JPS60243016A - Anti-influenza virus agent - Google Patents

Abstract

PURPOSE:To provide an anti-influenza virus agent containing 3,5-dicaffeoylquinic acid as an active component. CONSTITUTION:The objective anti-influenza virus agent contains the compound of formula I (both R may be H, or one R is caffeoyl and the other is H) [white powder; specific rotation, [alpha]D<20>=-158.2 deg. (c=0.55, MeOH)[. Dose: 2-20mg/kg daily in 2-3 divided doses by oral administration. It can be used in the form of tablet, syrup, capsule, suppository, powder, granule, injection, etc. The compound of formula I can be prepared by extracting the seed of sunflower seed with methanol, and purifying the extract by counter-current fractionation.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anti-influenza virus agent. One relates to an anti-influenza virus agent containing a compound represented by a cafnioyl group: H 2 as an active ingredient.

Cold epidemics occur every year in both urban and rural areas. However, prevention of influenza virus infection, which is the main cause of the common cold, must rely on vaccines, and in that case, it is difficult to do so without knowing the prevalent virus species. However, effective and safe drugs for colds caused by the influenza virus have not yet been widely available, and the only treatments currently available are symptomatic treatment with antipyretic and antiinflammatory analgesics and secondary infection treatment with antibiotics. Therefore, it is desired to develop a drug that is truly effective and safe for the common cold.

In view of the above circumstances, the present inventors aimed to find substances that can be used for the treatment of common cold among the ingredients of Japanese and Chinese medicine that have been used for many years, and in particular to find compounds that exhibit effective medicinal effects against influenza viruses. We have been conducting extensive research with this goal in mind. As a result, the above-mentioned formula (I)
We discovered that the compound represented by has an anti-influenza virus effect, and as a result of investigating and examining its safety etc.
The present invention was completed by confirming that the anti-influenza virus agent has remarkable efficacy against the common cold.

In the above formula (I), two R are hydrogen atoms, and R at the 5th position is a caffnioyl group,
3, 5. in which R at the 4th position is a hydrogen atom; -Cicaffeoylquinic acid is a known compound as described in the following literature, and is a substance that widely exists not only in Japanese and Chinese medicine but also in the plant kingdom.

chlorogenic acid
): THE MERCK NDKX-TENTHED
I: Tl0N, Page 3013.5. -dicaffeoylquinic acid (3,5,-dicaffe-○ylqui
nic acid): Dictionary of Intermediate Dogs (Supplementary Edition) Chemical Component Index 442 Monocyclocinic acid is a currently commercially available compound, which can be purchased, for example, from Aldrich in the United States. Also, 3,5. −',; Caffeoylquinic acid is derived from sunflower (
It is known that it can be obtained by purifying the methanol extract of Helianthus annuus) seeds by subjecting it to a countercurrent distribution operation (K, L, Mikol
ajczak, C.R., Sm1th, Jr., and
1. A.

Wolff Jpirnal Of Agriculture
ural and, FoodChemistry7.
Vol, 18. No], ]27-32, (
1970) l. Also, 3,5. - Another method for producing cicaffeoylquinic acid is as shown in the following specific example.

Specific example: Honeysuckle (Lonicera japonica TH) of the Caprifoliaceae family.
LONICERAE, which is the flower bud of UNB,
20 t of methanol was added to 3 kg of FSO8) powder, and the mixture was extracted under reflux under heating for 6 hours. After cooling, the extract was filtered.

Further, 15 t of methanol was added to the residue and extracted under heating under reflux for 4 hours. After cooling, the extract was filtered through JF5. The same operation was repeated 9 more times, the extracts obtained from a total of 3 extractions were combined, methanol was distilled off under reduced pressure, and methanol extract 1
．． 13Kg was obtained. Next, this methanol extract 350
~4007 was divided into three portions, each of which was dissolved in 1.5 t of water, distributed three times with 1.5 t of ether, and then the aqueous layer was further partitioned three times with 1 t of n-butanol. This n-
N-butanol was distilled off from the butanol layer to obtain n-butanol extract k with a total amount of 221.7. 97.4f of this n-butanol extract was subjected to preparative high performance liquid chromatography [equipment: Waters prep LC/s
system 500A+ column: manufactured by Waters
Prep PAK-500/C18 (5x 30z)
2, detector: differential refractometer (R1:), mobile phase solvent: water-methanol (2:1), mobile phase flow rate: 150 m1/m
in] and the n-butanol extract was added to the mobile phase solvent 10
After injecting the solution dissolved in 0 mg in two doses, 40
The fraction eluted between minutes and 50 minutes (fraction name).

LBu 3 ) was separated and the solvent was distilled off, and LBu 3 was
．． You get 9q.

LBu 3 was subjected to preparative high performance liquid chromatography again (the equipment, conditions, etc. were the same as above, except that the number of columns used was one), and the part eluted 12 to 30 minutes after injection was analyzed. (Fraction name: LBu33) was fractionated and the solvent was distilled off to obtain q and 4q of LBu 33.

Next, LBu 33 is about 2.3 W ~ 2.5! Divide into 4 times of i' each time, and apply each to column chromatography using Sephadex LH-20 manufactured by Farmana (column size: 27wnφX 85 ynm),
Elute with water-ethanol (7:3) and reduce the solvent to about 2J!
.

Fraction 1 was collected from the fraction eluted after the flow, and the solvent was distilled off to obtain a total of 6.17 g of white powder.

The properties of the substance obtained here are FD-MAEI, U
V absorption, R absorption, ``H-NMR, 13C-NMR
3,5. -Identified as cicaffeoylquinic acid.

The physical property values are shown below.

3.5. -Cicaffeoylquinic acid properties: white powder specific rotation C (fJ'i, 0 (c = 0.55. MeoH
): -15B, 2°FD-MAS vector (m/z): 517 (M"+1) UV absorption vector λmax, 332 nm (e ~ 287
00 ) IR absorption spectrum/l/I/ max ((
yH-") 3100-3600.1700.1690
．． 1600.15201H,-NMR spectrum/l/
(in C,,D5N')δI)pm:3.02(
4H, m), 4.68 (IH, da, J=5.31z
), 6.18 (2H,m), 6.48 (2H
, cl, J=16Hz), 6.8~7.4 (6H
), 7.86 (I H, d.

J = 1.6 Hz), 7.94 (I H, d,
J = 16FIz) 13C-1jMRs, 2 ctors (
in CD30D) δppm: 36.0(t),
37.8(t), 70.9(d), 72.0(d
).

72.6(d>, 74.8(S), 115.1(d
), 1. , j5.3(d), 115.4(d),
11.5.6(d), 116.6('1).

1.23.0((1), ], 23.1((1), 127
．． 8(S), 127.9(s), 146°6(S), 1
47.0(d), 147.1(d).

149.3(8), 149.4(S), 168.5(S
), 1G8.900.177.4(s) Next, an experimental example will be shown showing that the compound of formula (I) shown above exhibits an antiviral effect, and its safety will be described.

Experimental Example 1 (About the influence on the survival rate of animals infected with influenza virus) Influenza virus (A/PR/8/34/HON] strain) was administered to cLdY SPF mice (♀: 1 group).
The amount equivalent to LD8o was intranasally infected. The above formula (I
) was orally administered once a day for 5 consecutive days starting one day before infection, and the condition of the mice was observed for 14 days using survival as an indicator. The results are shown in Table 1 and FIGS. 1 and 2. In addition,
A commercially available chlorogenic acid was used by dissolving it in sterile purified water, and sterile purified water was administered as a control. Further, 3,5°-cicaffeoylquinic acid was prepared by the same method as in the specific example and dissolved in 0.5% ethanol, and 0.5% ethanol was administered as a control.

(H2O), 3 g, 3 Chlorogenic acid 0.002 9 35.3 11.1 0.02 9 35.311.3 Control (0.5% C2H50H) 6 9.83.5-Cicaffeoylquinic acid\ 0.002 8 14.3 ], 0.6 ・0.02
11 35.7 11.8 Experimental Example 2 (About the effects on the lungs of animals infected with influenza virus) (Influenza virus (A/PR/8/34/HON 1 strain) was administered to ldY strain SPF mice (10 mice in group 91).
The amount corresponding to LD8o was infected through the nose. The above formula (I
) was orally administered once a day for 5 consecutive days starting 1 day before infection, and 4 days after infection, the mice were sacrificed and the lungs were removed. The lung index was calculated from the weight. (As the infection progresses, the influenza virus causes complications such as pharyngobronchitis and viral pneumonia, causing considerable inflammation in the lungs, making gas exchange difficult, and leading to death. Reducing the lung wet eJ amount (lung weight) means improving the symptoms of influenza virus infection, and at the same time, the value of the lung index, which is the ratio of body weight to lung weight, is lower than that of the control. (This also means improvement in symptoms of influenza virus infection.) The results are shown in Table 2.

In addition, sterile purified water was administered to the control group.

Table 2 Control 0 1.359±0.196 - Chlorogenic acid 0.002 1.169±0.316. 14.00.
02 1.240±0.247 8.8*1:m index=
"ri,=<#:!i(5') x 1o.

Mouse body weight (1) Lung index of drug administration group *2: Lung index inhibition rate - (1 - group. - 1 group. index)
As shown in the results of x 100 or more, the drug of the present invention can significantly suppress death of rats due to influenza virus infection. In addition, it is known that chlorogenic acid does not cause any death even when administered intraperitoneally to rats at 400 to 2437 mg.
OLOGYAND PHARMACOLOGY'36
.

227-237 (1976) l, also the formula (Il
This compound is found in many plants that are used as food on a daily basis, so it is highly safe. Furthermore, chlorogenic acid and 3,5. - No deaths were observed even when LOOOmg/kg of cicaffeoylquinic acid was orally administered to mice. From the above, it was confirmed that the compound used in the present invention has lower acute toxicity than the effective dose for anti-influenza virus activity, is highly safe, and is extremely useful as an anti-influenza virus agent.

Next, from the data obtained in Experimental Examples 1 and 2, the compound used in the present invention is administered at a daily dose of about 2'
It seems appropriate to take an amount equivalent to mg to 20 μg/kg orally in 2 to 3 divided doses a day.

The compounds used in the present invention can be used in the form of pharmacologically acceptable salts.

The compound represented by the above formula (Il) or its salt can be used alone or in combination with other pharmaceutically active ingredients.For formulation, excipients, binders, lubricants, lubricants, etc. may be used as appropriate. It can be formulated into dosage forms such as tablets, syrups, capsules, suppositories, powders, granules, and injections using agents, flavoring agents, disintegrants, or solubilizing agents.

Next, an example of manufacturing the preparation will be shown.

Production Example 1 Commercially available chlorogenic acid 209'' is finely powdered, and lactose 1
757 and magnesium stearate 57;
This mixture was compressed into tablets with a diameter of 2 mm using a single-shot Slang tablet press.
A slug tablet having a size of 1 Jmm and a weight of about 2.37 mm is made, which is crushed with an oscillator, sized, and sieved to obtain granules with good cut-to-blade mesh particles. This granule contains 100 m9 of chlorogenic acid in 12.

Production Example 2 3, 5 obtained by the same method as the above production example
．． - Add and mix 100 f of cicaffeoylquinic acid, 907 microcrystalline cellulose, and magnesium stearate IOf, and compress this mixture into tablets with a single-shot tablet machine to form tablets with a diameter of 9.
M, tablets weighing 200 cm are prepared.

This drug is 3.5% in one ship. -〇6 phenylphohydric acid 10
Contains 0■.

Production Example 3 Commercially available chlorogenic acid 10? is dissolved in 1 ton of distilled water for injection according to a conventional method for manufacturing injections, and after being strengthened with sodium chloride, the mixture is sealed in an ampoule.

This injection contains 10 m7 of chlorogenic acid in 1 ml.

Production Example 4 3 + obtained by the same method as the above production example
5.) Cafnioylquinic acid 1007 is made into a fine powder,
Capsules were obtained by filling 200 μm into hard capsules.

This capsule contains 3,5. - Contains 200 my cicaffeoylquinic acid.

Production Example 5 10 g of commercially available chlorogenic acid is dissolved in as little water as possible, and 5 W/' of Orange Essence and simple syrup are added thereto to make a syrup of 1 OOWl.

This syrup contains 100 m9 of chlorogenic acid in 1 me.
contains.

[Brief explanation of drawings]

Figure 1 shows the results of Experiment 1 mentioned above (effect of chlorogenic acid on survival rate of influenza virus infected mice).
FIG. 2 similarly shows the results of Experimental Example 1 (effect of 3,5.-cicaffeoylquinic acid on survival rate of mice infected with influenza virus). In FIGS. 1 and 2, ↓ indicates administration of the test drug orally, and influenza virus infection was performed on the day corresponding to O of the number of days after infection on the horizontal axis. Patent applicant Jun Tsumura Co., Ltd. Attorney Tendo Patent attorney Takao Minami ′

Claims (1)

[Scope of Claims] An anti-influenza virus agent characterized by the formula: (In the formula, both R's are hydrogen atoms, or one of them is a cafnioyl group: H.