Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
lOS042~
This invention relates to a pharmaceutical preparation
including sapogenin from roots of the l~elleborus species and a
pharmaceutically acceptable carrier.
The roots and rootstocks of Helleborus species contain
a mixture of saponins the principal sapogenin of which has the
structure of a spirost-5,25~27)-diene-1~, 3~ -triol ~formula I)
, 3
CH~ ; ~` ~ CH ( I )
El ", .
H10
According to Helv. Chim. Acta 54, 1707 (1971), pulverized
rhizomes (rhizomes are underground, more or less thickened root-
ing stems, which can be clearly distinguished from roots by the
presence of scale-like lower leaves and by their structure) and ¦ -
roots of Helleborus species are coarsely pulverized, taken up in
water and left to autoferment for 22 days in order to obtain the
above sapogenin. The residue is subsequently filtered and
extracted with aqueous alcohol. The residue of the alcoholic
extract is chromatographed on silica gel. The sapogenin frac-
tions are recrystallized from methanol-acetone. The pure sapo-
i , ,.
;~ yenin (m.p. 236-240C) is finally obtained by preparatory thin-
layer chromatography in the system diisopropyl ether ethanol
; (92:8).
It has been found that this principal genin of the sapo- F
~;~ genin mixture - which is contained in the roots and rhizomes of
Helleborus species - can be obtained in a simpler and faster way
: if the roots and rhizomes of Helleborus species or drugs or ~-
extracts obtained therefrom are treated with an enzyme or enzyme .
- 30
mixture, the important active components of which are cellulase
(3.2.1.4), hemicellulase or ~-glucosidase (3.2.1.21). Accordiny
- A~
105U427
~o J.-E. Courtois alld Mme. Bui Khac Diep ~Annales pharmaceuti~ues
francaise 23, 1965, no. 9-10, pp.533-459) hemmicellulase is a
mixture of
~-galactosidase (3.2.1.22)
g-mannanase (3.2.1.25)
and
~-mannosidase (3.2.1.25)
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The treatment with an enzyme is carried out in the usual
manner. The temperature is suitablY kept between 20 and 50~C.
A temperature between 30 and ~QC i9 particular]y favourable.
In the above process, the comminuted roots and rhizomes
of ~elleborus species can be used directl~ or an extract obtained
in the usual manner or a drug obtained from Helleborus species,
treated and pulverized in the usual manner, can be used. The
treatment can be carried out in solution, in suspension or in
the form of a mash. Thus, for example, the enzyme can be added
to the autolyzate of roots and rhizomes of Helleborus species.
The autolysis or autofermentation can be effected by comminuting
parts of fresh plants and, if necessary, after adding water allow-
ing the material thus obtained to stand for a lengthy period
(e.g., 2 to 25 days) at a temperature of 18 to 60C, while stirr-
ing occasionally. Parts of dried plants can also be mixed with
water and subjected to autofermentation as long as their ferment-
ative activity is maintained. The enzyme can be added at the
start of the autofermentation process or even during said process.
However, it is also possible first to produce an
extract in a known manner from the roots and rhizomes or from a
drug produced therefrom and treat it with the enzyme, if required,
after the usual preliminary purification. For this purpose, the
roots and rhyzomes or the drugs are-extracted with alcohol or
., .". ".
~ alcohol-water mixtures containing a maximum of 50~ water. The
- extract thus obtained is subse~uently shaken with an organic
solvent such as an aromatic hydrocarbon, halohydrocarbon or a
mixture of a halohydrocarbon and alcohol such as for example,
chloroform-ethanol. If mixtures of a halohydrocarbon and alcohol
are used, then the ratio of the mixtures is preferably 2:1. It
is favourable to pre~purify the organic extract thus obtained.
This type of preliminary purification is carried out in
- 2
:
~OS042'7
the usual manner. However, it may~also be carried out by chroma-
tography on a silica geI, the silica gel being a synthetically
produced, highIy porous a~orphous silica, in the form of hard
particles having a granular size of 0.15 to 10 mm. A granular
size of 0.15 to 0.30 mm is particularl~ favourable. The water
content of this silica can be up to 10~. The specific surface
area can he up to 650 sq m per gram. It usually is approximately
4000 sq m per gram. The powder density can be up to 650 g per
litre. A powder density of 450 to 500 g per litre is favourable.
Suitable eluants for the silica-gel chromatography of the
i organic extract of the roots and rhizomes of ~elleborus species
include aliphatic halohydrocarbons, mixtures of aliphatic halo-
hydrocarbons with aliphatic alcohols, esters o~ aliphatic acids
with aliphatic alcohols, ester-alcohol mixtures, ester-alcohol-
water mixtures, benzene, halogen benzenes, alkyl benzenes, alkyl
benzene-alcohol mixtures, ester-pyridine mixtures, halohydrocar-
bon-pyridine mixtures, halohydrocarbon-alcohol-pyr dine mixtures,
ester-pyridine-water mixtures, mixtures of benzene, halogen
benzenes and alkyl benzenes with pyridine, aliphatic ketones,
ketone-water mixtures, ketone-benzene mixtures, ketone-benzene-
acetic acid mixtures, etc. Of course, other mixtures with the
` above components are also possible. The optimum mixing propor-
tion of the components must always be determined in a separate
preliminary test.
The aliphatic li~uids mentioned above are the usual lower
liquid agents used as solvents. In this connection, halogen means
fluorine, chlorine or bromine, preferably fluorine or chlorine.
By alkyl benzenes are meant the liquid agents with lower alkyl
radicals. Examples are toluene, ethyl henzene, xylenes.
The pre-purified saponin fraction, which is completely
free from solvents, is then incubated in water with the enzyme.
.
~ .
~05(~4'~
Toluene can be a~ed (usually in small amounts) to the incubate
as a protection against bacterial decomposition.
For the process, commercial cellulase, hemicellulase
and ~-glucosidase preparations can be used. Likewise, it is also
possible to use preparations of the enzymes produced from fungi,
microorganisms (Trichoderma viride), protozoa, bacterial insects,
plants, intervertebrates such as snails (edible snails~ and worms
by means of the processes known for this purpose [see The Enzymes
1, part 2 (1951) page 729 ff; Ullmann's Enzyklopadie der technis-
chen Chemie, 3rd edition (1956) page 391 ff].
The enzymes such as, for example, the cellulase, are
obtained by precipitating the culture medium or the aqueous
Mycelium extract of the fungi with alcohol, acetone or salt. The
crude enzyme can then be further purified, for example, on alu-
minium oxide. L
The preparations to be used must be as fresh as possible t
without being stored for a lengthy period. Stored preparations
must be kept cool and dry. Enzyme preparations or enzyme con-
centrates produced from fungi, for example, Aspergillus species
such as Aspergillus niger, ~spergillus flavus, Aspergillus
oryzae, Aspergillus fumigatus, Aspergillus nidulans are parti-
- culary suitable. Preparations or enzyme concentrates produced
from Aspergillus niger are particularly favourable.
-; The activity of the enzymes must be determined by pre-
liminary tests and the optimum conditions for the reaction and k
quantitative relations must also be determined. Enzyme concen-
trates which, for example, consist primarily of cellulase, hemi-
cellulase or e-glucosidase or of mixtures of such enzymes are
particularly favourable. Frequently, such preparations also con- r
30 tain related enzymes such as pectinase (3.2.1.15), amylase
(3.2.1.1), acid protoase (3.4.23.6), xylanase (3.2.1.32), cello-
biase (3.2.1.21), glucoamylase (3.2.1.3), endopeptidase (3.4.21.16),
5., '
lipase (3.1.1.3), and pectin
- 4 -
~ . . . . . . .
1050427
exopolygalacturonase. It is also favourable if the enzvme pre-
parations according to the invention also contain enæymes having
a macerating effect (for example, ~ectin glvcosidase~. Examples
are highIy purified products obtained from Aspergillus species
and containing primarily cellulase as hemicellulase as well as
pectinase, amylase and acid protease (cellulosin preparations).
The enzymatic reaction isusuallycompleted after two days. If
the activity of the enzymes is high, then the reaction is comple-
ted earlier, but, if the activity of the enzymes is low, then the
treatment is likely to require a much longer period.
The enzyme can be mixed with the substrate either as such
or in aqueous solution.
The optimum pH value for the treatment with the enzymes
is from approximately 4.5 to 4.7. It is favourable to stir the
incubate or to ~eep it in motion in some other manner.
Acid proteases contain cellulosin preparations.
The amount of the enzyme added depends on the activity of
the enzyme and on the substrate used. The enzyme can be added,
for example, in substantial excess. For example, if a crude
saponin fraction which is pre-puri~ied as described hereinbefore
is used, then an addition of 5 to 100~ of the enzyme preparation
concerned is required depending on the degree of purity, which is
determined by thin-layer chromato~raphy. For example, if the
drug or the roots and rhizomes of Helleborus species are reacted
directly, then an amount from 1 to 10~, relative to the drug or
to the roots and rhizomes should be sufficient.
The crude sapogenin obtained after the enzyme treatment
can be further processed by means of the usual process describedin
-~ ~heliterature~ For example, the process described in Helv. Chim.
~,~
Acta 54, page 1707 (chromatography on SiO2 with a granular size
of 0.05 to 0.2 mm) is suitable for this purpose.
_ 5
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1~)504Z~
Howe~er! a purification (~or example, by chromatography)
of the crude sapoqenin with the aid of a silica gel having a
grain size of 0~05 to 0,2 mm is particularly favourable. Lower
halogenated hydrocarbons such as dichloromethane and chloroform,
to which a lower aliphatic alcohol such as methanol or ethanol
can be added, are suitable as eluants, the amount of alcohol
increasing from 1%.
The sapogenin thus obtained can be recrystallized once
more from propanol/water.
10By means of the above process it is possible to isolate
the Helleborus sapogenin in substantially larger amounts than by
way of the preparatory thin-layer chromatography.
The known Helleborus species, for example, Hellevorus
foetidus L., Helleborus multifidus Vis., Helleborus niger L.,
Helleborus adorus Waldst. et Kit., Helleborus orientalis Lam.,
Helleboris purpurascens Waldst. et Kit., Helleborus viridis, are
suitable starting materials.
Nothir~was previously known concerning the physiological
effect of the principal sapogenin from the roots and rhizomes of
Helleborus species. It has now been discovered that the princi-
pal genin produces from Helleborus saponin has a curative effect
on ulcers. Moreover, a muscle-relaxing effect and an effect
influencing the central nervous system have also been detected.
The ulcer-protective effect is evident, for example, from the
table hereafter:
Daily Dose Ulcer-Protective Effect
:
50 mg per kg 58
100 mg per kg 78
200 mg per kg 80
30These tests were carried out on the indomethacin-induced
- ulcer of rats using the method of WilheImi which was modified by
- 6
.. . .. . . : . . :
~504'~7
Jahn and Adrian [see Arzneimittelfor~chung 19 (1969) page 45 ff].
The determination was carried out in the ~ollowing manner:
Albino rats of the strain SIV 50 (S. Ivanovas, Kissle~g/
Allgau) which had an initial weight of 250 to 300 g were kept in
wire cages (Ebeco, type 3, double width) in rooms, the temperature
of which was kept between 20 and 22C, on a standard diet
(Altromin ~). After withholding food for 48 hours, the animals
are given 20 mg of indometh~cin in 1.5% tragacanth (l ml of
tragacanth solution per 100 g of rat) per kg of rat, applied
intragastrally. One hour thereafter the animals are given the
test substance orally. The rats continue to stay sober (water
ad libitum).
; 24 hours after administration if indomet~a-cin, the
animals are killed by means of CO2. The stomachs are resected,
opened along the large curvature and rinsed under running water.
The ulcerative changes appear as dark punctiform or ~-
striated spots on the mucosa. The evaluation is carried Ollt
macroscopically, using the method of MUNCHOW [Arzneimittelfor-
schung 4 (1954) page 341].
Therefore, the present invention provides a pharmaceu-
tical preparation containing compound I as the active ingredient,
if required, mixed with other pharmacologically or pharmaceuti-
cally active substances. The medicines can be produce using
`; conventional pharmaceutical adjuvant substances.
~ For example, substances which are recommended and/or
,:
listed in the following references from the literature as adju-
vant substances for pharmacy, cosmetics and related fields are
.; .
;~ suitable as this kind of fillers and adjuvent substances:
Ullmann's Encyklop~die der technischen Chemie, Vol. 4 (1953),
- 30 page 1 to 39; Journal of Pharmaceutical Sciences, Vol. 52 ~1963),
- page 918 ff, H`.v. Czetsch-Lindenwald, Hilfsstoffe f~r Pharmazie
,~
; - 7
,~ .
.
... ~ . ,
105042~
und angrenzende Gebiete; Pharm. Ind ~ No. 2, 1961, page 72 ff;
Dr. H.P. Fiedler, Lexikon der Hilfsstoffe f~r Pharmazie,
Kosmetik and angrenzende Gebiete~ Cantor RG., ~ulendorf i. W~rtt.
1971.
Examples are gelatin, natural sugar such as sucrose or
lactose, lecithin, pectin, starch (for example, cornstarch),
tylose, talc lycopodium, silica ~for example, colloidal silica),
cellulose, cellulose derivatives (for example, cellulose ethers
in which the cellulose-hydroxy groups are partially etherified
with lower saturated aliphatic alcohols and/or lower saturated
aliphatic oxy-alcohols, for example, methyl-oxy-propyl cellulose),
stearates, magnesium and calcium salts of fatty acids, containing
12 to 22 carbon atoms, particularly of the saturated ones (for
example, stearates), emulsifiers, oils and fats, particularly
vegetable oils (for example, peanut oil, caster oil, olive oil,
sesame oil, cottonseed oil, corn oll, mono-, di- and trigl~cer-
ides of saturated fatty acids C12H24O2 to Cl8H36o2
mixtures), pharmaceutically compatible monohydric or polyhydric
alcohols and polyglycols such as polyethylene glycols as well as
derivatives thereof, esters o~ aliphatic saturated or unsaturated
fatty acids (2 to 22 carbon atoms, particularly 10 to 18 carbon
atoms) with monohydric aliphatic alcohols (1 to 20 carbon atoms)
or polyhydric alcohols such as glycols, glycerin, diethylene
glycol, petnaerythrite, sorbite, mannite, etc., which, if required,
can also be etherified, benzyl benzoate, dioxolanes, glycerin
formals, glycol furoles, dimethyl acetamide, lactamides, lactates,
ethyl carbonates, silicones (particularly medium-viscosity
dimethyl polysiloxanes) and the like.
For example, water or physiologically compatible organic
. 30 solvents are suitable for the production of solutions such as,
for example, ethanol, 1,2-propylene glycol, polyglycols and their
~;
, ' . .
- 8
.
. ., .~ . . - . :
10504Z7
derivatives, dimethyl sulphoxide, fatty alcohols, triglycerides,
partial esters of glycerin, para~fin and the like.
For the production of the preparations, Known and conven-
tional dissolving intermediaries can be used~ Suitable dissolving
intermediaries are polyoxy-ethylated fats, polyoxy-ethylated
oleotriglycerides, linolized oleotriglycerides~
Examples of oleotriglycerides are olive oil, peanut oil,
castor oil, sesame oil, cottonseed oil, corn oil [see Dr. H.P.
Fiedler "Lexikon der Hilfsstoffe f~r Pharmazie, Kosmetik und
- 10 angrenzende Gebiete" (1971), page 191 to 195].
In this connection, "poly-oxylated" means that the sub-
stances concerned contain polyoxy-ethylene chains the degree of
polymerization of which is usually between 2 and 40 and particu-
larly between 10 and 20. Such polyoxy-ethylated substances can
be obtained, for example, by reaction of the corresponding
glycerides with ethylene oxide (for example, ~0 moles of ethylene
oxide per mole of glyceride)~
;,,
Moreover, the addition of preservatives, stabilizers,
buf~er substances, taste corrigents, antioxidants and complexing
agents (for example, ethylenediaminetetraacetic acid) and the like
is possible. If required, the pH may be adjusted, for the sta-
bilization of the active substance molecule, to approximately
3 to 7 with physiologically compatible acids or buffers. In
general, a pH as neutral as possible to weakly acid (to pH 5)
is preferred.
~ or example, sodium metabisulphite, ascorbic acid, gallic
acid, gallic alkyl ester, butyl hydroxy anisole, nor-dihydro
guaiaric acid, tocopherols as ~ell as ~ocopherols + synergists
, .:,.
(substances which bind heavy metals by complexing action, for
example, lecithin, ascorbic acid, phosphoric acid) are used as
antioxidants~ The addition of the synergists substantially
,.:'
.
, - g _
:
~OS04~7 ~ ~
increases the antioxygenating action of the tocopherols.
Suitable preservatives include sorbic acid, p-hydroxy
benzoic esters (for example, lo~er alk~1 ester~, benzoic acid~
sodium benzoate, trichloro isobutyl alcohol, phenol, cresol,
benzethonium chloride and formalin derivatives.
The pharmacological and galenic handling of the compounds
according to the invention is carried out according to conven-
tional standard methods. For example, the active ingredient and
adjuvant substances or fillers are properly mixed by stirring or
homogenizing (~or example, by means of colloid mills, ~all mills),
usuall~ at a temperature of 20 to 80C, prefera~ly from 20 to
50C.
The medicines can be applied orally, parenterally, rect-
ally, vaginally, perlingually or locally. The addition of other
active medical substances is also possible.
The compound shows a good antiulcerogenic effect on the
indomethacin ulcer of rats and on the histamine-induced ulcer of
guinnea pigs.The antiulcerogenic effect is comparable to the
effect of the known drug biogastrone.
The testing was carried out on histamine-induced ulcers
of guineapigs by means of the method of Eagleton and Watt (Peptic
Ulcer, Pfeiffer, Munksgaard, Copenhagen, printed in Denmar~ of
Aarhus Stiftsbogtrykkerie A/S ~rhus ISBN 8716003225). In this
test, the test substances are given orally to male guinae pigs
' (weighing 300 to 400 g each) after withholding food for 24 hours
and drinking water for 12 hours. One hour after administering
i the substance, the animals are injected with histamine diphos-
;~ phate (dose: 5 mg/kg). The histamine diphosphate is applied
intraperitoneally. Three hours after the histamine injection the
animals are killed by means of CO2. The stomachs are resected
and cut open along the small curvature and inverted over the
,
- 10 ~
10504Z7
testing finger, The ulcers axe evaluated according to M~nchow
(Arzneimittelforschung 4, 341, 1954~
The computed control index must be o~ the order of 5 to
15.
The lowest effective dose in the animal test mentioned
hereinbefore is, for example, 50 mg per kg orally. For example,
50 to 500 mg per kg orally is suitable as a general dose range
for the above effect (animal test as above).
The compound according to the invention is indicated for
Ulcus ventriculi, Ulcus duodeni, gastritis and duodenitis.
The pharmaceutical preparations usually contain between
1 and 50% of the active component according to the invention.
They can be given, for example, in the form of tablets,
capsules, dragees or in a liquid form. Oily or alcoholic solu-
tions and emulsions are suitable as li~uid forms of application.
Tablets containing between 50 and 500 mg of the active substance
or solutions containing between 0.5 and 10% thereof are preferred
forms of application. For oral medicines, the single dose of the
active component according to the invention can be between 50 and
500 mg. For example, three times daily 1 to 5 tablets containing
from 50 to 500 mg of active substance can be recommended.
In mice, the acute toxicity of the compound according to
the invention [expressed by the LD 50 mg/kg; method according to
Miller and Tainter; Proc. Soc. Exper. Biol. a. Med. 57 (1944)
Z61] is above 6000 mg per kg for oral application. ~-
The invention will now be described in greater detail by
means of the following examples, which describe pharmaceutical
preparations containing the principal sapogenir. of roots and
rhizomes of the Helleborus species, and a process for extracting
such sapogenin.
:
;
~5V4Z7
Example 1 (Tablets)
_
50 g of the principal sapogenin of Helleborus are mixed
with 50 g of highIy dispersed silica, 10 g of cornstarch and
120 g of lactose. The powder is granulated with a solution of
5 g of methyl-oxy-propyl cellulose in approxi~atel~ 160 ml of a
30% ethanol. The dried granulate is mixed with 21 g of corn-
starch, 18 g of talc, 125 g of microcrystalline cellulase and
1 g of magnesium stearate, whereupon the mixture is pelletized
in a known manner. One tablet, which weighs 400 mg, contains
50 mg of sapogenin. `
Example 2 (Gelatin Capsules)
500 g of dimethyl pol~siloxane having a viscosity of
360 to 380 cp at 20C (silicone oil AK 350) are triturated with
20 g of highly dispersed silica. The triturate is processed on
a water bath with 130 g of isopropyl myristinate, 150 g of a
melted mixture of mono-, di- and tri-ylycerides of the saturated
' 12 242 C18H36O2, a commercial product known as
hard fat (Deutsches ~rzneibuch 7th edition) and 200 g of prin-
cipal sapogenin of ~elleborus. A homo~eneous paste is thus
obtained.
The paste is put into gelatin capsules in single doses
. . .
of 500 mg. One gelatin capsule contains 100 mg of sapogenin.
Example 3 (Coated Tablets, Dragees)
Tablets or dragee cores, which are produced corresponding
to Example 1, can be provided in a known manner with a film coat-
ing, which is soluble in the stomach or in the small intestine,
~` and which consists of suitable cellulose derivatives or other
polymeric film forming agents and suitable additives such as
`~ plasticizers, dyes, fla~oring substances, etc., or with a suitable
dragee coating.
, ,~
- 12
~,
.
1051~4Z7
Example 4 (Alcoholic Solution)
7.5 g o~ principal sapogenin of Helleborus are dissolved
with 0.5 g of cherry aroma in a 96% ethanol and the solution is
diluted with ethanol to lO0 ml. For the peroral application,
1 ml of the solution is mixed with approximately 50 to 100 ml
of liquid. l ml of solution contains 75 mg of sapogenin.
Example 5
100 kg of roots and rhizomes of Helleborus viridis are
comminuted, degreased with petroleum ether and then thoroughly
extracted with an 80% aqueous ethanol. The residue of the
ethanol extract is taken up in water, and the solution is shaken
with chloroform/ethanol (2:1). The chloroform-soluble components
are chromatographed with chloroform/ethanol (9:1) on a silica-
gel column, and the fractions are examined by means of thin-layer
chromatography:
adsorbent; silica gel for thin-la~er chromatography (silica gel
G of the firm ~f Merck sRD)
solvent: chloroform/methanol/water (35:25:10)
detection: anise aldehyde/sulphuric acid/acetic acid tl:l:100).
The fraction containin~ primarily a substance which can
be colored brown, has an average Rf value (approximately O.S0)
and is localized between des-glucohellebrin and helle~rin in the
chromatogram.
The residue of the combined fractions ~2621 g) is diss-
olved in 2.4 litres of methanol and 2.4 litres of warer with
reflux. Upon adding 24 litres of hot water, 4.8 litres of sol-
vent are distilled from the solution.
Upon cooling, the solution is treated with 240 g of a
; ~ commercial cellulase (R~hm and Haas) and lO0 ml of toluene, and
stored at 40C while shaking occasionally. The reaction is
completed after approximately 48 hours. The precipitate is
.
. ~
_ 13
~504'~7
filtered with suction, washed with hot water and dried (1100 g).
The dried precipitate is dissolved in methanol and
; dichloromethane, and the solution is chromatographed with
dichIoromethane~methanol (increasing methanol concentration) on
silica gel for the column chromatography with a granular size
of 0.2 to 0.5 mm.
The sapogenin fraction (736 g) is dissolved in 5 litres
of propanol-(l), and the solution is treated with 30 litres of
water. After 24 hours, the separated crystals are washed with
propanol/water and dried.
C27H405
m.p. = 238 to 240C
; [a] D = - 86.93C (c=l.l; pyridine)
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