compound 1¹H-NMR and¹³the C-NMR data are shown in Table 1.

Table 1. hydrogen and carbon spectra data for compound 1.

Compound 2: the powder was white. According to¹H，¹³The CNMR data are very similar to compound 1, except that a signal for 3,4, 5-trihydroxybenzoic acid is absent. Therefore, the compound 1 is also a trihydroxybenzoic acid substituted monoterpene glycoside compound. The structural formula of the compound 2 is shown in figure 2.

Test example 1: pharmacological Activity test

Plant pathogenic fungi are one of the major hazards to crops and one of the major causes of agricultural losses. According to the invention, the compounds have good inhibitory activity against plant pathogenic fungi by detecting the inhibitory action of the compounds 1 and 2 on the plant pathogenic fungi.

The test method comprises the following steps: microdilution method. The method is convenient to operate and can be used for large-batch drug sensitivity tests, and the dosage of the culture medium is small. Generally, MH culture medium is used, the prepared liquid medicine is diluted in a test tube in a two-fold decreasing concentration mode, the diluted liquid medicine is respectively filled in a 96-hole plate or directly diluted in the plate hole in a micro liquid adding device in a two-fold mode, then diluted bacterial liquid is inoculated, a row of holes are reserved for liquid medicine comparison, only culture medium and bacterial liquid are added in the row of holes to serve as bacteria comparison, after the test is finished, a micro stirrer is used for shaking and mixing uniformly, then the 96-hole plate is placed in an incubator at 28 ℃ for incubation for 18-48h, and the absorbance under 630nm is measured by a microplate reader.

Preparation of the plant pathogenic fungi strains:

respectively inoculating the activated 9 plant pathogenic fungi into sterilized LB culture medium, and placing the culture medium in a constant temperature oscillator for shake culture at 28 ℃ for 24 h.

Preliminary screening of the compounds:

diluting the cultured plant pathogenic fungi with culture solution with the dilution ratio of 1:500-1: 1000; quantitatively adding the diluted culture solution containing the bacteria into each hole of a 96-hole plate respectively; samples were prepared at 1mg/mL and dosed individually to each well. After the addition, the mixture is placed in a constant temperature incubator for constant temperature culture for 48h at 28 ℃, and then the absorbance of each hole is measured by a microplate reader at 630 nm. Test results show that the compound 1 and the compound 2 have obvious inhibitory activity on 9 plant pathogenic fungi (cabbage black spots, tobacco black shins, pepper anthracnose, rice flax leaves, kumquat husks, sugarcane pineapples, corn large spots, tea wheel spots and tribute citrus chain lattices).

Testing of minimum inhibitory concentration:

diluting the cultured plant pathogenic fungi with culture solution with the dilution ratio of 1:500-1: 1000; quantitatively adding the diluted culture solution containing bacteria into each first row of wells of a 96-well plate respectively, and diluting downwards for one time; each sample was then added to each row of wells separately. After the addition, the mixture is placed in a constant temperature incubator for constant temperature culture for 48h at 28 ℃, and then the absorbance of each hole is measured by a microplate reader at 630 nm. The results are shown in Table 2.

TABLE 2 minimum inhibitory concentration of trihydroxybenzoic acid-substituted monoterpene glycosides in melaleuca alternifolia against 9 plant pathogenic fungi

Test results show that the trihydroxybenzoic acid substituted monoterpene glycoside compounds have obvious and broad-spectrum activity of resisting plant pathogenic fungi. Both compound 1 and compound 2 showed broad spectrum antibacterial activity and most showed activity higher or comparable to the positive control. Wherein the overall antibacterial effect of the compound 1 is better than that of the compound 2, and the minimum inhibitory concentration of the compound 1 to cabbage black spots, pepper anthrax, kumquat shapi and tea leaf spots is as low as 12.5 ug/ml.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. The application of the trihydroxybenzoic acid substituted monoterpene glycoside compounds in resisting phytopathogens is characterized in that the trihydroxybenzoic acid substituted monoterpene glycoside compounds have the following structural formula:

wherein R is₁、R₂、R₃、R₄Are each independently selected from-H or 3,4, 5-trihydroxybenzoyl, and R₁、R₂、R₃、R₄At least one of them is 3,4, 5-trihydroxy benzoyl.

2. Use according to claim 1, wherein R is₁Is 3,4, 5-trihydroxybenzoyl, R₂Is 3,4, 5-trihydroxybenzoyl, R₃is-H, R₄is-H; the structural formula is as follows:

3. use according to claim 2, wherein R is₁Is 3,4, 5-trihydroxybenzoyl, R₂is-H, R₃is-H, R₄is-H; the structural formula is as follows:

4. use according to claim 1, wherein the phytopathogen is a fungus.

5. The use according to claim 4, wherein the plant pathogenic bacteria are cabbage black spot pathogenic bacteria, tobacco black shank pathogenic bacteria, pepper anthracnose pathogenic bacteria, rice flax leaf disease pathogenic bacteria, kumquat sand disease pathogenic bacteria, sugarcane pineapple disease pathogenic bacteria, corn northern leaf spot pathogenic bacteria, tea round spot pathogenic bacteria or alternaria gongganii.