JPS617290A - Triterpene-type saponin compound - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to triterpene saponin compounds.

In the process of searching for physiologically active substances contained in various plants, the present inventors discovered that certain compounds contained in camellia leaves have excellent antibacterial activity against harmful microorganisms such as various plant pathogens. The present invention was completed based on this knowledge.

That is, the gist of the present invention is [wherein R1 represents a hydrogen atom or an acetyl group, and R2 represents 1
A sugar chain consisting of a molecule of glucuronic acid, one molecule of glucose, and two molecules of galactose, and in which glucuronic acid is directly bonded to an aglycone. ] It consists of triterpene saponin compounds represented by

To explain the present invention to 1iP4111, the compound of the present invention represented by the above formula
and its specific rotation, IR absorption spectrum, and mass spectrum.

“H-NMR and”C! -NMR measurement results are as shown in Examples below. If the compound of the present invention is hydrolyzed, for example, in a mixture of dilute sulfuric acid and dioxane, cameliagenin (Oame
lliagenin) F [Refer to Proceedings of the 1988 Annual Meeting of the Pharmaceutical Society of Japan, p. 256 6G4-2 (1968)]
is obtained. Furthermore, from the results of thin layer chromatography and gas chromatography of the trimethylsilylated sugar moiety, the sugar moiety from which cameliagenin F was removed from the hydrolyzed product was found to contain one molecule of glucuronic acid, one molecule of glucuronic acid, and one molecule of glucuronic acid.
It was shown to consist of one molecule of glucose and two molecules of galactose.

Furthermore, among the compounds of the present invention, when a compound in which R8 in the forepaw CI) formula is an acetyl group is heated under reflux in pyridine and acetic anhydride, [R in formula 13 and R2
A diacetate compound in which both are acetyl groups is produced. This revealed that glucuronic acid was directly bonded to the aglycone, and the structure of the compound of the present invention was confirmed.

The compounds of the present invention can be obtained by, for example, shredding camellia leaves and extracting them using water or an appropriate organic solvent, and using the resulting extract with an appropriate adsorbent and eluent, as detailed in the Examples below. It can be isolated by fractionating it by column chromatography using a reagent, collecting an active fraction, and repeating the purification process.

As shown in the test examples below, the compound of the present invention exhibits excellent antibacterial activity against various plant pathogenic fungi such as tea anthracnose, tea leaf spot, rice blast, and rice sesame leaf blight. Therefore, it is expected to be used as a control agent for these harmful microorganisms. The present invention will be explained in more detail below with reference to Examples and Test Examples.

Example 1 3.2 kg of leaves of Camellia japonica were ground into pulp (2,120°C) in a mixer with 10 times the amount of water.
After heating for 5 minutes, filtration was performed to obtain an extract having a concentration of 25.6%.

3.24 each of this extract, 60 g in dry form, in Ol-form I)]]1iAE-cellulose-filled 5,0
The active ingredient was eluted by applying it to a cIILX 45.0 cm column and flowing 4 e of I M NaCl aqueous solution as an eluent. From this active ingredient aqueous solution containing Na (Jl), the active ingredient was taken as 1 n-butanol, -l, and concentrated to dryness under reduced pressure to form n-butanol, ethanol, and
It was redissolved in water (60:20:20) to obtain a solution at a total temperature of 400°C. 2, Ocrr filled with 150 g of silica gel (suspended in n-butanol) to 20 ml of this solution.
of n-butanol 2
401, then 500 ml of the above n-butanol/ethanol/water mixture was poured at a rate of 4 me 7 minutes, and 37
The mixture was divided into two portions, and 25 to 350 portions containing the active ingredient were collected and concentrated to dryness under reduced pressure. Add the above n-butanol/ethanol/water mixture 1. Dissolved in Qmt, each 100
μl was subjected to high performance liquid chromatography using a preparative octadecylsilane (ODS-Olg) column of 1.0 cnL x 25 crfL.
6 each using acetic acid (37.0:62.9:0.1) as the eluent. The compound of the present invention (forepaw [compound in which R1 in formula 13 is an acetyl group]) and compound B (forepaw [compound in which R1 in formula 13 is a hydrogen atom]) of the present invention were isolated.

These compounds were analyzed by various thin layer chromatography [silica gel (chloroform:methanol:water=65:3
5: 10. Lower layer: n-butanol: 1 acid: 4 water
The results of measurements by high performance liquid chromatography (ODS-01g, 5μ, methanol:water = 65:35) revealed that it was a pure product.

Note that the measurement of physiological activity during the separation and purification process in this example was carried out in accordance with the test methods described in Test Examples 1 and 2 below.

Specific rotation, IR spectrum, mass spectrum, "I(-NMR and 130-
The NMR measurement results were as follows.

Compound A: [(χ] name 5+2°(c O-5,0R40
H) i IR(K]3r) 3600-3200.1
710cm; fiIMs m/'L1191 (M
+2Na-E), 1169 (M+Na), 11
029(163+2Na), 867(M-325+2
Na), 727 (M-442+-Na), 565 (M-
604+Na), 365(0,IIH,.Ol,
7 Na) i'H-NMR (500RfH2
,P'Y-ds'D20'' 0.4:
0.05Id, +100℃) δ0.901 (3H,s
), 0.918(3)L s ).

1.00 (3H,s), 1.094 (3H,s)
, 1.209 (3H,s).

1.289 (3H, s), 1.296 (3H, s)
, 2.147 (3■1.s).

2.410 (IH, at, , T = 13,311z), 3
．． 026 (IH, dt, J = 13.31h), 3.2
84 (IH, m), 3.580 (IH, m), 3.69
2 (IH, m), 5.603 (IH, 6, J = 7.61
(z), 5.637 (IH, d.

J=7.6Hz), 5.678 (III, d,
J=3.711z) i”O-NMR(22,5MII
z, Py-aa' D20 =0.4: 0.1
5wLl.

+100°C) δ14.93. 16.34, 17
．． 33, 18.09.

20.38, 23.42, 25.82,
26.47, 27.87.

30.33, 30.51, 32.09,
33.03, 36.66.

38.53, 39.06, 40.05,
46.73, 47.73.

47.84, 52.41, 55.81,
61.55, 62.01.

63.83, 69.16, 69.39,
69.68, 70.68.

70.01, 72.85, 73.84,
74.66, 75.83.

76.36, 76.60, 77.00, 7
B, 70, 79.41.

80.48, 82.39, 89.77,
90.18, 100.37.

102.36. 104.35, 124.26
, 141.42, 171.86°175.03.
215.61.

Compound B: [aa1. -6°(co, 5. On'
60IJ) i IR(K]3r)3600~32o0
, 171ocTL-1; SIMS m/z 114.
9 (M+2Na-II), 1127 (M+Na
), 987 (Rt163+2Na, ), 825 (M-
325+2Na), 685(M-4421-Na),
523 (M 604+Na), 365 (0+JT9*
'H-NMR (50ON[Iz, py-d5+ +1
00'C) δ0.908 (311°s), 0.94
6(3)1. s), 0.996 (3E1.s),
1.113 (3B, 3).

1.219(3I-1', s), 1.27(1(
3H,s), 1.304(3H,s).

2.415 CITJ', dt, ,■=13
．． 3Hz), 3.00 (IH,brd,
J = 13Hz), 3.30 (In, m),
3.92 (IH, d, J=15.5Hz) i' 8
Q-NMR, (22,5Fllllz, Py-65,
+1.00'C) δ15.69.

δ7. ]6. J, s, o4. 18.9], ,
24.13, 27L35.

28.69, 31.04, 31.68, 3
2.79, 33.73.

37.42. Ko (9, 76, 40, 70, 48,
43, 53, 23.

56.45, fi2.48, 63.01,
70.33, 70.91.

71.15, 71.91, 73.73, 7
3.96, 74.60.

75.01, 75.42, 76.19, 7
6.30, 76.71.

78.00, 79.29, 81.9B,
84.03, 90.30.

101.66, 103.12, 105.05, 1
05. Visit, 124.50.

142.53, 175.79, 213.97° Compound A of the present invention was mixed with 7% sulfuric acid/dioxane (3:1
) After heating under medium-speed flow for 3 hours, the chloroform layer was separated from the aqueous layer, the solvent was removed, and the resulting residue was subjected to instrumental analysis and was found to be the same as the known cameliagenin r.

On the other hand, barium hydroxide was added to the aqueous layer after removing the chloroform layer to neutralize it, and the sugar portion was evaporated to dryness.
:1 :10) and allowed to stand at room temperature for 30 minutes to obtain a trimethylsilyl ether. Standard solutions of glucose and galactose at the same concentration were similarly trimethylsilylated, respectively, and the results of gas chromatography and GO mass spectrometry measurements were compared. It was identified as 7.

In addition, after heating Compound A under reflux in IN sulfuric acid/dioxane (1:1) for 1 hour, the sugar moiety was prepared by the above method, and thin layer chromamigraphy (Avicel, ethyl acetate, pyridine, acetic acid, The glucuronic acid was identified by comparing it with the glucuronic acid of the sample using water (5:5:1:3).

Furthermore, the compound A was mixed with acetic anhydride/pyridine (1:l).
When the mixture is heated under reflux for 1 hour, an acetate of the aglycone represented by the formula in which the sugar moiety is substituted with an acetyl group is obtained.

The facts are IR (KBr) 1735, 1710
am' iMS m/z 526;'H-NM
R(90MHz, CD(1!t8)δo, 87(91
(,brs), 0.96(3H,s), 0.98(
3H,s), 1.07(3,Tf,s), l
-16 (3H,s), 2.03 (3i4.s
), 2.05 (3H, s).

4.49 (iiL brt, J=8Hz), 5.
42 (LH, t, J=3Hz) etc., it is certain that
It was done. Therefore, it was confirmed that glucuronic acid was directly bound to the aglycone. [Chemical Ph
armaceutical Bulletin.

25, p. 1408 (1977)] Example 2 After freezing 7.5 to 9 leaves of Camellia japonica (Otome Camellia) in liquid nitrogen, they were crushed, and then extracted with 201 methanol. The obtained extract was concentrated under reduced pressure to remove methanol and obtain 600 g of extract.

The obtained extract was concentrated under reduced pressure to remove methanol.
001 extract was obtained. Divide this extract into 5 equal parts, 400
．． 5,6t1m×35.lit filled with silica gel.
Apply to 5 columns of 0cIIL and elute with ethyl acetate/ethanol (increasing methanol by 10% for each C31) as eluent, and collect the eluted fraction of ethyl acetate/methanol (60:40) containing the active ingredient. and concentrated. Next, this was applied to a silica gel packed column of the same capacity as above, eluted using chloroform/methanol/water (65:35:10, lower layer) as an eluent, and divided into 22 portions of 11 each. From this fraction, 9 to 120 fractions containing the active ingredient were collected and concentrated under reduced pressure.
．． A residue of 625, lit was obtained. This residue was dissolved in methanol and reprecipitated to obtain a precipitate of 2.1289.

The above precipitate was further prepared in a similar manner to form a 3.5 cm X i 00. Add chloroform/methanol/water (65:35:
10. The lower layer) was fractionated into 100 ml portions each as an eluent, and the compounds of the present invention A (a compound in which R1 in the front foot [I) formula is an acetyl group] and compound B (a compound in which RQ in the front foot [formula 13] is a hydrogen atom) were obtained. The husband was isolated.

Note that the measurement of physiological activity during the separation and purification process in this example was carried out in accordance with the test methods described in Test Examples 3 and 4 below.

Specific rotation i, IR of compound A and compound B obtained here
Spectrum, mass spectrum, "H-NMR and"
The 0-NMR measurement results were as in Example 1.

Test Example 1 Tea anthracnose fungus spore germination inhibition test 50 μl of the aqueous solution of Compound A in Example 1 at a predetermined concentration on a whole glass, and tea anthracnose cultured in Chaka culture medium (adult leaves of tea leaves directly high-pressure sterilized). Disease germs (Gloeospo)
50 μl of a suspension of S. rium theaesinensis) spores in a liquid potato sucrose medium was dispensed, mixed, and cultured in a greenhouse at 24° C. for 24 hours. The number of germinated spores and the state of germination were measured using a microscope.

The results were as shown in Table-1.

The degree of spore abnormality is 1 to 5 (5 is the strongest), which is the degree to which spores expand abnormally due to the action of compound A and hyphae do not grow normally.
Shown separately.

Test Example 2 Tea leaf spot fungus spore germination inhibition test Char leaf spot cultured on a whole glass with 501 tll of aqueous solution of Compound A in Example 1 at a predetermined concentration and chaka culture medium (adult leaves of tea leaves sterilized under high pressure) Disease germs (Pe5tal
otia longiseta) spores in a liquid medium of potato sucrose was dispensed, mixed, and cultured for 24 hours in a greenhouse at 24°C. The number of germinated spores (1) was measured using a microscope.

The results were as shown in Table-2.

(Note) A. The number of spore germination and spore germination rate indicate the number and rate of normal spore germination.

The degree of germ tube abnormality is determined by the action of Compound A, which causes germ tubes to become abnormally V-shaped and fail to grow normally.
Shown separately.

Test Example 3 Rice blast fungus spore germination inhibition test 50 μl of the aqueous solution of Compound A in Example 2 at a predetermined concentration was placed on a whole glass, and 1 of rice blast fungus (Pyricularia oryzae) cultured in an oatmeal medium was placed on a whole glass.
After dispensing and mixing 50 μl of a spore suspension containing % glucose, the mixture was cultured in a groove chamber at 27° C. for 12 hours, and the number of germinated spores was counted using a microscope.

The results were as shown in Table-3.

Table 3 (Note) A. Number of spore germination and spore germination rate indicate the number and rate of normal spore germination.

Test Example 4 Rice Sesame Leaf Blight Spore Germination Inhibition Test 50 μe of the aqueous solution of Compound A in Example 2 at a predetermined concentration was placed on a pole glass and a rice sesame leaf blight fungus (0OCchli-obolus m1) was cultured in a potato dextrose medium.
After dispensing 50 μe of a 1% glucose-containing spore suspension of P. yabeanus and mixing, it was heated in a groove chamber at 27°C for 12 hours.
After culturing for an hour, the number of germinated spores was counted using a microscope.

The results were as shown in Table-4.

Table-4 Show rate.

-15=

Claims (1)

[Claims] (1) The following formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R_1 represents a hydrogen atom or an acetyl group, and R_2
indicates a sugar chain consisting of one molecule of glucuronic acid, one molecule of glucose, and two molecules of galactose, and in which glucuronic acid is directly bonded to an aglycone. ] A triterpene saponin compound represented by