U.S. patent application number 11/993142 was filed with the patent office on 2009-10-15 for extracts and compounds from "agapanthus africanus" and their use as biological plant protecting agents.
This patent application is currently assigned to AGRARFORUM AG. Invention is credited to Johannes Christiaan Pretorius.
Application Number | 20090258097 11/993142 |
Document ID | / |
Family ID | 37488153 |
Filed Date | 2009-10-15 |
United States Patent
Application |
20090258097 |
Kind Code |
A1 |
Pretorius; Johannes
Christiaan |
October 15, 2009 |
EXTRACTS AND COMPOUNDS FROM "AGAPANTHUS AFRICANUS" AND THEIR USE AS
BIOLOGICAL PLANT PROTECTING AGENTS
Abstract
The invention relates to plant extracts, especially based on
species of the genus Agapanthus and combinations thereof with other
extracts deriving from other plants. The invention further relates
to the isolation, purification and identification of compounds in
these extracts. The plant extracts and the isolated substances show
significant antimicrobial activity, especially antifungal activity,
and bio-stimulatory efficacy, when applied to other plants in vitro
and in vivo, including under field conditions. The products
according to this invention are suitable to be used as plant
protecting agents for many crops and economic plants as an
alternative for chemical pesticides.
Inventors: |
Pretorius; Johannes Christiaan;
(Sooth, ZA) |
Correspondence
Address: |
ARENT FOX LLP
1050 CONNECTICUT AVENUE, N.W., SUITE 400
WASHINGTON
DC
20036
US
|
Assignee: |
AGRARFORUM AG
Bomlitz
DE
AGRARFORUM SA (PTY) LTD.
Universitas
ZA
|
Family ID: |
37488153 |
Appl. No.: |
11/993142 |
Filed: |
June 24, 2006 |
PCT Filed: |
June 24, 2006 |
PCT NO: |
PCT/EP2006/006104 |
371 Date: |
December 19, 2007 |
Current U.S.
Class: |
424/773 ;
424/725; 424/778; 514/26 |
Current CPC
Class: |
A01N 65/34 20130101;
A01N 65/00 20130101; A01N 65/38 20130101; A01N 65/42 20130101; A01N
65/28 20130101; A01N 45/00 20130101; A01N 65/30 20130101; A01N
65/08 20130101; A01N 65/20 20130101; A01N 65/06 20130101; A01N
65/40 20130101; A01N 45/00 20130101; A01N 2300/00 20130101; A01N
65/00 20130101; A01N 2300/00 20130101 |
Class at
Publication: |
424/773 ;
424/778; 514/26; 424/725 |
International
Class: |
A01N 65/40 20090101
A01N065/40; A01N 45/00 20060101 A01N045/00; A01P 3/00 20060101
A01P003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 30, 2005 |
EP |
05014247.0 |
Jun 30, 2005 |
EP |
05014265.2 |
Claims
1-38. (canceled)
39. A preparation suitable for biological plant protection based on
parts of plants from the species Agapanthus africanus in form of a
dry powder, obtainable by the following steps: (i) drying one or
more of the different aerial parts of the plant at 30-40.degree. C.
to the exclusion of sun light; (ii) grinding the dried plant
material to a grit size less than 0.1 mm, (iii) soaking the ground
material in methanol, thus forming a suspension/solution; (iv)
performing a stirred extraction of the suspension; (v) evaporating
the solvent without prior separation of the solid phase from the
soluble organic phase; (vi) soaking the evaporated solid phase
residue in ethanol and repeating steps (iv) and (v); and (vii)
drying the evaporated solid phase residue, thus obtaining a dry
powder.
40. A preparation according to claim 39, wherein the combined
aerial parts are used.
41. A preparation of claim 39, wherein the flowers are used.
42. A preparation according to claim 39, wherein, instead of aerial
parts, soil parts of the plant are used.
43. A preparation according to claim 39, further comprising solid,
pulverulent carrier materials or fillers, and optionally additives
that augment or regulate the effect of the preparation.
44. A preparation in form of an aqueous solution or suspension
based on a preparation according to claim 39.
45. A preparation of claim 44, wherein the concentration of the dry
powder is in the range from 0.25 g/l to 2 g/l.
46. A plant preparation comprising a first plant preparation
according to claim 39 and at least a second plant preparation in
form of a crude extract, dry powder or an aqueous suspension or
solution thereof, wherein said second plant preparation exerts an
additional plant protective effect on the plants or parts thereof
treated with the composition, is obtained by analogous process
steps as said first plant preparation, and derives from (i) a
species from the species Tulbaghia violacea, or (ii) a mixture of
species of the Pink family and Alfalfa species, wherein the
proportion by weight of the dried Pink species material is between
80 and 99%.
47. Use of a plant preparation according to claim 39 as a
biological plant protective agent.
48. Use of a plant preparation according to claim 47, wherein the
plant protective agent is an antifungal agent.
49. Use of a plant preparation according to claim 48, wherein the
antifungal agent inhibits mycelial growth of fungi.
50. Use of a plant preparation according to claim 48, for
preventing infection of crop by fungi in vivo under field
conditions.
51. Use of claim 50, wherein a plant preparation from the combined
aerial parts of Agaphanthus africanus is used.
52. Use of claim 47, wherein the biological plant protective agent
is a bio-stimulatory agent that induces systemic acquired
resistance (SAR) and exerts growth induction in plants or plant
parts treated with the agent.
53. Use of claim 52, wherein the activity or efficacy of said
preparation is higher than the sum of the activities or efficacies
of preparations based on the respective single components of the
aerial parts of Agaphanthus africanus.
54.
3-[{O-.beta.-D-glucopyranosyl-(1''-3')-.alpha.-L-rhamnosyl-(1''-2')}--
.beta.-D-glucopyranosyloxy]agapanthegenin or a derivative thereof,
wherein all sugar hydroxyl groups are acetylated
55. A composition suitable for biological plant protection
comprising a compound of claim 54 and the flavonoid compounds
5,7,4' tri-O-flavanone, 5,7,3',4'-tetra-O-acetylflavanone and
trans-4,2',4'-tri-O-acetylchalcone.
56. Use of the compound of claim 54 as antifungal agent that
inhibits the mycelial growth of fungi.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The invention relates to plant extracts, especially based on
species of the genus Agapanthus and combinations thereof with other
extracts deriving from other plants. The invention further relates
to the isolation, purification and identification of compounds in
these extracts. The plant extracts and the isolated substances show
significant antimicrobial activity, especially antifungal activity,
and bio-stimulatory efficacy, when applied to other plants in vitro
and in vivo, including under field conditions. The products
according to this invention are suitable to be used as plant
protecting agents for many crops and economic plants as an
alternative for chemical pesticides.
BACKGROUND OF THE INVENTION
[0002] Worldwide agriculture suffers, especially in developing
countries such as in Africa, from annual huge losses of crop and
other economic plants due to plant diseases. More than 30% of the
food, fiber, feed and energy produced in crop production systems
are destroyed by insects and diseases annually on a global scale.
These yield losses are high as a result of low-input production
systems due to the non-affordability of synthetic fungicides to
farmers in developing countries that depend on non-conventional
disease management practices often providing doubtful results.
[0003] In contrast, crop and plant producers in developed countries
rely largely on synthetic pesticides to control plant diseases. It
is an established fact that the use of synthetic chemical
pesticides provides many benefits to crop producers. These benefits
include higher crop yields, improved crop quality and increased
food production for an ever increasing world population. The
development of a wide range of chemicals with different
formulations has enabled man to control a wide range of plant
pathogens and substantially increased crop yields. More than a
decade ago crop producers spent nearly $20 billion on pesticides
and $150 million on other plant protection techniques, worldwide,
to control pests in general. The world market share of fungicides
alone was 20% in recent years whilst Europe accounted for 30% of
the market. However, the same level of pathogen control has not
been realized in developing countries, partly as a result of
pesticide technology not being accessible to most resource poor
farmers. Failure of modern approaches, technology and chemicals to
reach farmers in developing countries is solely the result of high
costs in relation to the value of the crops cultivated by these
farmers. Consequently, crops are routinely subjected to attack from
a wide spectrum of a diversity of pathogens and these farmers
constantly experience serious crop damage. Moreover, yield losses
are on the increase despite high pesticide usage, even in developed
countries. Furthermore, control of plant diseases is not easily
achieved with a single application of fungicide but requires
frequent applications during the crop-growing period. However,
synthetic pesticides may pose a couple of threats and hazards to
the environment, especially when improperly used by farmers in
developing countries who lack the technical skill of handling them,
and who fail to adopt to this technology easily. This may result in
undesirable residues left in food, water and the environment, and
may cause toxicity to humans and animals, contamination of soils
and groundwater and may lead to the development of crop pest
populations that are resistant to treatment with agrochemicals.
Especially sulfur and copper containing synthetic fungicides are
toxic to mammals, wildlife and many beneficial insects.
[0004] Furthermore, in Africa and the Near East, obsolete
pesticides have become a source of an additional great
environmental concern. Some stocks are over 30 years old and are
kept in poor conditions because of inadequate storage facilities
and lack of staff trained in storage management. Obsolete pesticide
stocks are potential time bombs. Leakage, seepage and various
accidents related to pesticides are quite common and widespread.
Additionally, that frequent application of fungicides has resulted
in fungal mutation and, subsequently new resistant strains (Khun,
1989, Pesticide Science 14:272-293), the combat of which usually
requires stronger pesticides with again stronger impacts on the
environment.
[0005] For all these reasons there is a considerable and increasing
consumer resistance especially in the developed countries,
initiated politically by the green parties, towards the use of
synthetic chemicals/pesticides especially, supplying a rationale
for a shift from chemical pesticides applications to the use of
naturally derived plant protecting agents in order to reduce the
pollution and health risk caused by pesticides.
[0006] As a result, research on the possible utilization of
biological resources and its application potential in agriculture
has become very relevant. A promising approach in this regard is
the use of natural plant products as an interesting alternative to
synthetic chemicals due to the apparent less negative impact on the
environment.
[0007] This especially applies to the search for environmentally
friendly bioactive naturally derived components and agents with,
for example, broad-spectrum antimicrobial activity.
[0008] Natural products from plants are expected to have a narrow
target range and highly-specific mode of action, to show limited
field persistence, to have a shorter shelf life and present no
residual threats. They are generally safer to humans and the
environment than conventional synthetic chemical pesticides and can
easily be adopted by farmers in developing countries who
traditionally use plant extracts for the treatment of human
diseases.
[0009] A further rationale for exploring the use of plant extracts
or natural products as biological pesticides more extensively can
be found in the plant itself. Plants have evolved highly specific
chemical compounds that provide defense mechanisms against attack
by disease causing organisms, including fungal attack, microbial
invasion and viral infection (Cowan, 1999, Clinical Microbiology
Reviews 12:564-582). These bioactive substances occur in plants as
secondary metabolites, and have provided a rich source of
biologically active compounds that may be used as novel
crop-protecting agents. In nature some plants have the potential to
survive very harsh environmental conditions. This has initiated the
postulate that such plants might be utilized as sources for the
development of natural products to be applied in agriculture by man
as natural herbicides, bactericides, fungicides or products in
crude or semi-purified form. Secondary plant metabolites are
distinct from primary metabolites in that they are generally
non-essential for the basic metabolic processes such as respiration
and photosynthesis. They are numerous and widespread, especially in
higher plants and often present in small quantities (1-5%) as
compared to primary metabolites (carbohydrates, proteins, lipids).
Secondary metabolites are probably produced when required in the
plant system and are synthesized in specialized cell types.
Ecologically, secondary metabolites play essential roles in
attracting pollinators, as adaptations to environmental stresses
and serve as chemical defenses against insects and higher
predators, micro-organisms and even other plants
(allelochemicals).
[0010] Abiotic stress such as nutrient limitation, light intensity,
water stress and others has been considered to trigger the
formation of secondary metabolites. A biotic stress related type of
plant-pathogen interaction involves the production of metabolites
as part of a plant defense arsenal against microbial invasion and
is considered disease determinants. Secondary metabolites with
anti-microbial properties include terpenoids (e.g. iridoids,
sesquiterpenoids, saponins), nitrogen- and/or sulphur containing
(e.g. alkaloids, amines, amides), aliphatics (especially long-chain
alkanes and fatty acids) and aromatics (e.g. phenolics, flavonoids,
bi-benzyls, xanthones and benzoquinones).
[0011] Another related area of organic farming systems is the
potential to apply natural plant extracts as either plant growth
regulators or bio-stimulants. Many natural plant compounds have
been identified that affect the growth and development of plants.
Secondary metabolites from plants may show also bio-stimulatory
activities in plants, other plants included. Probably the most
effective compound to enhance crop yield, crop efficiency and seed
vigour has been identified as a brassinosteroid (Mandava, 1988,
Plant Physiology Plant Molecular Biology 39:23-52).
Brassionosteroids have also been identified as bio-stimulatory
substances from a plant extract mixture deriving from a specific
Pink species and a specific Alfalfa species (EP 1 051 075 B1). An
elevated interest therefore exists to identify natural plant
compounds with the ability to manipulate plant growth and
development over a short period, e.g. a growing season. An
additional consideration is that plants whose extracts, for example
show antimicrobial and/or bio-stimulatory properties, could be
cultivated as alternative agricultural crops for serving as sources
of active compounds in the production of natural pesticides or
plant growth regulators.
[0012] Although plants are a valuable source for the development of
new natural products with the potential to be used for disease
management in organic crop production systems only a small number
of plants has been investigated for possible use in plant disease
control in agriculture. However, related to this relatively small
number of investigated plants a relatively large number of
scientific research activities has been done during the last couple
of years. Some of them are listed as follows: [0013] It was shown
(Pretorius et al., 2002, Annals of Applied Biology 141:117-124)
that mycelial growth inhibition was obtained with extracts from two
species of the subclass Liliidae, namely Aristea ecklonii and
Agapanthus inapertus. The crude extract of A. ecklonii performed
best of all extracts as it totally inhibited the mycelial growth of
all seven of the plant pathogenic test organisms and outperformed
the inhibition by a broad spectrum synthetic fungicide
(carbendazim/difenoconazole). Crude extracts of A. inapertus showed
complete inhibition of four and strong inhibition of the remaining
three plant pathogenic fungi. [0014] Plant seeds also contain
compounds with antimicrobial properties. Seed extracts of 50 plant
species, belonging to different families, were evaluated for their
ability to inhibit the growth of Trichoderma viride in vitro
(Bharathimatha et al., 2002, Acta Phytopathologica et Entomologica
Hungarica 37:75-82). Of the various seed extracts, that of
Harpullia cupanioides (Roxb.), belonging to the family Sapindaceae,
displayed very high antifungal activity. [0015] The natural plant
product Milsana.RTM., extracted from the giant knotweed (Reynoutria
sacchalinensis), is probably best known (Daayf, 1995, Plant Disease
79:577-580). The product has been reported to control powdery
mildew, caused by Sphaerotheca fuliginea, in long English cucumber
under greenhouse conditions and also showed broad spectrum activity
against powdery mildew of tomato, apple and begonia as well as
downy mildew of grapevine and rust of bean. [0016] Extracts from
the leaves and seed kernels of the neem tree (Ume et al., 2001, The
science and application of neem, meeting proceedings, Glasgow, U.K.
April-2001. Pp. 33-37) were tested for antifungal activity against
the plant pathogenic fungus Sclerotium rolfsii [Corticium rolfsii].
All the extracts showed some effect against different growth stages
of the fungus, but the effects were fungistatic rather than
fungitoxic. [0017] Amadioha (2002, Archives of Phytopathology and
Plant Protection 35:37-42) evaluated the antifungal activities of
the different extracts of A. indica. The oil extract from seeds as
well as water and ethanol leaf extracts of the plant were effective
in reducing the radial growth of Cochliobolus miyabeanus in culture
and in controlling the spread of brown spot disease in rice. [0018]
A study directed towards identifying bio-stimulatory properties in
plant extracts was performed by Cruz et al. (2002, Acta
Horticulturae 569:235-238) by treating the roots of bean, maize and
tomato with an aqueous leachate of Callicarpa acuminate. The
aqueous extract of C. acuminata inhibited the radical growth of
tomato but had no effect on root growth of maize or beans. [0019]
According to Singh et al. (2001, Journal of Crop Production 4:121),
allelochemicals isolated from some plants show strong
bio-herbicidal activity at high concentrations, but at low
concentrations these extracts can promote crop seed germination and
seedling growth, hence showing a potential to be applied as
bio-stimulatory agents or growth promoting substances in
agriculture. [0020] Extracts from some lucerne cultivars had a
stimulatory effect in terms of seed germination as well as root and
hypocotyl growth, whereas others showed the direct opposite effect,
confirming that crop plants can also be affected by plant extracts
aimed at controlling weed growth (Tran and Tsuzuki, 2002 Journal of
Agronomy and Crop Science 188:2-7). [0021] Leksomboon et al. (2001,
Kasetsart Journal, Natural Sciences 35:392-396) demonstrated the
antibacterial effect of leaf and other aqueous extracts of Hibiscus
sabdariffa, Psidium guajava, Punica granatum, Spondias pinnata and
Tamarindus indica against Xanthomonas axonopodis, the casual agent
of citrus canker under both laboratory and field conditions. [0022]
Another natural product, carvone, derived from dill and caraway
seed, has been developed to inhibit the growth of storage pathogens
and to suppress sprouting of potatoes in the warehouse (Moezelaar
et al., 1999, In: Modern fungicides and antifungal compounds II,
Intercept Limited, p. 453-467). Carvone is currently marketed as
Talent.RTM. in the Netherlands. [0023] In European patent EP 1 051
075 a preparation of a combination of species of the Pink family
and species of Alfalfa is described (ComCat.RTM.) which reveals
within a specific ratio a synergistic bio-stimulatory effect.
ComCat.RTM. has demonstrated consistent plant growth enhancement
and physiological efficiency in the treated plant's utilization of
available nutrients. ComCat.RTM., which enhances the health of
vegetables, flowers and agricultural crops, is not a fertilizer
substitute but, instead, it is a biological enhancer which
stimulates the plant to more properly utilize available nutrients.
Moreover, it activates and induces allelopathy and disease
resistance in the treated plant and stimulates greater production
of sugars, which are the building blocks for cellulose and fruiting
bodies. The result is a more productive, healthier plant with
stronger plant stalks, better flowering and greater fruit biomass
(Agraforum: Germany, 2002, Technical data sheet).
SUMMARY OF THE INVENTION
[0024] The invention provides extracts and preparations based on
species of the genus Agapanthus, preferably Agapanthus africanus,
which elicit a significant antimicrobial, preferably antifungal
activity in vitro and in vivo, even under field and glasshouse
conditions. Moreover, these extracts elicit a significant
bio-stimulatory activity, expressed, above all, by an increased
growth metabolism. Extracts or preparations from the aerial parts
of the plants show a higher efficacy as compared to the soil parts
of the plant. Furthermore, extracts or preparations from the
combined aerial parts of the plants (flowers, leaves, stalks) show
a higher antifungal and bio-stimulatory efficacy as compared to the
sum of extracts or preparations from the single components of the
aerial parts, indicating that synergism is participated in the
involved biological processes. Furthermore, combined extracts or
preparations from species of the genus Agapanthus and the species
Tulbaghia violacea show a higher antifungal and bio-stimulatory
efficacy as compared to the extracts or preparations of the single
species and let assume the existence of a synergistic process.
[0025] The invention provides, in addition, compositions of
combinations of extracts or preparations of different plant
species. These combinations comprise-preparations from species of
the genus Agapanthus, preferably A. africanus, and other plant
species, preferably garlic species, most preferably from Tulbaghia
violacea (wild garlic). Alternatively, according to the invention,
a preparation from species of the genus Agapanthus is combined with
a preparation of a mixture of species of the Pink family and
Alfalfa species, preferably in a specific ratio. In another
embodiment of the invention provides combinations of species of the
genus Agapanthus with Tulbaghia violacea and a mixture of species
of the Pink family and Alfalfa species. These combinations elicit
an increased and synergistic plant protective activity, preferably
an antifungal and bio-stimulatory activity, as compared to the
corresponding single-component preparations.
[0026] The invention provides finally at least four compounds
isolated and purified from said extracts/preparations, which also
show significant plant protecting activity, especially antifungal
activity, when applied to other plants in vitro and in vivo, field
cultivation included. These four compounds are:
3-[{O-.beta.-D-glucopyranosyl-(1''-3')-.alpha.-L-rhamnosyl-(1''-2')}-.bet-
a.-D-glucopyranosyloxy]agapanthegenin, 5,7,4' tri-O-flavanone,
5,7,3',4'-tetra-O-acetylflavanone and
trans-4,2',4'-tri-O-acetylchalcone.
[0027] The preparations according to the invention can be provided
as crude extracts or as dried powder dependent on the process of
their manufacture. The preparations may comprise additionally,
especially for use in field cultivation, solid preferably
pulverulent fillers or carrier materials according to the state of
the art. Moreover, the preparations according to the invention may
comprise conventional additives that augment or modulate the effect
of the preparation.
[0028] The preparations according to the invention can be provided
also in a liquid, preferably aqueous form, which can be uses as a
spray, and thus can be easily atomized on the areas under
cultivation. In such solutions or suspensions the extracts and
preparations of the invention reveal their full plant protecting
activity in a concentration range between 0.25 g (extract/powder)/1
to 2 g/l, preferably from 0.5 g/l to 1 g/l. With respect to the
antifungal activity of the preparations, the term "full plant
protecting activity" means 100% inhibition of the mycelial growth
of a typical fungal plant pathogen compared to a standard reference
pesticide.
[0029] The invention also provides processes for the manufacture of
the crude extracts and dry powder preparation based on extraction
of the plants or plant parts with organic polar solvents, such as
methanol or ethanol or mixtures thereof.
[0030] The invention finally provides a process of isolating,
purifying and identifying substances from said extracts which show
significant antifungal and bio-stimulatory activity in diseased
plants in vitro and in vivo.
[0031] In more detail the invention provides: [0032] A preparation
suitable for biological plant protection based on plants or parts
of plants from the genus Agapanthus, preferably the species A.
africanus, in form of a crude extract, whereby said preparation is
obtainable by the following process steps: [0033] (i) drying the
plant material preferably at 30-40.degree. C. preferably to the
exclusion of sun light; [0034] (ii) grinding the dried plant
material to a grit size between 0.2-2 mm; [0035] (iii) soaking the
ground material in a polar organic solvent selected from the group
consisting of methanol and ethanol, thus forming a
suspension/solution [0036] (iv) performing a stirred extraction of
the suspension and separating the supernatant from the solid phase;
[0037] (v) repeating step (iii) and (iv) at least one additional
time; preferably two times, [0038] (vi) combining the soluble
organic phases of step (iv) and removing the organic solvent by
preferably vacuum evaporation at 30-40.degree. C., thus obtaining
the crude extract residue. [0039] Alternatively, a corresponding
preparation in form of a dry powder, obtainable by the following
steps: [0040] (i) drying the plant material at preferably
30-40.degree. C., preferably to the exclusion of sun light; [0041]
(ii) grinding the dried plant material to a grit size less than 0.1
mm, [0042] (iii) soaking the ground material in methanol or
ethanol, preferably methanol, thus forming a suspension/solution;
[0043] (iv) performing a stirred extraction of the suspension;
[0044] (v) evaporating the solvent without prior separation of the
solid phase from the soluble organic phase; [0045] (vi) soaking the
evaporated solid phase residue in ethanol or methanol, preferably
ethanol and repeating steps (iv) and (v); and [0046] (vii) drying
the evaporated solid phase residue, thus obtaining a dry powder.
[0047] A corresponding preparation, wherein one or more of the
different aerial parts (flowers, leaves, stalks) of the plants are
used, preferably the flowers. [0048] A corresponding preparation,
wherein the combined aerial parts (flowers plus leaves plus stalks)
are used; said preparation is showing an additional (synergistic)
effect as compared to the over-all effect of the single components
of the aerial parts of Agapanthus. [0049] A corresponding
preparation, wherein the soil plant parts are used. [0050] A
corresponding preparation comprising:
3-[{O-.beta.-D-glucopyranosyl-(1''-3')-.alpha.-L-rhamnosyl-(1''-2')}-.bet-
a.-D-glucopyranosyloxy]agapanthegenin, and/or 5,7,4'
tri-O-flavanone and/or 5,7,3',4'-tetra-O-acetylflavanone and/or
trans-4,2',4'-tri-O-acetylchalcone. [0051] A corresponding
preparation comprising
3-[{O-.beta.-D-glucopyranosyl-(1''-3')-.alpha.-L-rhamnosyl-(1''-2')}-.bet-
a.-D-glucopyranosyloxy]agapanthegenin, and 5,7,4' tri-O-flavanone,
and 5,7,3',4'-tetra-O-acetylflavanone and
trans-4,2',4'-tri-O-acetylchalcone. [0052] A corresponding
preparation, which further comprises solid, pulverulent carrier
materials or fillers and/or additives that augment or regulate the
effect of the preparation. [0053] A corresponding preparation in
form of an aqueous solution or suspension, which can be easily
sprayed and distributed on fields and areas under cultivation, in
which the plants to be protected are cultivated. [0054] A
corresponding, wherein the concentration of crude extract or the
dry powder is in the range from 0.25 g/l to 2 g/l, preferably from
0.5 g/l to 1 g/l. [0055] A composition comprising a first plant
preparation as specified above and second plant preparation in form
of a crude extract, dry powder or an aqueous suspension or solution
thereof, wherein said second plant preparation exerts an additional
or even synergistic plant protective effect on the plants or parts
thereof treated with the composition. [0056] A corresponding
composition comprising as second plant preparation a preparation
deriving from garlic species, preferably Tulbaghia violacea,
whereby said second preparation is obtained by analogous process
steps as said first preparation. [0057] A corresponding composition
comprising as second plant preparation a preparation deriving from
a mixture of species of the Pink family and Alfalfa species,
wherein preferably the proportion by weight of the dried Pink
species material is between 80 and 99%, and said second plant
preparation is obtained by analogous process steps as said first
plant preparation. [0058] A composition comprising (i) said first
plant preparation, (ii) said second plant preparation, and (iii) a
third plant preparation deriving from a mixture of species of the
Pink family and Alfalfa species, wherein preferably the proportion
by weight of the dried Pink species material is between 80 and 99%,
whereby each preparation is in form of a crude extract, dry powder
or an aqueous suspension or solution thereof, and said second and
third plant preparation exert an additional plant protective effect
on the plants or parts thereof treated with the composition. [0059]
The use of a preparation/composition as described above as a
biological plant protective agent. [0060] The use of a
preparation/composition as described, wherein the biological plant
protective agent is an antimicrobial agent, preferably an
antifungal agent, which preferably inhibits or reduces the mycelial
growth of fungi and is enabled to prevent plants, preferably crop,
from infection by fungi under field conditions. [0061] The use of a
preparation/composition as described above as a bio-stimulatory
agent, which exerts growth induction [0062] The use of a
preparation/composition as described above as a bio-stimulatory
agent, which induces systemic acquired resistance (SAR) in plants
or plant parts treated with the agent. [0063] The corresponding
uses, wherein the applied Agapanthus preparations derive from the
combined aerial parts of Agapanthus. [0064] The corresponding use,
wherein the activity or efficacy of said preparation is higher than
the sum of the activities or efficacies of preparations based on
the respective single components of the aerial parts of Agapanthus.
[0065] A process for the preparation of a crude extract or a dry
powder preparation or aqueous suspensions or solutions thereof from
Agapanthu as defined above, comprising the following steps: [0066]
(i) drying plant material from Agapanthus at 30-40.degree. C.,
preferably to the exclusion of sun light; [0067] (ii) grinding the
dried plant material to a grit size between 0.1-3 mm, preferably
between 0.2-2 mm; [0068] (iii) soaking the ground material in a
polar organic solvent, such as methanol or ethanol, preferably
90-10% methanol or ethanol or mixtures thereof, thus forming a
suspension/solution; [0069] (iv) performing a stirred extraction of
the suspension and separating the supernatant from the solid phase;
[0070] (v) repeating step (iii) and (iv) at least one additional
time; preferably two times, [0071] (vi) combining the soluble
organic phases of step (iv) and removing the organic solvent by
preferably vacuum evaporation at 30-40.degree. C., thus obtaining
the crude extract residue; [0072] and in the case of the
preparation of an aqueous preparation; [0073] (vii) suspending the
resultant crude extract in water in a suitable concentration
preferably in a range between 0.1 g/1 and 2 g/l, more preferably
between 0.5 g/l and 1 g/l. [0074] A compound of formula I,
[0074] ##STR00001## [0075] wherein R.dbd.H or acetyl. [0076] A
corresponding compound isolated from a preparation as described,
wherein said compound is
3-[{O-.beta.3-D-glucopyranosyl-(1''-3')-.alpha.-L-rhamnosyl-(1''-2')}-.be-
ta.-D-glucopyranosyloxy]agapanthegenin. [0077] A corresponding
composition suitable for biological plant protection comprising a
compound of formula I or, more specifically,
3-[{O-.beta.-D-glucopyranosyl-(1''-3')-.alpha.-L-rhamnosyl-(1''-2')}-.bet-
a.-D-glucopyranosyloxy]agapanthegenin and at least one flavonoid
compound selected from the group consisting of 5,7,4'
tri-O-flavanone, 5,7,3',4'-tetra-O-acetylflavanone and
trans-4,2',4'-tri-O-acetylchalcone. [0078] The use of said isolated
compounds as plant protective agent, wherein the plant protective
agent is preferably an antifungal agent that inhibits the mycelial
growth of fungi. [0079] An alternative process for the preparation
of a crude extract or a dry powder preparation or aqueous
suspensions or solutions thereof derived from Agapanthus as defined
above comprising the steps [0080] (ia) drying the plant material
from Agapanthus at preferably 30-40.degree. C., preferably to the
exclusion of sun light; [0081] (iia) grinding the dried plant
material to a grit size less than 0.1 mm, [0082] (iiia) soaking the
ground material in a first polar organic solvent, preferably
90-100% methanol, using 1.0-3.0 ml/g dry weight of the ground plant
material, thus forming a suspension/solution; [0083] (iva)
performing a stirred extraction of the suspension; [0084] (va)
evaporating the solvent without prior separation of the solid phase
from the soluble organic phase; [0085] (via) soaking the evaporated
solid phase residue in a second polar organic solvent, preferably
90-10% ethanol, using 1.0-3.0 ml/g dry weight of the ground plant
material, and repeating steps (iva) and (va); [0086] (viia) drying
the evaporated solid phase residue, thus obtaining a dry powder;
and in the case of the preparation of an aqueous preparation,
[0087] (viii) suspending/solving the resultant dry powder in water
in a suitable concentration, preferably in a range as indicated
above.
DETAILED DESCRIPTION OF THE INVENTION
(A) General Definitions
[0088] Above and below terms and expressions are used which have
according to the understanding of the this invention the following
meanings:
[0089] The term "plant protecting agent" or "plant protective
agent" means, if not otherwise specified, any kind of synthetic or
natural agent, product, extract, composition that is effective in a
broad sense for the protection and health of a plant against
infection and damages by pathogens in vitro and/or in vivo. The
term includes agents, products, extracts, compositions or single
isolated components of extracts which may show a couple of
different biological activities and/or properties, such as
antimicrobial, antiviral, antifungal, and bio-stimulatory
activity/efficacy, growth inducing/promoting activity (with respect
to the plant to be protected), growth inhibitory activity (with
respect to the plant(s) competitive to the plant to be protected),
systemic and/or immunological acquired resistance
inducing/promoting activity, and allelopathy inducing/promoting
activity.
[0090] The term "biological plant protection" means according to
the invention, if not otherwise specified, that the protection of a
plant is achieved by naturally occurring or naturally derived
substances or sources preferably from plants, and not by synthetic
or chemical means or agents, which do not occur in nature,
preferably plants or part of plants.
[0091] The term "biological plant protecting (protective) agent" is
thus, consequently a plant extract, a plant preparation, a
composition based on plants or parts thereof, or an agent isolated
from a plant extract/preparation/composition, which all show
significant efficacy against a plant pathogen in vitro and/or in
vivo. This term includes also chemically synthesized compounds
which are structurally and functionally identical with the isolated
naturally derived compound, but excludes expressively chemically
synthesized pesticides and related compounds having no natural
derived counterpart.
[0092] The term "pesticide" means according to the invention, if
not otherwise specified, not naturally derived or occurring,
synthetic compounds, agents or compositions which have plant
protecting efficacy.
[0093] The term "plant pathogen" means a compound or composition or
living material, such as a microorganism (including viruses), which
causes disease or damage to the plant. In a narrower scope of the
invention the term is focused to pathogenic microorganisms
including metabolic products of these microorganisms.
[0094] The term "antimicrobial" according to the invention
encompasses an efficacy or activity against microorganisms,
including viruses, bacteria and fungi, that reduces or eliminates
in vitro and/or in vivo the (relative) number of active
microorganisms which attack the plant or parts thereof to be
protected. Thus, the term includes the terms "antiviral",
"antibacterial", and "antifungal". An "antimicrobial agent"
according to the invention is a biological plant protecting agent
as specified above, which prevents or reduces infections or damages
of a plant caused by a pathogenic microorganism.
[0095] The term "antibacterial" means according to the invention an
activity or efficacy (e.g. of an agent or extract, etc.), that
reduces or eliminates the (relative) number of active bacteria. An
"antibacterial agent" according to the invention is a biological
plant protecting agent as specified above, which prevents or
reduces in vitro and/or in vivo infections or damages of a plant
caused by a pathogenic bacterium.
[0096] The term "antiviral" means according to the invention an
activity or efficacy (e.g. of an agent or extract, etc.), that
reduces or eliminates the (relative) number of active viruses. An
"antiviral agent" according to the invention is a biological plant
protecting agent as specified above, which prevents or reduces in
vitro and/or in vivo infections or damages of a plant caused by a
pathogenic virus.
[0097] The term "antifungal" means according to the invention an
activity or efficacy (e.g. of an agent or extract, etc.), that
reduces or eliminates the (relative) number of active fungi. An
"antifungal agent" according to the invention is a biological plant
protecting agent as specified above, which prevents or reduces in
vitro and/or in vivo infections or damages of a plant caused by a
pathogenic fungus. The antifungal activity may lead to the
inhibition of mycelial growth as well as spore germination of
fungi.
[0098] The term "bio-stimulatory" means according to the invention,
if not otherwise specified, an activity or efficacy which
stimulates, increases or improves many different processes in the
plant or plant parts, such as improved generation of growth
promoting substances like sugars and amino acids, improved adequate
supply of cells with available nutrients and growth regulators,
enhanced cell metabolism, improved cell decontamination, enhanced
immune defense, promotion of growth and yield, induction of
systemic acquired resistance (SAR), inhibition of growth and yield
of competing plants (allelopathy). The bio-stimulatory activity can
be caused by agents, plant extracts and compositions including
metabolic compounds synthesized by the plant to be protected after
induction of their synthesis by said bio-stimulatory agent. A
"bio-stimulatory agent" according to the invention is a biological
plant protecting agent as specified above, which shows the
above-specified bio-stimulatory properties in a plant treated with
this agent in vitro and/or in vivo.
[0099] A "plant growth regulator" is a compound or a mixture of
substances either natural or synthetic, that modifies or controls
one or more specific physiological processes within a plant. If the
compound is produced within the plant it is called a plant hormone
e.g. auxins, gibberellins, abscisic acid and ethylene.
[0100] "SAR" (Systemic Acquired Resistance) occurs in a plant or
parts thereof according to the invention if it shows induction or
enhancement of activity of defense or protection related enzymes
(PR-proteins). Such enzymes include, for example, peroxidase,
.beta.-1,3-glucanse and NADPH oxidase.
(B) Plant Description
[0101] Agapanthus is originally indigenous to South Africa. Studies
on its distribution indicated that the evergreen species of
Agapanthus grows wildly from the south-western Cape eastwards into
Natal and further North. It is also grown in Europe, America,
Australia, New Zealand and South America.
[0102] The taxodermic classification of Agapanthus africanus
is:
TABLE-US-00001 . Division: Magnoliophyta Class: Liliopsida
Subclass: Lilidae Family: Amaryllidaceae Subfamily: Lilidaceae
Genus: Agapanthus Species: Agapanthus africanus (A. umbelattus)
[0103] The genus Agapanthus (L.) Hoffmg (Alliaceae) may be divided
into two groups according to the type of flowers they bear namely
those with flowers having short tubes with perianth segments
spreading out widely, and those with long tubes and perianth
segments that do not spread much. The genus is sometimes also
divided into evergreen or deciduous types. A. africanus (synonym A.
umbellatus) is the evergreen one with flowering stems of about 60
cm in length and deep blue flowers with a darker stripe down the
center of each petal. It grows 30 to 60 cm in height and has
shorter, fewer and more leathery leaves than the subspecies A.
praecox (orientalis). It also has much fewer flowers, usually about
12 to 18, in a smaller head than that of A. praecox and flowers
from December to March. There is also a rare white form, A.
walshii.
[0104] A. africanus can be cultivated. It is a perennial with a
large root system that enables it to go without water for long
periods of time. As the root volume increases from season to season
and give rise to new plants spontaneously, roots can also be used
to multiply the plant as a cultivation practice. Eventually the
plants begin to suffer through being overcrowded. For this reason
the clumps which they form should be lifted every few years and
divided. A. africanus grows in any kind of soil. To obtain good
results in poor soil, it may be necessary to prepare trenches of
approximately 30-45 cm deep and incorporate compost and manure.
Although the plants are drought tolerant, they flower better if
watered regularly during spring and summer when flower formation is
at its peak.
(C) Microorganisms
[0105] Six common South African plant fungal pathogens are chosen
to test for the fungitoxic properties of the plant extracts. These
fungal pathogens included Botrytis cinerea Pers.:Fr.
(Hyphomycetes), Fusarium oxysporum Schlechtend.:Fr. (Hyphomycetes),
Sclerotinia rolltsii Sacc. (Agonomycetes), Rhizoctonia solani Kuhn
(Agonomycetes), Botryosphaeria dothidea (Moug.: Fr.) Ces. & De
Not. (Loculoascomycetes) and Pythium ultimum Trow (Oomycetes).
Plant pathogenic bacteria used in this study include Agrobacterium
tumefaciens Smith and Townsend, Clavibacter michiganense
Spieckermann pv. michiganense Smith, Erwinia carotovora pv.
carotovora Jones, Xanthomonas campestris Pammel pv. phaseoli Smith,
Ralstonia solanacearum Smith and a human bacterium Moraxella
catharrhalis.
(D) Screening of Crude Extracts from Agapanthus Africanus for In
Vitro Antimicrobial and Biostimulatory Activity
(1) General
[0106] Crude extracts of different plant parts of Agapanthus
africanus are screened in vitro against six plant pathogenic fungi,
six plant pathogenic bacteria as well as one human bacterium (see
below. The plant parts are dried, ground and extracted with
preferably methanol as specified in more detail in the
Examples.
[0107] In another approach preparations comprising extracts/dry
powders from Agapanthus africanus in combination with respectively
produced extracts from wild garlic (Tulbaghia violacea) are tested
with respect to their plant protecting activities.
[0108] A standard chemical, Carbendazim/Difenoconazole is used as a
positive control. Screening activities are performed using a disk
diffusion method. To determine the bio-stimulatory activity of
crude extracts, two methods are applied. Firstly, the effect of the
extracts on the respiration rate of a monoculture yeast cells is
measured using a specially manufactured respirometer. Secondly,
radish seeds are used to ascertain the influence of crude extracts
on seed germination as well as root and coleoptile growth in
seedlings. In both techniques, ComCat.RTM. a commercial
biostimulant (EP 1051075) is used as a positive control.
(2) Antimicrobial Properties of A. Africanus Crude Extracts
[0109] Crude methanolic extracts of all different plant parts of A.
africanus significantly (P<0.05) inhibit the mycelial growth of
all test fungi, in vitro, at a concentration of 1 mg/ml (FIG. 1)
compared to the standard fungicide, used as a positive control.
[0110] The root extract completely inhibits mycelial growth of B.
cinerea, S. rolfsii, R. solani and B. dothidea, in vitro, and shows
a degree of control against F. oxysporum (77%) and P. ultimum
(64%). A crude leaf extract shows a similar inhibitory effect
against S. rolfsii, R. solani (FIG. 1) and B. dothidea, slightly
lower against B. cinerea (97%) and F. oxysporum (73%) but is
equally effective against P. ultimum as was the root extracts (FIG.
1). Extracts from the stalk also completely inhibit mycelial growth
of S. rolfsii and B. dothidea but are slightly less effective
against B. cinerea (87%) and F. oxysporum (73%) (FIG. 1).
[0111] To sum up, a preliminary assessment of the inherent
potential of crude extracts from A. africanus, based on in vitro
results, indicates that B. cinerea, S. rolfsii, R. solani and B.
dothidea are most sensitive to treatments with extracts from all
plant parts. P. ultimum, and to a lesser extent F. oxysporum, are
more resistant to treatment with the crude extracts (Table 1).
TABLE-US-00002 TABLE 1 In vitro antifungal activity of crude
extracts from different organs of A. africanus % Mycelial Growth
Inhibition for different fungi Plant Plant Botrytis Fusarium
Sclerotium Rhizoctonia Botryosphaeria Pythium extract organ cinerea
oxysporum rolfsii solani dothidea ultimum Plant X Root 100a .+-. 0
75c .+-. 4 100a .+-. 0 100a .+-. 0 100a .+-. 0 63d .+-. 2 Leaves
94ab .+-. 2 71c .+-. 2 100a .+-. 0 94ab .+-. 3 100a .+-. 0 64d .+-.
3 Stalks 90b .+-. 3 74c .+-. 2 100a .+-. 0 99a .+-. 1 100a .+-. 0
63d .+-. 2 Flower 98a .+-. 2 78c .+-. 5 100a .+-. 0 100a .+-. 0
100a .+-. 0 60d .+-. 4 *Standard 100a .+-. 0 50e .+-. 2 51e .+-. 3
52e .+-. 2 67d .+-. 5 7f .+-. 2
[0112] The flower and the aerial part crude extracts also show
almost the same inhibitory effect against all tested fungi, as it
is the case for other crude extracts (FIG. 1). However, aerial part
crude extracts are more effective in inhibiting the mycelial growth
of P. ultimum (79%) than other plant part extracts when tested
separately (FIG. 1). In all cases the crude extracts out perform
the standard fungicide. However, none of the extracts
exhibit-antibacterial activity against any of the plant pathogenic
bacteria tested.
(3) Biostimulator Properties of A. Africanus Crude Extracts
[0113] In order to establish whether crude extracts of A. africanus
plant parts possess inherent biostimulatory properties, their
effect on the respiration rate of a monoculture yeast cells are
first determined in vitro over a three hour period. Compared to
both a water and a positive control (ComCat.RTM.), similar
respiration rates are observed when extracts of different plant
parts are tested separately (FIG. 2). However, the aerial part
crude extract increases the respiration rate of yeast cells
significantly over the first two hours (FIG. 2).
[0114] Subsequently, the in vivo effect of crude extracts on the
germination of radish seeds as well as seedling growth is
determined. Crude extracts of the flowers, flower stalks, leaves
and the aerial part crude extract significantly (P<0.05)
increase seed germination by 21%, 18%, 16% and 6%, respectively,
compared to the water control (Table 2).
[0115] Although most extracts seem to have a stimulatory effect on
the germination of radish seeds in vitro, only the leaf and flower
extracts show a significant stimulatory effect on root growth of
the seedlings. Root, flower stalks and the aerial part crude
extract, on the other hand, significantly inhibit root growth
compared to the water control (Table 2).
TABLE-US-00003 TABLE 2 The effect of crude extracts from different
plant parts of A. africanus on the germination of radish seeds as
well as seedling growth Root Coleoptiles Plant extracts Germination
(%).dagger. length (mm) length (mm) Roots 62.67 .+-. 10c 30.62 .+-.
15.50d 24.01 .+-. 7.69bc Leaves 71.33 .+-. 11.5ab 43.14 .+-. 13.61a
24.64 .+-. 4.12abc Flowers stalks 72.67 .+-. 5.78a 38.40 .+-.
8.78bc 26.40 .+-. 4.05ab Flowers 74.78 .+-. 5.96a 43.09 .+-. 9.5a
26.71 .+-. 3.07a Aerial part 65.22 .+-. 4.56bc 35.81 .+-. 7.29c
23.07 .+-. 2.37c Comcat 63.11 .+-. 3.45c 40.31 .+-. 7.96ab 22.12
.+-. 2.37c Water (distil) 61.67 .+-. 2.37c 41.06 .+-. 7.46ab 23.10
.+-. 2.33c .dagger.Values designated with different letters, within
a column, indicate significant differences at the 5% level (P <
0.05) according to Duncan's multiple range procedure.
[0116] Both the flower stalk and flower extracts significantly
increase coleoptile growth of radish seedlings in comparison to the
water control. The commercial biostimulant, ComCat.RTM., used as a
positive control, has no significant effect on either seed
germination or seedling growth.
(4) Antifungal Properties of A. Africanus Crude Extracts Combined
with Extracts from Tulbaghia Violacea
[0117] A crude extract or a dry powder of wild garlic (T. violacea)
is prepared analogously to the methods described here for species
of the genus Agapanthus. The extracts or dried powders are mixed in
a 1:1 ratio and aquous solutions are applied in different
concentrations varying from 0.25 mg/ml to 2 mg/ml.
[0118] It is interesting to note that a 50:50 mixture of the two
extracts, applied at 0.5 mg/ml, shows total control of the six test
fungi (Table 3), whereas in comparison hitherto, applying
separately the two-fold concentration (1 mg/ml) of the A. africanus
preparation or the T. violacea preparation, inhibition of the
mycelial growth of the test fungi is not complete. Even a
concentration of 0.25 mg/ml of a combined extract/dry powder
preparation (1:1) leads to an over-all inhibition of the same
fungus system of more than 90%, indicating that significant
synergism is effective in the combination system. The same effect
is observable with other plant-protecting agents.
TABLE-US-00004 TABLE 3 In vitro antifungal activity of crude
extracts from the above ground parts of A. africanus (X) and T.
violacea (Y) used together in a 1:1 ratio and applied at 0.5 mg/ml.
Extract Mix % Mycelial Growth Inhibition for different fungi
(50:50) Plant Botrytis Fusarium Sclerotium Rhizoctonia
Botryosphaeria Pythium Fungus material cinerea oxysporum rolfsii
solani dothidea ultimum Plant X + Above 100a .+-. 0 100a .+-. 0
100a .+-. 0 100a .+-. 0 100a .+-. 0 100a .+-. 2 Plant Y ground
(50:50) parts Standard 100a .+-. 0 70c .+-. 3 68c .+-. 4 38 .+-. 2
87bc .+-. 3 4f .+-. 1
[0119] Standard broad spectrum fungicide;
Carbendazim/difenoconazole (Eria@) [0120] Different letters
following values indicate statistical significant differences.
(5) Summary Results
[0121] Results indicate that none of the plant extracts from A.
africanus shows any antibacterial activity. However, crude extracts
or dry powders of all different plant parts of A. africanus
significantly (P<0.05) inhibit mycelial growth as well as spore
germination in all test fungi, indicating a strong antifungal
activity of the preparations according to the invention. Root and
flower extracts as well as an extract of the aerial part crude
extracts show significantly higher antifungal activity than
extracts from leaves and stalks. Among the tested fungi, Pythium
ultimum, and to a lesser extent Fusarium oxysporum, shows a degree
of tolerance towards all extracts. This is especially significant
in light of the experience that mycelial growth inhibition by
fungicides is more difficult to accomplish than inhibition of spore
germination. The average inhibitory effect of the plant extracts
against the test fungi ranges between 59 and 100%. Of these the
aerial part crude extract (leaves, stalks and flowers combined) is
highest (92%), emphasizing the broad-spectrum fungicidal potential
of the extract. Moreover, the potent anti-fungal activity shown by
this combined extract indicate a synergistic effect of different
active substances and support the assumption of differential
accumulation of bioactive compounds in different organs of plants.
An extract from the aerial part crude extract of A. africanus as
well as a flower extract significantly enhances the respiration
rate of a monoculture yeast cells and all plant part extracts
enhance the germination of radish seeds, thus indicating that a
bio-stimulatory in vitro activity is effective. The aerial part
crude extract increases the respiration rate of a monoculture yeast
cell substantially compared to the separate plant part extracts as
well as to both the water control and the positive control,
ComCat.RTM.. The same effect is not observed when the different
plant part extracts were tested separately. All the crude extracts
of different plant parts of A. africanus as well as the aerial part
crude extract increased the germination percentage of radish seeds
indicating a stimulatory effect. It is possible that one or more
active substance contained in the crude extracts could have had a
stimulatory effect on one or more of the respiratory enzymes, most
probably regulatory enzymes, or even storage material mobilization.
Combined extracts/dry powders based on preparations from two or
more plants having plant protecting properties, wherein at least
one is a species of the genus Agapanthus, show over a broad range
antifungal and bio-stimulatory efficacy at least in vitro based on
synergistic effects.
(E) In Vivo Antimicrobial and Bio-Stimulatory Effects of
Prepeparations from Agapanthus
(1) General
[0122] Mycosphaerella pinodes (Berk & Blox.) Vesterger, is a
major constraint to field pea (Pisum sativum L.) production and is
the most destructive and widespread disease throughout the field
pea growing areas of the world. All aerial parts of the pea plant
are susceptible to infection while growth, yield and seed quality
are all adversely affected. The fungus infects pea seedlings as
they emerge causing girdling stem lesions that reduce field pea
populations and increase lodging. Later it also causes necrotic
lesions on leaflets and stipules and, in exceptional circumstances,
abscission of the leaflets. M. pinodes is spread via pycnideospores
throughout the season. After germination of spores, the fungus
grows over the plant surface for some distance before forming an
apersorium and penetrating the cuticle. Symptoms are characterized
by brown to purplish, coalescing lesions on aerial tissue. Crude
extracts of flowers, roots, leaves and the aerial plant parts are
subsequently tested under greenhouse conditions against
Mycosphaerella pinodes, the cause of black spot or Ascochyta blight
in peas. Fourth internode leaves are removed from four week old pea
plants, placed on moist filter paper in petri dishes and inoculated
with a M. pinodes spore suspension 30 min before and after
treatment with the extracts. The control of Ascochyta blight by
different concentrations of the crude extracts from different plant
parts of A. africanus is measured in terms of lesion size over a 6
day period at 20.degree. C. in a growth cabinet.
(2) In Vivo Antifungal Activity of Preparations of Agapanthum Under
Glasshouse Conditions
[0123] In the in vivo screening antifungal trial, using pea leaves
inoculated with M. pinodes spores either before or after treatment
with the plant extracts, the extract of A. africanus inhibits
completely spore germination of M. pinodes at a concentration near
1 mg/ml, when the extract is applied before spore inoculation. This
indicates that application of A. africanus on crops as a
preventative measure has potential in the agricultural
industry.
TABLE-US-00005 TABLE 4 In vivo antifungal activity of crude
extracts from the above ground parts of A. africanus against
Mycosphaerella pinodes on pea leaves Extract Mean lesion Treatment
concentration size (mm) % Inhibition Extract sprayed 2 mg ml.sup.-1
0 100% on leaves first and 1 mg ml.sup.-1 0 100% spore inoculation
0.5 mg ml.sup.-1 2.37 81% followed 30 min 0.25 mg ml.sup.-1 4.07
68% later *Fungicide 0 100% standard 12.8 -- Spores only Leaves
inoculated 2 mg ml.sup.-1 0 100% with spores first 1 mg ml.sup.-1
0.26 98% and extracts 0.5 mg ml.sup.-1 3.8 70% sprayed on leaves
0.25 mg ml.sup.-1 5.29 59% 30 min later *Fungicide 0 100% standard
12.8 -- Spores only *Standard broad spectrum fungicide;
Carbendazim/difenoconazole (Eria.sup..COPYRGT.)
[0124] Treatment of detached pea leaves with crude extracts of
different plant parts of A. africanus, both before and after
inoculation with M. pinodes spores, results in significant
differences among extracts, extract concentration and method of
inoculation in suppressing lesion development (Table 4). Among
extracts the aerial plant part extract is most effective in
suppressing lesion development, caused by M. pinodes on detached
pea leaves, especially when applied before spore inoculation. The
aerial plant part extract is also effective at the lowest
concentration (MIC=0.5 mg/ml) compared to other extracts. When this
extract is applied after spore inoculation, suppression of lesion
development on pea leaves is also statistically significant
compared to other extracts although complete suppression is
observed only with the highest concentration of 2 mg/ml (Table
4)
[0125] In comparison the flower extract performs second best in
terms of lesion development suppression both when applied before or
after inoculation (MIC between 1 and 2 mg/ml) The root extract
completely inhibits lesion development only at a concentration of 2
mg/ml when applied before inoculation. Although complete
suppression of lesion development is not observed with the 2 mg/ml
concentration when the root extract is applied after spore
inoculation, the degree of suppression is statistically significant
compared to the untreated control, except at 0.25 mg/ml (Table 4).
The leaf extract fails to suppress lesion development completely
both when applied before and after spore inoculation but, in both
cases, the degree of suppression obtained is significant compared
to the untreated control, except at 0.25 mg/mg (Table 4).
[0126] Crude extracts of different plant parts of A. africanus
suppress in vivo lesion development on detached pea leaves to
variable degrees depending on the concentration applied as well as
the time of inoculation. Of these the aerial part crude extract is
most effective at all concentration levels tested, compared to the
other plant part extracts, both when applied before and after
inoculation of detached pea leaves with M. pinodes spores. The
flower extract also shows significant suppression of lesion
development at a relative low concentration. As the aerial plant
part extract contains compounds from flowers, flower stalks and
leaves, the possibility of different active substances contained in
the different parts showing a synergistic effect in either
inhibiting spore germination or mycelial infection or both is not
excluded.
[0127] The ability of the aerial plant extract as well as the
flower extract to completely suppress lesion development even when
applied after inoculation of detached pea leaves, is especially
significant considering that the standard fungicide failed to do
so.
[0128] Treatment of detached pea leaves with root and leaf extracts
is less effective in preventing M. pinodes infection at lower
concentrations when applied both before and after spore is
inoculation compared to the aerial plant part and flower extracts.
The necrotic lesions measured on pea leaves treated with root and
leaf extracts at concentrations lower than 1.0 mg/ml are similar to
that measured on control leaves inoculated with spores only.
However, when applied to detached pea leaves before spore
inoculation, both extracts still show significant suppression of
lesion development. Interestingly, the aerial plant part extract,
of which leaves formed the largest portion, performs best overall.
A possible synergistic effect between compounds contained in
flowers, flower stalks and leaves in enhancing the fungicidal
properties of A. africanus again is assumed. In terms of the
potential to develop a natural product from A. africanus, the fact
that the root extract is less effective than the aerial plant part
extract underlines its exclusion and implies non-destructive
collection.
[0129] The present study confirms that, especially a combined crude
extract of aerial plant parts of A. africanus at a concentration of
0.5 mg/ml and lower, has the potential to be applied as both a
preventative or corrective measure against infection of pea plants
by M. pinodes spores. There are strong indications that the extract
possesses significant potential as a corrective broad spectrum
antifungal agent. In conclusion, the efficacy of different plant
part extracts of A. africanus varies in suppressing lesion
development on detached pea leaves caused by M. pinodes in vivo.
The aerial plant part extract is most effective, especially when
applied before spore inoculation and at a relatively low
concentration of 0.5 mg/ml. However, application at higher
concentrations after inoculation with M. pinodes spores shows
complete inhibition of spore germination or infection or both.
Importantly, none of the extracts causes phytotoxic yellowing or
necrosis on detached pea leaves even at the highest concentrations
applied.
(3) Phytotoxic Effects of Preparations from Agapanthus on Pea
Leaves Under Glasshouse Conditions
[0130] The in vivo phytotoxicity rating of the aerial plant parts,
flower, root and leaf crude extracts of A. africanus, in terms of
its interaction with and potential to induce necrosis in pea
leaves, reveals that the crude extract is not phytotoxic even at
the highest concentration tested (Table 5a, b) and the symptomless
effect of the extract is similar to that of the water and standard
fungicide controls. All plant part extracts of A. africanus as well
as the standard fungicide control differs significantly from the
leaf necrosis induced by the M. pinodes spore suspension.
TABLE-US-00006 TABLE 5a Mean foliar phytotoxicity symptom rating on
a six-category scale following direct inoculation of fourth node
pea leaflets with the highest concentration of crude aerial plant
part, flower roots and leaf of A. africanus. Mean foliar
phytotoxicity symptom Plant extracts applied as foliar treatments
Concentration at 2 mg/ml Aerial plant parts 0.0b Flowers 0.0b Roots
0.0b Leaves 0.0b Standard fungicide 0.0b Spore suspension 4.2 .+-.
0.8a
TABLE-US-00007 TABLE 5b Phytotoxic effect of crude extracts from A.
africanus plants on pea leaves. Mean Lesion size Extract (mm)
indicating Treatment Concentration phytotoxicity Crude extract only
2 mg ml.sup.-1 0 1 mg ml.sup.-1 0 0.5 mg ml.sup.-1 0 0.25 mg
ml.sup.-1 0
[0131] None of the different plant part extracts of A. africanus
show any phytotoxic effect on detached pea leaves even at the
highest concentration applied.
(4) Control of Sorghum Covered and Loose Smuts by an Aerial Part
Crude Extract of Agapanthus Africanus Under Field Conditions
[0132] Sorghum (Sorghum bicolor L. Moench) is an important source
of food in many non-developed countries and serves as staple food
for the majority of people. It is predominantly grown in
small-scale production systems under a wide range of environmental
conditions. However, production of sorghum is less than 1.0 ton/ha
due to various reasons. Sorghum covered kernel (fSporisorium sorghi
Link, G. P. Clinton) and loose kernel smuts (Sporisorium cruenta
Kuhn, A. A. Potter) are major factors that account for low yields.
Both diseases occur frequently where sorghum is grown without
treating seeds against these two pathogens.
[0133] Treatment of sorghum seeds with an aerial part crude extract
of A. africanus before planting completely (100%) (P<0.05)
reduce the incidence of both covered smut (Table 6a) and loose smut
(Table 6b) compared to the corresponding untreated controls, and in
both cases compared favourably with the synthetic fungicide
Thiram.
TABLE-US-00008 TABLE 6a Effect of an aerial part crude extract of
A. africanus on the percentage covered kernel smut disease
incidence under field conditions. Mean plant % mean smut Yield
Treatments population incidence (ton ha.sup.-1) Aerial plant
extract 171 .+-. ?a 0b 3.0a Thiram 175a 0b 2.6ab Control 173a 5a
1.6b
TABLE-US-00009 TABLE 6b Effect of an aerial part crude extract of
A. africanus on the percentage loose kernel smut disease incidence
under field conditions. Mean plant % mean smut Yield Treatments
population incidence (ton ha.sup.-1) Aerial plant extract 175 .+-.
?a 0b 2.9a Thiram 175a 0b 2.1ab Control 175a 18a 1.3b
[0134] Values designated with different letters differed
significantly (P<0.05) according to Duncan's Least Significant
Difference (LSD) statistical procedure.
[0135] Inoculation of pre-planted sorghum seed with covered or
loose smuts spores, without also treating the seeds with either
Thiram or the crude A. africanus extract (untreated controls),
significantly decreases the final yields (Tables 6a, b). In the
case of covered smut the yield loss is 46.7% and, in the case of
loose smut, 55.2%. However, in both cases, there is no significant
difference in yield between plots treated with either Thiram or the
A. africanus crude extract. Probably due to the high standard
deviation, no significant difference in yield between the Thiram
treated and untreated controls can be observed in both cases.
[0136] The percent covered and loose smuts incidences are
negatively correlated (R.sup.2=-0.92 and -75 respectively) with
sorghum grain yield indicating the negative impact both smut
diseases had on the yield.
(5) The Effect of A. Africanus Extracts on the Defense Mechanism of
Plants (SAR)
[0137] Plants (e.g. wheat and sunflower) elicit, when treated with
an extract of A. africanus and another reference plant (Tulbaghia
violacea) according to the invention, a significant activation of
PR-proteins such as NADPH oxidase, peroxidase and
.beta.-1,3-glucanse. Wheat plants treated with the A. africanus
extract show strong induction in NADPH oxidase activity after 6 h
reaching the highest activity at 9 h (112%) over the previous
sampling time. Activity remained high for up to 48 h (FIG. 4).
Sunflower reacts to treatment with the A. africanus extract in the
sense that two peaks in NADPH oxidase activity can be observed. The
first peak is reached 6 h after treatment with an increase in
activity of 61% over the previous sampling time while the second
peak in activity is reached 48 h after treatment with an increase
in activity of 333% over the previous sampling time (FIG. 5). From
these results it seems that in the C4 plant, wheat, activity
induction by treatment with the A. africanus extract is more
pronounced than in sunflower, a C3 plant. Wheat treated with the A.
africanus extract shows a significant induction (100%) in
peroxidase activity 24 h after treatment and this activity is
maintained over the test period (FIG. 6). In the case of sunflower
the A. africanus extract induces peroxidase activity significantly
especially after 48 h and 96 h (FIG. 7). For A. africanus the
induction is 212% after 48 h and 230% after 96 h. The sunflower
control, however, shows a slight increase in peroxidase activity
over the 96 h period indicating some natural resistance. Agapanthus
extracts induce defense mechanisms in wheat and sunflower plants.
These extracts induce localized acquired resistance, the
accumulation of PR-proteins by gene activation and ultimately
systematic acquired resistance. The fact that the extracts induce a
defense response in both the wheat and sun flower samples indicate
that the extracts are responsible for the induction of a general
broad-spectrum defense response. The extract-induced increase in
defense related enzyme activities was lower, but comparable to the
increase obtained during infection with resistant cultivars
(F) Isolation, Purification and Identification of Antifungal
Compounds from Root and Aerial Plant Part Extracts of Agapanthus
Africanus
(1) General
[0138] Although information about the chemical analysis of
different plant parts of A. africanus is scanty, initial attempts
was made by Takeda et al. (1955, Chemistry Abstract 50) who
isolated and identified the compound yuccagenin from the roots.
Others (Stephen, 1956 Journal Chemistry Society 1167.; Mathew et
al., 1957 Journal Chemistry Society 262), working with several
unspecified species of Agapanthus, reported the new spirostan
sapogenin, agapanthagenin. Subsequently, in addition to the
previously reported compounds, Gonzalez et al. (1973 Phytochemistry
13:627-631) isolated and identified two new spirostan sapogenins
from the root system of A. africanus. Most previous studies
concentrated on the isolation and identification of natural
compounds from the root parts of A. africanus, but the relationship
between these compounds and antimicrobial activity has not been
established. Moreover, virtually nothing is known about the
chemical composition of the aerial plant parts as well as their
fungicidal properties.
(2) Antimicrobial Activity of Liquid-Solid Extractions of the Roots
and Aerial Plant Parts
[0139] The semi-purified fractions of different plant parts of A.
africanus, obtained by means of liquid-solid extraction, differ
significantly in inhibiting the mycelial growth of F. oxysporum
(Table 8). The semi-purified extract of the roots, contained in
diethyl ether, and both the ethyl acetate and dichloromethane
extract of the aerial plant parts, significantly (P<0.05)
inhibit mycelial growth of F. oxysporum compared to the hexane
extract (Table 8). The diethyl ether root fraction showed the
highest inhibition (62%) compared to the ethyl acetate and
dichloromethane fractions that showed similar inhibition effects
(51%).
[0140] In case of the combined aerial plant part extract, both the
ethyl acetate and dichloromethane semi-purified liquid-solid
extracts are most active and completely inhibit the mycelial growth
of F. oxysporum (Table 7). This is statistically significant
compared to the antifungal activity of both the hexane and diethyl
ether fractions and compared favorably with the standard fungicide,
Carbendazim/difenoconazole. Mycelial growth inhibition of F.
oxysporum by semi-purified fractions of the aerial plant parts is
also significantly (P<0.05) higher than that of the roots (Table
7).
TABLE-US-00010 TABLE 7 Antifungal activity of semi-purified
liquid-solid extractions of the roots and aerial parts of A.
africanus against Fusarium oxysporium Mycelial growth inhibition
(%).dagger. Plant part Solvent Roots Aerial parts Hexane 24.3d 5.4c
Diethyl ether 62.3b 13.7b Ethyl acetate 51.1c 100a Dichloromethane
51.3c 100a Fungicide 100a 100a .dagger.Values designated with
different letters within a column indicate a statistically
significant difference at the 5% level (P < 0.05) according to
Duncan's multiple range procedure.
[0141] The recovered yields of the root and aerial plant part
liquid-solid extractions are presented in Table 8. Despite its low
activity, the hexane solvent system provides high amounts of
semi-purified residue of the roots and aerial plant parts ranging
between ca. 4.3 to 5.4% while the diethyl ether solvent system
provides ca. 3% and 2% from the roots and aerial plant parts,
respectively. The ethyl acetate solvent system yields approximately
ca. 1% residues in both the root and aerial part extracts while the
recovered yield from the dichloromethane solvent system is less
than ca. 1% in both cases.
TABLE-US-00011 TABLE 8 Residual yield recovered from A. africanus
root and aerial plant part extracts obtained by means of
liquid-solid extraction with a series of solvents, after drying at
35.degree. C. Crude root extract Crude aerial part extract Solvents
(268.5 g) (368.83 g) Hexane 14.5 g 15.89 g Diethyl ether 7.6 g 5.96
g Ethyl acetate 3.09 g 4.33 g Dichloromethane 0.36 g 0.45 g The
original dry mass of crude extracts is indicated in brackets.
[0142] The diethyl ether extract of the root and the ethyl acetate
as well as the dichloromethane fractions of the aerial parts show
the highest (>50%) antifungal activity against F. oxysporum.
However, as the recovery of compounds in the dicholoromethane
fraction is extremely low (Table 8), only the ethyl acetate
fraction of the aerial parts, together with the diethyl ether
fraction of the roots, are chosen for further activity directed
column chromatography fractionation.
(3) Activity Directed Column Chromatography Fractionation of the
Most Active Liquid-Solid Extracts
[0143] After collecting 300 column chromatography fractions of the
diethyl ether root extract, every third fraction is spotted on a
Q-TLC plate and developed with butanol:acetone:methanol (7:2:1;
v/v) in order to obtain TLC profiles used as an indicator for
combining fractions with similar profiles. In this way 17 combined
column fractions can be obtained from the root extract of which
nine showed high mycelial growth inhibition (65-97%) against F.
oxysporium (Table 9). After treating the ethyl acetate aerial part
fraction in the same way, 20 combined column chromatography
fractions are obtained of which six were active in inhibiting the
mycelial growth of F. oxysporium by more than 50% (Table 9).
[0144] Although nine of the combined column chromatography root
fractions show high mycelial growth inhibition against F. oxysporum
at a relatively low concentration of 625 .mu.g/ml (w/v), only
fraction 13 is used for further purification by means of
preparative thin layer chromatography (PTLC) due to the extremely
low recovery of the other fractions. In the case of the aerial
plant parts, only column fraction 14, that showed complete mycelial
growth inhibition against the test organism at the low
concentration of 125 .mu.g/ml (w/v), was further purified (Table
9).
TABLE-US-00012 TABLE 9 Antifungal activity of combined fractions
obtained from the most active root and aerial plant part
liquid-solid extracts following column chromatography against F.
oxysporum. Combined column % mycelial growth inhibition of Plant
part fraction number F. oxysporum Roots 8 76 9 67 10 93 11 97 12 83
13 83 14 74 16 65 17 71 Aerial plant parts 7 58 8 60 14 100 17 57
18 55 19 53 Only the most active combined column chromatography
fractions are shown.
(4) Preparative Thin Layer Chromatographic (PTLC) Purification of
Active Compounds from Column Chromatography Fractions
[0145] Following preparative thin layer chromatography purification
of active column fraction number 13 obtained from the root, 12
purified P-TLC fractions can be recovered, of which fraction 9 is
most active (95%) against F. oxysporum (Table 10). In the case of
active column fraction number 14 obtained from the aerial parts,
three purified P-TLC fractions are recovered following washing with
acetone, of which fraction number 1 shows complete mycelial growth
inhibition against F. oxysporum (Table 10).
TABLE-US-00013 TABLE 10 Antifungal activity of P-TLC fractions
obtained from root and aerial plant parts against F. oxysporum.
Mycelial growth inhibition (%) P-TLC fractions against F. oxysporum
Fraction 9 of the root 95 Fraction 1 of the aerial plant parts
100
[0146] After controlling the most active P-TLC fractions of both
the root and aerial parts for purity, by obtaining Q-TLC profiles
after acetylating the molecules and acidifying the mobile phase
with 1N HCl, the root fraction consists of four compounds (Table
11). By means of acidified P-TLC separation, these four compounds
are purified and tested for antifungal activity. All four compounds
are highly active. The active P-TLC fraction of the aerial parts
proves to contain only one pure compound that is active (Table 11).
All five of these pure compounds are subsequently subjected to
nuclear magnetic resonance (NMR) spectroscopy in order to elucidate
their molecular structures.
TABLE-US-00014 TABLE 11 Antifungal activity of pure compounds
obtained from the most active P-TLC root and aerial plant part
fractions, following acidification, against F. oxysporum. %
mycelial growth inhibition of Plant part Compound number F.
oxysporum Roots 1 100 7 87 8 93 9 97 Aerial plant parts 1 100
(5) Identification of Active Compounds Purified from Roots and
Aerial Parts of A. Africanus by Means of Nuclear Magnetic Resonance
(NMR) Spectroscopy
[0147] Based on the .sup.1H NMR spectra the single antifungal
substance derived from the combined aerial plant parts of A.
africanus provides a novel compound, saponin (1). Exactly the same
saponin (1) can be identified as one of the four active substances
derived from the roots of A. africanus together with three known
flavonoids, 5,7,4'-trihydroxyflavanone (7),
5,7,3'4'-tetra-O-acetylflavanone (8) and
trans-4,2',4'-Tri-O-acetylchalcone (9). Structural elucidation can
be achieved via spectroscopic methods (1D NMR and 2D NMR) FAB and
EI-MS spectrometry, and chemical methods such as hydrolysis.
(6) Saponin (1) Isolated from Both the Roots and Aerial Parts of A.
Africanus
[0148] The methanol extract from the roots and aerial plant parts
of A. africanus yields compound (I) as a light brown precipitate in
relatively large amounts. To obtain an acceptable level of purity,
the fractions are washed repeatedly with acetone. This, and the
highly insoluble nature of compound (1) (FIG. 3A), invariably lead
to substantial losses, prohibiting reliable quantification. Due to
the complexity of the .sup.1H NMR spectrum of the non-derivatised
saponin, the peracetate derivative (2) (FIG. 3B) is used in the
structural elucidation. FAB-MS shows the [M+H].sup.+ ion at m/z
448, consistent with the molecular formula C.sub.27H.sub.44O.sub.5
of the aglycone with the molecular mass of 448.
(7) Isolation and Identification of Flavones
[0149] In addition, flavanones (compound 7) and (compound 8) can be
isolated from the roots after acetylation of fractions nine of the
diethyl ether extract by means of P-TLC chromatography.
Characteristic of these compounds is the presence of the 3-CH.sub.2
[(two doublets of doublets, .delta. (3.00-3.15) and (2.70-2.85)]
and the 2-H [(doublets of doublets, .delta. (5.00-6.00)] in their
.sup.1H NMR spectra.
[0150] Besides the sapogenin compound isolated from the root parts
of A. africanus, by far the most frequently encountered flavanone,
naringenin (5,7,4'-trihydroxyflavanone), was identified
##STR00002##
[0151] It is common as a free phenol, occurs with a wide variety of
glycosylation patterns, has been isolated in all of its possible
O-methylated forms and is susceptible to various C-alkylation
processes (Batterham et al., 1964). Naringenin can be isolated
after acetylation and P-TLC separation as the 5,7,4'-tri-O-acetyl
derivative (7).
[0152] Additionally, 5,7,3'4'-tetra-O-acetylflavanone (8) can be
isolated after acetylation and PLC separation from fraction number
nine of the root part.
##STR00003##
[0153] Moreover, an additional compound,
trans-4,2',4'-Tri-O-acetylchalcone, Isoliqiuritigenin can be
isolated as a peracetate derivative (9) after acetylation and PLC
separation from fraction-number nine of the roots. This compound is
found in many leguminous plants (Roux et al., 1962, Biochemical
Journal, 82:324).
[0154] 5
##STR00004##
(8) Discussion
[0155] Subsequent activity directed purification of only the most
active fractions, using column and preparative thin layer
chromatography followed by nuclear magnetic resonance (NMR)
spectroscopy, and fast atom bombardment (FAB-MS), reveals the
universal presence of a novel saponin (1) with strong antifungal
activity at a concentration of approximately 125 .mu.g/ml (100-150
.mu.g/ml). Additionally, three flavonoids 5,7,4'-tri-O-flavanone
(7), 5,7,3'4'-tetra-O-acetylflavanone (8) and
trans-4,2',4'-Tri-O-acetylchalcone (9), showing strong antifungal
activity at a concentration of about 625 .mu.g/ml (570-650
.mu.g/ml), can be purified from A. africanus roots.
[0156] Semi-purified extracts of the roots and aerial parts of A.
africanus obtained by means of solid-liquid extraction with hexane,
diethyl ether, ethyl acetate and dichloromethane solvent systems in
this order of increasing polarity, variety in antifungal activity.
The diethyl ether extraction of the roots is most active in
inhibiting the mycelial growth of F. oxysporum, used as test
organism in the activity directed purification protocol, while the
ethyl acetate extraction of the aerial parts is most active.
Although the hexane extraction removes most compounds from both the
roots and aerial parts, it is comparably active in both cases.
[0157] Following column fractionation of the active liquid-solid
extractions, the Q-TLC profiles show diverse chemical constituents
in the roots and the aerial plant parts of A. africanus while the
latter extract contains comparatively more compounds. However, four
active compounds can be isolated from the diethyl ether root
extract while only one active compound can be detected in the ethyl
acetate aerial part extract. Despite the lower number of active
substances in the aerial parts, following different extraction
procedures, this fraction is more active in inhibiting the mycelial
growth of the the test fungus, F. oxysporum.
[0158] Compounds purified from the roots and aerial plant parts of
A. africanus can be identified by means of .sup.1H-NMR and
.sup.13C-NMR spectroscopy. The major compound predominantly
isolated from both the roots and aerial plant parts is a novel
steroidal saponin with a three sugar chain attached at the C3
position of ring A in the aglycone moiety. The compound can be
identified as
3-[O--.beta.-D-glucopyranosyl-(1''-3')-.alpha.-L-rhamnosyl-(1''-2')-.beta-
.-D-glucopyranosyl oxy]agapanthegenin. Previously, a C-3
monoglycosylated saponin, with the same aglycone, was isolated from
the root system of A. africanus by both Stephen (1956) and Gonzalez
et al. (1974). However, the saponin identified in this study is
different with respect to the additionally attached three sugar
chain. Sapogenins with a three sugar chain attached are of sporadic
occurrence. Additionally, three flavonoids with notable fungicidal
activity, 5,7,4'-tri-O-flavanone, 5,7,3'4'-tetra-O-acetylflavanone
and trans-4,2',4'-tri-O-acetylchalcone, can be isolated from the
roots of A. africanus. Each individual purified flavonoid showed
significant in vitro mycelial growth inhibition against F.
oxysporum.
[0159] The in vitro and in vivo antifungal activity observed with
crude and semi-purified extracts of different plant parts of A.
africanus seems to be related to the presence of the major
compound, a steroidal saponin in all plant parts, as well as the
presence of the three flavonoids 5,7,4'-tri-O-flavanone,
5,7,3'4'-tetra-O-acetylflavanone and
trans-4,2',4'-Tri-O-acetylchalcone in the root system.
[0160] In summary, all four compounds in their pure forms provide
an acceptable level of efficacy in inhibiting the mycelial growth
of the test fungus in vitro and hold great promise to be applied as
one or more natural products in integrated disease management
systems in vivo. However, due to a possible synergistic effect of
the combined compounds, the application of either a crude or a
semi-purified extract might be considered. Importantly, in light of
the fact that A. africanus is a perennial, an extract of the
combined aerial parts might possess the most potential to be
developed into a natural product as harvesting of the above soil
parts is non-destructive. This implies that the potential exists
for A. africanus to be cultivated as a new crop and to serve as a
source for a natural fungicide with broad spectrum control of
economically important plant pathogens, especially to small-scale
farmers who have no access to synthetic chemicals.
FIGURE LEGENDS
[0161] FIG. 1: In vitro inhibitory effect of crude extracts from
different plant parts of A. africanus on the mycelial growth of
various fungi. Vertical bars indicate standard deviations. Bars
designated with different letters indicate significant (p<0.05)
differences between means according to Duncan's multiple range
procedure. Y-axis: mycelial growth inhibition (%). (1)=root;
(2)=leaves, (3)=Flower stalks; (4)=Flowers; (5)=above ground plant
parts; (6)=reference fungicide
[0162] FIG. 2: The effect of crude extracts from different plant
parts of A. africanus on the respiration rate of a monoculture
yeast cells. Vertical bars indicate standard deviations. X-axis:
time (min); Y-axis: respiration rate (cm.sup.3 CO.sub.2
release).
1=roots, 2=flower stalks, 3=above ground plant parts, 4=water,
5=leaves, 6=flowers, 7=ComCat.RTM.
[0163] FIG. 3A: Sructure of novel saponin (1):
[0164]
3-[{O--O-D-glucopyranosyl-(1''-3')-.alpha.-L-rhamnosyl-(1''-2')}-.b-
eta.-D-glucopyranosyloxy]agapanthegenin.
[0165] 1: R.dbd.H; 2:R=Ac
[0166] FIG. 3B: Structure of the aglycone (3; agapanthegenin), the
glucosylated sapogenin (5) and their respective O-acetyl
derivatives (4 and 6).
[0167] 3: R.dbd.H; 4: R=Ac; 5: R=glucose; 6: R=acetyl glucose
[0168] FIG. 4: NADPH oxidase activity pattern in wheat treated with
an Agapanthus extract and a Tulbaghia extract (as reference)
according to the invention in dependency of the time after
treatment.
[0169] FIG. 5: NADPH oxidase activity pattern in sunflower treated
with an Agapanthus extract and a Tulbaghia extract (as reference)
according to the invention in dependency of the time after
treatment.
[0170] FIG. 6: peroxidase activity pattern in wheat treated with an
Agapanthus extract and a Tulbaghia extract (as reference) according
to the invention in dependency of the time after treatment.
[0171] FIG. 7: peroxidase activity pattern in sunflower treated
with an Agapanthus extract and a Tulbaghia extract (as reference)
according to the invention in dependency of the time after
treatment.
EXAMPLES
Example 1
Preparation of Crude Extracts
[0172] Dried plant material was powdered, using a Retsch SM2000
cutting mill and soaked in 100% methanol (v/g) at a ratio of 2 ml
g.sup.-1 dry weight on a roller mill overnight and the supernatant
subsequently decanted. This was repeated five times. The combined
suspensions were filtered twice, first under vacuum through a
double layer of Whatman filter paper (No. 3 and No. 1) and then by
gravity through a single sheet of Whatman No. 1 filter paper. The
methanol was removed from the clear supernatant by means of vacuum
distillation at 30-35.degree. C. using a Buichi Rotary Evaporator.
The remaining aqueous solution was referred to as the crude
extract.
Example 2
Preparation of a Dry Powders
[0173] Instead of the preparation of a crude extract according to
Example 1, the preparation of a dry powder is also applicable. The
implication for it is that a considerable reduction in production
costs might be achieved and that more hectares of cultivated land
can be treated with the product in this form. The preparations of
Example 1 and Example 2 show almost identical qualitative and
quantitative results with respect to their plant protecting
activity/efficacy. Plant material is dried at 35.degree. C.,
preferably in a drying oven. Dried plant material is first ground
to a course powder, using a Retsch SM2000 cutting mill, and
subsequently to a fine powder using a special mill than can grind
to particles smaller than 100 micron to prevent clogging in a
nozzle spray system. The powder is soaked in 100% methanol or
ethanol (v/g) at a ratio of preferably 2 ml/g dry weight on a
roller mill for 48 h and the bulk of the methanol decanted before
the remaining methanol is allowed to evaporate on a large surface.
Subsequently, the powder is treated with 100% Ethanol for 24 h in
exactly the same way as with methanol. The final product is in the
form of a wettable powder that is applied at a rate of preferably 1
g/l and at approximately 300-600 liters per hectare.
Example 3
Screening for Antifungal Properties
[0174] A modified agar dilution method (Rios et al. 1988, Journal
of Ethnopharmacology 23:127-149) was used for determining the
inhibition of mycelial radial growth of the test organisms by the
plant extracts. All plant pathogenic test fungi were cultured on 2%
(rn/v) malt agar, prepared according to the specifications of the
manufacturers, and autoclaved for 20 min at 121.degree. C. On
cooling to 45.degree. C. in a waterbath, 300 .mu.l of a 33% (m/v)
Streptomycin solution was added to the basal medium for controlling
bacterial growth. Dried material of each plant extract was
dissolved in 100 ml sterile distilled water and amended in the agar
to yield a final concentration of 1 mg/ml. Working in a laminar
flow cabinet, the medium was poured into 90 mm sterile plastic
Petri dishes, to a thickness of 2-3 mm, and allowed to set The
center of each test plate was subsequently inoculated with a 5 mm
size plug of 7-10 day old cultures, for each of the pathogens
separately. A plate containing only the basal medium served as
control. Additionally, a plate containing a standard fungicide,
carbendazim/difenococnazole (Eria.RTM.-187.5 g/1 EC), at 1 .mu.g/ml
was used as a positive control against each test organism
separately to determine the effectiveness of the extracts by
comparison. Plates were incubated for four days at 25.+-.2.degree.
C. in a growth cabinet. Each assay was performed in triplicate.
Radial mycelial growth was determined after four days by
calculating the mean of two perpendicular colony diameters for each
replicate. The measurement included the assay wells (March et al.,
1991, Zentralbladtfur Mikrobiologie 146:291-295; Pfaller et al.,
1992, Antimicrobial Agents and Chemotherapy 36:1805-1809) and was
expressed as percentage mycelial growth inhibition by calculating
according to the formula of Pandey et al (1982, Zeitschrift
Pflanzenkrankheit und Pflanzenschutz 89:344-349):
(dc-dt)/dc.times.100, where dc=average diameter of the fungal
colony of the negative control and dt=average diameter of the
fungal colony treated with the extracts. The data reported were
pooled from the two experiments.
Example 4
Screening for Antibacterial Properties
[0175] A modified agar diffusion method (Caceres et al., 1993,
Journal of Ethnopharmacology 38:31-38) was used. Plate count agar
(PCA, Biolab) was prepared in the same way as malt agar used in the
antifungal screening tests except that the plant extracts were not
suspended in the agar. Mother cultures of all plant pathogenic
bacteria were sustained on nutritional agar (Caceres et al., 1991,
Journal of Ethnopharmacology 31:193-208, Rasoanaivo et al., 1993).
Overnight soup cultures of the test bacteria were initially
prepared separately in sterile 1% (w/v) nutrient broth (Biolab)
solutions at 30.degree. C. (Meyer et al, 1995). One hundred .mu.l
of each of these bacterial suspensions were subsequently separately
transferred to 90 mm Petri dishes containing the sterile PCA agar
and evenly streaked on the surface using sterile swabs. Petri
dishes were divided into four quarters and a hole, 6 mm in
diameter, plunged into the agar of each quarter by means of a
sterile cork borer. 50 .mu.l of the 1 mg/ml crude stock solution
extracts were transferred into the holes in the agar. The plates
were equilibrated at 4.degree. C. for 1 h to allow the extracts to
diffuse into the agar before incubation commenced. In this way the
development of clear inhibition zones was optimized. Plates were
incubated for three days at 25.degree. C. for all plant pathogenic
bacteria, but at 35.degree. C. for M. catharrhalis. Each assay was
performed in duplicate. Inhibition zones were measured using a
digital caliper.
Example 5
In Vitro Screening for Bio-Stimulatory Properties of Crude
Extracts
[0176] Two methods were applied to determine the biostimulatory
potential of the organ crude extracts of A. africanus.
Method 1: Manometric Method for Determining the Effect of Crude
Extracts on the Respiration Rate of a Monoculture Yeast Cells
[0177] A specially constructed glass respirometer with a short
bulged section (reservoir) to contain the yeast cells and a long
calibrated tube, closed at the top end to collect CO.sub.2 gas, was
used in determining the effect of the A. africanus crude extracts
on the respiration rate of a monoculture yeast cells. Dry baker's
yeast (0.8 g) was placed in the reservoir of the respirometer.
Subsequently, 70 ml of each of the plant extracts, previously
prepared at a concentration of 0.5 mg/ml and containing 5 mg/ml
glucose to serve as respiratory substrate for the yeast cells, was
added to the respirometer. The apparatus was tilted sideways to
release air bubbles trapped in the dry baker's yeast and placed in
a water bath pre-heated to 29.degree. C. ComCat.RTM., a commercial
biostimulant, was used as a positive control at 0.5 mg/ml (optimum
concentration according to the manufacturers; Agraforum, Germany,
2002) and distilled water as a second control. CO.sub.2 release by
the yeast cells was measured in cm.sup.3 at 30 minute intervals
over a three hour incubation period by reading the released gas
volume from the calibrated tube. Tests were performed in
triplicate.
Method 2: the Effect of Different Organ Crude Extracts of A.
Africanus on the Percentage Germination of Radish Seeds and
Subsequent Seedling Growth
[0178] Two sheets of special germination paper (30.times.30 cm)
were used to test the effect of each plant crude extracts of A.
africanus on the germination of radish seeds as well as the
subsequent seedling growth. A line, 10 cm from the top, was drawn
on the one sheet and 20 radish seeds spaced evenly on the line. A
second sheet of germination paper was placed on top of the first
and moistened with either 0.5 mg/ml solutions of the crude
extracts, distilled water (negative control) or 0.5 mg/ml solution
of ComCat.RTM. (positive control). Both sheets of paper were rolled
up longitudinally and placed upright in Erlenmeyr flasks containing
either crude extract, distilled water or the ComCat.RTM. solution
and kept at 25.degree. C. in a growing chamber in the dark. Seed
germination as well as coleoptile and root lengths were determined
at 24 h intervals over a 96 h incubation period. Tests were
performed in triplicate.
Example 6
Statistical Analysis of Data
[0179] Analysis of variance (ANOVA) was performed on the data,
using the SAS (1999; SAS/IML software; Version 6; SAS Institute)
program, to identify differences between treatments. Duncan's
multiple range (DMR) procedure for comparison of means (Steele
& Torrie, 1980, Principles and procedures of statistics,
2.sup.nd Edition. New York: McGraw Hill.) was applied to separate
means (P<0.05).
Example 7
Isolation of Mycosphaerella Pinodes
[0180] M. pinodes was isolated from diseased leaves and stems of
various winter cultivars of field pea at the time of senescence.
Collections of the infected plant material were made from the
central and south eastern pea-growing areas of Ethiopia. Pieces of
the diseased tissues were surface sterilized for 1 minute in 96%
(v/v) ethanol, 3 minutes in a 3.5% (v/v) NaCl solution (Moussart et
al., 1998, European Journal of plant Pathology 104:93-102) and 30
seconds in 96% (v/v) ethanol. The tissues were subsequently
aseptically transferred to corn meal agar amended with streptomycin
(0.3 ml/l) in 9 cm Petri dishes and incubated at 20.+-.1.degree. C.
in a growth chamber. Isolates initially obtained from the plant
material were then grown on Coon's medium (Ali et al., 1978,
Australian Journal of Agricultural Research 29:841-849) consisting
of 4 g maltose, 2 g KNO.sub.3, 1.2 g MgSO.sub.4, 2.7 g
KH.sub.2PO.sub.4 and 20 g agar. Cultures were incubated for 14 days
to obtain pycnidiospores. To obtain an isolate derived from a
single uninucleate cell, a suspension of pycnidiospores was
streaked on 15% water agar, incubated overnight at 20.+-.1.degree.
C. and examined under a dissecting microscope (80.times.
magnification). A germ tube arising from one cell of a
pycnidiospore was severed and transferred to Coon's agar (Clulow
& Lewis, 1992, Plant Pathology 41:362-369). Six isolates of M.
pinodes were obtained. All isolates from a single-spore and
cultures were maintained on Coon's agar slants and stored in the
dark at 5.degree. C.
Example 8
Preparation of a M. Pinodes Spore Suspension
[0181] Oat meal agar was prepared by gently heating 30 g of oats in
1 litre distilled water for 1 h, stirring frequently, and
subsequently filtering through a fine sieve upon which the volume
was readjusted to 1 litre. Twenty g of technical agar and 0.1 g
Keltane AP was added to the filtrate to yield a 2% (m/v) agar
concentration. The agar was autoclaved for 15 min, poured into
Petri dishes and allowed to cool off before inoculation of three
oatmeal plates with M. pinodes mycelia. Plates were incubated in a
12 h photoperiod incubator at 20.degree. C. for 14 days, to ensure
the production of pycnidiospores. To prepare the inoculum (spore
suspension), sterile distilled water was added to the 14-day-old
cultures dislodging spores gently with a sterile glass rod. The
suspension was subsequently filtered through four layers of cheese
cloth in order to remove the mycelia and the concentration of
pycnidiospores was determined by means of a haemocytometer. The
pycnidiospore concentration was adjusted to 1.times.10.sup.5 spores
per ml (Nasir & Hoppe, 1997, Annals of Applied Biology
18:32-33) with sterile distilled water prior to the inoculation of
pea leaves.
Example 9
In Vivo Assessment of Crude Extract Phytotoxicity
[0182] Pea seeds were planted in plastic pots in Bainsvlei soil and
grown in a glasshouse (minimum temperature 18.degree. C.). Four
weeks after planting, when the leaflets on the third and fourth
nodes were fully expanded, three fourth node leaflets per replicate
were removed from the plants, placed on Schleicher and Schull No.
595 filter paper and moistened with 4 ml of sterile distilled water
in 9 cm Petri dishes. 30 .mu.l of each of a 0.25, 0.5, 1.0 and 2.0
mg/ml solution of the crude extract were placed separately on each
of the three leaves per Petri dish and replicated three times.
Treatment of the leaves with water and a standard fungicide
(Carbendazim/difenoconazole) served as controls. Petri dishes
containing the treated leaflets were incubated at 20.degree. C. in
a day/night incubator programmed for a 16 h day cycle while 2 ml
sterile distilled water was added daily to keep the filter paper
moistened. Six days after treatment, phytotoxicity symptoms were
assessed on leaves using a six-category scale [0=symptomless;
1=<5% necrotic flecks; 2=>5% necrotic flecks; 3=<50% of
inoculated area necrotic; 4=50-100% of inoculated area necrotic;
5=necrosis spreading beyond inoculated areas] based on stereo
microscopic observations (Clulow et al., 1991, Mycological Research
95: 817-820).
Example 10
In Vivo Assessment of Crude Extract Antifungal Properties Under
Glasshouse Conditions
[0183] Fourth node pea leaflets were obtained and sustained on
moist filter paper in Petri dishes as described for the
phytotoxicity assessment test. In vivo control of M. pinodes spore
infection of the leaves by different concentrations (0.25, 0.5, 1.0
and 2.0 mg/ml) of the aerial plant parts, roots, leaves and flowers
of A. africanus was followed in two ways namely, by inoculating the
leaves with 15 .mu.l of a spore suspension (1.times.10.sup.5
spores/ml; Nasir & Hoppe, 1997, Annals of Applied Biology
18:32-33) 30 min before applying the different concentrations of
the crude extract separately, and the other way around. A standard
fungicide, carbendazim/difenoconazole, currently used against
Ascochyta blight in peas, as well as leaves inoculated only with
the spore suspension, served as controls. Three leaves per Petri
dish represented a replicate and the experiment was performed in
triplicate. Petri dishes containing the differently treated leaves
were incubated at 20.degree. C., the optimal temperature for M.
pinodes spore germination in a day/night incubator as illumination
is necessary for spore germination (Roger & Tivoli, 1996,
Mycological Research 100:304-306). After incubation for six days
the foliar lesions were measured and leaf damage compared to that
of the controls.
Example 11
Seed Treatment
[0184] Different lots of sorghum seeds were artificially inoculated
with either covered (Sporisorium sorghi) or loose (Sporisorium
cruentum) kernel smuts spores at the rate of 5% (w/w) before
application of seed treatments. An aerial crude extract of A.
africanus was suspended in water at a rate of 2.0 g/1. Sorghum seed
lots of 90 g each were treated with 15 ml of the crude extract by
mixing thoroughly in a small plastic bag 24 h before planting. A
standard synthetic seed dressing fungicide, Thiram (65 W), was
applied in the same way at the rate of 0.25% (w/w) per Kg seed and
served as a positive control. Sorghum seeds artificially inoculated
with both loose or covered smuts spores, but were not treated with
the extract or synthetic fungicide, served as a second control.
Example 12
Field Trial
[0185] A field trial was conducted under irrigation at Melkassa
Research Centre, Ethiopia during 2003. Plots were arranged in a
randomised complete block design and treatments were replicated
three times. Treated sorghum seeds were planted by hand in five
rows, leaving 0.75 cm between rows, in 18.75 m.sup.2 plots.
Standard fertilizer was applied and plots were kept at field
capacity by means of furrow irrigation. Disease incidence was
recorded as percentage infected plants. Grain yield was determined
on the whole plot.
Example 13
Activity Directed Liquid-Solid Extraction
[0186] Dried methanolic crude extracts of the roots (268.5 g) and
aerial parts (368.83 g) of A. africanus were fractionated by means
of liquid-solid extraction using hexane (DC=2.0), diethyl ether
(DC=4.3), ethyl acetate (DC=6.0) and dichloromethane (DC=8.6) as
solvents at a ratio of 2 ml/g crude extract. Extraction was
repeated more than 20 times with fresh solvent for each step by
shaking vigorously on a mechanical shaker for 10 min. The four
fractions were collected separately and evaporated to dryness under
vacuum at 35.degree. C. by means of a Buichi rotavapor. The mass of
recovered dry material was determined for each fraction. In order
to establish the success of the fractionation process, a thin layer
chromatography (TLC) profile was obtained for each fraction with a
0.5 mm Silica Gel 60 plate using chloroform:methanol:water
(80:20:10) as mobile phase. The mycelial growth inhibitory activity
potential of each semi-purified extract was subsequently
established using F. oxysporum as test organism.
Example 14
Activity Directed Column Chromatography Fractionation
[0187] The most active extractants obtained from the liquid solid
extraction procedure were further fractionated using column
chromatography. A column (2.6.times.46 cm) packed with either
Sephadex LH20 (Pharmacia) for the root extracts or Silica gel
(0.25; Merck, Darmstadt, Germany) for the aerial parts was
employed. The residue of the root (7 g) was eluted successively
with ethanol (100%) followed by methanol (100%) and a
methanol:water (50:50 v/v) mixture. The column chromatographic
residue of the aerial plant parts (5 g) was eluted with a gradient
solvent system of methanol:chloroform (15:85, 20:80, 25:75, 30:70
and 40:60 v/v). Elution was adjusted at a flow rate of 3
ml/min.
[0188] Approximately 120 ml of the root and aerial part eluent were
collected. Those column chromatographic fractions that showed
similar Q-TLC profile patterns were combined separately. Mycelial
growth inhibition of F. oxysporum was used to identify active
column chromatographic fractions for further purification of the
active compounds by means of preparative thin layer
chromatography.
Example 15
Preparative Thin Layer Chromatography (PTLC)
[0189] The most active combined column chromatography fractions
were further purified by means of preparative thin layer
chromatography (PTLC) using Silica gel F 1500/LS (1 mm) plates.
Fifteen mg of each of the active column fractions were dissolved in
50 .mu.l methanol (100%) and loaded onto the plate by streaking
evenly over the baseline with the aid of a glass capillary tube.
This was repeated 10 times on 10 different plates to separate
compounds from a total of 150 mg of each of the active fractions.
The plates were dried in front of a fan between streaking and then
developed in a saturated chamber using a chloroform:methanol:water
(80:20:10 v/v) solvent system as mobile phase. Detection of
compounds was done under UV-light at 254 and 365 nm (Wagner and
Bladt, 1996, Plant Drug Analysis. A thin layer chromatography
atlas. Second edition. Springer, Berlin). Individual compounds were
isolated by scraping off the detected zones of the sorbent layer
from the plates using a spatula and transferred to Eppendorff
vials. The compounds were recovered from the Silica by elution with
methanol (100%), followed by centrifugation for five minutes at
12000 r.p.m., and tested for antifungal activity after the methanol
was removed by drying at 35.degree. C. in an oven.
Example 16
Qualitative Thin Layer Chromatography (Q-TLC)
[0190] Only the most active isolated compounds were again tested
for purity in an original analytical thin layer chromatography
(TLC) system (Mikes and Chalmers, 1979, Laboratory handbook of
chromatographic and allied methods. Ellis Horwood Ltd., London)
using Silica gel 60 F.sub.254-aluminium backed and pre-coated
plates. Ten to 15 mg of each sample were loaded onto the plates at
the baseline and developed in a saturated chamber using either
chloroform:methanol:water (80:20:10 v/v) or toluene:acetone:ethyl
acetate (7:2:1 v/v; Wagner and Bladt, 1996) as solvent systems.
After drying the plates in a stream of air, compounds were either
detected under UV-light at 254 and 365 nm or the plates were
stained with 5% (v/v) ethanolic H.sub.2SO.sub.4 or 1% (m/v)
Vanillin (1 g in 100 ml H.sub.2SO.sub.4; Wagner and Bladt, 1996).
Non-pure compounds were again subjected to preparative TLC
acidified with 1% (v/v) HCL until pure compounds were obtained.
Only pure compounds that showed the highest antifungal activity
were subjected to nuclear magnetic resonance (NMR) spectroscopy in
order to identify them and to elucidate their molecular
structures.
Example 17
Nuclear Magnetic Resonance (NMR) Spectroscopy
[0191] To identify the most bioactive compounds purified from the
roots and aerial plant parts and elucidate their molecular
structures, isolated compounds were washed repeatedly with acetone
to obtain an acceptable level of purity. Subsequently, the
compounds were submitted to nuclear magnetic resonance spectroscopy
(.sup.1H NMR). NMR-spectroscopy was performed on a Bruker 300 MHz
DRX 300 spectrometer at 296K (23.degree. C.) with tetramethylsilane
(Si(CH.sub.3).sub.4; TMS) as the internal standard. The solvents
used were deuteriochloroform (CDCl.sub.3), or deuterioactetone
[(CD.sub.3).sub.2 CO] as indicated. Chemical shifts were reported
in parts per million (ppm) on the 6-scale and coupling constants
were given in Hz. The following abbreviations were used: s=singlet,
d=doublet, dd=doublet of doublets, m=multiplet, br=broadened,
t=triplet. All FAB mass spectra were recorded on a VG 70-70E
double-focusing mass spectrometer. Circular dichroism (CD) spectra
were recorded on a Jasco J-710 spectropolarimeter with methanol as
solvent. Structural elucidation was achieved via spectroscopic
methods (1D NMR and 2D NMR spectrometry), FAB and EI-MS as well as
chemical methods, such as hydrolysis. Due to the complexity of the
.sup.1H NMR spectrum of the non-derivatised sapogenin, a peracetate
derivative (2; FIG. 3A) was used in the structural elucidation.
This was achieved via spectroscopic (NMR) and spectrometric (MS)
methods, as well as hydrolysis.
* * * * *