NZ622493B2 - Method for producing dry extracts - Google Patents
Method for producing dry extracts Download PDFInfo
- Publication number
- NZ622493B2 NZ622493B2 NZ622493A NZ62249312A NZ622493B2 NZ 622493 B2 NZ622493 B2 NZ 622493B2 NZ 622493 A NZ622493 A NZ 622493A NZ 62249312 A NZ62249312 A NZ 62249312A NZ 622493 B2 NZ622493 B2 NZ 622493B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- dry plant
- nasal
- sinupret
- plant extract
- dry
- Prior art date
Links
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Classifications
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Abstract
The disclosure relates to a method for producing dry extracts of plants and to pharmaceutical preparations containing the same, more particularly phytopharmaceuticals, which contain at least one ethanolic/aqueous extract of a plant (drug), the plants being selected from the group consisting of: Rumicis herba; Verbena officinalis; Sambucus nigra; Primula veris; and Gentiana lutea and mixtures thereof. The disclosure further relates to a pharmaceutical for treating inflammatory and/or infectious diseases of the nose and throat area and/or the nasal sinuses, as well as the use thereof. cis herba; Verbena officinalis; Sambucus nigra; Primula veris; and Gentiana lutea and mixtures thereof. The disclosure further relates to a pharmaceutical for treating inflammatory and/or infectious diseases of the nose and throat area and/or the nasal sinuses, as well as the use thereof.
Description
METHOD FOR PRODUCING DRY EXTRACTS
Description
The invention relates to a method for ing dry
plant ts and to pharmaceutical preparations
containing the same, in particular
phytopharmaceuticals, which contain at least one
ethanolic/aqueous t of a plant (drug), wherein
the plants are selected from the group consisting of:
Rumex acetosa L., Rumex acetosella L., Rumex
folius L., Rumex patientia L., and Rumex crispus
L., (referred to hereinafter by the collective term
“Rumicis herba”); Verbena officinalis; Sambucus nigra;
Primula veris; and Gentiana lutea and mixtures thereof.
The invention also relates to a pharmaceutical for
treating matory and/or infectious diseases of the
nose and throat area and/or of the nasal sinuses, and
to a dietary supplement, and also to the use f.
The above medicinal plants are known as secretolytic
agents in the case of infections of the upper airways,
in particular in the case of sinusitis. Here, each
individual drug contributes its share to the unique
efficacy of the composition:
The root of Gentiana lutea (Gentian root) is generally
used for medicinal purposes. Various secoiridoid
glycosides with expectorant effect are found inter alia
among the ingredients.
The leaves and stem of the aforementioned Rumex types,
referred to hereinafter as “Rumicis herba” (Sorrel
herb), are generally used for medicinal purposes.
Flavonoids and various tanning agents are found herein
as ingredients with nflammatory effect, which
additionally positively support the body’s own
defences.
The collective term “Rumicis herba (Sorrel herb)” is to
be understood to mean a mixture of the following
Rumex acetosa L., synonym: Lapathum a SCOP,
synonym: Lapathum pratense LAM, synonym: Acetosa
pratensis MILLER; Rumex acetosella L, synonym: Rumex
infestus ; Rumex folius L., synonym
um obtusifolium MOENCH, synonym Lapathum
obtusantum MONTAD, synonym Rumex actus WALLR, synonym
Rumex silvestris WALLR;
Rumex patientia L., synonym Rumex olympicus ,
synonym Lapathum hortense MOENCH;
Rumex crispus L.;
Rumex thyrsiflorus FINGERH., synonym Acetosa
thyrsiflora FINGERH, synonym Rumex acetosa subsp.
auriculatus WALLR.
The leaves and stem of Verbena officinalis (Verbena
herb) are preferably used for medicinal purposes and
contain d glycosides, phenylethanoid glycosides
and flavonoids as primary ingredients, whereby
expectorant and antiviral effects are achieved.
The leaves of Sambucus nigra (Elder , Sambucus
nigra leaves) are typically used for medicinal
purposes, of which the contents contain various
flavonol ides and which contains sambunigrin, a
cyanogenic glycoside, as a primary ingredient, which
have an expectorant and antiviral effect (Grabovac, A.
and Ullmer, A., Österreichische Apotheker-
Verlagsgesellschaft m.b.H (Austrian Pharmacists’
Publishing House), 2003).
Flowers and sepals of Primula veris (Primula ,
Primula veris leaves) are used for medicinal purposes.
The ingredients comprise triterpene saponins and also
phenol glycosides such as primulaverin. They have an
expectorant effect and fight against viruses. The
ingredients act as a mild secretolytic agent and
expectorant in the case of treatment of respiratory
diseases.
The combination of the aforementioned medicinal plants
is known as a secretolytic agent under the trade name
Sinupret®, which is ered for the applicant and
has been available on the market for approximately 75
years. The medicinal plants used in Sinupret® are
selected, tested and further processed in a targeted
manner. The m quality of the pharmaceutical
attained hereby is ed by the manufacturer, the
company BIONORICA, by optimized propagation and
harvesting strategies and also by strict quality
control.
The composition forming the basis of Sinupret® is
preferably effective in the case of mation and
also infections of the throat, nose and ear , and
is particularly suitable for the treatment of acute and
chronic sinusitis and/or rhinosinusitis.
Both acute and c sinusitis are common. In three
of four cases, the sinusitis develops as a result of a
cold that s to the nasal sinuses and is
accompanied by inflammation of the mucous ne. The
airways reach from the main nasal cavity to the various
sinuses as far as the pulmonary alveoli. The nasal
sinuses include the frontal sinus, the ethmoid sinus,
the sphenoid sinus and the maxillary sinus. All of the
aforementioned bone cavities are lined with mucous
membrane and open out via narrow openings (the ostia)
into the main nasal cavity.
The surface of the airways is coated with a protective
mucus, to which dirt particles and pathogens, such as
viruses, bacteria or funguses, which infiltrate
together with the inhaled air, remain adhered. The
mucus ns antibodies, which attack the
infiltrating substances and make them harmless. So that
the foreign substances can be flushed out from the
body, the mucus is generally transported away with the
aid of the cilia of the ciliated epithelium in the
ion of the throat, where it can be swallowed. In
order to fend off infection-induced atory
diseases, the mucous membrane must have unhindered
protective and cleaning mechanisms. In order to
transport away the mucus charged with pathogens, the
unhindered function of the cilia is indispensible, said
cilia transporting the mucus further as a result of
undulating movements. With infection and with
inflammatory processes of the upper airways, the
function of the protective and cleaning mechanisms of
the mucous membrane is limited.
For example, viruses such as rhinoviruses, adenoviruses
or coronaviruses trigger inflammatory reactions of the
mucous membranes, whereby the mucous ne becomes
swollen and produces increased mucus. This initially
s in aqueous and then viscous mucus flow. Over
the course of inflammation of the nasal mucous
membrane, the ostia of the sinuses may swell and impair
or even prevent the discharge of the mucus. This leads
to a blockage in the sinuses associated with viscous
mucus, which leads to a deterioration of function or
loss of function of the cilia. This tely causes a
deterioration of the cleaning mechanism of the mucous
membrane.
Such a microenvironment promotes the rapid increase of
the ubiquitous microorganisms. Over a relatively long
period of time, these unfavourable conditions, such as
a swollen mucous ne and cilia conglutinated by
viscous mucus, this may lead to c tis,
resulting in permanent damage to the mucous membrane
and to the ciliated lium. Pathogens relevant to
WO 26830
the airways, which are also to be understood in
particular to include ENO-relevant germs, which settle
in the mucus, for e include lococcus
aureus, Staphylococcus epidermidis, Streptococcus
pyogenes, Streptococcus pneumoniae, Streptococcus
mutans or Haemophilus influenzae.
In the event that the upper airways become
conglutinated as a result of viscous mucus, the
ingredients of the used composition (Gentiana lutea :
Rumicis herba : a officinalis : Sambucus nigra :
Primula veris = 1:3:3:3:3) induce the formation of
fresh, thin mucus, whereby the above-described process
of expectoration and the transporting away and also
reduction of the inflammatory symptoms is achieved and
a healing process of the nasal mucous membrane is
ted. Sinupret® gently causes the re-establishment
of the self-cleaning power of the airways and
aneously develops a strong antimicrobial .
Sinupret® is characterized by its good compatibility,
composition established by BIONORICA and dosing, which
rarely cause side effects in patients, and, in
addition, no interactions with other pharmaceuticals
are known.
Dry plant extracts are described in general and dry
plant extracts formed from aqueous/ethanolic extracts
are known.
Dry plant extracts can be produced for example in large
amounts in accordance with the technical ng of
EP0753306.
However, there is a considerable need to provide a new
Sinupret® dry plant extract, which has an advantageous
improved effect of the plant combination.
The he Arzneimittelbuch (DAB or German
Pharmacopoeia) 2010 establishes minimum content s of
ients for drug quality, such that increased
efforts have to be made for constant and improved
quality. Extraction and drying methods specifically
tute a bottleneck for a sufficient quality of
harmaceuticals.
Based on this prior art, the object of the present
ion is therefore to provide an improved method
for producing a dry plant extract and also an improved
dry plant extract as such, which contains at least one
ethanolic/aqueous extraction step, n the plants
are selected from the group consisting of: Rumex
acetosa L., Rumex acetosella L, Rumex obtusifolius L.,
Rumex patientia L., and Rumex crispus L., red to
hereinafter and in the claims by the collective term
“Rumicis herba”); Verbena officinalis; Sambucus nigra;
Primula veris; and/or Gentiana lutea and mixtures
thereof.
Surprisingly, an improved dry plant extract could be
obtained by means of a twofold extraction process,
wherein, in a first step, an aqueous/ethanolic
extraction took place, and, in a second step, an
aqueous extraction took place.
Surprisingly, the content of advantageous active
ingredients in existing active ingredient e in
the respective overall extract can be enriched after
drying by means of this method according to the
invention, such that an improved pharmacological
efficacy of the obtained dry plant extract is achieved.
The invention therefore relates to a method for
producing dry plant extracts, said method comprising
the following steps:
a.) alcoholic/aqueous extraction of Rumicis
herba, a officinalis, us nigra,
Primula veris and Gentiana lutea,
b.) separation of the supernatant,
c.) second aqueous extraction of the residue from
a.),
d.) separation of the supernatant,
e.) ing of the obtained atants from
b.) and d.),
f.) drying of the supernatants and provision of
the dry plant extract.
The improvement according to the method lies in the
fact that a r efficacy is achieved compared to a
conventional simple aqueous-ethanolic extract (see the
examples). This is surprising since a linear
extrapolation would have been expected at best as a
result of a change of the solvent for extraction (for
example if aqueous/ethanolic extraction is carried out
exclusively or if aqueous extraction is carried out
exclusively).
In particular, the method according to the invention
allows an improved dosing, such that an increased
curative effect can be achieved with the same dose.
The invention therefore likewise relates to a dry plant
extract, which is obtained or is obtainable in
accordance with a method according to the invention
(hereinafter dry (plant) extract according to the
invention, Sinupret dry extract (TE)).
In a further preferred embodiment of the method
according to the invention, the ratio of na lutea
: s herba : Verbena officinalis : Sambucus nigra
: Primula veris is 1:3:3:3:3, in each cas e +/- 0.3 to
0.5 (for example 1 : 3.2 : 2.9 : 2.5 : 3.2 : 3).
WO 26830
Furthermore, it is preferable for a plant ng of
all plant (drugs) to be provided in a batch, wherein
the ratio of plant (drugs) is as mentioned above.
Furthermore, it is preferable for the presented plant
(drugs) to be cleaned and cut.
In a further preferred embodiment of the method
according to the invention, the aqueous/alcoholic
extraction agent in step a.) has a content 40 : 60
(v/v) to 60 : 40 (v/v), in particular 41 : 59 (v/v) or
50 : 50 (v/v) of water/alcohol. Ethanol is preferred,
however methanol and propanol or mixtures thereof are
also included. Furthermore, the use of 96 % ethanol is
preferred.
In accordance with the ion, the extraction in
steps a.) and c.) is also d out at 20 to 40 °C,
wherein the extraction is carried out in steps a.) and
c.) in 2 to 8 h.
Within the meaning of this invention, a “separation of
the supernatant” can be carried out continuously or
tinuously after extraction in accordance with the
invention by means of draining, decantation,
filtration, screening or a separation method known to a
person skilled in the art.
Furthermore, it is preferable for the drying ing
to step f.) to be carried out under vacuum at 30 to 60
°C, in particular at 40 to 50 °C, preferably in a
vacuum agitated dryer. The dry extract according to the
ion has a residual ethanol content of at most 0.5
Further suitable drying methods according to the
invention will be explained:
Dry plant extracts are produced conventionally, n
a plant material is extracted with the aid of a solvent
or solvent mixture, for example by means of maceration
or percolation, and, after tion of the extraction
residue, the ed fluid extract or the obtained
re is compressed until dry.
Conventional drying methods are sed in accordance
with the invention and include fluidized bed drying or
the compression to a thick or spissum extract and
subsequent vacuum band drying or tray drying of said
spissum extract.
Conventional methods for producing dry plant extracts
according to the invention via a fluid extract (or
liquid plant extract) or a re may likewise be
considered; wherein, after uent distillation of
the solvents, what is known as a spissum extract
(viscose t) is obtained, to which auxiliary
agents and/or additives, such as lactose,
polyvinylpyrrolidone, sucrose, silicon dioxide, etc.,
are often added. This moist, viscose mass is then
introduced into tray cabinets or dryers for desired dry
extract preparation.
A method that is very often used in dry extract
tion is what is known as the vacuum band drying
method. In this case, the spissum extract is brought to
dry extract preparation after preliminary drying via a
downdraft vaporizer.
A drying method by means of a fluidized bed dryer
requires temperatures between approximately 47 °C and
117 °C. The drying process in this method is carried
out under normal pressure conditions.
A gentle drying method for obtaining dry plant extracts
according to the invention is described in EP 0 753
306. In ance with the described method, the fluid
extract from the plant materials obtained in the
extraction is introduced in accordance with the method
according to the invention in a vacuum drying ,
preferably a vacuum ed dryer with a multibranched
stirrer extending through a cylindrical mixing
and drying chamber and having its own drive, and, where
necessary, provided with vapour filter, backwash
device, solvent condenser with aftercooler and
collection vessel, back-condenser and a process,
l and regulation unit, and optionally with
granulation s, and is dried in the dryer equipped
with a chopper extending over the overall depth of the
mixing and drying chamber and having blades rotating
through a comb-shaped stator at a supply and return
ature between 120 °C and 5 °C, an inner chamber
temperature between 10 °C and 80 °C, a filter
temperature from 15 °C to 55 °C, and a pressure between
0.5 1,000 mbar, and also a stirrer speed of rotation
from 0 and 10 rpm and a chopper speed of rotation
between 200 and 800 rpm. The vacuum drying systems used
according to EP 0 753 306 are sold for example by the
former companies Firmen Inox Glatt AG or Inox-Maurer AG
under the names “IUT” or “INOX”. Current manufacturers
and distributors e, for example, De Dietrich
s Systems GmbH, Mainz, Germany (Rosemund ®).
With this vacuum drying system, the fluid extract to be
dried is pumped from above into the mixing and drying
chamber in the batch method and is then subjected to a
A preferred vacuum drying system comprises the
following features, as are implemented for example in a
known IUT/INOX system (see above):
a.) A multi-branched stirrer extending through a
cylindrical mixing and drying chamber and having its
own drive and, depending on ements, vapour
filter, backwash device, solvent condenser with
ooler and collection vessel, back-condenser, a
process, control and regulation unit, and optionally
granulation nozzles.
b.) Furthermore, a chopper extending over the entire
depth of the drying and mixing chamber and having a
drive independent of the stirrer may be provided as
well as optionally a comb-like stator for increasing
the chopper effect.
C.) Furthermore, one or more nozzles may optionally be
provided in order to introduce the liquid plant extract
from a oir into the drying r, for example
as bed in WO2002073108.
The dry plant extracts obtained in this way are
processed further to form ceutical preparations.
The agent having an antimicrobial effect, containing
the dry plant t ing to the invention, can
thus advantageously be used in the treatment of
infections triggered by pathogens relevant to the
airways. The expectorant and anti-inflammatory effect
is supplemented by the additional antimicrobial effect.
An infection of the upper airways is thus confined or
even completely stopped in addition to the loosening of
the viscous mucus charged with pathogens, weakened by
killing and/or reduction of the proliferation of the
bacterial pathogens.
Due to the above-described invention, a patient
suffering from sinusitis and/or rhinosinusitis and/or
inflammation of the nasal sinuses for example, in
particular in the acute form in each case, is treated
in a gentle manner without the use of synthetic-
al ents.
The (pharmaceutical) composition of the present
invention having an antimicrobial effect is
particularly effective against pathogens relevant to
the airways, n it has demonstrated antimicrobial
efficacy specifically against gram-positive cocci, such
as Staphylococcus aureus, Staphylococcus aureus (MRSA),
Staphylococcus epidermidis, Streptococcus pyogenes,
Streptococcus niae and Streptococcus mutans,
against gram-negative bacilli, such as Haemophilus
influenzae, and against Enterobacteriaceae faecalis.
Efficacy t viruses is also achieved.
The galenic formulation of the antimicrobial agent can
be ed from the group consisting of: drops, juice,
syrup, tablets, dragées, capsules, retard formulations,
rectal or vaginal suppositories, ons, in
particular throat sprays and disinfection solutions;
ointments, emulsions, granulates, powders, nose sprays,
liquid or solid preparations for inhalation,
compresses, waddings, in particular wound and gum
dressings, tamponades, including for teeth, rinsing
solutions, in particular in combination with
logical and hyperosmolar concentrations of salts
or salt mixtures, preferably table salt, in particular
sea salt. Of course, the ation can contain
pharmaceutically conventional auxiliary agents.
The present invention therefore also relates to the use
or application of the antimicrobial agent according to
the invention containing a dry plant t according
to the invention for treatment of infections triggered
by pathogens relevant to the airways, and to the use
for production of a pharmaceutical, and also to a
ceutical as such.
In a further embodiment, the invention relates to a
pharmaceutical for use or application in the case of
sinusitis and/or inusitis and/or inflammation of
the nasal cavities, in particular in the acute form in
each case, in ular for treatment and prophylaxis
of sinusitis and/or rhinosinusitis and/or inflammation
of the nasal sinuses, in ular in the acute form
in each case.
Due to the ENO relevance of the tested pathogens, the
agent according to the invention is likewise
outstandingly suitable in any instance in which
bacteria can be fought ly and immediately locally
or lly.
The agent according to the invention can thus
preferably be used (in addition to the systemic
application) directly at the contaminated location, for
example in the form of a disinfection solution to be
biologically degraded 100 %, or of course also for
direct topical application to skin and mucous membranes
both in humans and in animals. ent forms of the
application are considered for this purpose. The
formulations are particularly le as solutions,
creams, ointments and emulsions in the dermatological
field of human and also veterinary medicine. Here, the
agent ing to the invention can be applied
directly to the diseased part of the skin and/or can be
used in the form of saturated compresses, waddings or
tamponades.
Of course, the application of the agent according to
the invention in the overall field of diseases of the
entire respiratory tract, in particular of the upper
airways, here preferably in the region of throat, nasal
and nasal sinus mucous membranes, is of ular
interest. Nasal rinsing, in particular in conjunction
with salts, for example in combination with a
physiological or hyperosmolar salt solution, is of
particular significance. In accordance with the
invention, a nasal spray containing the agent according
to the ion is also included.
The broad range of new application possibilities
reaches from tonsil paint solutions and ng
solutions in the case of pharyngeal infections to
powder inhalation preparations or nebulizer tion
preparations.
Further fields of application and indication comprise
wound and gum dressings, for example in the form of
cotton waddings or cotton yarn waddings, which are
ted with the agent according to the invention.
Ear rinses with solutions that contain the agent
according to the invention are also conceivable in the
case of infections of the ear canal.
The invention therefore likewise relates to a
pharmaceutical for use and ation of diseases of
the entire respiratory tract, in particular of the
upper airways, in particular in the region of the
throat, nasal and nasal sinus mucous membranes,
respiratory diseases, in particular mucoviscidosis
(cystic fibrosis), in particular the treatment and
prophylaxis thereof. Mucoviscidosis can particularly
advantageously be d, see Figures 7A and 7B.
A further preferred embodiment ns a dietary
supplement containing the agent according to the
invention, in particular in the form of a dietary
composition. Suitable foods or uffs, including
water, according to the invention are those as defined
(although not definitively) for example in regulation
(EC) No. 178/2002 of 28 January 2002, such as bakery
products and beverages and infant food preparations.
The dietary supplement according to the invention can
be mixed with a suitable physiologically compatible
r.
The pharmaceutical preparations according to the
invention can be produced in the form of dosage units.
This means that the ations may be present in the
form of individual parts, for e capsules and
ampoules, of which the active ingredient content of dry
plant extracts corresponds to a fraction or a multiple
of an individual dose. The dosage units may contain,
for example, 1, 2, 3 or 4 single doses or 1/2, 1/3 or
1/4 of a single dose. A single dose preferably contains
the amount of dry plant extract (active ingredient)
according to the invention that is administered in one
application and that usually corresponds to the entire
daily dose or one half, one third or one fourth of a
daily dose. A dosage of three times daily, preferably
in the form of a tablet, in particular in the morning,
afternoon and evening, possibly at meal times, is
preferred.
In a further red embodiment, the galenic
formulation of a m coated tablet can be selected,
as disclosed in EP1392337.
Non-toxic, inert pharmaceutically suitable carrier
substances are to be understood to include solid, semisolid
or liquid diluting agents, fillers and
formulation aids of any type, such as a) fillers and
diluting agents, for example starches, lactose, cane
sugar, glucose, mannite, dextrins, maltodextrin and
silicic acid, highly sed n dioxide, b)
binders, for e carboxymethyl cellulose, ose
powder, microcrystalline cellulose, alginates,
gelatines, polyvinylpyrrolidone, c) humectants, for
example glycerol, d) ing agents, for example
agar-agar, calcium carbonate and sodium carbonate, e)
dilution restrainers, for example paraffin, and f)
resorption accelerators, for example quaternary
ammonium compounds, g) wetting agents, for example
cetyl alcohol, glycerol earate, h) adsorption
agents, for example kaolin and bentonite, and i)
lubricants, for e talc, calcium stearate and
magnesium stearate, and solid polyethylene glycols or
mixtures of the substances listed under a) to i).
The tablets, dragées, capsules, pills and granulates
may be provided with the conventional coatings and
coverings, optionally containing opacity promoting
agents, for e such as, although not definitively,
hypromellose, rystalline cellulose, stearic acid,
titanium dioxide, and may also be composed such that
they release the active ingredient(s) only, or
preferably, in a certain part of the intestinal tract,
in a delayed manner where necessary, n polymer
substances and waxes for example may be used as
embedding matter.
Examples:
These examples are intended merely to n the
invention, without limiting the invention to these
Hereinafter, “dry extract (TE)” means the dry plant
extract produced according to the invention.
Example 1:
The ral activity of the dry extract according to
the invention was confirmed in a number of in vitro
tests.
In these tests, the general cell-damaging (cytotoxic)
effect of the dry extract according to the ion
compared to the known product et®
(alcoholic/aqueous) was initially tested. After
incubation of suitable cell lines (for example HeLa,
HEp-2) with different viruses over a period of one
hour, the infected cell lines were treated with
different concentrations, and the effect on the virus
proliferation was then measured.
The ral activity was determined quantitatively in
vitro via the detection of a cytopathogenic effect
(Adeno5 Virus), in a plaque ion assay (FluA,
HRV14, RSV) and in virus-specific enzyme immunoassays
(ELISA; Adeno5, RSV).
In the event of detection of a cytopathogenic ,
the virus-sensitive cells grown confluently were
infected with a defined virus solution (M.O.I.,
multiplicity of infection). After tion for one
hour, the virus inoculum was drawn off and the infected
cell layers were washed. The physiolo gical substance
concentrations were then added. The respective test
batches were cultivated until a 70-90 % cytopathogenic
effect (CPE), which presents itself as a destroyed cell
region, was observed under microscope in the untreated
virus controls. The area of the destroyed cell region
was defined as 100 % infection. Comparatively, the cell
areas of the respective test batches were evaluated so
that inhibiting effects of the substances to be
analysed can be shown as an inhibition percentage (%
inhibition).
In the case of the plaque reduction assay, the virussensitive
cells grown confluently were infected with a
defined virus solution (M.O.I., multiplicity of
ion). After incubation for one hour, the virus
inoculum was drawn off and the infected cell layers
were washed. The physiological substance concentrations
and also a solid medium component (agarose or
methylcellulose) were then added, ed by r
incubation. The infection area was delimited by the
solid component in the superimposed medium, such that a
series of ed cells (“plaque”) was produced. The
respective test batches were cultivated until the set
plaque number .) was observed under microscope in
the untreated virus controls. By fixing and dyeing the
2012/066212
cell layer, the virus plaque can be made visible as
light rings in the dark-coloured cell layers. The
plaque number was ined with the aid of image
processing systems. The plaque number of the untreated
control was defined as 100 % ion. By contrast,
the plaque number of the respective test batches was
evaluated such that inhibitory effects of the test
substance can be presented as an inhibition percentage
(% inhibition).
The virus tion was analysed by ELISA. Test strips
with antibodies against the specific viruses bind the
viruses found in the cell e supernatant of the
infected cell lines. In order to make the reaction
visible, a pathogen-specific detection antibody
labelled with peroxidase was introduced. After addition
of a ate/chromogen and also of hydrogen peroxide
and tetramethylbenzidine, a colour reaction takes
place. The intensity of the colouration was determined
photometrically and was proportional to the content of
virus antigen. The virus tion in the case of
infected and treated cells was analysed after infection
of virus-sensitive cells grown confluently with a
defined virus solution (M.O.I., multiplicity of
infection). After incubation for one hour, the virus
um was drawn off and the infected cell layers
were washed. The physiological substance concentrations
were then added. The respective test batches were
ated until a 70-90 % cytopathogenic effect (CPE)
was observed under microscope in the ted virus
controls. The newly synthesized viruses were located in
this stage in the cell culture supernatant. The
photometrically determined extinction values of the
untreated controls were defined as 100 % infection. By
way of comparison, the extinction values of the
respective test batches were evaluated, such that
inhibitory effects of the test substances can be
presented as an inhibition percentage (% inhibition).
A significant inhibition of the virus proliferation,
that is to say a reduction of the virus load, was
demonstrated in all tests (see Figure 1).
The dry extract according to the ion inhibits the
proliferation of human (FluA) and pig (pFluA) nza
viruses (flu s). Concentrations of 124.8 μg of
Sinupret/ml (FluA) and of 43.4 μg of Sinupret/ml
(pFluA) are sufficient in order to inhibit (reduce) the
virus (load) by 50 %. The et dry extract
according to the invention demonstrated a lower EC50
against virus strains HRV 14, Adeno5 and RSV, and
therefore accordingly a greater efficacy compared to
Sinupret® alcoholic/aqueous (see Figures 1 and 2).
Table 0: antiviral effect
Virus strain Sinupret Sinupret
alcoholic/aqueous dry extract
EC50 [μg/ml] EC50 [μg/ml]
HRV 14 73.1 50.5
Adeno 5 66.4 13.8
RSV 20.7 10.4
Example 2:
The anti-inflammatory activity of Sinupret could be
med in the animal model. For example, carrageeninduced
paw oedema in rats (Male Wistar Han rats, 220-
230g) was selected as the test model. In this model, it
was possible to examine the anti-inflammatory effect of
test substances by measuring their inhibitory effect on
the paw oedema or tis caused by carrageen. The
following were used as reference substances: (RS)[4-
(2-methylpropyl)phenyl]propanoic acid (Ibuprofen®) and
2-[1-(4-chlorobenzoyl)methoxymethyl-1H-indol
yl] acetic acid (indomethacin, “indo”). In the present
tests, groups of 10 rats in each case were treated
either with Ibuprofen®, indomethacin, Sinupret® dry
extract (SIN TR) or Sinupret ® drug mixture (SIN, as
commercially available) and were injected one hour
later with carrageen. The inhibition of the oedema
formation by the test and reference substances was
determined at various moments in time after carrageen
ion, wherein the paw volume of animals treated
only with carrageen was used as a l (vehicle =
blank control). The s of these tests are
presented in Tables 1 and 2 and in Figures 3 to 6 and
will be explained hereinafter.
Figure 3: In the een model, it was found that the
dry extract ing to the invention inhibits the paw
oedema induced by carrageen after just 30 minutes
following carrageen injection, and more specifically
inhibits said paw oedema to a greater extent than the
Sinupret drug mixture, where as Ibuprofen® still did
not show any anti-inflammatory effect at this early
moment in time. Even one and two hours after oedema
induction, the anti-inflammatory effect of the dry
extract according to the invention was still stronger
than that of Ibuprofen® and et® drug mixture
(SIN).
Table 1 shows the effect of Sinupret® (SIN) in the case
of een-induced pleuritis on the basis of the
inflammatory markers (PGE2, LTB4, TNF alpha, IL1 beta)
with measurement after 4 h.
Rats (10 per group) were each treated with 100 mg/kg or
500 mg/kg of SIN and by way of comparison with 5 mg/kg
of indomethacin and blank and were injected 1 hour
later with carrageen.
“Inflammatory cells” ates to PMN
(polymorphonuclear neutrophils)
accumulation/infiltration.
Statistics: average +/- SEM, n = 10, ** p< 0.01;
.001 vs vehicle (blank) (Tukey Test), p< 0.05 is
statistically significant.
A comparative illustration by means of graphs is
provided in Figure 4.
Table 2 shows the effect of Sinupret® dry extract (SIN
TR) in the case of carrageen -induced pleuritis on the
basis of the inflammatory markers (PGE2, LTB4, TNF
alpha, IL1 beta) with measurement after 4 h.
Rats (10 per group) were each treated with 100 mg/kg or
500 mg/kg of SIN TR and by way of comparison with 5
mg/kg of indomethacin and blank and were injected one
hour later with carrageen.
“Inflammatory cells” correlated with PMN
(polymorphonuclear neutrophils)
accumulation/infiltration.
Statistics: average +/- SEM, n = 10, ** p< 0.01;
***p<0.001 vs vehicle (blank) (Tukey Test), p<0.05 is
statistically icant.
A comparative illustration by means of graphs is
provided in Figure 4.
Figure 5 shows the effect of et® drug mixture
(SIN) and of Sinupret dry extract (SIN TR) on the
expression of COX-2 protein in the rat lung.
Rats (10 per group) were each treated with 100 mg/kg or
500 mg/kg of SIN or SIN TR and by way of comparison
2012/066212
with 5 mg/kg of indomethacin and blank and were
injected 1 hour later with carrageen. A western blot
was carried out in each case with 30 μg of protein from
rat lung (homogenized) on 10 % SDS polyacrylamide gel
and COX-2 was ed.
Figure 6 shows the effect of Sinupret® drug mixture
(SIN) and Sinupret dry extract (SIN TR) on cytokines.
Rats (10 per group) were each treated with 100 mg/kg or
500 mg/kg of SIN or SIN TR and by way of comparison
with 5 mg/kg of indomethacin and blank and were
injected 1 hour later with carrageen. The inflammatory
markers IL1 beta and TNF alpha were ined 4 h
after carrageen injection.
Statistics: average +/- SEM, n = 10, ** p< 0.01;
***p<0.001 vs e (blank) (Tukey Test), p<0.05 is
tically significant.
Conclusion:
The Sinupret dry extract (SIN TR) according to the
invention demonstrated, at least for PGE2, a
ularly advantageous significant inhibition of
PGE2 formation (30 %; p<0.01; Figure 4C, Table 2)
compared to Sinupret® drug mixture (SIN). Furthermore,
the Sinupret dry extract (SIN TR) according to the
invention is more effective at lower dosage compared to
the known Sinupret® drug mixture (SIN).
Example 3:
The following example shows that the dry extract
according to the invention, with topical use, activates
chloride secretion, most likely via the tion of
CFTR. In addition, the dry extract according to the
invention stimulates the ciliary beat frequency.
Material: cell culture: human ial epithelial
cells (HBE) were acquired from Lonza (Walkersville, MD)
and were expanded with bronchial epithelial cell growth
medium (BEGM) from Lonza (Walkersville, MD). 250 mg of
dry extract according to the invention were dissolved
in 1 ml of 50 % ethanol and were treated by ultrasound
at 35 kHz for 30 s with subsequent centrifugation
at 3,000 g for 10 minutes at room temperature. The
supernatant was suctioned off. Amiloride (Sigma,
St.Louis, MO) was dissolved in distilled deionized
water and diluted 1,000 times. Forskolin (Kaliokemm,
EMD, San Diego, CA) was dissolved in DMSO and diluted
1,000 times.
An Ussing chamber (Physiology ments San Diego,
CA, USA) was used, containing Transwell inserts
(Corning Life Sciences) with entation at 37
degrees and a monolayer with a voltage terminal of 0
volts (VCC 600) (Physiology Cal Instruments San Diego,
CA, USA) after setting of the forward resistance.
Transwell filters were placed in the solution at 37
degrees and the solution was infused continuously with
95 % oxygen to 5 % CO2. The transepithelial resistance
(RT) was ed using a computer program (Physiology
Instruments San Diego, CA, USA) at 640 ms, bipolar 10
mV potential via the monolayer measured in accordance
with Ohms Law. By definition, a positive result is an
anion ion or cation absorption. The experiments
were repeated at least 3 times in HBE cell cultures.
Used olyte solution (in mM): 120 NaCl, 25 NaHCO3,
3.3 , 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2 and 10
glucose.
The cilial beat ncy (CBF) measurements were
carried out in accordance with a method according to
Woodworth et al (Woodworth, BA, Zhang S, Tamashiro E,
Zinc increases ciliary beat frequency in a m
dependent manner, Am J Rhinol Allergy 24: 6-10, 2010).
The images were created by Leica Microsystems, Inc.,
Bannockburn, IL with a 63x lens (model A 602f-2, high-
speed monogram digital video camera, Basler AG,
Ahrensburg, Germany). The dry t according to the
invention was examined for transepithelial electrolyte
transport. The dry extract was applied in increasing
concentration amounts to the basolateral surface of HBE
cells in the Ussing r, before subsequent addition
of amiloride and forskolin.
In Figures 7a and 7b, the dry extract according to the
invention demonstrates a change of the transepithelial
short-circuit current (ISC) (7A) after addition of
amiloride and forskolin, such that a ependent
rise in the cilial beat frequency (CBF) measurements
can be observed (7B), which is accompanied by a
de ion secretion.
These results prove the advantageous use of the dry
extract according to the invention, for example by
means of a nasal spray, since ed mucociliary
nce (MCC) can be achieved.
Conclusion: The dry extract according to the invention
is suitable for the treatment of respiratory diseases,
in particular for the treatment of mucoviscidosis
(cystic fibrosis).
Example 4:
The nflammatory effect of the Sinupret dry
extract according to the invention and Sinupret drops
(alcoholic/aqueous (“Sinupret OD”)) was examined as
follows.
Carrageen-induced paw oedema (see above): The test
animals (n=8/group) were weighed in the fasted state
and the basal volume of the rear paw was measured. The
animals were fed the test substances (10 mL/kg of body
mass) (Sinupret dry extract (TE): 5 mg/kg, equivalent
to the amount of drug mixture in 1 mL of drops/kg; 50
mg TE/kg corresponding to 1 times the human equivalent
dose (1 x HED); Sinupret drops ret OD): 1 mL
OD/kg, corresponding to 1 times the human equivalent
dose (1 x HED); 2.5 mL OD/kg, equivalent to the amount
of drug mixture in 50 mg TE/kg; thacin: 20 mg/kg
as a positive control; 10 % v/v ethanol as vehicle
control). After 60 s, the animals were provoked
by a subcutaneous injection of carrageen (0.1 mL of a 1
% w/v on in physiological table salt solution)
into the left rear paw (in a plantar manner). Paw
volumes were ined in all animals by means of
plethysmography before (-1 h) and after carrageen
injection (study 1: +1 h (Figure 8), study 2: +15 min,
+30 min, +1 h (Figure 9)). The inhibition percentage of
paw swelling vs. e control was calculated
individually for each test animal.
The results are shown by way of comparison in Figure 8
(inhibition of inflammation after 1 hour).
Figure 8: A: et TE and Sinupret OD were each
administered in a dose equivalent to 1 times the human
equivalent dose. Sinupret TE has a quicker and stronger
anti-inflammatory effect than Sinupret OD. The
inhibition of inflammation by Sinupret TE, not by
Sinupret OD, is comparable after 1 h with the
inhibitory effect of the known antiphlogistic
indomethacin.
B, C: The dose administered here of Sinupret TE and
Sinupret OD is comparable based on the respective
amount of drug mixture (DM) used for production (B:
23.6 mg/kg, C: 223 mg/kg). Sinupret TE has a quicker
and stronger anti-inflammatory effect compared to
Sinupret OD. The inhibition of inflammation produced by
et TE, not by Sinupret OD, is comparable after 1
h with the inhibitory effect produced by the known
antiphlogistic indomethacin.
The results are shown by way of ison in Figure 9
(inhibition of inflammation after +15 min, +30 min, +1
h).
Figure 9: the administered dose of Sinupret TE and
Sinupret OD is comparable based on the respective
amount of drug mixture used for production (23.6 mg of
drug mixture/kg). Sinupret TE is characterized by an
r and stronger onset of action compared to
et OD. Sinupret OD only inhibits mation
from 30 min. Sinupret TE inhibits inflammation
atively strongly and quickly over the entire
period of time tested in a manner comparable to the
known antiphlogistic indomethacin.
Example 5:
The following example describes specific marker
compounds from the multi-substance mixture.
Implementation:
All samples were extracted immediately after the
individual extraction steps, were filtered and were
analysed by mass spectrometry in a concentration of 600
mg/L (in 30 vol. % MeOH). Methylparaben was used as an
internal standard. The samples were processed twice and
analysed twice. The ing parameters and devices
were used:
MS: 5600 Triple ToF (ABSciex); HPLC: Agilent 1290
re: Analyst TF 1.5.1, MultiQuant 2.1.1,
MarkerView 1.2.1
Stationary phase: Zorbax RRHD Eclipse Plus C18, 2.1 x
50 mm, 1.8 μm
LC method:
Step Time Flow rate Eluent Eluent
(min) (μL/min) A B
0 0.00 600 95.0 5.0
1 1.00 600 95.0 5.0
2 6.00 600 69.0 31.0
3 10.00 600 0.0 100.0
4 11.00 600 0.0 100.0
11.10 600 95.0 5.0
6 13.00 600 95.0 5.0
A = 0.1 % of formic acid in H2O; B = acetonitrile
MS method:
Scan Type negative
TOF MS
Duration 10.997
mins
Cycle Time 0.2750
secs
GS1 (Spray Gas): 70.00
GS2 (Turbo Gas): 55.00
CUR (Curtain Gas): 25.00
TEM (GS 2): 500.0
ISVF (Ion Spray Voltage 4500.0
Floating):
CAD (Collision Gas): 6
TOF Masses (Da): 130-2000
Accumulation Time (sec): 0.25
Time Bins to sum: 4
DP: -100.0
CE: -10.0
Table 3: selective enrichment and depletion of
teristic ingredients
m/z = 399.2 m/z = 540.3 m/z = 279.2
RT = 0.5 min RT = 8.4 min RT = 9.6 min
Lab batch 1st extraction (59 vol.% EtOH) 8 % 2879 % 737.2 g (±11.7g)
2nd tion (water) 224 % 61 % 0.0 g
total extract 100 % 100 % 622.3 g (±6.0 g)
Production 1st extraction (59 vol.% EtOH) 16.5 % 896 % 745.5 g (±19.6 g)
2nd extraction (water) 67 % 104 % 0.0 g
batch total extract 100 % 100 % 119.9 g (±12.2 g)
In so far as quantified via reference standards, the
quantities of ingredients are specified as absolute
content in [g]. ise, the content of ingredients
is specified in relation [%] to the respective total
extract (after step f.)). The data originate from a lab
batch A1 and a production batch P1. The signals were
detected with negative ionisation.
Table 4: ingredients with typical ment in the
first extraction step with increase by subsequent
aqueous tion.
m/z = 401.1 m/z = 463.1 m/z = 623.2
RT = 2.6 min RT = 3.7 min RT = 3.9 min
Lab batch 1st extraction (59 vol.% EtOH) 500.7 g (±6.4 g) 122.0 g (±2.3 g) 575.6 g (±6.4 g)
2nd tion (water) 152.3 g (±3.6 g) 11.9 g (±0.4 g) 91.2 g (±0.1 g)
total extract 655.4 (±3.3 g) 134.2 g (±0.8 g) 657.6 g (±17.4 g)
Production 1st extraction (59 vol.% EtOH 704.5 g (±6.1 g) 127.6 (±3.0 g) 527.0 g (±12.5 g)
2nd extraction (water) 70.0 g (±0.03 g) 9.0 g (±0.02 g) 42.3 g (±1.7 g)
batch total extract 788.2 g (±11.8 g) 138.1 g (±0.7 g) 571.1 g (±20.2 g)
In so far as quantified by reference standards, the
quantities of ingredients are specified as absolute
content in [g]. Otherwise, the content of ingredients
is specified in relation [%] to the respective total
extract (after step f.)). The data originate from the
lab batch A1 and from the production batch P1. The
s were detected with negative ionization.
Patent
Claims (16)
- Claims 1. A method for producing dry plant extracts, said method comprising the ing steps: 5 a.) alcoholic/aqueous extraction of Rumicis herba, Verbena officinalis, Sambucus nigra, Primula veris and Gentiana lutea, b.) separation of the supernatant, c.) aqueous extraction of the e, 10 d.) separation of the atant, e.) combining of the obtained supernatants from b.) and d.), f.) drying of the supernatants and provision of the dry plant extract.
- 2. The method according to Claim 1, wherein the ratio in step a.) of Gentiana lutea : Rumicis herba : Verbena officinalis : Sambucus nigra: Primula veris is 1:3:3:3:3, in each case +/- 0.3 to 0.5.
- 3. The method according to Claim 1 or 2, wherein, in step a.), the aqueous/alcoholic extraction agent is 40 : 60 (v/v) to 60 : 40 (v/v), in particular 41 : 59 (v/v), or 50 : 50 (v/v).
- 4. The method according to one of the preceding claims, characterized in that ethanol, in particular 96 % ethanol, is used as alcohol. 30
- 5. The method according to one of the preceding claims, characterized in that the plants in a.) are provided together in a batch.
- 6. The method ing to one of the preceding 35 claims, characterized in that the tion in steps a.) and c.) is carried out at 20 to 40 °C.
- 7. The method according to one of the preceding claims, characterized in that the extraction in steps a.) and c.) is carried out in 2 to 8 h. 5
- 8. The method according to one of the preceding claims, characterized in that the drying process according to step f.) is carried out under vacuum at 30 to 60 °C, in particular at 40 to 50 °C. 10
- 9. The method according to one of the preceding claims, characterized in that the drying process according to step f.) is carried out in a vacuum ed dryer. 15
- 10. Dry plant extracts obtained in accordance with one of Claims 1 to 9.
- 11. Pharmaceutical preparations containing dry plant extracts according to Claim 10, where applicable 20 ing suitable r substances, in particular in the form of dragées, tablets, filmcoated tablets, powder, capsules or liquid dilutions, in particular drops, juices or syrups, ointments, emulsions, granulates, powders, nasal 25 , liquid or solid preparations for inhalation, compresses, wound and gum dressings, tamponades, tonsil paint solutions, gargling solutions or rinsing solutions. 30
- 12. A pharmaceutical containing a dry plant extract according to Claim 10 or a ceutical preparation according to Claim 11.
- 13. A pharmaceutical containing a dry plant extract 35 according to one of Claims 10 to 12 for use or application as an cterial, antiviral or anti-inflammatory agent.
- 14. A pharmaceutical containing a dry plant extract according to one of Claims 10 to 12 for use or application in the case of sinusitis and/or rhinosinusitis and/or mation of the nasal 5 sinuses, in particular in acute form in each case, in particular for the treatment and prophylaxis of tis and/or inusitis and/or inflammation of the nasal sinuses, in particular in acute form in each case.
- 15. A pharmaceutical containing a dry plant extract according to one of Claims 10 to 12 for use and application with diseases of the entire atory tract, in particular of the upper 15 airways, in particular in the region of the throat, nose and nasal sinus mucous membranes, and respiratory diseases, in particular mucoviscidosis (cystic fibrosis), in particular for the treatment and prophylaxis of diseases of the entire 20 respiratory tract, in particular of the upper airways, in particular in the region of the throat, nose and nasal sinus mucous membranes, and atory diseases, in particular mucoviscidosis (cystic fibrosis).
- 16. A dietary supplement containing a dry plant extract according to Claim 10. Inhibition of virus eration (human and pig flu viruses) WO 26830
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11178206.6 | 2011-08-19 | ||
EP11178206A EP2559438A1 (en) | 2011-08-19 | 2011-08-19 | Method for manufacturing dry extracts |
EP11193734.8 | 2011-12-15 | ||
EP11193734 | 2011-12-15 | ||
EP12170125 | 2012-05-30 | ||
EP12170125.4 | 2012-05-30 | ||
PCT/EP2012/066212 WO2013026830A1 (en) | 2011-08-19 | 2012-08-20 | Method for producing dry extracts |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ622493A NZ622493A (en) | 2016-06-24 |
NZ622493B2 true NZ622493B2 (en) | 2016-09-27 |
Family
ID=
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