JPH02270824A - Reverse transcriptase inhibitor - Google Patents

Abstract

PURPOSE:To obtain an activity inhibitor of reverse transcriptase of retrovirus, having high safety, low side effects, inhibiting and suppressing intracellular multiplication of retrovirus, containing spice and a herb extract as active ingredients. CONSTITUTION:1 pt.wt. spice or herb is extracted with 10-1,000 pts.wt. distilled water it 50-100 deg.C for 0.5-5 hours, the extract is used as an active ingredient and prepared by a conventional procedure to give the aimed substance. The extraction can be carried out by using a water-miscible organic solvent such as methanol, ethanol, acetone or ethyl acetate alone or a mixture thereof with water or n-hexane, chloroform, ether, etc. Spice widely used in general food processing and cooking is used as the spice and plants having well-known medicinal effects and fragrance are cited as the herb.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a retrovirus reverse transcriptase inhibitor containing extracts of spices and herbs as active ingredients. This reverse transcriptase inhibitor is used to suppress and prevent the intracellular proliferation of retroviruses by inhibiting the activity of reverse transcriptase (RNA-dependent DNA polymerase) of retroviruses that infect living or animal cells. be done.

Technical background Retroviruses have a diameter of 11 mm and have RNA as their genes.
It is a spherical virus of around 00n. Infection occurs when the viral glycoprotein envelope binds to and fuses with host cell receptors, allowing viral RNA and reverse transcriptase to enter the cell. and own RN
The means of self-replication is to use A as a template and use the host's gene synthesis system to synthesize DNA using reverse transcriptase.

Retroviruses host various animals such as humans, mice, guinea pigs, birds, pigs, and monkeys, and cause sarcoma, leukemia, and carcinoma. In particular, in humans, LAV (
Lymphoadenabsi associitenodovirus) and H
Human immunodeficiency virus (HIV), including TLV (human tea lymphotropink retrovirus)
is known to cause severe immunodeficiency.

To date, methods for preventing and treating human immunodeficiency virus infection and onset include neutralizing antibodies, anti-idiotype antibodies, blocking of virus binding with dextrin sulfate, and 3-azido-2,3-dideoxythymidine (AZT).
) and glycyrrhizin derivatives, etc., and research has been conducted on the inhibition of DNA synthesis using reverse transcriptase inhibitory effects. In addition to this, research has been conducted on methods of inhibiting virus binding using soluble CD4, which is a genetically engineered version of the cell receptor CD4, and on the use of α-interferon to suppress virus budding. It is still possible to manufacture industrially to this day, and
No effective therapeutic agent with high safety and few side effects has been developed.

Is the unique DNA of the virus completely unrelated to host cell functions used to prevent and treat the onset of retroviruses? The most effective method is to inhibit the Ji production process, ie, the activity of reverse transcriptase. AZT is currently widely used for HIV treatment. It is a nucleoside analog and a type of reverse transcriptase inhibitor. However, A.Z.
When T is used in the amount necessary for treatment, it can cause anemia, white blood cells, neutrophils,
It is known to cause serious side effects such as a decrease in platelets.

For these reasons, there has been a strong desire to develop a reverse transcriptase inhibitor that can be manufactured industrially, has few side effects, and has minimal toxicity.

Apart from these, a good reverse transcriptase inhibitor is cD
It goes without saying that it can also be effectively used as a research reagent in molecular biology such as NA synthesis, cloning, and probe preparation.

Problems that Japan is Trying to Solve In view of the above situation, the inventors of the present invention have developed a method for producing heat from food materials that are widely used in daily diet, which are natural materials with high hygiene safety and few side effects. The aqueous extract was examined for its inhibitory activity against reverse transcriptases derived from various retroviruses.

As a result, they discovered strong reverse transcriptase inhibitory activity in extracts of various spices and herbs, leading to the completion of the present invention.

Therefore, by using extracts of spices and herbs used in foods as active ingredients, the present invention provides
An object of the present invention is to provide an inhibitor of retrovirus reverse transcriptase activity that can be industrially advantageously produced and has fewer side effects.

Means for Solving Problems In the present invention, spices used as raw materials are generally used for food processing and cooking, and are flavored spices made from plant seeds, fruits, rhizomes, leaves, flowers, bark, etc. , refers to a food material that has a flavor that is irritating to the sense of smell, imparts flavor to food and drink, and increases appetite.

Herbs refer to aromatic plants (referred to as aromatic herbs or scented herbs) that have been handed down for their medicinal properties, and are obtained in the same manner as described above, and their distinction from spices is not necessarily clear.

Examples of these spices and herbs found on the market are as follows.

Note that many of these are common names and are not necessarily indicated by accurate botanical names.

Hyakumi pepper (Myrtaceae, Pimentali)
, Anise (Apiaceae, Pimpjnel la genus), Ajowan.

Agrimonia (Rosaceae, genus Agrimonia), mebouki (Lamiaceae, genus Ocimum), laurel (Laurinaceae, genus Laurus), Pai leaves (Laurinaceae, La
urus genus), .

Hayberry (Proteaceae, Myrica[), Helhenu, Hymeukyou (Umbelliferae, Carum genus),
Shōzuku (Zingiberaceae, Elettaria genus),
Catfish (Lamiaceae, genus Nepeta), celery seed (Apiaceae, genus Apium), Dutch honeysuckle (Umbelliferae, genus Nepeta)
Apium spp.), Cornflower (Asteraceae, Centa
urea), Chamomile (Asteraceae, Matricart)
a genus), Roman chamomile (Asteraceae, genus Anthemis), Japanese citron (Lauraraceae, genus Cinnamomum), yuzu (Rutaceae, genus Ci trus), clove (
Myrtaceae, Syzygium genus), Syzygium (
Coriandrum (Umbelliferae, genus Symphytum), Coriander (Umbelliferae, genus Coriandrum), Coriandrum (Umbelliferae, C
uminum genus), Anethum (Apiaceae, Anethum
genus), Eucalyptus (Myrtaceae, Euca I yp
tus spp.), Baswell
ia), fennel (Apiaceae, Foeniculu)
M genus), fenugreek (Fabaceae, Frjgonella genus),
Gardenia (Rubiaceae, genus Gardenia), garlic (Liliaceae, genus AAl11u), ginger (Zingiberaceae, genus Zingiber), hops (Moraceae, Ilumu
lus), wasabi radish (Brassicaceae, Armor
acia), hibiscus (Malvaceae, 1lybis)
(genus Cus), Salix spp.
s genus), Japanese pepper (Rutaceae, Xanthoxyl
um), Juniper (Cupressaceae, Juniperu
s genus), Alchemilla (Rosaceae, Alchemilla
genus), lavender (Lamiaceae, genus Lavendula)
, lemon balm (Lamiaceae, phylum Elissa genus), mellisosa (Lamiaceae, genus Mwl 1ssa), lemon balmna (Lamiaceae, phylum elissa),
Verbenaceae, Aloysia genus), lemongrass, lemon peel (Rutaceae, Ci trus genus), Verbenaceae, Ti1ia genus), loquat (Rosaceae, Er
1obotrya genus), ruskbar, rubia (Rubiaceae, Ru former genus), Malva (Malvaceae, Malvaceae)
va genus), mallow (Malvaceae, Malva genus),
Marigold (Asteraceae, genus Calendula), Japanese oak (genus Origanum, genus Origanum), genus mustard (genus Brassica, genus Brassica), genus Myristica, genus Myristica), orange (genus Rutaceae, C
Ci trus genus), Chimpi (Rutaceae, Ci trus genus), Hanahasoka (Lamiaceae, Origanum genus),
Capsicum capsicum (Solanaceae, Capsica)
m genus), Capsicum (Solanaceae, Capsicam genus),
Dutch celery (Umbelliferae, Petroselium genus)
, Patchouli (Lamiaceae, Pogos temon genus)
, Peonies (Paeoniaceae, genus Paeonia), Beni-Royal (Lamiaceae, genus Mentha), Blackberry (Familyaceae, genus Piper), Green besover (Familyaceae, genus Piper), White venopa (Familyaceae, genus Piper) ), No1 Nassho (Rutaceae,
Xanthxylum (genus Xanthxylum), Papaver (genus Mentha), Papaver
genus), red clover (Fabaceae, Trifolium
), rose buzz red, rose pink, rose red, rosemary (Lamiaceae, genus Rosemarinus), safflower (Asteraceae, genus Carthamus), saffron (Iridaceae, genus Crorus), salvia (Lamiaceae, genus 5alvia), Sandalwood (Salmonaceae, Sa
ntalum spp.), Seipori (Lamiaceae, 5ature
ia), sesame (Lamiaceae, Seamum genus), Oriental pepper (Lamiaceae, Mentha genus), magnolia (Magnoliaceae, Illicium genus), wheat grass (Asteraceae, Helchrysum genus), Lucian tarragon (Asteraceae, Altemisia genus) , Estragon (Asteraceae, Al temisia genus), Thymus (Lamiaceae, Thymus genus), Turmeric (Zingiberaceae, Carum genus), Vanilla (Fabaceae, Vanill)
a genus), wasabi (Brassicaceae, Wasabia genus), white oak (Fagaceae, Qnercus genus), mugwort (Asteraceae, Altemisia genus), yarrow (Asteraceae, Ac
11ea), five-spice powder (a mixture of cinnamon, cloves, chimpi, fennel, chives, star anise, and veliper), bouquet garni (a mixture of oregano, rosemary, and tarabon), cloves (Myrtaceae, Syzygi)
u...genus), Nutmeg family, Curioaceae, Myristica genus).

In the present invention, spices and herbs prepared from these plants are treated with hot water, or with water-miscible organic solvents such as methanol, ethanol, acetone, and ethyl acetate alone or in mixtures with water, or with n-hexane, chloroform, and ether. Perform extraction using etc.

The amount of solvent and extraction conditions for this extraction may be determined as appropriate depending on the type of spices and herbs and the type of solvent used.

The results of testing the effects of the enzyme inhibitor according to the present invention are shown below as examples.

Example Test Example +1.1 Add 1-1 of distilled water to 2 mg of each sample,
℃ for 1 hour to obtain a hot water extract. This extract was adjusted to pl+ 7.0 with an alkaline solution, and then filtered and sterilized with a 0.2 μm Millipore filter to be used as a sample.

Inhibition of reverse transcriptase activity was measured as follows. Specifically, using Po1y(8) (dT) + s as a template primer, Moloney murine leukemia virus-derived reverse transcriptase (M-MuLV) was added to the reaction solution composition for activity measurement shown in Table 1.
)]μl (1 unit), and at the same time add the above extract solution]/1
After diluting to 0.1/100 and 1/1000, 10% of each reaction solution was added. Poly(A)
(dT) + s as template primer, 3 at 37°C.
After reacting for 0 minutes, trichloroacetic acid was added to the reaction solution at a final concentration of 5%, and the mixture was incorporated into the insoluble fraction (3II).
Enzyme activity was determined by measuring the amount of TTP.

The results are shown in Table 2. In other words, many of the various spices and herbs tested were quite strong in inhibiting the activity of reverse transcriptase, and in particular, agrimoni, mebouki, porphyry, laurel, laurel, cloves, sagebrush, hops, and sardines. , lemon balm,
Even when 1/1000 amount of the extract is added, the enzyme activity is reduced by 22-79% for Bodaiju, Mayonarahanahasoriki, Patchouli, Peonies, Pennyroyal, Japanese Chrysanthemum, Poppy, Rose Buzz Red, Orchid Japonica, Thymus Civet, White Oak, and Bouquet Garni. I can see that it was inhibited.

Table 1 Reaction solution composition for reverse transcriptase activity measurement (llII)
TPP (50μCi/m1) 5μ
ρTPP (5mM) 1
0 μp-TrisllCI (pH 8,0) (0,5
M) 10μ1KCI (
0.5M) 10μpMg(OAc)z
(50mM) +0114! DTT
(30mM) 10p
lNon1det P-40 (1%) 1
0μ+! BS^ (IOmB/++1)
2 p lPo1y(^)(dT) +s
(0.8mg/mf) 10μβRTas
e (3001/300μp) 1μ
p11□0 12μp
Sample 10IiA100
μ1 Table 2 Inhibition activity of retrovirus reverse transcription enzyme inhibition by food spices and herb extracts (%) 1/10 1/100 ]/10001, control
0 0 02,
Hyakumi pepper 24 26
03, Anise Seed 7
8004, Ajowan seeds 3960
5. Agrimoni flowers, leaves, stems 100 7
2 456, Mebouki
97 72 367, Gesokeiju
Leaf 83 67 368, Laurel 97 78 4
09, Pay Leaves 39
15 010, Peiheli-9610061 11, Herhane 75
54 012, Seeds
93 16 013, Shozuku fruit 260014 Shozuku 86 35 0
15. Catfish leaves, flower spikes 100
60 016, celery seed
64 14 0I7 Dutch mitzvah 39 16 01
8. Cornflower petal 7900
19. Chamomile petal 77002
0 chamomile flower 100
12 021, Roman chamomile flower
100 49 022, Roman chamomile 766023, Ninokei cork periderm 70 53 0
24, Nisokei 5
8 58 025, Yuzu
Pericarp 262026, Clove
Bud 96 84 7527,
Chouji 93 95
028, Leaf and stem
99 97 5929, Coriander
Fruit 589030, Coriander
34 1.6
031, Bakin Seed 4300
32, Hakin 69 5
3 033, Inondo
72 19 034, Inondo
Fruits, leaves 46 11 035
, Eucalyptus 93
77 036, Nyukou seeds
6B 1.4 037, fennel seeds 87 17
038, Wikyo 1
1 14 039, Fenugreek
220040, Gardenia fruit 760041, Ginger
80042, hop
Flower 95 77 264
3. Wasabi radish 4840
44, Hibiscus 84
12 045 Willow Hatsuka
611046, Japanese pepper peel
90 73 4847, juniver fruit 95 23 048,
Lahenda flower 100 52
049, Lahenda-6900 50, Lemon Balm 100
97 7251, Merisosa
91 82 052, lemon balmuna leaf 94 20
053, lemongrass
400054, lemon peel peel
50055, Bodaiju leaves, flowers, fruits 9
2 64 4256, Bodaiju
94 47 057,
Loquat 97
50 058, Laxbar flower
470059, Madder root
520060, Usheniaoi
32 21 061, Mallow flower 299062, Marigold petal, bud 480°63, Mayonara leaf 99 79
4464, oran seeds 4
60065, Nikuzuku seeds
180066, Nikuzuku
84 89 067, Nikuzuku rind 90068, orange
Petals, leaves 8 0 069, Hanahanoka leaves, stems 100 71
5270, Hanahasoka 88
68 071, Orisroot
4 0 072, Bachuri
Leaves, stems 99 65 2273
, Peony petals 100
92 4974, Pennyroyal leaves, flowers, stems
97 100' 4275, Green Pesobar Fruit 163°76, Hana Sansho 94 63 2177
, Seiyou Hasoka 100
70 078, poppy petal
93 77 2879, poppy
Seed 110080, Red Clover Flower, Seed 100 50 081,
Rose buzz red flower 93 82
7982, rosehip
92 72 083, rose pink
92 82 084,
rose red 98 9
7 085, rosemary
95 35 086, Henibana 45 11
087, saffron flower 29
19 088, Salvia pistil 91 41 089, Salvia 96 25
090, Sandalwood xylem 1
23091, Sandalwood xylem 85
15 092, Savory-95560 93, Sesame seeds 89
41 094, Oranda leaf
98 88 2295, Danwikyo 89 41096,
Flowers, seeds 97 30
097, Lucian tarragon stem
99 47 098, Estrabon 95 35 099
．． Tachimusou stem 97
82 30100, Tachimusou
86 11 0101, turmeric 50 28
0I03. vanilla fruit 53
19 0104, Wasabi root, stem 1300105, White oak
Kibe 96 100 46106
, Mugwort 97
71 0107, Yarrow flower
100 44 0]08. Five spice powder 97 75 0
109, Puke Garni 97
97 43 Incidentally, all of these spices and herbs are used in normal food processing and cooking, and depending on the characteristics of their active ingredients, they can be fully utilized as ingredients for functional foods. It is.

Test Example (2) The extract and reaction composition obtained in Test Example (1) were used to compare the inhibitory activities against reverse transcriptases of different origins. Specifically, the inhibitory activity of the hot water extract against the reverse transcriptase RAV-II derived from Tosu-Raus-related virus, AMV derived from avian myeloid leukemia virus, and M-MuLV was investigated using four spices. . The results are shown in Table 3.

As is clear from Table 3, hot water extracts from various spices have been found to effectively inhibit reverse transcriptases derived from various retroviruses at low concentrations, and are widely used as antiviral agents. It can be said.

Table 3 Inhibitory activity of spice hot water extract against various reverse transcriptases Inhibitory activity (%) M-MuLV RAV-[I AMν
Clove 98 27 73 Lemon balm 100 44 73
Nutmeg -31,760-Zuresodo
98 34 81 Note) 1 of the hot water extract of each sample (extract obtained from 20 mg/mff1)
A 10% amount of the 0-fold dilution was added to the reaction composition solution.

Table 4 shows the results of an investigation using cloves regarding the effects of extraction conditions and solvent types on the inhibitory activity of these reverse transcriptases. 2.0 m of dry glue robe
Various solvents were added so as to give g/-, and extraction treatment was carried out under the conditions shown in the table. The obtained extract was diluted 100 times and added to the reaction composition solution in an amount of 10%.

As a result, the inhibitory activity against reverse transcriptase was expressed most strongly when hot water was extracted at 100°C for 11 hours.

However, activity was also observed in extracts using water-miscible organic solvents such as ethyl acetate, acetone, and methanol. Furthermore, even the extract using chloroform has 50% of the hot water extract.
% activity was observed, which may indicate the presence of multiple active ingredients with different properties in the extract.

Table 4

Claims (4)

[Claims] (1) A retrovirus reverse transcriptase activity inhibitor containing extracts of spices and herbs as active ingredients. (2) To prepare the extract, add 10 to 1000 parts by weight of distilled water to 1 part by weight of the spice or herb, and add 50 to 100 parts by weight of distilled water.
The retrovirus reverse transcriptase activity inhibitor according to claim 1, which is extracted by heating at a temperature of 0.5 to 5 hours. (3) The extract is one obtained by extracting spices or herbs using a water-miscible organic solvent such as methanol, ethanol, acetone, or ethyl acetate alone or in a mixture with water. An inhibitor of retroviral reverse transcriptase activity. (4) The retrovirus reverse transcriptase activity inhibitor according to claim (1), wherein the extract is obtained by extracting spices or herbs using n-hexane, chloroform, ether, etc.