U.S. patent application number 09/765075 was filed with the patent office on 2002-09-26 for use of tagetes minuta oil and its components as antiviral agents.
This patent application is currently assigned to COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH. Invention is credited to Joshi, Virendara Prasad, Ram, Raja, Sharma, Anupama, Singh, Bikram, Zaidi, Aijaz Asghar.
Application Number | 20020136789 09/765075 |
Document ID | / |
Family ID | 25072569 |
Filed Date | 2002-09-26 |
United States Patent
Application |
20020136789 |
Kind Code |
A1 |
Singh, Bikram ; et
al. |
September 26, 2002 |
Use of tagetes minuta oil and its components as antiviral
agents
Abstract
This invention relates to an antiviral composition for the
treatment of plant viruses comprising an effective amount of
Tagetes minuta oil, its active constituents, Z-.beta.-ocimene and
dihydrotagetone, or any mixture thereof. The invention also relates
to the use of Tagetes minuta oil, its active constituents
Z-.beta.-ocimene and dihydrotagetone, or any mixture thereof for
the treatment of plant viruses.
Inventors: |
Singh, Bikram; (Himachal
Pradesh, IN) ; Joshi, Virendara Prasad; (Himachal
Pradesh, IN) ; Ram, Raja; (Himachal Pradesh, IN)
; Sharma, Anupama; (Himachal Pradesh, IN) ; Zaidi,
Aijaz Asghar; (Himachal Pradesh, IN) |
Correspondence
Address: |
LADAS & PARRY
26 WEST 61ST STREET
NEW YORK
NY
10023
US
|
Assignee: |
COUNCIL OF SCIENTIFIC &
INDUSTRIAL RESEARCH
|
Family ID: |
25072569 |
Appl. No.: |
09/765075 |
Filed: |
January 18, 2001 |
Current U.S.
Class: |
424/764 |
Current CPC
Class: |
A01N 27/00 20130101;
A01N 35/02 20130101; A01N 65/12 20130101; A01N 65/00 20130101; A01N
35/02 20130101; A01N 35/02 20130101; A01N 65/12 20130101; A01N
27/00 20130101; A01N 27/00 20130101; A01N 35/02 20130101; A01N
2300/00 20130101 |
Class at
Publication: |
424/764 |
International
Class: |
A01N 065/00 |
Claims
We claim:
1. An antiviral composition for the treatment of plant viruses
comprising an effective amount of Tagetes minuta oil.
2. An antiviral composition for the treatment of plant viruses
comprising an effective amount of Tagetes minuta oil, and its
active constituents, Z-.beta.-ocimene and dihydrotagetone.
3. An antiviral composition for the treatment of plant viruses
comprising an effective amount of an active ingredient selected
from Z-.beta.-ocimene and dihydrotagetone.
4. Use of Tagetes minuta oil and its constituents, Z-.beta.-ocimene
and dihydrotagetone to inhibit growth poty and diantho virus
groups.
5. Use of Tagetes minuta oil to inhibit growth poty and diantho
virus groups.
6. Use of Tagetes minuta to inhibit the growth of Carnation Vein
Mottle (CaVMV) virus.
7. Use of Z-.beta.-ocimene to inhibit the growth of Carnation Vein
Mottle (CaVMV) virus.
8. Use of dihydrotagetone to inhibit the growth of Carnation Vein
Mottle (CaVMV) virus.
9. Use of Tagetes minuta to inhibit the growth of Carnation Ring
Spot Viruses (CaRSV).
10. Use of Z-.beta.-ocimene to inhibit the growth of Carnation Ring
Spot Viruses (CaRSV).
11. Use of dihydrotagetone to inhibit the growth of Carnation Ring
Spot Virus (CaVMV).
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of Tagetes minuta
oil and its components as antiviral agent. More particularly this
invention relates to identification of antiviral activity of
Tagetes minuta oil. This invention also relates to the use of the
compounds Z-.beta.-ocimene and dihydrotagetone present in Tagetes
minuta oil which are now found to inhibit Carnation Ring Spot
(CaRSV) and Carnation Vein Mottle Viruses (CaVMV).
BACKGROUND OF THE INVENTION
[0002] Carnation Ring Spot (CaRSV) and Carnation Vein Mottle
Viruses (CaVMV) are widespread in carnations and cause appreciable
amount of damage. Production of disease free plants and chemical
control of vectors are the methods employed for reducing disease
incidence in the field (Matthews R. E. F. 1991, Plant Virology,
Academic Press, San Diego, pp.835)
[0003] Carnation Vein Mottle virus (CaVMV) is a member of potyvirus
group, first reported from U.S.A. and is found all over the world
(Kissanis B., 1954, Nature 173: 1097). On natural hosts chlorotic
and darker green spots, flecks and mottling, flower breaking and
malformation symptoms are developed after infection. Virus is
transmitted mechanically, and also by aphid vectors.
[0004] Chenopodium amranticolor, Chenopodium quinoa and Silene
pendule are diagnostically susceptible hosts. On Chenopodium
amaranticolor chlorotic and necrotic local lesions appear whereas,
in Chenopodium quinoa, chlorotic lesions with systemic develop
after inoculation. Chenopodium quinoa, and Dianthus barbatus are
the maintenance and propagation host. It has RNA genone, which is
single stranded and virions found in all parts of the host plant.
(Morgan J. R., Verhoyen M. and Caneghem, G. V., 1996, Carnation
Vein Mottle Potyvirus, In-Viruses of plants, Description and lists
from VIDE database ed. Brunt A. A., Crabtree K., Dallwitz M. J.,
Gibbs A. J. and Watson L. CAB International pp 309-312).
[0005] Carnation Ring Spot Virus (CaRSV) first isolated from
Dianthus species from U.K. by Kassanis belongs to dianthovirus
group (Kassanis B., 1995, Ann. Appl. Biol. 43:103). CaRSV is found
all over the world wherever carnations are grown. Virus is
transmitted mechanically, and by grafting. Vector transmission is
by nematodes. The virus causes leaf mottling, ring spotting, plant
stunting, distortion, and flower distortion in host plants whereas,
in experimentally infected plants chlorotic and necrotic local
lesions, rings and flecks and occasional systemic symptom also
appear.
[0006] Chenopodium amaranticolor, Chenopodium quiona and Vigna
unguiculata are local lesion assay hosts. Virus can be maintained
on Dianthus barbatus, Nicotiana clevelandii and Phaseolus vulgaris.
Virions are isometric, non-enveloped 34 nm in diameter. Gonome of
virus consists of RNA, linear, single stranded. Virions are found
in all part of the host plant. (Termaine J. H. and Moran J. R.,
1996, Carnation Ring spot virus. In-Viruses of plants, Description
and lists for VIDE database ed. Brunt A. A., Crabtree K., Dallwitz
M. J., Gibbs A. J., and Watson L. CAB International pp
309-312).
[0007] The plant volatile oils have been recognised since antiquity
to possess biological activity and a number of plant extracts and
pure isolates have been mentioned as containing substances which
interfere with or inhibit infection of viruses. Some of the
compounds like galangin when used in concentrations ranging form
12-47 .mu.g/ml showed significant antiviral activity against HSV-1
and Cox B1 (Meger J. J. M., Afoloyan A. J., Taylor M. B., Erasmus
D., 1997, Antiviral activity of galangin isolated from the aerial
parts of Helichrysum aureonitens, J. Ethnopharma, 56:165). Plant
Hyptianthera stricta L. is used against Encephalitis causing
viruses pronounced inhibiting activity 75% and 50% CPE inhibition
at 62.5 .mu.g/ml and 15.6 .mu.g/ml against these two viruses
(Saxena G., Gupta P., Chandra K., Lakshmi V., 1997, Antiviral
activity of Hyptianthera shivta L. against encephalitis causing
viruses, Indian Drugs, 34:694). The essential oil of Melaleuca
alternifolia in concentration of 100, 250, 500 ppm was found to be
effective in decreasing local lesions of TMV on host plant
Nicotiana glutinosa (Bishop C. D., 1995, Antiviral activity of the
essential oil of Melaleuca alternifolia (Maiden & Betche) cheel
(Teatree) against Tobacco Mosaic Virus: J. Essen. Oil Res 7:641).
The chemical composition of essential oil for Senecio graveleopeus
was analysed by GLC-MS and different components like
isovaleraldehyde, .alpha.-pinene, sabinene, p-cymene,
terpinen-4-ol, .alpha. and .beta.-eudesmone were identified and
found to have antimicrobial activity against Microccus letus,
staphylococus aureus and antifungal activity against Candida
albicans. The MIC (Minimum Inhibitory Concentration) was 8.73,
10.91 and 2.13.times.10.sup.-2 mg/ml respectively against all the
three organisms. A number of compounds from different plant
extracts have antiviral activity (Perez C., Agnese A. M. Cabrere J.
L., 1999, The essential oil of Senecio graveoleus (Compositae):
chemical composition and antimicrobial activity test 66:91). A new
acelycated flavonol glycoside, quercetin exhibited IC.sub.50 values
of 18.1.+-.1.3 .mu.g/ml against HIV integrase (Kim J. H., Woo E.
R., Shin C. G., Park 1998, Acer okamotoanum and its inhibitory
activity against HIV-1 integrase. J. Natural Products 61:145).
Three new triterpene lactones lancilactones A, B, C together with
the known Kadsulactone A were isolated from stems and roots of
Kadsura lancilimba. Their structure with sterochemistries was
determined from mass and NMR. Compound 3 inhibited HIV replication
with an EC.sub.50 value of 1.4 mg/ml and a therapeutic index of
greater than 71.4 (Chen F. D., Zhang X. S., Wang K. H., Zhang Y.
S., Sun Q. Z., Cosentine L. M., and Lee K. H. 1999, Novel Anti-HIV
Lancilactone C and related Triterpense from Kadsura lancilimba J.
Natural Products 62:94). Salvia fructiosa oil extracted form aerial
parts was analysed by GC-MS. It contained 1,8 cineole (eucalyptol)
(47.48%) .alpha.+.beta. thujone (11.93%) and camphor (9.04%). The
essential oil of S. fructiosa and its isolated components, thujone
and 1,8 cineole, exhibited activity against 8 bacterial strains.
Camphor was almost inactive against 8 bacterial strain. Camphor was
almost inactive against all the bacteria tested. The essential oil
was almost inactive against all the bacteria tested. The essential
oil was bactericidal at {fraction (1/4000)} dilution; dilutions of
up to {fraction (1/10000)} decreased bacterial growth rates. The
essential oil of S. fructicosa and its three main components
exhibited cytotoxic activity against African Green Monkey Kidney
(vero) cells and high levels of virucidal activity against Herpes
Simplex Virus 1 (Sivropou A., Nikolaou K. E., Kokkini S. L. and
Arsenalics M., 1997, Antimicrobial, cytotoxic and antiviral
activities of Saliva fruiticosa essential oil. Journal of
Agriculture and Food chemistry, 45: 3197.) The essential oils and
their components exhibited inhibiting properties against viruses
(Deans S. G. and Waterman P. G., 1993, Biological activity of
volatile oils, in: Volatile oil crops, Hay R. K. M. and Waterman P.
G. Longman Scientific and Technical pp. 97) fungi (Baruah P.,
Sharma R. K. Singh, R. S. and Ghosh A. C. 1996, Fungicides activity
of some naturally occurring essential oils against Fusarium
monitiform, Journal of Essen. Oil Res 8:411) bacteria (Chalchal J.
C., Carry R. P., Menut C., Lamaty Li., Malhuret R. and Chopineau,
J., 1997, correlation between chemical composition and
antimicrobial activity VI, Activity of some African essential oils,
Journal of Essen. Oil Res. 9:67) malaria (Milnau G., Valentin A.,
Benoit R., Mallie M., Bastide J. M., 1997, in vitro antimalarial
activity of eight essential oils, Journal of Essen. Oil Res.
9:329). These are very few reports on effects of essential oils on
viruses or viral infection in either animals or plants.
[0008] Tagetes minuta L. (Asteraceae) grows wild and yields
essential oil having commercial value in perfumery and flavour
industry (Handa K. L., Chopra M. M., Nigam M. C., 1963, The
Essential Oil Res. 54:372). The essential oil produced from plants
has been chemically investigated (Chopra I. C., Nigam M. C., Kapoor
C. D. and Handa K. L., 1963, Indian Tagetes Oils, Soap Perfumes
Cosmetics, 36, 686; Razden T. K., Wanchoo R. K. and Dhar K. L.,
1986, Chemical composition and antimicrobial activity of the oil of
Tagetes minuta L. Perfum. Kosmet. 67:52: Villeirs F. J., Garbes C.
F. and Lasnvie R. N. 1971, synthesis of tagetones and their
occurrence in oil of Tagetes minuta, Phytochemistry, 10:1359;
Lawrence M., Powell R. H., Swith T. M. and Kranes S. W. chemical
composition of Tagetes minuta, Perf & Flav., Singh B., Sood R.
P. and Singh V 1992, chemical composition of Tagetes minuta L. from
Himachal Pradesh (India), Jour Essent. Oil Res. 4:525; Thapa R. K.,
Agrawal S. G., Kalia N. K. and Kapoor R., 1993, Changes in chemical
composition of Tagetes minuta at various stages of flowering and
fruiting, Jour, Essen. Oil Res; 5:375). The oil produced from
Tagetes minuta was reported to have hypotensive, branchodilatory,
spasmolytic, anti-inflammatory and tranquilizing properties
(Chandhoke N. and Ghatak B. J. R., 1969, Tagetes minuta;
Pharmacological action of the essential oil, Indian J. Med Res,
5:864); juvenile hormone mimicking activity (Saxena B. P. and
Srivastava J. B., 1973, juvenile hormone mimicking substances,
Indian J. Exp. Biol, 11:56) 5-E Ocimenone was reported to exhibit
mosquito larvaecideal activity (Maradufu A., Lubega R. and Dorn F.,
1978, Isolation of 5-E-ocimenone, A mosquito larvicides from
Tagetes minuta. J. Natu.Prod.41:183).
[0009] So far only synthetic compounds have been used against plant
viruses. Ribavirin (Virazole) (Lozoya-Saldana H., Dawson O. and
Murashige T., 1984, Effect or ribavirin and adenine arabinoside on
tobacco mosaic virus in Nicotiana tabacum L. var. xanthim tissue
cultures. Plant Cell Tissue Org. Cult., 3:41) Tiazofurin (Caner J.
Amelia V and Vicente M, 1984, Effect of tiazofurin on tomato plants
infected with tomato spotted wilt virus. Antiviral Res., 4:325) and
Pyrazofurin (Lerch B., 1987, on inhibition of plant virus
multiplication by ribavirin, Antiviral Res., 7:257). Synthetic
compounds which inhibit virus replication are found to be effective
against at least 16 plant viruses (Hansen A. J. 1989, Antiviral
chemicals for plant disease control, Critical Review in Plant
Sciences, 8: (1) 54). Approximately 1000 ppm ribavirin are needed
to inhibit local lesion development and to prevent infection with
susceptible viruses such as PVX. In callus culture, PVX was not
inhibited by 100 ppm ribavirin in the medium. However, when these
calli started to differentiate, 10 ppm were enough to prevent virus
spread into 90% of developing shoots.
[0010] PNRSV seems to be resistant to ribavirin (Hansen A. J. 1984,
Effect of ribavirin on green ring mottle saucan agent and necrotic
rings spot virus in Prunus species, Plant Dis. Rep. 68:216) and TMV
is much less susceptible than other viruses, except during the very
early replicative steps directly following inoculation (Dawson O.
and Lozoya--Saldana H., 1984, Examination of mode of action of
ribavirin against tobacco mosaic virus. Intervirology, 22:77)
Ribavirin is relatively or completely ineffective against BGMV, SSV
and CaMV (Kluge S. and Ortel C., Arch 1976, Priufing von virazol
auf vermehrung des gurkenmosaik-virus (cucumber mosaic virus) und
des nelken scheckungs-virus carnation mottle virus) Phytopathoi,
Pflanzenschutz, 14:219; Caner J, Amelia V., and Vicente M. 1984,
Effect of tiazofurin on tomato plants infected with tomato sported
wilt virus. Antiviral Res., 4:325). In some reports, tiazofurin
(Lerch B., 1987, on the inhibition of plant virus multiplication by
ribavirin Antiviral Res., 7:257) and Pyrazofurin are tested to see
antiviral effect on a range of plant viruses. Other synthetic
antivirals are purine-based analogs 8'-azaguanine (Matthews R. E.
F., 1954 Effects of some purine analogues on tobacco mosaic virus.
J.Gen Microbiol 10:521), adenine arabinoside (Lozoya-Saldana H. and
Dawson W. O. Rev. Mex. 1986, Effect de ribavirin adenina
arabinlosida sorbre el virus mosaico del tabac el virus moteado
clorotico del chicaro de vace in vivo. Fitopatrol. 3:38, 1985, Rev.
Plant Pathol 65:306) Uracils (Commoner B. and Mercer F. L. 1951,
Inhibition of biosynthesis of tobacco mosaic virus by thiouracil,
Nature (London), 168:113) 5-Azauracil (Cassells A. C. and Long R.
D. 1982, The elimination of potato viruses X Y, S and M in meristen
and explant cultures of potato in presence of virazole, Potato
Res., 25:165) and other large number of cyclic compounds and
non-cyclic azyne compounds (Schuster G., Heinisch L., Schulze W.,
Ulbright H. and Willitzer H., 1984, Antiphytovirole verbindungen
mit nich and zyklischer Azin-struktur Phytopathol. Z 111:97)
[0011] Some plant extracts have also been screened for their
antiviral activity but these extracts have not been exploited
commercially. Only a few reports are available in which essential
oils were used as antiviral agent. Most of this work done on
Tobacco mosaic virus (Bishop C. D, 1995. Antiviral activity of
essential oil of Melaleuca alternifolia (Maidan & Betche) cheel
(Tea tree) against tobacco mosaic virus. J. of Essen Oil Res., 1995
7:6,641).
[0012] The essential oil of Melaleuca alternifolia (100, 250 or 500
ppm) was sprayed on plants of Nicotiana glutinosa inoculated before
plants were inoculated with tobacco mosaic virus isolated form
infected tomato leaves. The essential oil treatment reduced lesion
number for at least 10 days post inoculation. (Rao G. D., Pandey A.
K., Shukla K., 1986, Essential oils of some higher plants vis-a-vis
some legume viruses. Indian perfumer 30:4, 483-486). Essential oils
of Ageratum conyzoides, Callistemon lanceolatus (C. citrinus),
Carum copticum (Trachyspermum ammi) Ocimum sanctum and Peperomia
pellucida were evaluated for inhibitory activity against cowpea
mosaic virus (CPMV), mung bean mosaic virus (MBMV), bean common
mosaic virus (BCMV) and southern bean mosaic virus (SBMV). Ocimum
sanctum at 3000 ppm gave the best inhibition of 89.6, 90, 92.7,
88.2% against CMV, MBMV, BCMV, and SBMV respectively. The other
oils also showed inhibitory activity against other viruses.
[0013] Another report showed 62% inhibition against tobacco mosaic
virus. The fresh hydrodistilled carrot leaves yielded 0.07%
essential oil, analyzed by GLC and TLC. Constituents were
identified by IR, NMR and mass spectra. Antifungal activity was
tested against Colletotrichum capsici and Sclerotium rolfsii,
antibacterial activity tested against E. Coli and Aeromonas sp. and
antiviral activity against tobacco mosaic tobamovirus. Twenty nine
compounds were identified and the major constituents were Sabinene
(10.93%) linalool (14.90%), linalyl acetate (8.35%), Carvone
(8.77%) of C. caprici and S. folfric by 36% and 80UV% respectively.
Aeromonas sp. and E. coli were inhibited at 20% and 16%
respectively (Khanna R. K., Sharma O. S., Singh A., Battacharya S.
C., Sen N., Sethi K. L. 1989, The essential oil from leaves of
Dacus carota Linn. Var Sativa. Proceedings of 11.sup.th
International Congress of essential oils, fragrances and flavours.
New Delhi India, Nov. 12-16, 1989 Vol 4 Chemistry analysis and
structure 1990, 173-176).
[0014] Tagetes minuta oil was found to be active against carnation
ring spot (CaRSV) and carnation vein mottle viruses (CaVMV). The
ingredients present in the oil namely dihydrotagetone and ocimene
when tested individually in pure form, were found to have enhanced
antiviral activity against two carnation viruses. The oil as such
and the bioactive consituent present in oil can be commercially
used as an natural and eco-friendly antiviral products.
[0015] After application of whole oil of Tagetes minuta and its
compounds (ocimene and dihydrotagetone) individually for antiviral
activity for two carnation viruses i.e. CaVMV and CaRSV following
results were observed. In case of whole oil which is applied on the
half leaf of the host plant Chenopodium amaranticolor in comparison
to control (Virus+Buffer only) applied on other half of leaf,
number of lesions were observed. Each concentration of whole
Tagetes minuta oil and its pure isolated components were applied on
10 leaves so that the average could be taken.
OBJECTS OF THE INVENTION
[0016] The main object of the present investigation is to evaluate
the antiviral activity of Tagetes minuta oil.
[0017] Another object of the present invention is to isolate and
characterize antiviral components from Tagetes minuta oil.
[0018] Still another object of the present invention is to provide
easy and convenient method to enrich Z-.beta.-ocimene and
dihydrotagetone from the tagetes oil by solvent-solvent
partitioning.
[0019] Yet another object of the present investigation is to
provide control measure for carnation and for other plant viruses
using natural products.
[0020] Yet another object of the present investigation is to
provide quick and efficient natural products to control Poty and
Diantho group viral infection.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0021] FIG. 1 is a gas chromatogram of Tagetes minuta oil.
[0022] FIG. 2 is a gas chromatogram of the acetonitrile
fraction.
[0023] FIG. 3 is a gas chromatogram of Z-.beta.-ocimene.
[0024] FIG. 4 is a mass spectrum representation of
Z-.beta.-ocimene.
[0025] FIG. 5 is the .sup.1H NMR of Z-.beta.-ocimene.
[0026] FIG. 6 is the .sup.13C NMR of Z-.beta.-ocimene.
[0027] FIG. 7 is the gas chromatogram of dihydrotagetone.
[0028] FIG. 8 is the mass spectrum representation of
dihydrotagetone.
[0029] FIG. 9 is the .sup.1H NMR of dihydrotagetone.
[0030] FIG. 10 is the .sup.13C NMR of dihydrotagetone.
SUMMARY OF THE INVENTION
[0031] Accordingly the present investigation provides
identification of antiviral activity of Tagetes minuta oil and its
components which comprises of (a) Hydrodistillation of Tagetes oil
(b) drying and storage of oil (c) fractionating the oil into the
hydrocarbon rich fraction and ketone rich fraction by
solvent-solvent partitioning (d) Isolation of dihydrotagetone and
ocimene by chromatographic techniques (e) Raising of host plant (f)
Application of whole oil and pure isolates in ppm concentration on
leaves of Chenopodium amaranticolor.
[0032] In an embodiment of the present invention Tagetes minuta oil
was obtained by hydro/steam distillation in laboratory/pilot
scale.
[0033] In another embodiment of the present invention the two major
constituents ocimene and dihydrotagetone were enriched in the oil
by solvent-solvent partitioning.
[0034] In yet another embodiment of the present invention, Tagetes
minuta oil was tested against two carnation viruses.
[0035] In yet another embodiment of the present invention,
dihydrotagetone was tested against two carnation viruses.
[0036] In yet another embodiments of the present invention, ocimene
was tested against two carnation viruses.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The essential oil of Tagetes minuta was produced by steam
distillation in pilot plant or by hydrodistillation on Clevenger
type apparatus (Clevenger J. F. 1928, J. Amer Pharm Assoc. 17:346)
when the crop is matured generally during the month of Sept - Dec
(India). Drying of the oil is generally done in anhydrous sodium
sulphate or sodium chloride. The oil may be stored in aluminium
containers or in amber coloured glass bottles, without leaving any
gap of foreign particles. The freshly distilled Tagetes minuta oil
contains ocimene 54.97%, and dihydrotegetone 32.58% 9Singh B., Sood
R. P. and Singh V., 1992, chemical composition of Tagetes minuta
oil from Himachal Pradesh (India) J. Essent. Oil Res. 4:1992)
[0038] For enrichment of two major constituents present in tagetes
oil, primary fractionation of hydrocarbons and ketones (ocimene and
dihydrtotagetone), it is subject to solvent-solvent partitioning
using n-pentane n-hexane may also be used and acetonitrile. Final
purification of these components is achieved by chromatographic
separation using Silica gel (60-120 mesh). The purity of the
compounds was checked with the help of TCL and GC. Other
components, tagetones and ocimenones could not be isolated in pure
form because these components get polymerise at faster rate.
[0039] The whole oil of Tagetes minuta and its pure components i.e.
ocimene and dihydrotagetone were tested individually with virus
cultures of CaVMV and CaRSV and applied on the leaves of
Chenopodium amaranticolor. In all the experiments each
concentration of three testing mixtures were applied on ten leaves
of Chenopodium amaranticolor palnt. The isolated compounds were
stored at 0.degree. C. for two months and the experiments were
repeated.
EXAMPLE 1
[0040] In 350 ml of Tagetes minuta oil, 250 ml of acetonitrile and
250 ml of n-pentane (may be replaced by n-hexane or n-heptane) were
added and mixture was shaken slowly in separating funnel. This was
allowed to stand for half an hour. After separating two layers, the
acetonitrile layer was washed three times with n-pentane (250 ml
each), the pentane fractions were combined and the solvent
evaporated i.e. n-pentane from pentane fraction and acetonitrile
form acetonitrile fraction. After analysing both the fractions by
Gas Chromatographic technique, following percentage of ocimene
(hydrocarbon) and dihydrotagetone (ketone) were observed. The
freshly distilled Tagetes minuta oil contained ocimene 54.97% and
dihydrotagetone 32.58%.
1 1. Pentane fraction Ocimene 62.5% Dihydrotagetone 24.62% 2.
Acetonitrile Fraction Ocimene 26.96% Dihydrotagetone 69.87%
[0041] The enrichment of dihydrotagetone was achieved form 32.5%,
present in freshly distilled oil to nearly 70% after
partitioning.
[0042] The acetonitrile fraction was subjected to the column
chromatography on Silica gel (60-120 mesh), run initially with
n-hexane and then with an increasing polarity with ethyl acetate
upto 2% to get pure dihydrotagetone. Confirmation of
dihydrotagetone was made by various analytical techniques like IR,
GC, GC-MS, .sup.1H & .sup.13C NMR spectroscopy and the odour
profile was checked by the internal faculty members.
[0043] Identification
[0044] The identification and structure confirmation was done with
the help of MS, IR,
2 .sup.1H--NMR and .sup.13C--NMR. Dihydrotagetone Molecular Formula
C.sub.10H.sub.18O m/e (%) 154 (10), 97 (30), 85 (100), 69 (50) 57
(65), 55 (35), 53 (18), 44 (15), 41 (60) .sup.1H--NMR 4.89-5.02 (2H
m, H-1), 5.64-5.78 (1H, m, H-2) (ppm, CDCl.sub.3) 2.68-2.75 (1H, m
H-3), 2.09-2.47 (4H, m, H-4, 6) 1.11-1.26 (3H, d J = 6 Hz, H-8),
1.02-0.89 (6H, d, J = 6 Hz, H-9, 10) .sup.13C--NMR 112.49, (C-1),
142.64 (C-2), 23.97 (C-3), 207.9 (ppm, CDCl.sub.3) (C-4), 51.94
(C-5), 49.4 (C-6) 32.75 (C-7), 32.75 (C-7), 22.23 (C-8) 19.8 (C-9),
22.23 (C-10). Ocimene Molecular Formula C.sub.10H.sub.16 m/e (%)
136 (5), 121 (15), 1.5 (19), 93 (100), 91 (52), 79 (45), 65 (15),
53 (20), 41 (27) .sup.1H--NMR 1.55, (3H, s, H-8), 1.63 (3H, s,
H-10), (ppm, CDCl.sub.3) 1.81 (3H, s, H-9), 2.85 (2H, t, J = 7.5
Hz, H-5), 5.06-5.38 (4H, m, H-1, 4, 6), 6.73-6.87 (1H, m, H-2).
.sup.13C--NMR 113.83 (C-1), 134.04 (C-2), 132.34 (C-3) (ppm,
CDCl.sub.3) 130.0 (C-4), 26.85 (C-5, 122.92 (C-6) 132.34 (C-7),
18.07 (C-8) 26.03 (C-9) 20.09 (C-10)
EXAMPLE 2
Virus Culture
[0045] The Cultures of Carnation ring spot and Carnation vein motto
viruses were maintained both in natural host and on Phaseolus
vulgaris and Chenopodium quinoa which are maintenance and
propagation host of these viruses respectively. For virus culture,
leaves of fifteen days old individual virus inoculated host plants
with CaVMV and CaRSV were ground in phosphate buffer pH 7.5 and the
viruses were extracted separately in crude sap.
[0046] Screening of Antiviral Activity
[0047] Activity of the above volatile oils were tested against
Carnation ring spot and Carnation vein mottle virus in different
dilutions. Most of the tests were performed by using 0.5% and 2.5%
concentration of essential oils as the phytotoxic effect appeared
on Chenopodium amaranticolor leaves, at higher concentrations. The
0.5% and 2.5% concentration of essential oils were mixed with crude
sap containing each virus and incubated at room temperature for 24
hrs. After incubation, sap containing virus were inoculated
individually on bioassay host Chenopodium amaranticolor after
adding Cellite (as abrasive) to monitor the inhibitory effect.
Following results were obtained in different concentrations.
3TABLE 1a Treatment of CaVMV with 0.5% Tagetes minuta oil No. of
Lesions on Average No. of lesions S. No. Test Control Test Control
% inhibition 1 39 713 3.9 71.1 94.5 2. 46 657 4.6 65.7 92.2 3. 42
702 4.2 70.2 94.2
[0048]
4TABLE 1b Treatment of CaRSV with 0.5% Tagetes minuta oil No. of
Lesions on Average No. of lesions S. No. Test Control Test Control
% inhibition 1 67 761 6.7 76.1 91.3 2. 55 769 5.5 76.9 92.8 3. 65
770 6.5 77 91.5
[0049]
5TABLE 2a Treatment of CaVMV with 2.5% Tagetes minuta oil No. of
Lesions on Average No. of lesions S. No. Test Control Test Control
% inhibition 1 82 699 8.2 69.9 88.2 2. 76 648 7.6 64.8 88.2 3. 70
655 7.0 65.5 89.3
[0050]
6TABLE 2b Treatment of CaRSV with 2.5% Tagetes minuta oil No. of
Lesions on Average No. of lesions S. No. Test Control Test Control
% inhibition 1 73 582 7.3 58.2 87.45 2. 77 624 7.7 62.4 87.6 3. 75
612 7.5 61.2 87.7
[0051]
7TABLE 3a Treatment of CaVMV with 0.5% Dihydrotagetone No. of
Lesions on Average No. of lesions S. No. Test Control Test Control
% inhibition 1 38 141 4.22 15.66 73.04 2. 40 150 4.44 15.66 73.3 3.
45 152 5.00 15.70 70.3
[0052]
8TABLE 3b Treatment of CaRSV with 0.5% Dihydrotagetone No. of
Lesions on Average No. of lesions S. No. Test Control Test Control
% inhibition 1 32 115 4.22 15.66 72.1 2. 41 141 4.44 15.66 70.9 3.
42 150 5.00 15.70 72.0
[0053]
9TABLE 4a Treatment of CaVMV with 2.5% Dihydrotagetone No. of
Lesions on Average No. of lesions S. No. Test Control Test Control
% inhibition 1 31 183 3.44 20.33 83.0 2. 25 192 2.77 21.33 87.0 3.
38 200 4.20 22.22 81.0
[0054]
10TABLE 4b Treatment of CaRSV with 2.5% Dihydrotagetone No. of
Lesions on Average No. of lesions S. No. Test Control Test Control
% inhibition 1 82 155 2.8 15.5 81.8 2. 25 140 2.5 14.0 82.0 3. 40
195 4.0 19.5 79.4
[0055]
11TABLE 5a Treatment of CaVMV with 0.5% Ocimene No. of Lesions on
Average No. of lesions S. No. Test Control Test Control %
inhibition 1 66 228 6.6 22.8 71.05 2. 70 215 7.0 21.5 63.50 3. 65
233 6.5 23.3 72.1
[0056]
12TABLE 5b Treatment of CaRSV with 0.5% Ocimene No. of Lesions on
Average No. of lesions S. No. Test Control Test Control %
inhibition 1 60 198 6.0 19.8 69.6 2. 65 195 6.5 19.5 66.6 3. 58 180
5.8 18.0 67.7
[0057]
13TABLE 6a Treatment of CaVMV with 2.5% Ocimene No. of Lesions on
Average No. of lesions S. No. Test Control Test Control %
inhibition 1 43 262 4.3 26.8 83.58 2. 40 250 4.0 25.0 84.00 3. 38
265 3.8 26.5 85.66
[0058]
14TABLE 6b Treatment of CaRSV with 2.5% Ocimene No. of Lesions on
Average No. of lesions S. No. Test Control Test Control %
inhibition 1 40 205 4.0 20.5 80.4 2. 25 135 2.5 13.5 81.4 3. 35 185
3.5 18.5 81.0
[0059] When these pure isolated compounds were stored at 0.degree.
C. for two months, following results were obtained.
15TABLE 7a Treatment of CaVMV with DHT (stored at 0.degree. C. for
two months) 0.5% concentration 2.5% concentration S. No. Test
Control Test Control 1. 0 158 0 200 2. 0 170 0 185 3. 0 165 0
189
[0060]
16TABLE 7b Treatment of CaRSV with DHT (stored at 0.degree. C. for
two months) 0.5% concentration 2.5% concentration S. No. Test
Control Test Control 1. 0 141 0 195 2. 0 168 0 188 3. 0 166 0
190
[0061]
17TABLE 8a Treatment of CaVMV with Ocimene (stored at 0.degree. C.
for two months) 0.5% concentration 2.5% concentration S. No. Test
Control Test Control 1. 0 200 0 262 2. 0 250 0 285 3. 0 260 0
203
[0062]
18TABLE 8b Treatment of CaRSV with Ocimene (stored at 0.degree. C.
for two months) 0.5% concentration 2.5% concentration S. No. Test
Control Test Control 1. 0 210 0 267 2. 0 235 0 282 3. 0 216 0
213
[0063]
19TABLE 9a Percentage Inhibition of CaVMV with Three Treatments
Compo- Ist Treatment Compo- IInd Treatment IIIrd Treatment nent
0.5% 2.5% nent 0.5% 2.5% 0.5% 2.5% Whole 93.8 88.5 Ocimene 70.2
84.3 100 100 oil Dihydro- 72.2 83.6 100 100 tegetone I Treatment:
With Whole oil II Treatment: With Both the compounds after
Extraction III Treatment: With both the compounds after storing at
0.degree. C.
[0064]
20TABLE 9b Percentage Inhibition of CaRSV with Three Treatments
Compo- Ist Treatment Compo- IInd Treatment IIIrd Treatment nent
0.5% 2.5% nent 0.5% 2.5% 0.5% 2.5% Whole 91.5 87.5 Ocimene 67.9
80.9 100 100 oil Dihydro- 71.6 81.06 100 100 tegetone I Treatment:
With Whole oil II Treatment: With Both the compounds after
Extraction III Treatment: With both the compounds after storing at
0.degree. C.
[0065] The main advantages of the present invention are:
[0066] 1. Tagetes minuta plant grows wild in the hilly areas like
Himachal Pradesh, Jammu and Kashmir, Uttar Pradesh, North Eastern
States of India, and cultivated as commercial Tagetes oil crop
hence easily available in bulk quality.
[0067] 2. The oil and pure isolates are natural products and hence
no threat to environment.
[0068] 3. Application of oil and pure isolates ensure quick and
efficient recovery from viral infections.
[0069] 4. It also helps in the plant virus management.
[0070] 5. Tagetes crop grows wild and can be distilled in rich
pockets/places with prototype distillation unit.
[0071] 6. A cheap, eco-friendly and easily available anti-viral
natural product.
* * * * *