Abstrict
The invention relates to the use of inulin, oligofructose or their
derivatives as functional ingredients in functional food, functional
feed, OTC and pharmaceutical composition to prevent mammary carcinogenesis
or treat breast cancer.
Claims
We claim:
1. Method of preventing mammary carcinogenesis in a mammal susceptible
to mammary carcinogenesis which comprises the administration to
said mammal of a sufficient amount of a composition comprising at
least one physiologically active ingredient selected from the group
consisting of inulin, oligofructose and mixtures thereof.
2. Method according to claim 1, wherein the said physiologically
active ingredient has a negatively modulating effect on mammary
carcinogenesis.
3. Method according to claim 2, wherein said physiologically active
ingredient has one or more of the following effects: slows down
the kinetic appearance of malignant breast tumors, lowers the incidence
of breast cancer, lowers the intensity of breast cancer, lowers
the yield of tumors, lowers the degree of malignancy of the breast
cancer, lowers the degree of invasiveness of malignant breast cancer,
reduces metastasis of breast cancer.
4. Method according to claim 1, wherein said inulin comprises native
chicory inulin.
5. Method according to claim 1, wherein said oligofructose is obtained
by enzymatic hydrolysis of native inulin from chicory, J. artichoke
or dahlia.
6. Method according to claim 1, wherein the composition further
comprises an ingredient that actively destroys malignant breast
tumor cells.
7. Treatment method of breast cancer in a mammal, which comprises
the administration to said mammal of a sufficient amount of a composition
comprising at least one physiologically active ingredient selected
among the group consisting of inulin, oligofructose and mixtures
thereof.
8. Method according to claim 7, wherein the said physiologically
active ingredient has a negatively modulating effect on mammary
carcinogenesis.
9. Method according to claim 7, wherein said physiologically active
ingredient has one or more of the following effects: slows down
the kinetic appearance of malignant breast tumors, lowers the incidence
of breast cancer, lowers the intensity of breast cancer, lowers
the yield of tumors, lowers the degree of malignancy of the breast
cancer, lowers the degree of invasiveness of malignant breast cancer,
reduces metastasis of breast cancer.
10. Method according to claim 7, wherein said inulin comprises
native chicory inulin.
11. Method according to claim 7, wherein said oligofructose is
obtained by enzymatic hydrolysis of native inulin from chicory,
J. artichoke or dahlia.
12. Method according to claim 7, wherein the composition further
comprises an ingredient that actively destroys malignant breast
tumor cells.
13. Method according to claim 7, wherein said physiologically active
ingredient is administered in a pharmaceutically acceptable carrier
or in a food or feed.
14. Method according to claim 7, wherein said physiologically active
ingredient is administered in a pharmaceutically acceptable carrier
on in a food or feed.
15. Composition for the prevention of mammary carcinogenesis or
treatment of breast cancer which comprises at least one physiologically
active ingredient selected from the group consisting of inulin,
oligofructose and mixtures thereof, which further comprises chemotherapeutic
products capable of actively destroying malignant breast tumor cells.
Description The present invention relates to the use of the functional ingredients
inulin, oligofructose or their derivatives for the manufacture of
functional food, functional feed, OTC and pharmaceutical compositions
for the prevention of mammary carcinogenesis or treatment of breast
cancer. The invention also relates to functional food, functional
feed, OTC and pharmaceutical compositions comprising said functional
ingredients and to the method of prevention or the method of treatment
resulting from the use of these functional ingredients.
In industrialised countries the second cause of death after heart
disease is cancer. Particularly lung, breast and colon cancer predominate
in those countries.
Cancer is the result of an uncontrolled local proliferation of
cells with invasion of adjacent normal structures. Metastasis occurs
when the cancer spreads via bloodstream or lymphnodes or within
a body cavity.
Most patients are women but male breast cancer occurs with 1% the
frequency of female breast cancer. Domestic mammals such as dogs,
horses, etc. are equally susceptible to mammary cancer.
Human breast cancer also known as mammary cancer (and interchangeably
used in the text herewith) is a disease which can result from several
factors such as ionising radiation, diet, familial history or exposure
to genetic mutagens. Since the determination of the initiation of
the breast carcinogenic process is difficult, it is equally difficult
to know the exact agents causing the disease.
Although the exclusion of risk factors is a generally accepted
approach to avoid the onset or the appearance of putative tumors
after the latency period is ended, it is clearly insufficient.
As ideas on the pathogenesis of the disease and on the impact of
different factors such as enumerated herein above are continuously
evolving, there is still a need for new but also convenient methods
to prevent or treat breast cancer patients.
Moreover, there is an urgent need for malignant breast tumor prevention
due to the high medical costs involved once an individual becomes
a cancer patient. Above that, any possible preventive habit which
could be used to avoid or at least retard the disease should be
investigates.
In L. A. Cohen et al., J. Nat. Cancer Inst. 83(7), (1991), p 496
it is described how dietary fiber in a high-fat diet is found to
be protective against breast cancer. A supplemental soft white wheat
bran exerted an inhibitory effect on the promotional phase of N-methylnitrosourea
(MNU) induced breast carcinogenesis in rats when supplemented to
a high-fat diet but not when added to a low-fat diet. The researchers
speculated that dietary fiber acts by decreasing the digestibility
of fat, thus mimicing a low-fat condition.
P. D. Cooper et al., Molecul. Immunol. 23(8), (1986), p 895 describes
the activation of the alternative pathway of complement by a specific
polymorphic form of dahlia inulin named gamma-inulin. Gamma-inulin
is formed by >8000 to 10 000 MW polymers (degree of polymerisation
52 to 65) and is insoluble in diluted suspension at 37.degree. C.
It is known that an activator of the alternative pathway of complement
can have a potential non-specific anti-tumor effect. As such, intraperitoneally
injected gamma-inulin is shown to prolongate the survival of melanoma
bearing mice (P. D. Cooper et al., Molecul. Immunol. 23(8), (1986),
p 903) but the timing of the treatment is very critical.
The patent application JP 60/89427 describes an inulin extract
from the roots of a specific member of the Campanulaceae, Platycodon
grandiflorum A.DC. used to treat tumor cell bearing mice. Platycodon
grandiflorum comprises sapogenins, known for their pharmaceutical
properties.
SUMMARY OF THE INVENTION
The present applicant has now found that inulin, oligofructose
and their derivatives have properties of value as functional ingredients
in the prevention of mammary carcinogenesis or treatment of breast
cancer, comprised in a functional food or in a pharmaceutical composition.
More specifically, these functional ingredients negatively modulate
mammary carcinogenesis.
The present applicant has demonstrated that they slow down the
kinetics of appearance of breast tumors, that they lower the intensity
of malignant breast cancer and the yield of all tumors. Unmistakable
indications exist that they also lower the degree of invasiveness
and reduce metastasis of malignant breast cancer. It is necessary
to verify again if the incidence of breast cancer and the duration
of the latency period is influenced by these functional ingredients.
Some indications exist as to the degree of malignancy of the appearing
breast tumors, which appears to be lowered by the said functional
ingredient.
Therefore the present invention allows to manufacture a composition
destines for the prevention of mammary carcinogenesis or treatment
of breast cancer. The composition can be a functional food, a functional
feed, an OTC or a pharmaceutical composition. The functional ingredients
inulin, oligofructose or their derivatives are used in the manufacturing
thereof. The present invention also allows to prevent mammary carcinogenesis
of treat breast cancer by the administration of the said functional
ingredient according to the invention to a mammal in an amount sufficient
to exert its effect, in particular by negatively modulating carcinogenesis.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a typical HPAEC chromatogram for native chicory
inulin.
FIG. 2 shows HPLC analysis of inulin fraction.
FIG. 3 shows CGC chromatogram from silylated inulin.
FIG. 4 illustrates a CGC chromatogram of treated native chicory
inulin.
FIG. 5 illustrates the tumor incidence (number of rats bearing
mammary tumors in one group) according to Example 1.1.
FIG. 6 illustrates the yield or total number of malignant tumors
in one group according to Example 1.1.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Negative modulation of breast carcinogenesis is defined as any
prevention of mammary carcinogenesis that has at least one of the
following effects:
it allows to lengthen the latency period;
it slows down the kinetics of appearance of malignant breast tumors;
it lowers the incidence of breast cancer;
it lowers the intensity of breast cancer;
it lowers the degree of malignancy of appearing breast tumors;
it lowers the degree of invasiveness of malignant breast cancer;
it reduces metastasis of malignant breast cancer.
Incidence of breast cancer is defined as the number of individuals
bearing mammary tumors in one test group.
Intensity of breast cancer is the mean number of malignant mammary
tumors per individual bearing tumors.
Yield is the total number of, eventually malignant, tumors in one
test group.
Tumors can be classified as benign or malign as far as the degree
of malignancy is concerned.
Parameters for calculating the reduction of invasiveness and metastasis
are expressed as metastasis incidence and incidence of tumors in
other organs or tissues.
Modulation of breast carcinogenesis has, as defined herein, no
direct relevance to multiphase/multistep carcinogenesis. Its effect
can only be demonstrated by following the kinetics of cancers as
they appear and/or by recording the incidence and yield of histologically
characterised cancers. This terminology should broaden the view
on breast carcinogenesis. The negative modulating properties of
the functional ingredients according to the invention are particularly
interesting in the contest of the effects of more systematic procedures
or experimental conditions such as chronic exposure to chemicals,
dietary imbalances or surgery that disrupts metabolic or proliferative
breast homeostasis. Even though such procedures are not essential
for making an otherwise induced process fully carcinogenic, they
can still influence its pathogenesis by creating conditions that
speed up the kinetics of its development to malignancy and consequently
increase or decrease the incidence and/or yield of cancer.
The inulin used according to the invention is a carbohydrate belonging
to the group of polysaccharides named fructans. Fructans are compounds
in which fructosyl-fructose linkages constitute the majority of
the linkages. In inulin most linkages are of the beta-D-(2.fwdarw.1)
fructosyl-fructose type. Most but not necessarily all inulin molecules
contain a glucose moiety at the non-reducing end of the chain. Glucose
is linked as in sucrose by an alpha-1.fwdarw.beta-2 linkage.
It was shown that one of the most suited types of inulin used according
to the invention, independent of its origin, is basically a polydispersed
mixture of very slightly beta-(2.fwdarw.6) branched beta-(2.fwdarw.1)fructan
molecules. As most of the molecules have a terminal glucose unit,
this inulin can be presented by the general formula GFn (G=glucose,
F=fructose, n varies from 2 up to over 60 with a maximum of 72).
The present applicant observed that this inulin comprises a small
fraction of molecules with general formula Fm that do not contain
any glucose at all. The corresponding general chemical formula is
thus as follows: ##STR1##
GFn are non reducing molecules, all the fructose units are in the
furanose form. Fm molecules are reducing. As suggested by A. D.
French, J. Plant Physiol. Vol. 134, (1989), p 125, the reducing
fructose residues are predominantly in the pyranose form when dissolved
in D.sub.2 O and when the beta-D-(2.fwdarw.6)fructofuranosyl is
absent. This was confirmed by NMR analysis by the present applicant
to be the case for inulin particularly suited to be used according
to the invention namely native chicory inulin.
In the contest of the present invention, "native" shall
mean inulin or chicory inulin which prior to tis use is extracted
from inulin comprising plants with hot water, taking precautions
to inhibit plant-own inulinase activity and to avoid acid hydrolysis.
The extraction process does not essentially change the molecular
structure or the polydispersed composition of inulin. Native inulin
can be extracted from J. artichoke, chicory, dahlia etc.
After starch, inulin is one of the most abundant polysaccharide
found in nature. As it is present in cereals (e.g. wheat, barley),
vegetables (e.g. onion, leek, garlic, asparagus, salsify) and fruit
(e.g. bananas), it is naturally present in our diet.
Degree of polymerisation (DP) is defined as the number of monomer
units in one molecule. The average degree of polymerisation (average
DP) of inulin depends on the plant source and on the moment of harvesting
this plant. As such, native dahlia inulin has an average DP of 20
and native chicory inulin of 11. The percentual distribution of
the different molecules can be presented as follows:
______________________________________ DP* 2-9 10-20 21-40 >40
______________________________________ chicory 31 24 28 17 dahlia
12 18 21 40 J. artichoke 52 22 20 6 ______________________________________
*Pranzik, J. Chromato. 348, (1985), p 187
Two inulin crops are very well suited for commercial exploitation,
namely, Jerusalem artichoke (Helianthus tuberosus) and chicory (Cichorum
intybus). These plant sources, and the type of inulin they contain,
have been studied extensively in the past several decades.
The prefered native inulin of the present invention is chicory
inulin. Its average DP varies strikingly in function of the harvesting
date. In chicory roots, inulin functions as an osmoregulator increasing
cold resistance when broken down. Early in the harvesting season,
native chicory inulin has the highest DP. FIG. 1 gives a typical
HPAEC chromatogram for native chicory inulin suited for the invention
and with an average DP of 11,7 and a maximum DP of 72, obtained
with a Carbopac PA1 column, a Dionex 4500i with PAD detection and
a 100 mM NaOH/100 to 400 mM NaAc elution gradient. The average DP
is determined by the fructose to glucose ratio after complete hydrolysis
with SP230 Novozym inulinase at pH 4.5, 30 min at 60.degree. C.
By means of HPLC analysis (see FIG. 2) it is only possible to differentiate
between DP5.sup.+ (the integrated sum of DP5 and higher DP molecules),
DP4, DP3, saccharose (GF), F2, G, and F. The DP4 and DP3 fractions
are coelutions of GFn and Fm compounds.
CGC, as HPAEC analysis allows to differentiate between GFn and
Fm molecules. As shown in FIG. 3, this method allows to determine
oligomers with a DP up to 10 due to a temperature programming starting
at 105.degree. C. with a 10.degree. C. increase per min, up to 440.degree.
C. Helium is used as carrier gas at a constant flow rate of 9 ml/min.
The native inulin sample is first dried together with phenyl-beta-D-glucopyranoside
as internal standard. After treatment with hydroxylamine, the sugars
are derivatised with trimethylsilylimidazole and the volatile derivatives
are extracted with isooctane. The determination of the response
factor is based upon the analytical results of maltodextrin.
CGC allows to make quantitative analysis of the polydispersed native
inulin mixture up to DP 12.
At present, HPAEC is a handsome method to qualitatively characterise
inulin but since no standards are available in sufficient quantities,
no response factors can be determined and no quantitative measurements
could be obtained so far.
Native chicory inulin as particularly preferred in the present
invention is a 1 to 2% branched molecule. In the same way 4 to 5%
branched inulin molecules were observed in native dahlia inulin.
The type of linkage and the occurrence of branching is checked by
permethylation as described by S. I. Hakamori, J. Biochem. vol 55,
(1964) and followed by reductive cleavage and in situ acetylation.
The method is based on procedures described by I. Ciucanu et al.,
Carboh. Res. Vol. 131(2), (1984), p 209; P. Mischnick et al., Carboh.
Res. Vol 185(1), (1989), p 113 and J. G. Jun, Carboh. Res. Vol 163(2),
(1987), p 247. This technique allows to differentiate 4-linked aldopyranosyl
groups from 5-linked aldofuranosyl compounds as the ring structure
of each monosaccharide remains intact. By ionic hydrogenation of
all glycoside linkages of the methylated polysaccharide one gets
partially methylated anhydro alditols that are acetylated in situ.
FIG. 4 shows a CGC chromatogram of native chicory inulin treated
as such.
A commercially available product comprising native chicory inulin
and corresponding to particularly prefered type of inulin suited
for the present invention, is Raftiline.RTM. ST or GR (Raffinerie
Tirlemontoise, Belgium). The average DP of Raftiline.RTM. ST or
GR ranges from 5 to 14, more specifically from 8 to 12, and particularly
an average DP between 9 and 11 is prefered. More than 50% of the
molecules in Raftiline.RTM. ST have a DP between 2 and 20 of which
more than half have a DP lower than 10. The dry weight of Raftiline.RTM.
is on average composed of 92% of native inulin, 2% of monosaccharides
(G,F) and 6% of disaccharides (saccharose). Raftiline.RTM. is poorly
soluble: at high concentrations a temperatures above 85.degree.
C. is needed to completely dissolved it. At room temperature an
1% Raftiline solution precipitates and the precipitation accelerates
and becomes more complete when the solution is cooled down.
On the other hand, Raftiline.RTM. becomes more soluble in hot water
but then the pH of the solution becomes an important factor. When
the solution is too acidic (pH under 4,5) Raftiline.RTM. is partially
hydrolysed into his monomers. When the pH is too high (above 6.5)
the solution tends to colour. Fibruline.RTM. standard (Cosucra,
Belgium) is another commercially available native chicory inulin
comprising product that can be used according to the invention.
Equally prefered for this invention is a polydispersed inulin essentially
free of its mono- and disaccharides. Such a functional ingredient
can be obtained according to the process described in PCT/BE93/00072,
included herein by reference. Raftiline.RTM. LS (Raffinerie Tirlemontoise)
is a commercially available product comprising such inulin.
Instant inulin that can be used for this invention, is native inuline
dried such that inulin can be added to high dry weight products.
The patent application PCT/BE94/00019 describes an instant inulin
production method and is included herein by reference. Raftiline.RTM.
ST gel comprising instant inulin is commercialised by Raffinerie
Tirlemontoise, Belgium.
It can be foreseen to use polydispersed inulin whose average DP
is modified compared to the native inulin. This can be obtained
either by physically separating off the low DP fractions of the
polydispersed mixture, by selectively purifying specific DP fractions,
by synthesing longer inulin chains etc. Examples of such inulin
compositions are inulin I 2255, I 3754, I 2880 respectively comprising
modified average DP inulin prepared from native chicory, dahlia
and J. artichoke inulin (Sigma, USA) or polyfructans prepared as
described in EP 532 775.
Fructooligosaccharides with DP up to 20 are called, by definition,
oligofructose. These saccharides are well soluble in water (up to
80% DW solutions are stable). Oligofructose can be composed of a
few percentages of FM to roughly equal accounts of Fm or GFn molecules.
Onion comprises mainly native oligofructose from which it can be
extracted. Oligofructose can be produced from inulin by partial
hydrolysis, enzymatic or acidic.
An oligofructose particularly of interest when used according to
the invention can be obtained by partial enzymatic hydrolysis of
native inulin from chicory, J. artichoke or dahlia. The DP of the
molecules in this polydispersed mainly beta-(2.fwdarw.1)fructan
varies between 2 and 8. The average DP ranges from 3 to 6, preferably
3,5 to 5.
The enzymatic hydrolysis can be executed with an endo-inulinase
obtained from Aspergillus, Penicillum or Bacillus. An example is
described by B. E. Norman, Denpun, Kagaku 36(2), (1989), p 103.
A chicory inulin containing extract is partially purified by means
of carbonation and filtration. Filtered clarified juice is evaporated
up to a concentration suited for the enzymatic treatment, prior
to entering the final purification step. The transformation of inulin
into oligofructose is achieved by means of an endo-inulinase which
is added to the juice and which performs its hydrolytic action.
The resulting product is evaporated in order to prevent microbial
development. This crude oligofructose is the feed stock for the
final purification process carried out by means of demineralisation
ion exchange columns.
A final purification of the oligofructose is achieved by an activates
carbon treatment which allows to physically adsorb some minor compounds
which would not be eliminated in the preceding purification steps.
After removing the activated carbon by means of filtration and a
final purification on a mixed bed resin, the thus obtained very
pure syrup is filter sterilized, prior to evaporation to a commercially
defined concentration.
A particularly preferred oligofructose comprising product useful
in the present invention is Raftilose.RTM. (Raffinerie Tirlemontoise,
Belgium). It is a commercially available product comprising oligofructose
obtained by hydrolysing partially purified native chicory inulin
using an endo-inulinase.
The different commercially available Raftilose.RTM. compositions
of Raffinerie Tirlemontoise, Belgium, are on average composed as
follows:
______________________________________ oligo- RAFTILOSE fructose
glucose sucrose fructose ______________________________________
L30 60 7.5 2.5 30 L60 12 28 60 L85 7 85 L95 5--- 95 P95 5--- 95
______________________________________
Another method of preparing the functional ingredients according
to the present invention is the reaction of fructosyl transferase
with sucrose to bound fructose monomers to the sucrose. For example
GB 2,105,338 describes such a process of preparation. A commercially
available product is Neosugar.RTM. (Beghin-Say, France).
Branched oligofructoses can also be used as functional ingredients
in the present invention, they consist of a main chain and at least
one side chain and comprise mainly fructose units. The main chain
contains from 2 to 15 units and all the branching points are on
fructose units. The side chains can be branched also, resulting
in additional side chains. WO 91/13076, incorporated herein by reference,
in the name of Raffinerie Tirlemontoise, Belgium, describes the
production of branched oligosaccharides which can be used according
to the invention.
The process described in WO 91/13076 can equally be used to produce
branched inulin which can equally be used as a functional ingredient
in the present invention.
From another point of view, the present invention relates to functional
food comprising the products used for their beneficial effects in
the prevention of mammary carcinogenesis and the treatment of breast
cancer as indicated herein above.
A functional food is any food product considered to be edible and
comprising one or more food ingredients of which it is known that
they provide the consumer with a physiological benefit including
for example the prevention of disease, the treatment of disease,
the activation of the biorhythm or the immune system.
Normally food has two purposes: feeding and sensorial pleasure.
Functional food combines this with a supplementary physiological
benefit. Functional food is not a pharmaceutical product and is
different from a food product which naturally comprises a food ingredient
known to have a physiological benefit. Functional food shall be
designed such that when applied in a normal diet, the benefit on
health and disease is obtained. Therefore the food ingredient with
physiological benefit, defined herein as functional ingredient,
is added in an amount which is significant from a preventive or
therapeutic point of view.
Depending on the type of functional food, the functional ingredient
is added depending on factors such as daily intake, food law regulations,
organoleptic appreciations, sensorial pleasure, dose-related side
effects, such as diarrhea etc. It will be evident for the man in
the art to design and produce proper functional foods once he is
aware of the functional ingredient properties of a food ingredient.
The purpose of the functional food being such that this food product
combines physiological benefit with good feeding and organoleptic
pleasure.
Functional food thus conveniently allows to acquire the necessary
and beneficial amounts of a functional ingredient without the need
to take pills or syrups comprising the said ingredient.
Compared to food products naturally comprising one or another beneficial
food ingredient, a functional food allows the consumer to obtain
a higher concentration without the need to recur to an imbalanced
daily diet which would occur when significant amounts are to be
taken in. As only frequent intake can ensure that a functional ingredient
exercises his effect of preventing or treating disease, the consumer
can decide whether to take the same functional food regularly or
to diversify his diet, depending on the consumer or patients feeding
mode and wishes.
Functional food looks like everyday beverages, bakery, prepared
meals, confectionary, dairy, dressings, spreads. etc. but comprises
a functional ingredient. The functional ingredient can either be
added as a supplement or added to replace one or several of the
normal ingredients.
Functional feed is administered to non-human mammals.
As a well known example of a familiar dietary component with functional
ingredient properties one can cite calcium which can be added to
everyday dairy products for example although they naturally comprise
calcium. Calcium rich functional food can claim a beneficial effect
on the prevent of osteoporosis.
Other familiar food ingredients with functional ingredient properties
are certain dietary fibres reducing the risk of colon cancer for
example, or oligosaccharides lowering serum triglyceride levels
in hyperglycemic conditions or improving the gut flora, or gut bacteria
activating the immune system.
Inulin, oligofructose and their derivatives according to the invention
are food ingredients which behave as dietary fibre in that first
they are not digested in the small intestine as no human digestive
enzyme exists that can break down the beta(2.fwdarw.1)-linkages,
secondly because they enhance the passage through the gut. It is
known that inulin as well as oligofructose are prebiotics. In other
words that they are food ingredients known to modify the composition
of endogenous gut microflora (N. Delzenne et al., Lebensm-Wiss u.
Technol. 27, (1994), p 1) and especially stimulate the gut Bifidobacteria.
They have reduced caloric value, can modify lipid metabolism (N.
Delzenne et al., Am J. Clin. Nutr. 57 (suppl), (1993), p 820S) and
increase absorption of minerals such as Ca, Mg, Fe, Zn, and Cu.
The present invention exploits a thus far unknown beneficial physiological
effect of inulin, oligofructose and their derivatives combined with
the advantage of dealing with readily, safe and non toxic known
food ingredients.
The functional ingredients according to the invention may be incorporated
into food, feed or pharmaceutical products when it is in powder,
liquid or cream form, according to processes readily known by the
man in the art.
As an example WO 93/06744 in the name of Raffinerie Tirlemontoise,
Belgium, enclosed herein by reference, describes a Rafticreaming
process that can be used to prepare functional food products comprising
inulin. The same process can be used to prepare creamy or inulin
cream comprising OTC or pharmaceutical compositions.
Both the preferred products, Raftiline and Raftilose, are commercially
available in powder form and can therefore be added to for example
powdered food or tables.
The OTC or pharmaceutical compositions suited for oral administration
are the ones known for such form of administration, for example,
tables, (coated or non coated), pills, capsules, solutions or syrups.
By way of exception it can be conceived to administer the products
according to the invention rectally. Then, the prepared composition
is presented in the form of suppositories.
The pharmaceutical compositions are prepared according to the methods
generally applied by pharmacists and may include solid or liquid,
non-toxic and pharmaceutically acceptable vehicles. The inclusion
in galenic mediums can be equally be forseen.
The percentage of active product according to the invention can
vary within very large ranges, only limited by the tolerance and
the level of acquaintance of the patient to the product. The limits
are particularly determined by the frequency of administration.
The criteria of tolerance establishing the limits are comparable
with those for functional food or feed.
The present invention is illustrated by the following examples,
without limiting it.
Rat mammary carcinogenesis as used in the examples is a well established
model, closely mimicking the human disease, and allows to verify
whether breast carcinogenesis can be manipulated by treatment of
the host.
Especially the induction of breast cancer in Sprague Dawley rats
by the target specific N-methylnitrosourea (MNU) carcinogen induces
tumors with a latency period between 8 and 21 weeks and with an
almost 100% final breast cancer incidence in untreated rats.
MNU can be given in a single 25 to 50 mg/kg body weight dose through
subcutaneous injection. The susceptibility of the mammary gland
to this specific carcinogen is age-related and should therefore
be used for initiation between the age of 45 to 60 days of age.
That is the age of sexual maturity. The Sprague Dawley rats used
are known to be particularly susceptible for these testings.
As it is known that high-fat conditions increase breast carcinogenesis,
the diets fed to the rats must supply sufficient fat for normal
development but without excess.
Calorie consumption is another factor influencing breast tumor
incidence. Therefore the diet fed to the control group should be
iso-caloric compared to the one fed to the test group and equal
amounts should be consumed by both the control and the test animals.
EXAMPLE 1
Negative Modulation of Mammary Carcinogenesis
1.1 tumor induction method
30 Sprague Dawley female rats, 45 days old and weighing +/-100
g, are randomly divided into 4 groups. The animals of groups A and
B (9 per group) receive a subcutaneous injection of 0,9% NaCl containing
50 mg/kg body weight N-methylnitosourea (Serva, 30802) (MNU). The
animals of groups C and D (6 per group) which are considered as
controls are injected with the same volume of 0,9% NaCl solution.
One week later, the powder diet AO3-UAR (INRA, France) of the animals
of groups B and D is supplemented with 5% w/w Raftilose P95 (Raffinerie
Tirlemontoise, Belgium) (OF). The concentration of OF in the diet
is increased to 10% the second week and is finally 15% after the
third week and until the end of the experiment. The animals of groups
A and C receive successively the powder diet containing a supplement
of 1%, 2,5% and 5% starch. This is an isocaloric diet as compared
to the OF supplemented diet. Food and water are available at libitum.
Parameters monitored weekly are: body weight gain, diet and water
consumption, fecal excretion. The size, number and position of putative
mammary tumors are assessed by palpation.
At week 27, rats are anesthesised with diethylether and killed
by exsanguination. The organs (liver, kidneys, lungs, mammary glands)
are macroscopically examined in situ and then weighed. The tumors
are described (localisation, aspects) and measured. Tissue samples
are taken and fixed in formalin for 3 weeks, then embedded in paraffine
for further hematoxylin-eosin coloration and histological examination.
1.2. Results during the experiment
The diet consumption was not significantly different between the
different groups. The dry weight of fecal excretion was not significantly
different in OF treated rats as compared to rats receiving starch.
During the experiment according to example 1.1, the size, number
and position of the tumors were evaluated using palpation and recorded
as represented in FIGS. 5 and 6; they give the tumor incidence (the
number of rats bearing mammary tumors in one group) and the yield
(total number of malignant tumors in one group). It can be seen
from these data that the incidence and yield of mammary tumors is
always lower in rats fed OF instead of starch.
1.3. Results at the end of the experiment
After the sacrifice at week 27, the tumors are macroscopically
analysed and all the organs are checked for the putative presence
of lesions. Histological examination allows to classify the tumors
as benign or malignant. The incidence of metastases and tumors in
other organs or tissues is noted. As shown in table 1, in the control
group A, where 7 out of 9 rats are bearing tumors: 19 malignant
mammary cancers, consisting mainly of low or mid-differentiated
adenocarcinoma and 2 renal fibrosarcoma. One of those rats has an
epidermal, cystic papilloma of salivary glands with no signs of
malignity. The examination of the organs after sacrificing the animals
revealed that the number of rats bearing cancer, the incidence,
was the same for group B (MNU/OF), 7 out of 9 are bearing tumors.
But the total number, or yield, of low or mid-differentiated adenocarcinoma
is lower in group B than in group A: only 12 mammary adenocarcinoma
are diagnosed, but neither renal tumors nor metastasis can be detected.
The total volume of mammary tumors is almost 50% higher in the control
group. The mean volume of mammary tumors is more or less the same
in both groups, but lower in OF fed animals. The mean number of
malignant mammary tumors per rat bearing cancer, the tumor intensity,
at the time of sacrifice was lower in group B (MNU/OF) namely 1,7
compared to almost a double intensity in the control group A (MNU/starch),
namely 3.
TABLE 1 ______________________________________ Tumor Volume Malignant
(cm.sup.3) Mammary of mammary Be- adenocar- inten- Metas- tumors
Rats nign cinoma other yield sity tasis Total Mean ______________________________________
Control- 1 19 2 21 3 2 132 6,9 fed OF fed 0 12 0 12 1,7 0 73 6,1
______________________________________
From the above it can be concluded that addition of oligofructose
to the diet negatively modulates mammary carcinogenesis by slowing
down the kinetics of appearance of breast tumors. Although the incidence
looks the same, the mammary tumor intensity is lower.
EXAMPLE 2
Negative Modulation with Oligofructose, Inulin and Pectin
2.1. tumor induction method
45 days old female Sprague Dawley rats weight +/-100 g are purchased
from Charles River, Germany. They are housed by 3 in suspended stainless
steel cages under temperature and humidity control with a 12/12
h light/dark cycle. Food and water are available at libitum. After
their arrival, the rats are adapted to new housing conditions during
1 week. At the age of 52 days the rats are initiated for mammary
carcinogenesis by a single subcutaneous injection of 50 mg/kg body
weight MNU (Serva, 3080 2) in 0,9% NaCl. 3 days after initiation,
a period necessary to recover from MNU toxicity, the rats of the
ad hoc experimental groups are given access to a semi-synthetic
DIET (INRA, France) comprising 65% maize starch, 5% cellulose, 3%
maize oil, 3% palmoil (not hydrogenated), 22% pure casein, 1,2%
vitamines, 0,8% minerals and 0,13% methionin supplemented with 5%
OF, inulin (Raftiline.RTM.) or pectin for 5 days. The next five
days a supplement of 10% of the same non digestible oligosaccharides
or dietary fibre is given. Finally, until the end of the experiment,
they will receive experimental diets supplemented with 15% OF, inulin
or pectin.
Parameters examined during the test are: body weight starting from
the adaptation period, palpation, measurement and description of
the mammary tumors once every 2 weeks, starting 6 weeks after MNU
injection, diet and water consumption and 24 h faecal excretion
once every 6 weeks starting 6 weeks after MNU injection.
After sacrificing the animals the following parameters are determined:
mammary tumor incidence, mammary tumor intensity and yield, mammary
tumor diameters, determination of types of mammary tumors (benign
or malignant), metastases incidence, incidence of tumors in other
organs and tissues. Statistical analysis of the results are done
according to relevant procedure.
EXAMPLE 3
Effect of Oligofructose on Initiation of Breast Carcinogenesis
The experimental protocol according to example 1.1 and 2.1 illustrates
the protective effects of inulin and oligofructose on the promotion
(phase II) and progression (phase III) of breast carcinogenesis.
In order to investigate the effect of oligofructose and inulin
on all phases of breast carcinogenesis, including phase I of initiation,
the following protocol is followed.
Sprague Dawley rats of 37 days of age are given a semi-synthetic
diet (INRA, France) supplemented with 5% OF for 5 days, followed
by 5 days of a 10% supplemented and subsequently 15% supplemented
diet. Only then the rats are initiated with MNU at a dose of 50
mg/kg body weight in 0,9% NaCl and fed the 15% supplemented diet
until the end of the experiment. The same parameters are being controlled
as in example 2.1.
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