Note: Descriptions are shown in the official language in which they were submitted.
co~zz~x~s o~ NFOLZCN~raE DERZVATZ~s r~zTa raosPaoLZrzos,
TaE asp Ts~REOF ANn pa~RM~.cEaTIC~. ANn cos~~IC ~o~
TIONS CONTAINING THE~3.
The present invention relates to complexes of
extracts from Krameria triandra Ruiz et Pav. and other
plants of the Eupomatia genus, as well as some phenol
constituents thereof of neo-lignane "or nor-neolignane
nature, with phospholipids; to the processes for the
preparation of said extracts and complexes, and to
pharmaceutical compositions containing them:
The extracts from roots of Krameria triandra had
been used widely in the past as medicaments, thanks to
both the astringent property thereof, due to the
presence of Ratania-tannin, and the antiinflammatory,
antidiarrhoeal and antibacterial properties connected
with other components; some of those characteristics are
reported on Martindale, ~~The extrapharmacopeia~~, 28th
Ed. (1982) and on other documents (Canizzaro, Boll. Soc.
Ital. Biol. Sperim., 1, 22, 1964; and V. Hoppe,
Drogenkunde Bdl Walter De Gruyter ed., 1975). Two
components of neolignane nature have been described as
sunscreens for possible cosmetic use (Stahl, Planta
Med., 42, 144, 1981). The first remarks on the medicinal
properties of said plant go back to the Peruvian people
empiricism, which used the roots for the oral cavity
hygiene (~~raiz para los dientes~~), which use spread
rapidly also in other countries in more recent times.
British patent 2.184.353A claims the antibacterial
and antimycotic properties of extracts from Krameria
triandra and of some components, called Ratania-phenols,
- 2 -
for which the use by the topical route is envisaged for
the treatment of acne, dermatomycosis and decubitus ul-
cers attributable to infections caused by aerobic and
anaerobic strains. A renewed interest in Krameria trian-
dra derivatives came out from Italian preliminary sear
ches, the results of which, related to the antibacterial
and antimycotic activities, were reported in the 1st
Int. Symposium on Organic Chemistry of Medicinal Natural
Products, Shanghai, and also in a degree thesis at the
University of Milan.
From the above cited literature data, the antibac-
terial and antimycotic activities turn out to be essen-
tially related to Ratania-phenols of neolignane nature;
said compounds showing in vitro a remarkable antimicro-
bial activity which, as we could ascertain, almost com-
pletely disappears after administration of the compounds
by the systemic route, since the phenol groups easily
undergo oxidation, or other enzyme degradations can
occur. Topical treatments with total or purified
extracts also show to a considerable degree the same
drawbacks of the systemic route, thus decreasing the
effectiveness of the active principles.
Now, it has surprisingly been found that, by reac
ting these substances with phospholipids in aprotic sol
vents, to generate the lipophilic complexes disclosed
hereinafter, the same antimicrobial activities of the
free active principles can be obtained in vitro, with a
simultaneous remarkable improvement in the in vivo anti-
microbial, antiinflammatory arid antiradical activities.
zn order to prepare the above mentioned complexes,
either the main components of the Ratania-phenol class,
- 3 -
such as Eupomatenoid 6 or the compound 2(2,4-dihydroxy-
phenyl)-5-propeny:Lbenzofuran, which is among the main
responsibles for the biological activity, or purified
extracts standardized in these active principles have
been used, the latter being prepared chiefly by
extracting the roots with chlorinated solvents such as
methylene chloride, chloroform, dichloroethane, etc. and
partitioning the concentrate. between an aliphatic
alcohol and an aliphatic or aromatic hydrocarbon.
ZO Generally the roots are extracted with methylene
chloride, the percolate is concentrated to small volume
and the residue is dissolved in 90°~ aqueous methanol and
counter-extracted with n-hexane; the methanol phase,
after appropriate dilution with water, is counter-
l5 extracted with a chlorinated solvent which extracts the
active principles; the chlorinated organic phase is
concentrated to small volume and the residue is
insolubilized with n-hexane.
According to a preferred object of the present in
20 vention, in order to maintain the stability of the ac
tive principles during the extraction processes, the fi
nely ground roots are extracted with carbon dioxide un
der hypercritical conditions, working at a temperature
of 40°C and under a pressure of 120 bars; after the ex
25 traction under said conditions, the vegetable material
is further extracted with carbon dioxide added with ace-
tone, at a temperature of 45°C and under a pressure of
200 bars; the residue from this second extraction, after
evaporating off the gas, is dissolved in methylene chlo-
~0 ride, dehydrated, decolorized on charcoal and
insolubilized in hexane.
~Q~~~3'~
- 4 -
From the above prepared extracts, the single pure
components can be isolated, using chromatography techni-
ques on silica gel, working on the her se products or,
preferably, on the acetyl derivatives thereof, which are
subsequently deprotected under controlled conditions.
The above cited extracts contain about 50% Eupo-
matenoid 6 and 25% 2(2,4-dihydroxyphenyl)-5-propenyl-
benzofuran, as determined by g.as chromatography or by
I~PLC. These substances, at the pure state, and the ex-
tracts containing them have been tested in vitro for the
antiradical, antibacterial and antimycotic activities
thereof, both as the free compounds and as the complexes
thereof, and in vivo for the antiinflammatory and anti-
microbial activities.
The obtained data are reported in Tables I, II and
III.
The in vitro antimicrobial activity (M.I.C, expres-
sed in mcg/ml) was evaluated in agar culture medium
Isosensitest agar pxoid on Gram-positive and Gram-nega-
tive microorganisms from both collection and hospital
isolation; Difco blood agar was used for streptococci,
whereas Sabouraud maltose agar Difco was used for yea-
sts, moulds and dermatophytes, by incubating the various
inocula according to standard procedures. The results
are reported in the following Table I.
_ 5 _
fable I
Antimicrobial activity of Kra~ria active principles and of the
related canplexas.
Microorgar..a.sm M.I.C.mcg/m1
I II III IV V VI
Staphylococ.aureus 0.0120.78 1.563.12 1.560.78
~nith
Staphylococ.aureus 0.0240.78 1.561.56 1.563.12
9144
Staphylococ.aureus 50 0.78 1.563.12 1.563.12
F2*G
Staphylococ.aureus 0.0120.78 1.563.12 1.563.12
6538P
Staphylococ.aureus 3.12 0.78 0.783.12 1.561.56
OSCB*
Staphylococ.aureus 0.78 0.78 1.561.56 1.561.56
FBF*
Streptococ.pyogenes 0.0120.39 0.783.12 3.121.56
68
Streptococ.pyogenes 0.0120.39 0.783.12 0.650.65
68/24
Streptococ.faeca7.is 0.19 1.56 3.123.12 6.256.25
6057
Streptococ.faecalis 0.39 3.12 6.256.25 6.256.25
99/85
Streptococ.salivarius0.78 6.25 0.7812.5 1.5612.5
71/24
Streptococcus. mutans0.0120.78 0.781.56 1.563.12
60/21
Stxeptcccc.pyogenes 0.19 0.78 0.780.78 1.566.25
65/57
Streptoccc.mitis 77/2310.78 1.56 1.561.56 0.783.12
Sarcinia lutea 9341 0.0120.39 0.780.39 1.563.12
Fscherichia coli 120 0.39 >200 50 >200 100 100
Salmonella typhi 6/120.19 >100 25 >100 50 >100
Salmonella errteritidis0.78 200 50 >200 100 >100
2 Neisseria meningitides0.0121.56 0.781.56 1.563.12
5
Klebsiella pneuie >200 >200 12.5>200 12.5>100
- 6 -
Table I (continued)
Microorganism M.I.C. mcg/ml
I II III TV V VI
Candida albicans G1 3.12 6.25 25 6.25 6.25 >200
Aspergillus niger 6.25 6,25 12.5 6.25 12.5 100
Trichophyton mentag. 0.39 1.56 1.56 1.56 1.56 25
Trichophyton tonsurans 0.19 0.78 1.5~ 1.56 0.78 12.5
Microsporum cams L/55 0.78 1.56 0.78 3.12 1.56 12.5
Substances
I = ampicillin for bacteria and miconazole for fungi.
II = Eupomatenoid 6.
III = 2(2,4-dihydroxyphenyl)-5-propenylbenzofuran.
IV = Complex of Eupomatenoid with phosphatidylcholine.
V = complex of 2(2,4-dihydroxyphenyl)-5-propenylbenzo
,,,v i ~L
furan c~hi~ phosphatidylcholine.
VI = Standardized Krameria extract.
The antiinflammatory activity by the topical route
was evaluated in the mouse by the Croton oil test, ac
cording to the procedures reported in Agents and Ac
tions, 17, 347-49, 1985. The results are summarized in
the following Table II.
7 _
Table II
Antiinflammatory activity the Krameriaextract,
of of
the active components thereofand of the related
com-
plexes in the Croton oil in the mouse,at the maxi-
test
s mum edema (6 hours).
Substances Dose/mcg/ear Edema mg % reduction
Controls --- .7.410.2 ---
Eupomatenoid 6 56 1.50.2 78.5 **
28 4.50.1 40.2
Ratania-phenol* 56 1.90.2 73.7 **
28 4.70.2 34.8
Krameria extract 70 1.80.3 75.0 **
Phospholipid 157 6.50.3 10.2
78 6.20.2 14.0
Eupomatenoid/
complex 1M:11'~1 96 1.90.2 73.7 **
Krameria extr./
Complex lp:2p(w/w) 105 1.60.3 78.2 **
________________________________________________________
* 2(2,4-dihydroxyphenyl)-5-propenylbenzofuran.
Distearoylphosphatidylcholine.
P<0.01 Student t.
The antiradical activity was evaluated using
DPPH
as the competitor, accordingto proceduresdescribed
in
literature. Following Table obtained
III reports the re-
sults.
~~~1~3'~
_8-
Table III
Antiradical activity of a Krameria standardized extract
and of two components thereof.
Substances Conc.mcg/ml % destroyed DPNH
Eupomatenoid 6 6.6 -75.7
2.8 . -40.8
Ratania-phenol* 6.6 -80.5
2.8 -46.4
Krameria extract 10 -72.4
5 -41.6
Quercetin 6.2 -68.2
* 2(2,4-dihydroxyphenyl)-5-propenylbenzofuran.
In order to prepare the complexes with phospholi-
pids, pure soy phosphatidylcholine or standardized
mixtures of vegetable phospholipids with titres from 90°6
to 100°,~ or pure synthetic phospholipids having saturated
or unsaturated acyl chains and choline or ethanolamine
as the basic portion, were selected as the complexing
agent s .
The formation of said complexes is ascertained by
means of 1H-NMR, 13C-NMR, 31P-NMR spectroscopies and so
lubility tests. As regards solubility, substances which
are insoluble in aprotic solvents such as aromatic
hydrocarbons, become easily soluble after complexation;
in the lIi-NMR spectrum the signals of complexed
substances undergo a strong broadening, so as they can
no more be evidenced in the spectra; in the 13C-NMR
spectrum the signals of the complexed substance, as well
".
_ g _
as those of the choline and glyceryl portions of the
phospholipid, can no more be recorded; the phosphorus
nucleus itself undergoes a band broadening, which
indicates it is involved in the complex formation; in
both the 1H and the 13C spectra, substantially only the
lipid chain signals appear, even showing some
immobilization. The above data prove the interaction
between the phospholipid polar head and the active sites
of the complex, whereas the lipid chains are not
i0 involved, since they are free to rotate and can wind the
complexed molecule giving it a marked lipophilia.
This winding clearly results in a particular steric '
configuration when the complex is dispersed in an
aqueous medium, giving stability to the molecules them-
selves.
The complexes of the present invention are insolu-
ble in water and cannot form in this medium, therefore
associations of the same products with phospholipids or
liposomal preparations thereof do not give the same bio-
logical results; the complexes of the invention, due to
dipolarity reasons, in aqueous medium give rise to
micellar microdispersions remarkably similar to the
liposome structures. The novel structures according to
the invention show, in comparison with the same
structures in the free form, a different in vivo
bioavailability which involves an increase in the
specific activity as well as a longer lasting action by
the topical route (skin and accessible mucosae).
For this class of substances, phospholipids are a
selective carrier allowing the molecules to have both an
improved crossing of the horny layer of the skin and a
202~.~~'~
- 10 -
better interaction with cell and bacterial walls. The
molecules in the free form are restored and retained in
the action site for a longer time. The presence of a
surfactant, i.e. the phospholipid, in the molecule, al-
lows to obtain a higher adhesion of the product itself
to the surfaces it comes into contact with. This aspect
is of paramount importance in cosmetic and pharmaceuti-
cal formulations intended for the treatment of the oral
cavity, in which contact times are of course very short;
the opportunity to increase the adhesion of the product
to mucosae or to the tooth surface and to make the va-
rious molecules interact with cell structures is of
basic practical importance and it is an object of the
present invention.
The dosages of the complexes of the invention can
range widely, depending on the intended therapeutical
purposes, and they can vary from 0.001 to 1°,6. The com-
plexes of the present invention can be incorporated in
the traditional pharmaceutical forms, such as ointments,
fluid and thick 0/W or W/0 emulsions, solutions, chewa-
ble tablets, dusting powders, or in form of plasters or
medicated gauzes. Said preparations can be used in hu-
mans or in animals for the treatment of superficial in-
fected inflammatory processes, in torpid sores and in
all the phlogistic conditions of the oral cavity; more
particularly, as far as the oral cavity is concerned,
they are an effective protection against the formation
of dental plaque, when administered in form of medicated
toothpaste, collutory or gel. In the cosmetic field,
said complexes can be used for the treatment of acne and
as deodorant, antidandruff and personal hygiene pro-
- 11 -
ducts .
The following non limiting examples further illu-
strate the invention.
EXAMPLE 1
Preparation of a standardized Krameria triandra extract.
kg of finely ground roots of Krameria triandra are
extracted 4 times under mild reflux and nitrogen atmo-
sphere with 4 volumes of methylene chloride; the combi-
ned extracts are concentrated to small volume under nor-
10 mal pressure. The concentrate is taken up into 5 1 of
90°,6 aqueous methanol and the solution is extracted 3 ti-
mes with 1.5 1 of n-hexane; the hexane phase is discar-
ded since it contains no active principles whereas the
methanol phase is concentrated to 1.2 1; this concen-
trace is extracted 3 times with 1.5 1 of methylene chlo-
ride. The hydroalcoholic phase is discarded, whilst the
chloromethylene extracts are pooled and treated with 25
g of decolorant charcoal in the presence of anhydrous
sodium sulfate as the dehydrating agent; the decolorized
chloromethylene solution is concentrated to small volume
and the concentrate is poured into 1.5 1 of hexane, un-
der. strong stirring. The precipitate is filtered and
dried under vacuum overnight at 40°C, to obtain 300 g of
a product containing about 50°,~ Eupomatenoids and 27%
2(2,4-dihydroxyphenyl)-5-propenylbenzofuran.
EXAMPLE 2
Preparation of Eupomatenoid 6 and 2-(2,4-dihydroxyphe-
nyl)-5-propenylbenzofuran.
100 g of an extract prepared according to the pro
cedures reported in Example 1 are chromatographed on a
column containing 1.5 kg of silica gel, which has pre
- 12
viously been equilibrated with a toluene/ethyl acetate
9:1 mixture, eluting the products with the same solvent
mixture; the fractions showing a similar composition by
thin layer chromatography are pooled and concentrated to
dryness. The more abundant fraction, containing Eupoma-
tenoid 6 (54 g) is crystallized from an hexane-isopropyl
ether mixture, thus recovering the pure compound; analo-
gously, the fraction containing the benzofuran deriva-
tive is crystallized so as to isolate also this com-
pound, the physico-chemical and speetroscopical charac-
teristies of which are the same as those reported in li-
terature.
EKAMPLE 3
Preparation of a purified Krameria extract by means
of carbon dioxide under supercritical conditions.
1.5 kg of finely ground roots of Krameria are ex-
tracted continuously in a suited reactor, fitted with
heating and cooling mantle, with carbon dioxide under
supercritical conditions, in two steps. A first step is
carried out at a temperature of 35°C and under a pres-
sure of 110 bars; in the evaporator temperature and
pressure are 25°C and 50 bars, respectively. Two hours
after, the extracted material is unloaded from the
condenser and carbon dioxide containing 1.5% acetone is
circulated in the extractor. The first extract,
containing only lipophilic substances with no activity,
is discarded, whereas the second extract is collected,
which is obtained by increasing temperature to 45°C and
pressure to 200 bars. The collected extract in the
condenser is dissolved in isopropyl ether, the solution
is dried over sodium sulfate and subsequently
- 13 -
concentrated to small volume. The concentrate is poured
into n-hexane . an abundant precipitate forms which,
upon drying, weighs 46 g and has the same composition as
that of the product obtained in Example 1, but it has a
markedly lighter colour.
EXAMPLE 4
Preparation of the complex of the standardised extract
of Krameria with soy phosphatidy~lcholine.
1.5 g of a purified Krameria triandra extract are
dissolved together with 3 g of soy phosphatidylcholine
in 30 ml of methylene chloride and heated to mild reflux
for 2 hours; the chloromethylene solution is concentra
ted to dryness under vacuum at 30°C until the solvent is
completely evaporated off. The 1H-NMR spectrum of the
obtained product shows protonic signals at 0.8 ppm of
aliphatic methyls of the lipid chains and CH2 signals
between 1.5 and 2.8 ppm, a very broad N-CH3 signal at
about 3 ppm, signals of hydrogens on the double bond of
aliphatic chains at 5.5 ppm and very broad, unresolved
signals of aromatic protons of the Ratania-phenols at 6-
8 ppm. In the 13C-NMR spectrum, the signals characteri-
stics of Ratania-phenols between 102 and 135 ppm are
absent or unresolved, and the signals of the carbons of
phospholipid glycerin portion are also absent or extre-
melt' broadened, as well as those of the choline portion.
EXAMPLE 5
Preparation of the complex of Eupomatenoid 6 with soy
phosphatidylcholine.
2.8 g (O.OI mole) of Eupomatenoid 6 are dissolved
together with 7.8 g (0.001 mole) of pure soy phospha
tidylcholine in 30 ml of dioxane free from peroxides and
- 14 -
the solution is freeze-dried; 10.5 g of a white product
are obtained, having m.p. 98-101°C and spectroscopic
characteristics in conformity with those of a complex.
1H-NMR (C6D6 solution): strong broadening of aromatic
protons at 6-8 ppm; broadening of the N-CH3 signal at
3.3 ppm. The characteristic signals of lipid chains
appear at 0.5-2 ppm. 13C-NMR (C6D6 solution) . absence
of the signals of carbons of Eupomatenoid at 105-140 ppm
and also of the lipid signals at 60-80 ppm.
EXAMPLE 6
Preparation of the complex of 2-(2,4-dihydroxyphenyl)-5-
propenylbenzofuran with distearoylphosphatidylcholine.
2.78 g of 2-(2,4-dihydroxyphenyl)-S-propenylbenzo-
furan are dissolved in 50 ml of dioxane together with
7.95 g of distearoylphosphatidylcholine and heated under
strong stirring for 3 hours at SO°C, under argon atmo
sphere. When dissolution is completed the whole is
freeze-dried, to obtain a white-beige product having
spectroscopic characteristics corresponding to those of
a complex.
EXAMPLE 7
Preparation of a standardized extract of Eupomatia lau-
rina.
10 kilograms of finely ground roots of Eupomatia
laurina are extracted under weak reflux in a nitrogen
atmosphere with 4 volumes of methylene chloride for four
times and the pooled extracts are concentrated to a
small volume at ordinary pressure. The concentrate is
then dissolved in 2 1 of 90 per cent aqueous methanol
and the resulting solution is extracted three times with
1.5 1 of petroleum ether.
- 15 -
The petroleum ether phase not containing the active
principles is discarded and the methanolic phase is
concentrated to 0.9 1. The latter concentrate is diluted
with 300 ml of water and extracted three times with 0.8
1 of methylene chloride. The hydroalcoholic phase is
discarded and the pooled methylene chloride extracts are
treated with 25 g of decolorizing charcoal in the
presence of anhydrous sodium sulphate as dehydrating
agent. The decolorized methylene chloride solution is
concentrated to a small volume and the concentrate is
poured under vigorous shaking into 1 1 of hexane. After
filtration of the precipitate and overnight drying under
vacuum at 40°C, 190 g are obtained of a product which
contains as a major component Eupomatenoide 6.
EXAMPLE 8
Preparation of the complex between the standardized
extract of Eupomatia laurina and soy-bean phosphatidyl-
choline.
g of purified extract of Eupomatia laurina are
20 dissolved together with 40 g of soy-bean phosphatidyl
choline in 300 ml of methylene chloride and heated under
weak reflux for 2 h. The methylene chloride solution is
concentrated to dryness under vacuum at 30°C until the
solvent has been completely removed. 'rhe 1H-NMR spectrum
of the final product shows protonic singnals at 0.8 ppm
originating from the aliphatic methyl group of the
lipidic chains, CH2 signals between 1.5 and 2.8 ppm, a
very broadened N-CH3 signal at around 3.4 ppm, signals
from hydrogens on double bonds of aliphatic chains at
S.S ppm and very broadened unresolved signals of the
aromatic protons of phenols Eupomatenoide 6 like
- 16 -
substances between 6.2 and 8 ppm. In
the carbon spectra
it is remarkable the absence or the lack
of resolution
of the typical signals of the Eupomatenoide
like
substances between 102 and 135 ppm and absence or
the
the extreme broadening of the signals the carbons
of
belonging to the glycerine and choline
portions of the
phospholipid. This complex is freely
soluble in the
chorinated solvents and in the vegetal,
oils.
EXAMPLE 9
Preparation of a toothpaste containing complex of
the
the standardized Krameria extract.
100 g contain
Complex of Rrameria standardized extract0.1 g
Sodium carboxymethyl cellulose m.v. 1.0 "
Sorbitol 70% 20.0 '
Glycerin 18.0 "
Colloidal silica 15.0 "
Titanium dioxide 2.5 "
Maize starch 2.0 "
Sodium laurylsulfate 2.0 "
Sodium laurylsarcosinate 30% s. a. 1.0 "
Flavouring composition 1.0 '
Purified water q.s.to 100.0 "
EXAMPLE 10
Preparation of chewable tablets containingthe complex
of Krameria standardized extract.
Each 1 g tablet contains
Complex of Krameria standardized extract5.0 mg
Methyl cellulose m.v. 2.0 "
Colloidal silica g.0 ~~
Magnesium stearate 10.0 "
-- 17 _
Mannitol 250.0 "
Glycamil (monoammonium glycyrrhizinate)5.0 "
Flavours 20.0 "
Saccharose (compressible) 700.0 mg
EXAMPLE 11
Preparation of a medicated cream containing
the complex
Eupomatenoid/phosphatidylcholine.
100 g of an O/W emulsion contain
Complex Eupomatenoid/phosphatidylcholine0.1 g
PEG-8 C12-18 alkyl ester 10.0 "
Glyceryl stearate and PEG-8 stearate 2.5 "
Isopropyl myristate
Preservative 0.1 "
Glycerin 5.0 '
Perfumed composition 0.5 "
Purified water q.s.to 100.0 g
EXAMPLE 12
Preparation of an aqueous gel containingthe complex
of
the Krameria standardized extract
100 g of gel contain :
Complex of standardized Krameria extract0.1 g
PEG-6 caprylic/capric glycerides 10.0 "
Ethoxylated oleyl alcohol 5.0 "
Carboxyvinyl polymer 1.5 "
Preservatives 0.3 "
Perfumed composition 0.2 "
Sodium hydroxide 0.3 "
Purified water q.s.to 100.0 "
EXAMPLE 13
Preparation of a lipogel containing complex the
the of
Krameria standardized extract.
- 18 -
100 g of gel contain
Complex of standardized Krameria extract 0.1 g
PEG-45 dodecylglycole copolymer 4.0 "
Hydroxystearate 4.0 "
Polyisoprene 37.5 "
Isopropyl myristate 3g.9 n
Silicone oil 350 cps 3.5 "
Hydrogenated castor oil ~ 3.5 "
Colloidal silica 3.0 "
Polysorbate 80 3.0 "
Perfumed composition 0.5 g
EXAMPLE 14
Preparation of a dusting powder containing the
complex
of the Krameria standardized extract.
100 g of powder contain
Complex of Krameria standardized extract 0.1 g
Colloidal silica 2.p "
Micronized hydrogenated castor oil 49.0 "
Maize starch 48.9 "
