U.S. patent application number 10/081195 was filed with the patent office on 2002-10-17 for antibacterial composition comprising oenostacin from oenothera biennis.
This patent application is currently assigned to Kumaran & Sagar. Invention is credited to Gupta, Vivek Kumar, Kumar, Sushil, Santha Kumar, Tiruppadiripuliyur Ranganathan, Shukla, Yogendra Nath, Singh Khanuja, Suman Preet, Srivastava, Anil.
Application Number | 20020150635 10/081195 |
Document ID | / |
Family ID | 24675399 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020150635 |
Kind Code |
A1 |
Shukla, Yogendra Nath ; et
al. |
October 17, 2002 |
ANTIBACTERIAL COMPOSITION COMPRISING OENOSTACIN FROM OENOTHERA
BIENNIS
Abstract
The present invention provides a pharmaceutical composition for
treating bacterial infections in patients comprising an effective
amount of 3,5-dihydroxy-4-pent-4'-enoyl-1'-oxymethylbenzoic acid
(Oenostacin) isolated from Oenothera biennis.
Inventors: |
Shukla, Yogendra Nath;
(Lucknow, IN) ; Santha Kumar, Tiruppadiripuliyur
Ranganathan; (Lucknow, IN) ; Srivastava, Anil;
(Lucknow, IN) ; Singh Khanuja, Suman Preet;
(Lucknow, IN) ; Gupta, Vivek Kumar; (Lucknow,
IN) ; Kumar, Sushil; (Lucknow, IN) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
Kumaran & Sagar
|
Family ID: |
24675399 |
Appl. No.: |
10/081195 |
Filed: |
February 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10081195 |
Feb 22, 2002 |
|
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|
09666770 |
Sep 21, 2000 |
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6365197 |
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Current U.S.
Class: |
424/725 |
Current CPC
Class: |
A01N 37/40 20130101;
A61K 31/235 20130101 |
Class at
Publication: |
424/725 |
International
Class: |
A61K 035/78 |
Claims
What is claimed is:
1. A pharmaceutical composition for treatment of an antibacterial
infection, comprising an effective amount of Oenostacin in
combination, admixture, or associated with a pharmaceutically
acceptable carrier, diluent or excipient thereof.
2. A composition according to claim 1 wherein the Oenostacin is an
extract obtained from the plant Oenothera biennis.
3. A composition according to claim 1 wherein the concentration of
Oenostacin is in the range of 1-200.mu.ml.
4. A composition according to claim 1 wherein the concentration of
Oenostacin is in the range of 10-90% by weight.
5. A composition according to claim 1 wherein the composition is
effective against bacteria selected from streptococci,
staphylococci and Pseudomonas aeruginosa.
6. A method of treating a bacterial infection in a patient in need
thereof, comprising administering to the patient a
bacterial-treating effective amount of Oenostacin.
7. A method according to claim 6 in which the bacterial infection
is caused by bacteria selected from streptococci and
staphylococci.
8. A method according to claim 7 in which the bacterial infection
is endocarditis.
9. A method according to claim 7 in which the bacterial infection
is caused caused by S. epidermidis.
10. A method as claimed in claim 6 wherein the concentration of
Oenostacin in the composition is 1-200.mu.g/ml.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of Invention
[0002] This invention relates to the use of a compound
3,5-dihydroxy-4-pent-4'-enoyl-1'-oxymethylbenzoic acid (Oenostacin)
isolated from the roots of the plant Oenothera biennis as an
effective antibacterial agent active against bacteria such as
staphylococci, streptococci and the like. The invention also
provides pharmaceutical compositions comprising
3,5-dihydroxy-4-pent-4'-enoyl-1'-oxymethylbenzoic acid (Oenostacin)
useful for treatment of skin infections such as endocarditis in
humans caused by Staphylococcus epidermidis.
[0003] 1. Summary of the Related Art
[0004] The antimicrobial era has reached a point where the
emergence of resistant microbes is accelerating while the pace of
discovery of new drugs seems decelerating (Science 157:1064-1073).
Until recently, drugs or combination of drugs have not been able to
overcome the problem of resistance. Few novel chemical entities
have been brought to the market during the past decade to address
this problem, as most of the new drugs are derivatives of older
compounds. Many of these have increased activity or a broader
spectrum of activity or improved pharmacological properties but can
only temporarily overcome the problem of resistance. Thus, the
isolation of natural products from sources such as various species
of plants has become a basis for identifying new class of
antimicrobial compounds. These compounds are being investigated for
treatment of infections caused during seemingly uncomplicated
hospital treatment procedures such as catheterization, insertion of
intrauterine contraceptive devices and intravenous injections, for
example.
[0005] Inflammation of the endocardium, i.e., the tissue lining of
the cavities of the heart, is called endocarditis. Infective
(infectious) endocarditis may be due to infection by a range of
microorganisms such as Haemophilus sp., Staphylococcus aureus, S.
epidermidis (especially in patients with prosthetic valves),
Streptococcus faecalis, Neisseria gonorrhae, Candida, etc.
Infective endocarditis may be acute (e.g, when due to streptococci
or gonococci) or sub-acute when due to viridans streptococci or
fungi. Infection occurs through the circulatory system and
organisms may gain access to the blood stream during dental
treatment, catheterization, insertion of intrauterine contraceptive
devices or intravenous injections, for example. The symptoms
associated with such infections include fever, malaise, heart
murmurs, weight loss, clubbing of fingertips and embolism. Late
symptoms of sub-acute bacterial endocarditis include vasculitis,
petechial rash and Osler's nodes. In addition, damage to heart
valves may lead to heart failure. Endocarditis may also occur as a
complication of other infectious diseases.
[0006] Oenothera biennis (Onagraceae) is a genus of herbs and
undershrubs distributed mainly in temperate America together with
some species occurring in the tropics. Some of the species
including O. biennis have been introduced into Indian Gardens (J.
Med. Arom Plant Sci., 20:1998, 432). The oil from seeds of O.
biennis (Evening Primrose) is known to be a rich dietary source of
.gamma.-linolenic acid required for the formation of prostaglandins
and related hormones (Z Phytother, 4:1983, 531). The seeds are
reported to contain fatty acids (J. Am Oil Chem Soc, 61:1984, 540)
and sterols (Riv Ital Sastanze Grasse, 53, 1976,25) while the
leaves contain flavonoids (Phytochemistry, 6:1967, 317) and
Oenothein A (Chem Pharm Bull, 39:1991, 1157). However, no compounds
have been reported from the roots of O. biennis. Recently, certain
specific activity is observed in the root extracts and this
prompted a systematic activity-directed fractionation to isolate
pure compounds. This systemic activity-directed fractionation of
root extracts resulted in the identification of ten compounds, four
of which are novel and reported in the Indian Journal of Chemistry,
Vol. 38B, 1999, pages 705-708.
SUMMARY OF THE INVENTION
[0007] It is therefore an object of the invention to provide a
compound that has potent activity as an antibacterial agent.
[0008] It is further an object of the invention to provide a
pharmaceutical composition comprising a potent antibacterial
agent.
[0009] Yet another object of the invention is to provide a method
for the treatment of bacterial infections, especially diseases
caused by bacteria.
[0010] Other objects and further scope of applicability of the
present invention will become apparent from the detailed
description given hereinafter. It should be understood, however,
that the detailed description and specific examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent
to those skilled in the art from this detailed descriptions
[0011] Accordingly, the present invention provides the compound
3,5-dihydroxy-4-pent-4'-enoyl-1'-oxymethylbenzoic acid (Oenostacin)
as an antibacterial agent (Indian Journal of Chemistry, Vol. 38B,
1999, pages 705-708, incorporated herein by reference). This
chemical compound has the molecular formula C.sub.13H.sub.14O.sub.6
and the structural formula: 1
[0012] In a preferred embodiment, Oenostacin is active against
streptococci and staphylococci especially Staphylococcus
epidermidis, which causes skin infections/endocarditis in humans.
Hence, the present invention provides an antibiotic isolated from
the roots of the plant O. biennis for use against S. epidermidis
infection.
[0013] The invention further provides a pharmaceutical composition
having antibacterial activity, comprising Oenostacin in
combination, admixture, or associated with a pharmaceutically
acceptable carrier, diluent or excipient thereof
[0014] The invention also provides a method of treating a bacterial
infection, and preferably a bacterial skin infection, comprising
administering to a patient with such an infection an effective
amount of Oenostacin.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Accordingly, the invention provides a novel antibacterial
composition comprising an effective amount of Oenostacin
(3,5-dihydroxy-4-pent-4'-enoyl-1'-oxymethylbenzoic acid) obtained
from the roots of Oenothera biennis in combination, admixture, or
associated with a pharmaceutically acceptable carrier, diluent or
excipient thereof.
[0016] In an embodiment of the invention, the
3,5-dihydroxy-4-pent-4'-enoy- l-1'-oxymethylbenzoic acid is an
extract obtained from the plant Oenothera biennis (Indian Journal
of Chemistry, Vol. 38B, 1999, pages 705-708).
[0017] The concentration of Oenostacin in the composition is in the
range of 1-200.mu.g/ml, or in the range of 10-90% by weight.
[0018] The antibacterial composition is effective against bacteria
selected from streptococci, staphylococci and Pseudomonas
aeruginosa, and preferably streptococci and staphylococci. The
antibacterial composition may further be used for the treatment of
endocarditis in humans. Acute endocarditis is an inflammatory
disease of the endocardium i.e., the internal lining of the human
heart and may be caused by the staphylococci and gonococoi
bacteria, for example. Among staphylococci, S. epidermidis is one
of the major etiological agents of this disease. The infections
occur mainly in-patients with prosthetic valves.
[0019] Thus, the antibacterial composition may be used for the
treatment of infections caused by S. epidermidis.
[0020] Using a screening program aimed at detecting biomolecules
from plant sources, which can specifically act against S.
epidermidis, the present inventors have now discovered that the
ethanolic extract derived from the roots of a plant called 0.
biennis contains the compound
3,5-dihydroxy-4-pent-4'-enoyl-1'-oxymethylbenzoic acid, which
possesses antibacterial activity. Oenothera biennis is a genus of
herbs and under-shrubs; its species mainly distributed in temperate
America together with some species occurring in the tropics. Some
of the species such as 0. biennis have been introduced into the
Indian gardens. The seeds of the plant are rich source of fatty
acids and sterols required for the formation of prostaglandins and
other hormones. A systematic activity-directed fractionation of
root extracts resulted in the identification of ten compounds, four
of which are new (Indian Journal of Chemistry, Vol. 38B, 1999,
pages 705-708).
[0021] In a preferred extraction process, the air-dried powdered
roots of O. biennis are extracted with MeOH. Water is then added
and the extract is then fractionated successively into n-hexane,
ethyl acetate (EtOAc) and n-BuOH. Silica gel column chromatography
of the n-hexane and EtOAc fraction yields many known compounds,
such as gallic acid (CIM 790), and a new compound (CIM 791) further
identified by NMR. This compound is found to possess strong
activity against Staphylococcus epidermidis and is named
Oenostacin. Table 1 describes the general procedure followed for
the bioactivity-guided fractionation of the isolate active
compounds of O. biennis. Here, bioactivity-guided fractionation is
employed to isolate the active principle involved in the inhibition
of S. epidermidis growth.
1TABLE 1 ACTIVITY GUIDED FRACTIONATION OF Oenothera biennis 2
[0022] Each extract and fraction or sub fraction and or isolated
pure compound is simultaneously tested for its antibacterial
property using S. epidermidis culture in disc diffusion assay.
[0023] Silica gel chromatography of the ethyl acetate fractions of
the methanol extractives of the root yielded compounds 7-10. CIM
791 has the following physical properties: IR spectrum, bands for
COOH (1692 cm.sup.-1), double bond (1612 cm.sup.-1), COOCH.sub.3
(1730 cm.sup.-1) and phenol (PhOH) (3512 cm.sup.-1); UV maxima at
215 and 278 nm; [M+] ion in the mass spectrum of 3 at m/z 266 is in
agreement with the molecular formula C.sub.13H.sub.14O.sub.6. The
further identification properties and the NMR spectral data of this
compound is described in Indian J Chem., 38B, 1999, 705,
incorporated herein. Table 2 depicts the 1-R- mass spectral
structure of Oenostacin.
2TABLE 2 3
[0024] CIM 791 is tested for its minimal inhibitory concentration,
which is a measure of the growth inhibitory potential of the
compound and determined against S. epidermidis by two-fold broth
dilution technique.
[0025] In the methods of the present invention, a pharmaceutical
composition can be administered either orally, rectally,
parenterally (intravenously, intramuscularly or subcutaneously),
intracisternally, intravaginally, intraperitoneally,
intravesically, locally (powders, ointments or drops), or as a
buccal or nasal spray.
[0026] Compositions intended for oral use may be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions may contain one
or more agents selected from the group consisting of sweetening
agents, flavoring agents, coloring agents and preserving agents in
order to provide pharmaceutically elegant and palatable
preparations. Tablets contain the active ingredient in admixture
with non-toxic pharmaceutically acceptable excipients that are
suitable for the manufacture of tablets. These excipients may be
for example, inert diluents, such as calcium carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets may be uncoated or they may be
coated by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monosterate or glyceryl distearate may be
employed.
[0027] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0028] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents may be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions may also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one
or more sweetening agents, such as sucrose or saccharin.
[0029] Oily suspensions may be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavoring agents may be added to
provide palatable oral preparations. These compositions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0030] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, may also be present.
[0031] Pharmaceutical compositions of the invention may also be in
the form of oil-in-water emulsions. The oily phase may be a
vegetable oil, for example olive oil or arachis oil, or a mineral
oil, for example liquid paraffin or mixtures of these. Suitable
emulsifying agents may be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monoleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monoleate. The emulsions may also contain sweetening and flavoring
agents.
[0032] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitor or sucrose. Such
formulations may also contain a demulcent, a preservative and
flavoring and coloring agents. The pharmaceutical compositions may
be in the form of a sterile injectable aqueous or oleaginous
suspension. This suspension may be formulated according to the
known art using those suitable dispersing or wetting agents and
suspending agents which have been mentioned above. The sterile
injectable preparation may also be sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil may be
employed including synthetic mono-or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0033] Compositions suitable for parenteral injection may comprise
physiologically acceptable sterile aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, and sterile powders for
reconstitution into sterile injectable solutions or dispersions.
Examples of suitable aqueous and nonaqueous carriers, diluents,
solvents, or vehicles include water, ethanol, polyols
(propyleneglycol, polyethyleneglycol, glycerol, and the like),
suitable mixtures thereof, vegetable oils (such as olive oil), and
injectable organic esters such as ethyl oleate. Proper fluidity can
be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersions and by the use of surfactants.
[0034] These compositions may also contain adjuvants such as
preserving, wetting, emulsifying, and dispensing agents. Prevention
of the action of microorganisms can be ensured by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, for example sugars, sodium
chloride, and the like. Prolonged absorption of the injectable
pharmaceutical form can be brought about by the use of agents
delaying absorption, for example, aluminum monostearate and
gelatin.
[0035] Compositions for rectal administrations are preferably
suppositories which can be prepared by mixing the compounds of the
present invention with suitable nonirritating excipients or
carriers such as cocoa butter, polyethyleneglycol or a suppository
wax, which are solid at ordinary temperatures but liquid at body
temperature and therefore, melt in the rectum or vaginal cavity and
release the active component.
[0036] Dosage forms for topical administration of a compound of
this invention include ointments, powders, sprays, and inhalants.
The active component is admixed under sterile conditions with a
physiologically acceptable carrier and any preservatives, buffers
or propellants as may be required. Ophthalmic formulations, eye
ointments, powders, and solutions are also contemplated as being
within the scope of this invention.
[0037] The compound of the present invention can be administered to
a patient at dosage levels in the range of about 0.1 to about 1,000
mg per day. For a normal human adult having a body weight of about
70 kg, a dosage in the range of about 0.01 to about 100 mg per
kilogram of body weight per day is sufficient. The specific dosage
used, however, can vary. For example, the dosage can depend on a
number of factors including the requirements of the patient, the
severity of the condition being treated, and the pharmacological
activity of the compound being used. The determination of optimum
dosages for a particular patient is well-known to those skilled in
the art.
[0038] The disclosures in this application of all articles and
references, including patents, are incorporated herein by
reference.
[0039] The invention is illustrated further by the following
examples, which are not to be construed as limiting the invention
in scope or spirit to the specific procedures described in
them.
EXAMPLE 1
[0040] Extraction of Oenostacin
[0041] The air-dried and powdered roots (4.7kg) of O. biennis are
extracted with methanol (MeOH) (7.times.8.5 L) and the combined
extract is concentrated in vacuo to 500 mL. Water is added and the
extract then fractionated successively into n-hexane (5.times.600
mL, 65 g); EtOAc (5.times.600 mL,52 g) and n-BuOH (5.times.600 mL,
41 g). The MeOH extract and the various solvent fractions form the
initial material from which bioactivity guided fractionation is
performed.
EXAMPLE 2
[0042] Each of the solvent fractions obtained from the powdered
roots of O. biennis is tested for their antibacterial property.
This is performed by single disc diffusion assay as per Bauer et
al.,1966, (American Journal of Clinical Pathology 45:493-496) with
slight modifications. The discs are prepared (5 mm diameter made of
Whatman #3 filter paper) by impregnating 8 .mu.l of test compound
and placing them on pre-inoculated agar surface. A disc containing
only the solvent is used as the control. As evident from Table 3,
the EtOAc fraction show significant antibacterial activity against
Staphylococcus epidermidis and Streptococcus mutans. This indicates
the presence of antibacterial substance in the EtOAc fraction of O.
biennis roots. These results form the basis of further
experimentation.
3TABLE 3 Antibacterial activity of solvent extracts from roots of
O. biennis. Diameter of the Zone of growth inhibition Extract @
(mm) against 400 .mu.g/disc S. epidermidis S. mutans P. aeruginosa
MeOH extract -- -- -- Hexane extract -- -- -- EtOAc extract 08 08
-- BuOH extract -- -- --
EXAMPLE 3
[0043] Tests of Antibacterial Activity
[0044] To isolate the antibacterial principle from the EtOAc
fraction, a portion (19 g) of this fraction is chromatographed in a
column over silica gel (800g), eluting with varying proportions of
chloroform (CHCL.sub.3) and MeOH to afford four compounds. Each 100
mL fraction collected is monitored by TLC. One compound (CIM 791)
is identified as 3,5-dihydroxy-4-pent-4'-enoyl-1'-oxymethylbenzoic
acid (Oenostacin) based on NMR spectral data mentioned in Indian J
Chem., Vol 38B, 1999, 705. Table 4 indicates that the EtOAc
fractions 3 and 4 possess the expected antibacterial activity
against the test bacterial strains. The EtOAc fraction 4 exhibits
growth inhibitory activity against Pseudomonas aeruginosa
indicating the presence of a specific chemical compound in this
fraction.
4TABLE 4 Antibacterial activity of different fractions of EtOAc
extract of roots of O. biennis. Diameter of the Zone of growth
inhibition Extract @ (mm) against 400 .mu.g/disc S. epidermidis S.
mutans P. aeruginosa EtOAc. Fract-1 -- -- -- EtOAc. Fract-3 08 09
-- EtOAc. Fract-4 15 09 10
EXAMPLE 4
[0045] In order to identify the antibacterial chemical entities
present in the EtOAc fraction 3 and 4 of O. biennis roots, the
fractions are further run on silica gel column chromatography again
eluting with varying proportions of chloroform and MeOH. Aliquots
of eluate are run on TLC to check for the desired purity of the
compound. The pure compounds thus obtained are analyzed for their
antibacterial properties by two-fold broth dilution method to
determine the minimal inhibitory concentration (MIC). The results
indicate (Table 5) that one compound, CIM791, has strong
antibacterial activity against Staphylococcus epidermidis since the
MIC is found to be 31.5 .mu.ml. Yet another compound, CIM-790, is
found to possess low inhibitory activity against Streptococcus
mutans (MIC=625,.mu.g/ml). The compounds CIM791 and 790 are
identified as 3,5-dihydroxy-4-pent-4'-enoyl-1'-oxymethylbenzoic
acid (Oenostacin) and gallic acid, respectively, using NMR spectral
data.
5TABLE 5 The minimal inhibitory concentration of Oenostacin against
bacterial strains Minimal inhibitory concentration (MIC) Compound
S. epidermidis S. mutans P. aeruginosa Gallic acid -- 625 mg/ml
1.25 mg/ml CIM-790 Oenostacin 31.5 g/ml 1.25 mg/ml -- CIM-791
[0046] The invention and the manner and process of making and using
it, are now described in such full, clear, concise and exact terms
as to enable any person skilled in the art to which it pertains, to
make and use the same. It is to be understood that the foregoing
describes preferred embodiments of the present invention and that
modifications may be made therein without departing from the spirit
or scope of the present invention as set forth in the claims. To
particularly point out and distinctly claim the subject matter
regarded as invention, the following claims conclude this
specification.
* * * * *