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BACKGROUND OF THE INVENTION
Steroidal sapogenins are known to be useful animal growth stimulators. For
example, McKeen et al. (U.S. Pat. No. 3,144,337), the disclosure of which
is incorporated herein by reference, disclose animal feeds containing
selected steroidal sapogenins that elicit a substantially greater growth
increase and feed efficiency response in animals, as compared to feeds
which do not contain sapogenins. The beneficial effect of steroidal
sapogenins as animal growth stimulants is further discussed by W.H. Hale
et al., 1961, Proc. Soc. Exp Biol Med. 106, P. 486-489, the disclosure of
which is incorporated herein by reference. McKeen et al. also report that
when steroidal sapogenins are concurrently administered with estrogenic
substances, such as diethylstilbestrol (DES), estradiol, and the like, the
animals exhibit growth responses well beyond those which would be expected
from the administration of estrogenic substances alone. However, because
substances such as DES are suspected to be carcinogens, and are not always
approved as animal growth stimulants, there is an increasing need for
materials which will allow efficient incorporation of steroidal sapogenins
into animal feeds.
Sapogenins are the acid hydrolysis products of saponins, which are
amphiphilic, steroidal or triterpenoid glycoside compounds. Crude extracts
containing saponins can be obtained from a wide variety of plant
materials. The steroidal saponins, and the steroidal sapogenins derived
therefrom, possess the growth stimulating characteristics discussed
hereinabove, are characterized by a glycosidic moiety, such as a 5- or
6-membered sugar moiety or a chain of sugar moieties, attached to a
steroid moiety by means of an ether linkage at the carbon-3 (C-3)
position. The structure of such a steroid moiety is exemplified by the
structures of the sapogenins smilagenin, hecogenin and tigogenin which are
schematically illustrated in FIG. 1. These structures are representations
of sapogenins which would result from hydrolysis of the ether linkage
between a glycoside moiety and a steroid moiety of a saponin molecule
corresponding to each of these sapogenins. Saponins can be hydrolyzed in
this way enzymatically or by exposure to acid.
Steroidal sapogenins are also useful as precursors for pharmaceutical
steroids, such as cortisone and various hormones. See e.g. Wall et al., J.
Biol. Chem. 198, 533-543; and Printy et al., U.S. Pat. No. 3,169,959.
For steroidal sapogenins to find wide use, an efficient, cost effective way
of producing them must be made available. The difficulty of economically
obtaining large quantities of steroidal sapogenins has apparently limited
their use as animal growth stimulators, and as steroidal precursors.
Although crude aqueous plant extracts containing substantial amounts of
steroidal saponins are easily obtained and are commercially available, it
has been difficult to isolate the desired steroidal sapogenins from these
sources. In addition to saponins, these crude extracts usually contain
substantial amounts of plant fats and non-saponin carbohydrates. When
these crude extracts are hydrolyzed with concentrated mineral acids, a
virtually unusable, sticky, gumlike reaction product generally results. It
is believed that the fats and free carbohydrates are responsible for
forming this material. Once such a reaction product has formed, it is very
difficult to efficiently extract the sapogenins therefrom.
Historically, saponins have been separated from fats and non-saponin
carbohydrates by extracting crude aqueous plant extracts with butanol.
This method is tedious, and can result in an oily gum which is difficult
to handle in an efficient manner and which is not a suitable substrate for
subsequent acid hydrolysis to produce sapogenins.
Another method of extracting saponins from plant material has been
disclosed involving extraction with hot aqueous ethanol or isopropanol
(85-95%). See Wall et al., 1952, J. Biol. Chem. 198, 533-543. The fats are
subsequently extracted with benzene, and the saponins are then extracted
from the aqueous alcohol solution with butanol. Following these steps, the
saponins can be acid-hydrolyzed to form sapogenins. The crude product,
however, still requires treatment with hot methanol potassium hydroxide to
remove phenolic and acidic substances.
Although Printy et al. (U.S. Pat. No. 3,169,959) have disclosed a method
for deriving smilagenin from a crude Agave extract without separating the
saponin from the fats and non-saponin carbohydrates prior to hydrolysis,
the yield was very low. Smilagenin is considered to be a particularly
desireable sapogenin, both for use as a pharmaceutical precursor, and as
an animal growth stimulator. Printy et al. extracted pulverized plant
material with warm water. The extract was clarified and then hydrolyzed
with mineral acid. The crude smilagenin precipitate was isolated and
further extracted with heptane to give a 1% yield of smilagenin.
Given the usefulness of steroidal sapogenins as animal growth stimulants
and steroid precursors, it will be appreciated that a need exists for a
readily obtainable source of steroidal saponins which is substantially
free of fats and non-saponin carbohydrates, and which is, therefore,
easily hydrolyzed to form steroidal sapogenins. It will be further
appreciated that a need exists for an efficient method of producing such
an intermediate.
BRIEF DESCRIPTION OF THE INVENTION
The present invention provides a composition of matter comprising a
crystalline saponin-containing complex, wherein the crystalline complex is
derived from a steroidal saponin-containing plant material. The
crystalline complex is preferably derived from saponin-containing plant
material of a species selected from the group of plants consisting of
Agave, Yucca, Dioscorea, Quillaja, Medicago and Cyamopsis, more preferably
from a species of Yucca, even more preferably from a species of Yucca
selected from the group consisting of Yucca gloriosa, Yucca mohavensis,
Yucca schidigera, Yucca brevifolia, Yucca augustifolia, Yucca glauca,
Yucca elata, Yucca whipplei, Yucca bacatta, Yucca carnerosana, Yucca
schottii and Yucca aloifolia, most preferably from Yucca schidigera.
The crystalline saponin-containing complex of the present invention yields
sapogenins in a yield of at least about 5% by weight when hydrolyzed with
aqueous mineral acid. Hydrolysis of a preferred embodiment of the
crystalline complex will yield a sapogenin, preferably smilagenin, in a
yield of at least about 5% by weight. The crystalline complex of the
present invention is substantially free of plant fats and non-saponin
carbohydrates. Because of its relatively pure form, the present
composition has utility as an intermediate in the production of
sapogenins, preferably smilagenin, which are useful for a variety of
purposes.
The present invention further provides a method of forming a crystalline
saponin-containing complex comprising forming a mixture of about 1-20% by
weight steroidal saponin-containing extract solids in a fluid comprising a
fat solvent, methanol and water in a volume ratio of about
0.1-20:0.1-20:1.0, so that the saponins, fats and non-saponin
carbohydrates are dissolved in said fluid. The mixture is allowed to stand
at ambient temperature and pressure for a period containing complex while
the fats and non-saponin carbohydrates remain substantially in solution.
In a preferred embodiment, the fat solvent is acetone. Preferably, the
present invention further provides the method above wherein evaporation of
the fluid in step (b) is constrained such that evaporation of the fluid
does not proceed at a rate or to an extent which causes the precipitation
of the fats and non-saponin carbohydrates which are dissolved in the
fluid. Preferably, evaporation of the fluid is constrained such that no
more than 50% of the original fluid volume evaporates prior to isolation
of the crystalline complex.
The present method further comprises isolating the crystalline
saponin-containing complex and then hydrolyzing it to convert at least
about 20% by weight of the saponins of the crystalline complex into
sapogenins. In a preferred embodiment, the crystalline complex is
hydrolyzed with an amount of aqueous mineral acid effective to convert at
least about 50% by weight of the saponins of the crystalline complex into
sapogenins.
The present invention further provides a crystalline saponin-containing
composition derived from steroidal saponin-containing plant material which
is prepared by a process comprising the following steps. A mixture is
formed consisting essentially of one volume of an aqueous plant extract
containing about 40% by weight of steroidal saponin-containing plant
extract solids, and four volumes of a solvent consisting essentially of
methanol, acetone and water in a volume ratio of about 1:1:1. The
saponins, fats and non-saponin carbohydrates derived from the plant
material are dissolved in the solution of methanol, acetone and water.
Preferably, any insoluble material is separated by filtration means. The
filtrate is placed in a container effective to constrain evaporation at
ambient temperature and pressure such that evaporation of the filtrate
does not proceed at a rate or to an extent which causes the precipitation
of the fats and non-saponin carbohydrates which are in solution. The
solution is then allowed to stand at ambient temperature and pressure for
a period of time effective to form saponin-containing crystals while
retaining the fats and non-saponin carbohydrates substantially in
solution.
The present invention provides a crystalline intermediate which is useful
for the production of sapogenins which are in turn useful as steroid
percursors and as animal growth stimulants. In the past, the isolation of
saponins has been difficult and time consuming. Given the relative
difficulty of obtaining a saponin fraction which is a suitable
intermediate for the hydrolytic production of sapogenins, the availability
of a crystalline saponin-containing complex which can be obtained by a
straightforward process will greatly improve the cost effectiveness of
sapogenin production. It is believed that the present invention provides
the first example of a crystalline saponin-containing complex which is
substantially free of fats and non-saponin carbohydrates and, therefore,
is a suitable intermediate for the hydrolytic production of sapogenins.
The crystalline complex isolated in Example V below has been shown by x-ray
diffraction analysis to have regularly repeating interatomic bond lengths
and bond angles which indicate that the complex does indeed have a
crystalline structure. This is confirmed by data from studies of the
crystalline complex using nuclear magnetic resonance (NMR) and infrared
(IR) spectroscopy as discussed hereinbelow.
In the context of the present invention, the term "substantially free of
fats and non-saponin carbohydrates" means that the crystals contain no
significant amount of fats and non-saponin carbohydrates either as
integral constituents or as impurities or contaminants. In the context of
the present invention a fat solvent is defined as a fluid in which fats
are highly soluble.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic illustration of the molecular structure of three
steroidal sapogenins, smilagenin, hecogenin and tigogenin. These
sapogenins are the glycoside-hydrolyzed aglucone moieties of the
corresponding steroidal saponins. Steroidal saponins would have glycosidic
groups attached by means of an ether linkage at the C-3 carbon atom. The
C-3 carbon atom is numbered in accord with standard steroidal
nomenclature.
FIG. 2 is a schematic illustration of the steroidal nucleus of the class of
sapogenins which are the glucose-hydrolyzed aglucone moieties of the
corresponding steroidal saponins. Steroidal saponins would have
saccharidic groups attached to the C-3 carbon atom. The rings are
designated A-F, and the ring carbon atoms are numbered in accord with
standard steroidal nomenclature.
FIG. 3 is a reproduction of an infrared (IR) spectrum obtained for a sample
of the crystalline saponin-containing complex of the present invention
isolated in Example V below.
FIG. 4 is a reproduction of a .sup.1 H-nuclear magnetic resonance (NMR)
spectrum of a sample of the crystalline saponin-containing complex of the
present invention isolated in Example V below.
FIG. 5 is a .sup.13 C-nuclear magnetic resonance (NMR) spectrum of a sample
of the crystalline saponin-containing complex of the present invention
isolated in Example V below.
FIG. 6 is a reproduction of an x-ray diffraction pattern of a sample of the
crystalline saponin-containing complex of the present invention isolated
in Example V below.
DETAILED DESCRIPTION OF THE INVENTION
Saponins and Sapogenins
Saponins are amphiphilic steroidal or triterpenoid glycoside compounds
which are widely found constituents of a variety of different plant
species. Saponins typically include glycosidic moieties which may comprise
one or more sugar moieties such as pentose, hexose, or the like, which are
linked to a steroid or triterpenoid group called a sapogenin. Saponins and
sapogenins are described in The Merck Index (9th ed., Windholz et al.,
Editors, Merck & Co., Inc., Rahway, N.J., 1976) at page 1084, the
disclosure of which is incorporated by reference herein.
There are two types of saponins: (a) glycosides of triterpenoid alcohols;
and (b) glycosides of a particular steroid structure comprising a
spiroketal side chain. Both types are soluble in water, methanol and
ethanol but insoluble in ether. Aglucones of saponins, called sapogenins,
are prepared by acid or enzymatic hydrolysis. Without attached sugar
moieties, sapogenins have the solubility characteristics of other steroids
and triterpenoids.
Steroidal sapogenins, which are the aglucone moieties of steroidal
saponins, can be produced by hydrolyzing the bond between the glycosidic
moiety and the steroidal sapogenin moiety of a steroidal saponin molecule.
This bond is typically an ether linkage which is easily hydrolyzed by a
number of methods which are well known in the art. As mentioned above, the
hydrolysis may be catalyzed by enzymes or by acid. Acidcatalyzed
hydrolysis is generally preferable. The most preferred method of
hydrolysis is via aqueous mineral acids, such as hydrochloric acid (HCl),
sulfuric acid (H.sub.2 SO.sub.4), nitric acid (HNO.sub.3), and the like.
The spiroketal steroid nucleus of the steroidal saponins is schematically
illustrated in FIG. 2. Rings E and F contain the same basic carbon
skeleton as the common animal steroids but lack the extra carbon atoms
found in most plant sterols. Glycosylation is generally at C-3. [A variety
of different monosaccharides such as glucose, galactose, xylose, and the
like, can be present at C-3, as can oligosaccharides made up of a variety
of different monosaccharides. Uronic acids can also be present.]
Many saponins found in plant extracts have been characterized. They include
digitonin, dioscin, gitonin, filiferine (A+B), Agave saponin E, agavoside
C, yuccoside, protoyuccoside, sarsasaponin, smilonin, kammonin, and the
like. Some of these saponins are listed in Table I below along with
respective results of research regarding their reported melting points and
elemental analysis.
TABLE I
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Melting Range
Elemental Analysis
(.degree.C.)
(% C,H)
______________________________________
Filiferine A 292 Not Reported
Filiferine B 319-308 Not Reported
Agave saponin E
304-308 Not Reported
Agavoside C 275 Not Reported
Yuccoside 276-278 Not Reported
Protoyuccoside
183-184 Not Reported
Sarsasaponin 241-245 C, 59.8; H, 8.0
Smilonin 235-237 C, 54.9; H, 7.7
Kammonin 310-315 C, 52.2; H, 7.3
______________________________________
The glycosidic moieties of saponin molecules are generally soluble in polar
solvents such as water, methanol, and the like, whereas the steroid
moieties of steroidal saponins are generally more soluble in nonpolar
solvents.
Although Applicants do not wish to be held to a theory of action with
regard to how the crystalline saponin-containing complex is formed,
Applicants believe that the methanol solvent portion of the methanol-water
solution associates with the steroidal saponins in such a way in that the
saponins are free to form hydrophobic bonds and to orient to one another
in such a way that a repeating crystalline complex structure is formed.
Applicants believe that when saponins are hydrated by water in an aqueous
solvent, the water prevents the steroid moieties of the saponin molecules
from interacting in such a way that hydrophobic bonds can be formed. When
methanol replaces water as the solvent in direct association with the
steroid moieties, and perhaps the saponin molecule generally, it is
believed that this association frees the steroid moieties of the saponins
to associate and to form hydrophobic bonds. These hydrophobic interactions
act to orient the saponin molecules in such a way that a crystalline
complex of saponin molecules is able to form.
Saponins are known to act as surfactants. Aqueous extracts of the Yucca
plant contain significant quantities of saponins. This extract has been
used as a non-photosensitive emulsifying agent by film manufacturers, and
as a surfactant to increase the rate and amount of moisture addition to
grain prior to dry rolling or steam flaking grain. Yucca extract is also
used to form the stable foam or "head" associated with draft root beer and
can also be used as a flavor and/or coloring agent. Although sapogenins
are believed to be of greater value than crude saponins, Yucca extract has
been used to improve the feed efficiency of feed fed to cattle and sheep
and has also been used to reduce odor caused by the excrement of swine and
poultry raised in confined systems.
Steroidal sapogenins have been identified as precursors for the synthesis
of cortisone and useful pharmaceutical hormones. Wall et al., 1952, Jour.
Biol. Chem. 198, 533-543. McKeen et al. (U.S. Pat. No. 3,144,337), the
disclosure of which is incorporated by reference herein, disclose the use
of steroidal sapogenins to increase the weight of gain for domestic
animals such as poultry, lambs, cattle, swine and the like, when fed feed
containing certain selected steroidal sapogenins. Such feeds elicit a
substantially greater growth increase and feed efficiency response in
animals than when feeds without sapogenins are used. McKeen et al. report
that there is a general improvement in the quality of the meat produced
from the animals which are treated in this manner. They further disclose
that the sapogenins employed are non-toxic in nature and preferably
selected from a group consisting of smilagenin, sarsasapogenin and
hecogenin.
Saponins can be found in a wide variety of plants. For example, they may be
found in plant material used for human foods such as soybeans, kidney
beans, mung beans, chick peas, asparagus, sugar beets, yams, peanuts, oats
and spinach. A review of saponins found in foods has been published by D.
Oakenfull (Food Chemistry, 1981, 6, 19-40) the disclosure of which is
incorporated herein by reference. Steroidal saponins are most common in
the plant families Liliaceae, Amaryllidaceae and Dioscoraceae. Plants
containing steroidal saponins include species of Agave, Yucca, Dioscorea,
Quillaja, Medicaqo, Cyamoosis. Preferred sources of steroidal saponins
include Agave americana, Agave variegata, Agave lecheguilla, Dioscorea
floribunda, Cyamoosis tetragonolobus, Medicago sativa, Yucca gloriosa,
Yucca mohavensis, Yucca schidigera, Yucca brevifolia, Yucca augustifolia,
Yucca glauca, Yucca elata, Yucca whipplei, Yucca bacatta, Yucca
carnerosana, Yucca schottii and Yucca aloifolia.
The composition of the present invention can be derived from any
steroidal-saponin-containing plant species. Preferred embodiments of the
composition of the present invention are derived from Agave and Yucca
species, preferably species of the Yucca plant, more preferably Yucca
schidigera, Yucca brevifolia, Yucca augustifolia, Yucca glauca, Yucca
elata, Yucca whipplei, Yucca bacatta, Yucca aloifolia, Yucca mohavensis or
Yucca gloriosa, most preferably Yucca schidigera.
The composition of the present invention may be derived from cells or cell
cultures having the genetic capability of providing the necessary cellular
mechanisms to produce steroidal saponins. These cells or cell cultures may
be of any derivation, microbial, plant, animal, or the like, so long as
they are capable of producing steroidal saponins. It is intended that
plants, plant materials and plant species, as they are defined herein,
will include cells or cell cultures having genetic material derived from
or fashioned after genetic material of plants.
Saponin-Containing Agueous Extract Solids
Saponin-containing aqueous plant extract solids are the materials remaining
when liquid plant extracts are completely dried, as in Example II below.
The saponin-containing aqueous extract solids used in the present
invention may be a finely divided powder or they may be suspended in a
fluid mixture.
Saponins can be extracted from plant materials in a variety of ways which
are well known in the art. Saponins are soluble in many fluids including
water, methanol, ethanol, butanol, isopropanol and the like. A simple
water extraction of saponin-containing plant material will yield an
aqueous saponin-containing extract. A crude saponin extract may also be
produced using methanol to extract plant materials previously extracted
with acetone or diethyl ether to remove lipids and pigments. Other methods
which can be used include extraction with a 4:1 ethanol-water solvent,
followed by subsequent defatting of the extract with benzene, and transfer
of the saponins to a butanol phase to substantially free the saponins of
proteins and sugars. Hot aqueous extractants can also be used.
Another method used primarily for isolating steroidal saponins involves
extraction with hot aqueous ethanol or isopropanol (75 to 95% by weight
alcohol). The aqueous alcohol extraction fluid is filtered and
concentrated and the fat-soluble material is removed by mixing the
extraction fluid with benzene. The saponins are subsequently extracted
from the aqueous phase with butanol.
For the purpose of preparing the compositions of the present invention, and
for use in the present method, a simple aqueous extract is preferred, but
any method of extracting saponins from the bulk of the plant material can
be used. In the preferred method, the plant material is pulverized or
otherwise fragmented. A preferred source of saponin-containing extract is
Yucca trees. The trees are cut up into logs and the logs, which contain
considerable moisture, are converted into chips. The chips are then soaked
in water and pressed to drive out aqueous saponin-containing fluids. The
aqueous extracts are collected and concentrated, as by evaporating the
water in an open air holding vat. A bacteriostat or bactericide, such as
copper sulfate or sodium benzoate or the like, is generally used to limit
bacterial growth in the extract while it is concentrated. Typically, the
saponin-containing plant extract is concentrated until it has a solids
content of about 20-60% solids by weight.
Saponin-containing aqueous Yucca extracts are commercially available from
Extract Distributors Processing (EDP) Inc., Porterville, Calif. This
product is available as a 40% solids extract of Yucca. Applicant believes
that this product is derived from Yucca schidigera.
Fat Solvents
The term "fat solvent" as used herein is intended to include any liquid
with which fat will associate when the liquid is mixed with water and
methanol. This solvent is preferably miscible or at least partially
miscible, in methanol and water. The fat solvent can be acetone, methyl
vinyl ketone, methyl ethyl ketone, N-methyl pyrrolidine, tetrahydrofuran
or the like. Preferably, the fat solvent is acetone. In the context of
this invention, two liquids which are "miscible" can mix to the extent
that both are found in a single liquid phase. A partially miscible liquid
mixes so that at least 1% by volume of the liquid can be found in a liquid
phase containing a larger percentage of the other liquid or liquids.
Formation of the Crystalline Saponin-Containing Complex
The crystalline saponin-containing complex is formed by forming a mixture
of about 1-20% by weight, preferably 5-15% by weight of
steroidal-saponin-containing extract solids in a fluid comprising a fat
solvent, methanol and water in a volume ratio of about 0.1-20:0.1-20:1.0,
so that the saponins, fats and non-saponin carbohydrates are dissolved in
the fluid. The mixture is then allowed to stand at ambient temperature and
pressure for a period of time effective to form the crystalline
saponin-containing complex, while the fats and non-saponin carbohydrates
remain substantially in solution. In a preferred embodiment, the fat
solvent is acetone. Preferably, insoluble residue in the mixture is
removed prior to allowing the crystals to form. In a more preferred
embodiment the mixture comprises methanol, acetone and water in a volume
ratio of about 0.2-10:0.2-10:1.0, more preferably about 0.25-5:0.25-5:1.0,
and most preferably about 0.5-2.0:0.5-2.0:1.0.
In a preferred embodiment, the crystalline saponin-containing complex is
formed by forming a mixture consisting essentially of about one volume of
an aqueous steroidal-saponin-containing plant extract containing about
5-75% by weight plant solids, preferably 20-60% by weight plant solids,
with about four volumes of a solvent consisting essentially of methanol,
acetone and water in a volume ratio of 0.5-2.0:0.5-2.0:1.0, such that the
saponins, fats and non-saponin carbohydrates derived from the plant
extract are dissolved in the liquid phase of the mixture. The liquid phase
is then allowed to stand at ambient temperature and pressure for a period
of time effective to form the crystalline saponin-containing complex,
while the fats and non-saponin carbohydrates remain substantially in
solution. In a preferred embodiment, insoluble residue in the mixture is
removed prior to allowing the crystals to form.
After the insoluble residue has been optionally removed, the solution is
preferably placed in a container which limits air flow to the surface of
the fluid, and/or to the air space immediately above the fluid surface,
such that evaporation of the fluid is constrained. This may be
accomplished by placing the solution in a flask such as an Erlenmeyer
flask, a round-bottom flask, or any suitable container which in some way
constricts air flow to the surface of any fluid contained therein or to
the air space immediately above the fluid. It is possible to put the
solution in a container which may be sealed for part or all of the time in
which the solution is contained therein. The object is to constrain
evaporation of the fluid such that evaporation does not proceed at a rate
or to an extent which causes the precipitation of the fats and nonsaponin
carbohydrates which are dissolved in the fluid. Many of the most suitable
containers for this purpose are described in the Corning Glassworks
Catalogue (The Lab Book; Corning Glassworks, Inc.; 1983, Corning, N.Y.)
which is incorporated herein by reference. The containers include
round-bottom flasks, Erlenmeyer flasks, volumetric flasks, reagent
bottles, and the like.
The solution is allowed to stand in the container at ambient temperature
and pressure for a period of time sufficient to form the crystalline
saponin-containing complex. However, any temperature and pressure which
will allow the formation of the saponin-containing complex crystals may be
used. The crystals are typically formed at atmospheric pressure and at
room temperature which may be from about 10-37.degree. C., preferably
about 20-30.degree. C., although higher and lower temperatures are not
known to prevent crystallization. The period of time necessary to obtain
crystals may be anywhere from about 1 to 200 days, preferably about 5 to
100 days, most preferably about 15-60 days.
In preferred embodiments, the evaporation is constrained to limit the
evaporation of the original solution to no less than about half of its
original volume. If evaporation is allowed to proceed too far beyond about
50% of the original solution volume, the product can be contaminated with
unwanted amounts of non-saponin carbohydrates and fats. Preferably, the
rate of evaporation is limited to about 1.0% a day, more preferably, about
0.1%.
In the most preferred embodiment, the crystalline saponin-containing
complex is prepared by a process comprising the following steps:
(a) forming a mixture consisting essentially of one volume of aqueous plant
extract containing about 20-60% by weight of steroidal-saponin-containing
plant extract solids, and four volumes of a solvent consisting essentially
of methanol, acetone and water in a volume ratio of about 1:1:1, so that
the saponins, fats and non-saponin carbohydrates derived from said plant
material are dissolved in solution;
(b) separating any insoluble material from the mixture by filtration to
yield a filtrate;
(c) placing the filtrate in a container effective to constrain evaporation
at ambient temperature and pressure such that evaporation of the filtrate
does not proceed at a rate or to an extent which cause the precipitation
of the fats and nonsaponin carbohydrates which are in solution; and
(d) allowing the filtrate to stand at ambient temperature and pressure for
a period of time effective to form saponin-containing complex crystals,
while substantially retaining the fats and non-saponin carbohydrates in
solution.
The formation and/or nucleation of the crystalline saponin-containing
complex can be aided in any suitable manner, such as by "seeding" the
solution with previously isolated complex crystals, by placing a rough
surface in the solution to aid nucleation, by mild agitation during the
period when the solution is allowed to stand, or the like.
When the crystals have formed, they may be collected in any suitable
manner. The solution may be poured or decanted off and then washed in cold
water and collected by centrifugation, vacuum filtration, or the like. The
crystals may be dissolved and recrystallized from similar solvent mixtures
as recommended for the initial crystallization or from any other suitable
solvent, such as methanol, alcohol, butanol, methanol and water, or the
like.
The saponin-containing crystals are typically white, although they may from
time to time be slightly off-white, or even have a slightly brown tint
which makes them a light tan color. When the crystals are heated, they
discolor in a range from about 270-280.degree. C., typical from about
272-278.degree. C., most typically from about 274-276.degree. C. When
further heated the crystals melt in a melting range of about
285-305.degree. C., more preferably about 290-300.degree. C. The melting
range is the range between the temperature at which the crystals begin to
melt and the temperature at which all of the crystals are melted as
further defined in Example V below.
Almost all pure elements and compounds are capable of forming crystals
because they are substantially homogeneous and, therefore, interact in a
repetitive manner on the molecular level. A perfect crystal is one in
which the crystal structure would be that of an ideal space lattice. No
such crystals are believed to exist. All real crystals have imperfections
which influenced the physical properties of the crystals. The purer the
crystal, the more regular the interactions and the more consistent the
melting range and other physical properties. Evidence that would indicate
that a solid is indeed a crystal, includes evidence that the solid has a
narrow melting range. Other evidence generally includes the availability
of clear spectrographic data for infrared (IR) spectroscopy, nuclear
magnetic resonance (NMR) spectroscopy, and x-ray diffraction analysis. In
each case, data which show relatively sharp and distinct peaks, are
generally considered to indicate that the sample is a relatively pure
substance. In the case of x-ray diffraction patterns, clear and distinct
peaks are generally considered to indicate that a crystalline structure
does exist, and that there are repeating structural interactions which
would only be seen if this sample was in fact crystalline in nature. Such
data has been collected for the present crystals and is included in the
Figures and Examples herein below.
In addition, elemental analysis of the crystals indicates that the crystals
contain about 54-58% by weight carbon (C), about 6-10% by weight elemental
hydrogen (H), and about 34-38% by weight elemental oxygen (O). In a
preferred embodiment, the crystals contain about 55-57% by weight C. The
analysis was done by complete combustion analysis, methods for which are
well known in the art.
Samples of the complex crystals were also analyzed for impurities by
thin-layer chromatography. The results indicated that the crystals are
substantially free of fats and non-saponin carbohydrates derived from the
plant. This means that the crystals contain very little if any fats and/or
non-saponin carbohydrates, preferably less than about 3% by weight, more
preferably less than about 1% by weight, and even more preferably less
than about 0.1% by weight.
In the thin-layer chromatography method used in Example XII, a sample
containing the crystals isolated in Example V gave a chromatogram which
showed the samples to be substantially free of plant fats and non-saponin
carbohydrates. Studies of impure saponin-containing extracts show a solid
streak from rf 0 through 1.0. The sample containing crystals from Example
V, however, showed several large spots corresponding to saponins which
appeared between rf 0.3 and rf 0.7. Above rf 0.7 where the fats are
characteristically located, and below rf 0.3 where the non-saponin
carbohydrates are characteristically located, there were substantially nc
spots. Although some faint spots did develop above rf 0.7 and below rf 0.3
indicating some impurities, the crystals were substantially free of fats
and non-saponin carbohydrates.
Further indications of the crystals' purity were manifest in the sharp
x-ray diffraction pattern shown in FIG. 6, as well as the precise spectral
data obtained by infrared (IR) (FIG. 3) and NMR analysis (FIGS. 4 and 5).
In each case, the well defined peaks are indicative of a consistent
composition which allows sharply defined data measurements.
Hydrolysis Procedure
The saponin-containing complex crystals can be hydrolyzed to form
sapogenins by a process catalyzed by acid or by enzymes. The sapogenins
are formed from the saponins incorporated in the complex crystals by
hydrolyzing the ether linkage between the sapogenin moiety and the
glycosidic moiety of each saponin molecule. This process can be catalyzed
by an enzyme which will hydrolyze such an ether linkage, but is preferably
catalyzed with acid, most preferably with an aqueous mineral acid, such as
hydrochloric acid (HCl), sulfuric acid (H.sub.2 SO.sub.4), nitric acid
(HNO.sub.03), or the like. The aqueous mineral acid may be from about 0.1
N to a concentrated aqueous solution of the acid. In the most preferred
embodiment, 4 N HCl is used.
Hydrolysis of the crystalline saponin-containing complex of the present
invention yields a product which contains about 1-70%, preferably about
5-40%, more preferably about 10-30% by weight of sapogenins. In the
preferred embodiment, the yield of a sapogenin, preferably smilagenin, is
about 1-50% by weight, preferably about 5-35% by weight, more preferably
about 10-25% by weight. This yield is determined by gravimetric analysis
(weighing the product) and by thin-layer chromatographic comparison to
known samples of smilagenin. In the most preferred embodiment, hydrolysis
of the crystalline saponin-containing complex from Yucca schidigera gives
a yield of about 14-18% smilagenin. Of the saponins of the crystalline
complex, acid hydrolysis is effective to convert at least about 20%,
preferably about 50%, more preferably about 75% into sapogenins.
The invention will be further described by reference to the following
detailed examples.
EXAMPLE I
Aqueous Saponin-Containing Yucca Extract
The plant material of a Yucca schidigera tree is cut up and pulverized. The
pulverized plant material is soaked in water for 24 hours and then pressed
to drive out the aqueous saponin-containing Yucca extracts. The aqueous
extracts are collected and concentrated by evaporation in an open-air
holding vat. A bacteriostat, sodium benzoate is added in an amount
effective to make a 0.01 molar solution of sodium benzoate in order to
limit bacterial growth in the extract fluid. The saponin-containing liquid
extract is concentrated until it has a solids content of about 40% by
weight.
EXAMPLE II
Saponin-Containing Yucca Extract Solids
The saponin-containing liquid extract of Example I is further evaporated
under ambient conditions until a dry powder remains.
EXAMPLE III
Methanol, Water, Crystallization
One volume of the aqueous saponin-containing extract of Example I
containing about 50% by weight solids is added to 4 volumes of a mixture
of reagent grade methanol and water in a volume ratio of 1:1. The
suspension is efficiently mixed by manually shaking the mixture in an
Erlenmeyer flask. The solution is then allowed to stand approximately
10-20 minutes and the insoluble residue is removed by vacuum filtration
through filter paper (Whatman Qualitative #1 --medium fast; Whatman, Inc.;
Clifton, N.J.) in a Buchner funnel. A clear brown solution resulted. This
solution is allowed to stand for 3 weeks at ambient temperature and
pressure in a 2000 ml Erlenmeyer flask (number 4980-2L; Corning
Glassworks, Inc.; Corning, N.Y.). The saponin-containing complex crystals
form as a white solid crystalline mass on the walls of the container.
After standing for 3 weeks the solution is poured off and the crystalline
complex is collected from the walls of the flask by loosening the crystals
with a stainless steel spatula. The crystals are washed into a Buchner
funnel and are collected and separated from the fluid by filtration means
using Whatman #1 filter paper. Evaporation during the 3 weeks is slowed by
the constricted neck of the flask such that the solution volume at the end
of that time is 80% of the original solution volume.
EXAMPLE IV
Methanol, Acetone and Water Solution
One volume of an aqueous saponin-containing Yucca extract containing about
40% by weight solids (Yucca Extract; EDP, Inc.; Porterville, Calif.) was
added to 4 volumes of a mixture of reagent grade methanol and acetone and
water in a volume ratio of 1:1:1. This mixture was mixed manually by
shaking the mixture in a liter round-bottom flask (number 4280-lL; Corning
Glassworks, Inc.; Corning, N.Y.). The solution was allowed to stand
approximately 10 minutes. The insoluble residue was then removed by vacuum
filtration of the fluid through filter paper (Whatman Qualitative #1
--medium fast; Whatman, Inc.; Clifton, N.J.) in a Buchner funnel. A clear
brown solution resulted. This solution was allowed to stand in a liter
round-bottom flask (number 4280-lL; Corning Glassworks Inc.; Corning,
N.Y.) at ambient temperature and pressure in a laboratory room for 3
weeks. Formation of the complex crystals was aided by "seeding" the
solution with previously isolated crystalline saponin-containing complex
derived from Yucca schidigera extracts. The saponin-containing complex
crystals formed as a white solid crystalline mass on the walls of the
container. Evaporation of the solution was such that the solution volume
at the end of this period of time was 75% of the original solution volume.
The solution was poured off after the 3 week period ended. The crystals
were loosened with a stainless steel spatula and removed by filtration
through filter paper (Whatman #1) in a 30 Buchner funnel.
EXAMPLE V
Methanol, Acetone, Water Crystallization
To 110 ml of a saponin-containing Yucca extract containing about 40% by
weight solids (Yucca Extract; EDP, Inc.; Porterville, CA) was added a
mixture of equal volu | | |
