U.S. patent application number 13/444765 was filed with the patent office on 2012-08-02 for method for enhancing nutrient absorption with astragalosides.
This patent application is currently assigned to NuLiv Holding Inc.. Invention is credited to Tsu-Chung Chang, Wen-Liang Chang, Hsiou-Yu Ding, Hang-Ching Lin, Tian Shung Wu.
Application Number | 20120196816 13/444765 |
Document ID | / |
Family ID | 42109154 |
Filed Date | 2012-08-02 |
United States Patent
Application |
20120196816 |
Kind Code |
A1 |
Lin; Hang-Ching ; et
al. |
August 2, 2012 |
METHOD FOR ENHANCING NUTRIENT ABSORPTION WITH ASTRAGALOSIDES
Abstract
The present application relates to a method for enhancing
absorption of a nutrient in a subject in need thereof with an
effective amount of an isolated astragaloside compound.
Inventors: |
Lin; Hang-Ching; (Taipei,
TW) ; Chang; Wen-Liang; (Taipei, TW) ; Chang;
Tsu-Chung; (Taipei, TW) ; Ding; Hsiou-Yu;
(Tainan, TW) ; Wu; Tian Shung; (Tainan,
TW) |
Assignee: |
NuLiv Holding Inc.
Taipei
TW
|
Family ID: |
42109154 |
Appl. No.: |
13/444765 |
Filed: |
April 11, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12424193 |
Apr 15, 2009 |
8197860 |
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13444765 |
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11426029 |
Jun 23, 2006 |
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12424193 |
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60694097 |
Jun 23, 2005 |
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Current U.S.
Class: |
514/26 |
Current CPC
Class: |
Y10S 514/909 20130101;
A61K 36/481 20130101; Y10S 514/923 20130101; A61P 3/00 20180101;
A61K 31/704 20130101; A61P 1/00 20180101; A61P 3/02 20180101 |
Class at
Publication: |
514/26 |
International
Class: |
A61K 31/704 20060101
A61K031/704; A61P 3/00 20060101 A61P003/00 |
Claims
1-27. (canceled)
28. A method for enhancing absorption of folate in a subject in
need thereof, comprising: identifying a human subject in need of
enhanced absorption of folate, and administering to the subject an
effective amount of an isolated astragaloside compound for
facilitating transportation of folate across gut cells of the
subject, wherein the subject is an elderly, a pregnant woman, a
nursing mother, or a patient having insomnia, depression, a
cardiovascular disease, or long-term pressure.
29. The method of claim 28, wherein the astragaloside compound is
an astragaloside compound of Formula (A): ##STR00019## wherein
R.sub.1 is selected from the group consisting of H, OH, O-acetyl,
O-xylopyranosyl, O-(2-acetylxylopyranosyl),
O-(3-acetylxylopyranosyl), O-(2,3-diacetylxylopyranosyl),
O-(2,4-diacetylxylopyranosyl),
O-xylopyranosyl-(1-2)-.beta.-D-glucopyranosyl, and
O-xylopyranosyl-(1-2)-.alpha.-arabinopyranosyl; R.sub.2 is selected
from the group consisting of H, OH, O-acetyl, O-glucopyranosyl, and
O-xylopyranosyl; R.sub.3 is selected from the group consisting of
H, OH, and O-acetyl; and R.sub.4 is selected from the group
consisting of ##STR00020##
30. The method of claim 29, wherein the astragaloside compound is
selected from the group consisting of astragaloside I of Formula I:
##STR00021## astragaloside II of Formula II: ##STR00022##
astragaloside III of Formula III: ##STR00023## astragaloside IV of
Formula IV: ##STR00024## isoastragaloside I of Formula V:
##STR00025## astragaloside VI of Formula VI: ##STR00026##
isoastragaloside II of Formula VII: ##STR00027##
cycloastragenol-6-O-.beta.-D-glucopyranose of Formula VIII
##STR00028##
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/426,029, filed Jun. 23, 2006, which claims
the benefit of the priority pursuant to 35 U.S.C. .sctn.119(e) of
U.S. Provisional Patent Application No. 60/694,097, filed Jun. 23,
2005. The contents of the prior applications are incorporated
herein by their entireties.
BACKGROUND OF THE INVENTION
[0002] From the study of the human digestive system, it has been
found that a huge variety of nutritional substances are obtained by
breaking down and digesting the food in the gastrointestinal tract.
The gastrointestinal tract is an important route by which the food
is digested and absorbed. With regard to absorption, the
nutritional substances, such as glucose, amino acids, vitamins and
other smaller molecules are absorbed along the entire tract, either
by diffusion or by specialized transport processes. Instead of
moving freely across the intestinal membrane to the blood stream or
lymph, most of these nutritional substances are transported by a
tightly regulated mechanism. Based on current understanding in cell
biology and physiology, the nutritional substances are transported
across the cells with specific transport proteins and channels
anchored on the cell membrane.
[0003] In the example of glucose transportation, almost all of the
cells have a carrier-mediated mechanism for the transport of
glucose from blood. For most cells, this transport occurs by
facilitated diffusion using one or more of the glucose transporters
(GLUT) in a family of facilitated glucose transporters. In these
cases, net glucose transport occurs as a result of an inwardly
directed chemical gradient for glucose. In a few cell types (e.g.
those of intestinal mucosa and renal proximal tubule), uptake of
glucose from an extracellular solution can occur against a gradient
of glucose in a so-called active transport mechanism, thereby
permitting net absorption of glucose from a tissue compartment
whose glucose concentration may be lower than that of the blood.
There are two ways in which a flow of energy can be coupled to
transporters. The primary active transport requires energy be
provided by adenosine triphophatase (ATPase). The secondary active
transport provides energy from the flow of ions from an area of
higher concentration to one of lower concentration.
[0004] According to the secondary active transport model described
above, Na.sup.+ binds to transport protein on the luminal side of
the cell causing conformational change of the transport protein,
which opens the binding site for glucose. Then, glucose binds to
the transport protein. The transport protein that is bound with
both Na.sup.+ and glucose is subjected to further conformational
change to allow entry of glucose and Na.sup.+ into the cells. This
active transport of glucose involves a direct physical coupling of
flows of Na.sup.+ and glucose, with the energy of the process being
derived from the inwardly directed gradient for Na.sup.+. Since the
transport event includes a net movement of charge (the cationic
Na.sup.+ ion with the non-electrolyte glucose), the driving force
for this uptake includes both the chemical gradient for Na.sup.+
and the potential difference across the membrane. As the glucose
gradually accumulates in the cell, it is subsequently transported
out to the blood vessel via a glucose concentration gradient by
facilitated diffusion. Similarly, other nutritional substances may
be absorbed with the transport mechanism described above.
[0005] Astragalus root (Radix Astragali) has been used as a
traditional Chinese medicine that mainly serves to invigorate the
function of the spleen and increase stamina and endurance.
Astragalus root (Radix Astragali) was found to enhance the immune
system and help the human body resist virus infections,
particularly in the lungs, by increasing production of interferon,
an immune factor that inhibits viral growth. Astragalus root has
been used as an adjuvant therapy in the treatment of colds and
influenza. Radix Astragali was also reported to have effects on
cardiovascular activity. Alcohol extracts of Radix Astragali
enhanced both the contractility and contraction amplitude of
isolated frog or toad hearts. Furthermore, astragalosides isolated
from Radix Astragali have been reported to exert a positive
inotropic effect on isolated rat hearts.
[0006] However, Astragalus membranaceus var. mongholicus has not
been implied in regulating nutrient absorption and transportation.
None of the study or research has focused on regulating the
nutrient absorption using saponin compounds purified from Chinese
herbal medicines, particularly Astragalus membranaceus var.
mongholicus.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention provides a method for enhancing the
absorption of a nutrient, such as glucose, an amino acid (e.g.
arginine or tryptophan), and a vitamin (e.g., folate), in a subject
in need thereof. This method includes the steps of identifying a
subject who needs up-regulation of nutrient absorption and
administering to the subject an effective amount of an isolated
astragaloside compound (e.g., from Astragalus membranaceus var.
mongholicus). "An effective amount" as used herein refers to the
amount of each active agent required to confer therapeutic effect
on the subject, either alone or in combination with one or more
other active agents. Effective amounts vary, as recognized by those
skilled in the art, depending on route of administration, excipient
usage, and co-usage with other active agents. Subjects in need of
this regulation include elderlies, juveniles, pregnant or
menopausal women, post-surgery patients, and patients suffering
from long-term pressure, abnormal metabolism (e.g., type II
diabetics), a weakened immune system (e.g., leukemia patients, HIV
carriers, and organ transplantation recipients), or other
diseases/disorders listed in Table 1 below.
[0008] The astragaloside compound, preferably isolated, is an
cycloartane compound of Formula
##STR00001##
wherein R.sub.1 is selected from the group consisting of H, OH,
O-acetyl, O-xylopyranosyl, O-(2-acetylxylopyranosyl),
O-(3-acetylxylopyranosyl), O-(2,3-diacetylxylopyranosyl),
O-(2,4-diacetylxylopyranosyl),
O-xylopyranosyl-(1-2)-.beta.-D-glucopyranosyl and
O-xylopyranosyl-(1-2)-.alpha.-arabinopyranosyl; R.sub.2 is selected
from the group consisting of H, OH, O-acetyl and O-glucopyranosyl,
O-xylopyranosyl; R.sub.3 is selected from the group consisting of
H, OH and O-acetyl; and R.sub.4 is selected from the group
consisting of
##STR00002##
[0009] The term "isolated astragaloside compound" used herein
refers to an astragaloside compound prepared by a synthetic method
or enriched from a natural source (e.g., Astragalus membranaceus
var. mongholicus). For example, an isolated astragaloside compound
is a preparation that contains at least 40% (e.g., at least 95%) of
the astragaloside compound by dry weight. Purity of an isolated
compound can be measured by, e.g., column chromatography, mass
spectrometry, high performance liquid chromatography (HPLC), NMR,
or any other suitable methods.
[0010] Preferably, the astragaloside compound used in the method of
this invention is selected from the group consisting of
astragaloside I of Formula I:
##STR00003##
astragaloside II of Formula II:
##STR00004##
astragaloside III of Formula III:
##STR00005##
astragaloside IV of Formula IV:
##STR00006##
isoastragaloside I of Formula V:
##STR00007##
astragaloside VI of Formula VI:
##STR00008##
isoastragaloside II of Formula VII:
##STR00009##
and cycloastragenol-6-O-.beta.-D-glucopyranose of Formula VIII:
##STR00010##
[0011] Additional features and advantages of the present invention
will be set forth in part in the description which follows, and in
part will be apparent from the description, or may be learned by
practice of the invention. The features and advantages of the
invention will be realized and attained by means of the elements
and combinations as described.
[0012] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0013] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings. For the purpose of
illustrating the invention, there are shown in the drawings
embodiments, which are presently preferred. It should be
understood, however, that the invention is not limited to the
precise arrangements and instrumentalities shown.
[0014] In the drawings:
[0015] FIG. 1 is a line graph showing the glucose uptake rates of
the Caco2 cells treated with the isolated astragaloside AS4 of
Formula IV of selected concentrations;
[0016] FIG. 2 is a line graph showing the arginine absorption rates
measured in the Sink-transport across to basolateral chambers when
the Caco2 monolayers were treated with the isolated astragaloside
AS1 of Formula I of selected concentrations;
[0017] FIG. 3 is a line graph showing the tryptophan absorption
rates measured in the Sink-transport across to basolateral chambers
when the Caco2 monolayers were treated with the isolated
astragaloside AS1 of Formula I of selected concentrations;
[0018] FIG. 4 is a line graph showing the folate uptake rates of
the Caco2 cells treated with the isolated astragaloside AS1 of
Formula I of a selected concentration.
DETAILED DESCRIPTION OF THE INVENTION
[0019] To better understand the present invention, the terms used
herein are explained in further detail. An astragaloside is defined
as a triterpene saponin compound extracted from Radix Astragali,
the dried root of Astragalus membranaceus (Fisch) Bunge and
Astragalus mongholicus Bunge (Fabaceae).
[0020] As used herein, the singular forms "a", "an", and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "a compound" includes a
plurality of such compounds.
[0021] The term "absorption" as used herein refers to uptake of a
nutrient via a passage through the intestinal epithelium and into
the blood or lymph.
[0022] The term "gut cells" as used herein generally include
enterocytes, mucosal cell, and cells of intestinal epithelium
responsible for nutrient absorption of the body.
[0023] The term "subject" as used herein refers to any animal,
preferably including humans, where absorption of nutrients occurs
across gut cells in the subject's gastrointestinal tract.
[0024] The present invention is based at least in part on the
unexpected discovery that a number of astragaloside compounds
enhance transportation of certain nutrients across a monolayer of
the gut cells lining the gastrointestinal tract. See Examples 1-4
below. Thus, this invention provides a method for up-regulating the
absorption of a nutrient with a astragaloside compound in a subject
in need thereof. Table 1 below provides examples of the particular
types of subjects who need enhanced absorption of particular
nutrients:
TABLE-US-00001 TABLE 1 Subjects Who Need Up-Regulation of
Absorption of Certain Nutrients Nutrient Subject In Need Glucose
Elderlies, athletes, alcoholics, juveniles, post-surgery patients,
malnutrition patients, and patients having digestive tract
disorders Arginine Juveniles, athletes, over-weight patients,
patients suffering from cardiovascular disease, a weakened immune
system, physical injury (e.g., burn trauma), and erectile
dysfunction Tryptophan Over-weight patients, patients suffering
from insomnia, a weakened immune system, and long-term pressure
Folate Elderlies, pregnant women, nursing mothers, and patients
suffering from insomnia, depression, cardiovascular disease, or
long-term pressure
[0025] The astragaloside compounds may be formulated into tablets,
pills, capsules, liquid formulations and powder to be orally
administered to the individual with nutrient absorption problem or
mal-absorption syndrome, which is an alteration in the ability of
the intestine to absorb nutrients adequately into the bloodstream.
In one embodiment of the preparation of the liquid formulation, one
or more of the astragaloside compounds may be dissolved in any
solvent, preferably in a co-solvent, to produce a liquid
formulation of the astragaloside compounds (such as, 10 mg of any
of the astragaloside compounds may be dissolved in one mL of
Transcutol.RTM. P [2-(2-ethoxyethoxy)ethanol]). Also, the
astragaloside compounds may be optionally mixed with other nutrient
factors, additives, stabilizing agents, carriers, binders and
fillers to produce dietary supplements, beverages, food, and animal
feeds for a subject in need of enhanced nutrient absorption. It may
be apparent to one skilled in the art in view of the present
disclosure to administer the astragaloside compounds in combination
or in a cocktail manner with other ginsenosides and astragalosides
to provide a synergistic or accumulative effect on the nutrient
absorption.
[0026] The astragaloside compounds may be prepared by any standard
methodology or known methods or knowledge in the art. According to
the invention, the isolated astragaloside compounds, e.g., from
Astragalus membranaceus var. mongholicus, include the
astragalosides. They may be isolated by other available extraction
and isolation methods known to those skilled in the art. For
example, the isolated astragaloside compounds may also be enriched
from other Chinese herbal plants or vegetation to provide the same
regulatory effect on nutrient absorption function. According to an
embodiment of the invention, the astragaloside compounds may be
obtained by a method comprising the steps of grinding the root of
Astragalus membranaceus var. mongholicus and extracting with
alcohol to produce an alcohol extract. The alcohol extract of
Astragalus membranaceus var. mongholicus may be separated and
purified to give seven known cycloartane compounds including
astragaloside I (hereinafter "AS1") of Formula I:
##STR00011##
astragaloside II (hereinafter "AS2") of Formula II:
##STR00012##
astragaloside III (hereinafter "AS3") of Formula III:
##STR00013##
astragaloside IV (hereinafter "AS4") of Formula IV:
##STR00014##
isoastragaloside I (hereinafter as IsoAS1) of Formula V:
##STR00015##
astragaloside VI (hereinafter "AS6") of Formula VI:
##STR00016##
and isoastragaloside II (hereinafter as IsoAS2) of Formula VII:
##STR00017##
[0027] The cycloartane compounds may be separated and purified with
silica gel and reversed phase chromatography. AS4 may further be
hydrolyzed using naringinase to obtain a metabolite, such as
cycloastragenol 6-O-.beta.-D-gluco-pyranose (hereinafter "AA") of
Formula VIII:
##STR00018##
[0028] In one embodiment, the absorption of glucose is enhanced by
facilitating the transportation of glucose across the gut cells of
the subject in need of the absorption enhancement with
administration of one or more of the above-described astragaloside
compounds at a concentration of about 0.001 .mu.M to about 5 .mu.M;
wherein the astragaloside compound is AS1 of Formula I, AS4 of
Formula IV, AS6 of Formula VI, IsoAS2 of Formula VII, or AA of
Formula VIII.
[0029] In another embodiment, the absorption of arginine is
enhanced by facilitating the transportation of arginine across the
gut cells of the subject in need of the absorption enhancement with
administration of one or more of the above-described astragaloside
compounds at a concentration of about 0.001 .mu.M to about 5 .mu.M;
wherein the astragaloside compound is AS1 of Formula I, AS2 of
Formula II, AS3 of Formula III, AS4 of Formula IV, IsoAS1 of
Formula V, AS6 of Formula VI, IsoAS2 of Formula VII, or AA of
Formula VIII.
[0030] In still another embodiment, the absorption of tryptophan is
enhanced by facilitating transportation of tryptophan across the
gut cells of the subject in need of the absorption enhancement with
administration of one or more of the above-described astragaloside
compounds at a concentration of about 0.001 to about 5 .mu.M;
wherein the astragaloside compound is AS1 of Formula I, AS2 of
Formula II, AS3 of Formula III, AS4 of Formula IV, IsoAS1 of
Formula V, AS6 of Formula VI, IsoAS2 of Formula VII, or AA of
Formula VIII.
[0031] In yet another embodiment, the absorption of folate is
enhanced by facilitating transportation of folate across the gut
cells of the subject in need of the absorption enhancement with
administration of one or more of the above-described astragaloside
compounds at a concentration of about 0.001 .mu.M to about 5 .mu.M;
wherein the astragaloside compound is AS1 of Formula I, AS2 of
Formula II, AS3 of Formula III, AS4 of Formula IV, IsoAS1 of
Formula V, AS6 of Formula VI, IsoAS2 of Formula VII, or AA of
Formula VIII.
[0032] The present invention is more specifically explained by the
following examples. However, it should be noted that the present
invention is not limited to these examples in any manner.
Example 1
Regulatory Effects of Isolated Astragalosides on Glucose Uptake
Cell Culture
[0033] To evaluate the effect of the isolated astragaloside
compound on the uptake of nutrient substances across the intestinal
lumen, Caco-2 cells were grown on permeable filter as an
experimental model. Caco2 cells originate from human colonic
adenocarcinoma and spontaneously differentiate into an
enterocyte-like phenotype after two weeks. The Caco-2 cell line,
derived from a human colorectal carcinoma, has been used as an in
vitro model system for studying drug absorption in gastrointestinal
tract. These cells form monolayers with well-developed
tight-junctions, and have been evaluated in details as an in vitro
model to study both transcellular transport of nutrients and drugs
in intestinal lumen.
[0034] Caco-2 cells were obtained from the ATCC (American Type
Culture Collection). The cells were maintained in Dulbecco's
modified Eagle medium (DMEM) containing 4.5 g/L glucose and 25 mM
Hepes, supplemented with 10% fetal calf serum, 100 U/mL penicillin
G and 10 .mu.g/L streptomycin. The medium was changed every second
day. The cells were routinely checked for Mycoplasma in monthly
intervals. Caco-2 cells were cultured on semi-permeable membranes
to differentiate into a highly functionalized epithelial barrier
with remarkable morphological and biochemical similarity to the
small intestinal columnar epithelium. The Caco-2 cell monolayers
could therefore be used to study the membrane transport properties
of many compounds. To trypsinize the cells, the culture dish was
washed once with phosphate-buffered saline (PBS) followed by adding
trypsine-EDTA for 10 minutes. The trypsinized cells were separated
and filtered into single cells using a 35-.mu.m strainer cap
(Falcon 2235) before being seeded for further experiments.
Cell Viability Assay
[0035] To investigate whether the isolated astragalosides were
toxic to the Caco2 cells, cell viability assay was carried out
using culture medium supplemented with 1% and 10% FBS,
respectively. The cells were seeded at a concentration of 5000
cells/well in a 96-well plate. To eliminate the boundary effect of
the cell growth, the cells were only seeded in 60 wells of the
middle area of the plate, whereas 36 wells at the surrounding area
of the plate were filled only with 100 .mu.L of PBS. Once the cells
were attached to the plate, the cells were incubated in medium
containing the isolated astragalosides at various doses (0, 1, 10,
20 and 50 .mu.M). After 3 days, the culture medium was replaced
with fresh medium containing the same compounds and incubated for 2
more days before the cells were assayed for cell viability.
[0036] The cell viability was determined by a Cell Counting Kit-8
(CCK-8, Dojindo Laboratories, Kumamoto, Japan) assay that is based
on redox reaction of NADH in the living cells with cell
proliferation reagent WST-8. WST-8 was reduced by dehydrogenases in
electron transport chain (ETC) of mitochondria in the cells to give
a yellow-colored formazan product, which was soluble in the tissue
culture medium. The amount of formazan dye generated by the
activity of dehydrogenases in the cells was directly proportional
to the number of the living cells. Therefore, a greater light
absorbance detected by ELISA reader at wavelength of 450 nm
indicated presence of a larger number of the living cells.
[0037] The CCK-8 assay was carried out by adding 10 .mu.L of the
CCK-8 reagent in each well of 96-format plate. The plate was then
covered with aluminum foil and further incubated for two hours
before measuring for absorbance at wavelengths of 450 nm by using
an ELISA reader.
Glucose Uptake Assay
[0038] Caco-2 cells (5.times.10.sup.4) were seeded in a 48-well
plate and maintained in culture medium (DMEM with 10% FBS, 1%
nonessential amino acids, L-glutamine, penicillin G (100 U/mL),
streptomycin (10 .mu.g/mL), and amphotericin B (2.5 .mu.g/mL) in a
37.degree. C. incubator for 10 days for the cells to differentiate.
The culture medium was changed once every two days. The cells were
then washed with PBS before replenishing with the culture medium
containing 5% FBS and various astragalosides at the indicated
concentrations (0.01, 0.1 and 1 .mu.M) for 48 hours. The Caco2
cells were washed out of remaining glucose with PBS and replaced in
the glucose buffer (80 mM NaCl, 100 mM mannitol, 20 mM Tris-HCl, pH
7.4, 3 mM K.sub.2HPO.sub.4, 1 mM CaCl.sub.2, 1 mg/mL BSA) for 1
hour. Glucose uptake was initiated by replacing the glucose buffer
with 0.2 ml of glucose buffer containing 2 .mu.Ci/mL of
.sup.14C-glucose and unlabeled cold glucose to give a final glucose
concentration of 25 mM. Glucose uptake was stopped by removing the
glucose buffer and washing with PBS at designated time intervals.
The cells were lysed in 0.2 mL of 0.2 N NaOH, and 20 .mu.L of the
cell lysate were transferred to the filter-bottomed UniFilter
plates (Perkin-Elmer, Wellesley, Mass., USA) and dried in a vacuum
oven at 37.degree. C. The bottom of the UniFilter plate was sealed
and 250 .mu.L of the counting solution were added into each well.
Adhesive plate sealers were used in place of the lids and
radioactivity of each sample was counted using the microplate
liquid scintillation counter (TopCount, Packard NXT, Packard
BioScience Company, Meriden, Conn., USA). The amount of glucose
accumulated in the cells was calculated and normalized to protein
concentration, and uptake rate was expressed as nanomoles of
glucose per minutes per milligram of cell protein (nmol/min/mg).
Protein concentration was determined by a standard Bicinchoninic
acid (BCA) protein assay. Nonspecific glucose uptake was measured
by adding 2 .mu.Ci of L-[.sup.14C]-glucose and subtracting from
each determination to obtain specific glucose uptake.
[0039] In the cell viability assay, the isolated astragaloside did
not generally affect growth of Caco2 cells at a concentration range
from 1 to 50 .mu.M except when the AS1 of Formula I at a
concentration of 10 .mu.M was administered to Caco2 cells.
Therefore, the isolated astragaloside was administered in the
subsequent glucose uptake test or folate uptake test at a
concentration range that did not cause cell toxicity. Preferably,
the isolated astragaloside was administered at a concentration
range of about 0.001 .mu.M to about 1 .mu.M.
[0040] From the glucose uptake assay shown in Table 2, it was found
that isolated astragalosides, such as AS1 of Formula I, AS4 of
Formula IV, AS6 of Formula VI, AA of Formula VIII, and IsoAS2 of
Formula VII had regulatory effects on the glucose uptake of the
Caco2 cells. The amount of glucose uptake was determined and
expressed as "nmoles for each mg of cell protein." As shown in FIG.
1, the Caco2 cells treated with AS4 of Formula IV showed a much
higher glucose uptake rate than the control group. The regulatory
effects of the isolated astragalosides on the glucose transport in
Caco2 cells are listed in Table 2 below, wherein the arrows that
point up represent the enhancing effect on the glucose uptake.
TABLE-US-00002 TABLE 2 Regulatory effects of astragalosides on
glucose uptake Uptake rate Compound (.mu.M) (nmol/mg/min)
Percentage (%) * Control 6.3720 .+-. 1.9290 100 -- AS1 1 6.2780
.+-. 1.9930 98.52 0.1 7.8260 .+-. 1.7510 122.82 .uparw. 0.01 9.3510
.+-. 1.1370 146.75 .uparw. AS4 1 9.0020 .+-. 1.8300 141.27 .uparw.
0.1 10.460 .+-. 2.6690 164.16 .uparw. 0.01 11.670 .+-. 2.5800
183.15 .uparw. AS6 1 7.8050 .+-. 1.1830 122.49 .uparw. 0.1 7.0070
.+-. 1.9470 109.97 .uparw. 0.01 7.6360 .+-. 1.7330 119.84 .uparw.
AA 1 9.2830 .+-. 2.1150 145.68 .uparw. 0.1 9.3460 .+-. 2.3210
146.67 .uparw. 0.01 11.450 .+-. 3.2760 179.69 .uparw. IsoAS2 1
6.1520 .+-. 2.4610 96.55 0.1 6.8210 .+-. 1.6630 107.05 .uparw. 0.01
7.3720 .+-. 2.5700 115.69 .uparw.
[0041] It is concluded that the absorption of glucose can be
enhanced by the astragaloside isolated from Astragalus membranaceus
var. mongholicus, including AS1 of Formula I, AS4 of Formula IV,
AS6 of Formula VI, AA of Formula VIII or IsoAS2 of Formula VII.
Example 2
Regulatory Effect of Isolated Astragalosides on Arginine
Absorption
Arginine Absorption Assay
[0042] In measuring transport of arginine across the Caco-2 cell
monolayer, both sides of the transwells were washed with arginine
incubation buffer consisting of: 137 mM NaCl, 10 mM Hepes, 0.3 mM
NaH.sub.2PO.sub.4, 0.3 mM K.sub.2HPO.sub.4, 5.4 mM KCl, 2.8 mM
CaCl.sub.2, 1 mM MgSO.sub.4, 10 mM glucose, adjusted to pH 7.4.
Then, the cell layer was preincubated in the incubation buffer at
37.degree. C. for 1 h. The volume of incubation buffer was 0.2 mL
and 0.9 mL in the apical and basolateral chambers, respectively.
The cells were replaced with fresh incubation medium in both
chambers prior to the transport experiment. The transport
experiment was initiated by replacing the incubation solution on
the apical side with solution containing 10 mM of L-arginine in
which 0.125 .mu.Ci/mL of L-[.sup.3H]-arginine was included. At
designated time intervals, 10 .mu.L-solution samples were removed
from the basolateral side and radioactivity of each sample was
counted using a microplate liquid scintillation counter (TopCount,
Packard NXT). During the experiment, when a 10 .mu.L-solution
sample was removed from the basolateral side every time, 10 .mu.L
buffer was supplemented to keep the volume constant. The uptake of
[.sup.3H]-mannitol was used to correct for nonspecific transport of
molecules across the monolayer membrane. Results were expressed as
the nanomoles of arginine transport across the Caco-2 cell
monolayers with respect to time in minutes (nmol/min).
[0043] From the arginine absorption assay results shown in Table 3,
it was found that isolated astragalosides, such as AS1 of Formula
I, AS2 of Formula II, AS3 of Formula III, AS4 of Formula IV, AS6 of
Formula VI, AA of Formula VIII, IsoAS1 of Formula V and IsoAS2 of
Formula VII had regulatory effects on the arginine transport across
the Caco2 cell monolayer. Referring to FIG. 2 and Table 3, the
arginine transport rate was increased when the Caco2 cell monolayer
was treated with AS1 of Formula I or AS2 of Formula II at a
concentration from 0.001 .mu.M to 0.1 .mu.M. The arginine transport
rate was increased when the Caco2 cell monolayer was treated with
AS3 of Formula III, AS4 of Formula IV, AS6 of Formula VI, AA of
Formula VIII, IsoAS1 of Formula V or IsoAS2 of Formula VII,
respectively, at a concentration from 0.01 .mu.M to 1 .mu.M. The
regulatory effects of the isolated astragalosides on the arginine
transport in Caco2 cells are listed in Table 3 below, wherein the
arrows that point up represent the enhancing effect on the arginine
transport.
TABLE-US-00003 TABLE 3 Regulatory effects of isolated
astragalosides on Arginine transport Compound (.mu.M) Transport
rate (nmol/min) Percentage (%) * Control 10.6855 .+-. 0.2523 100 --
AS1 0.1 15.7300 .+-. 1.1250 147.21 .uparw. 0.01 16.2324 .+-. 0.7215
151.91 .uparw. 0.001 14.2554 .+-. 0.5851 133.37 .uparw. AS2 0.1
17.2771 .+-. 1.6170 161.69 .uparw. 0.01 16.2358 .+-. 1.6190 151.94
.uparw. 0.001 14.6355 .+-. 1.2910 136.97 .uparw. AS3 1 15.8341 .+-.
1.0000 148.18 .uparw. 0.1 13.2858 .+-. 1.4110 124.33 .uparw. 0.01
13.0084 .+-. 1.0510 121.74 .uparw. AS4 1 17.2241 .+-. 0.3759 161.19
.uparw. 0.1 18.4575 .+-. 0.5955 172.73 .uparw. 0.01 16.7245 .+-.
0.2890 156.52 .uparw. AS6 1 13.5942 .+-. 1.2760 127.22 .uparw. 0.1
14.9986 .+-. 1.3200 140.36 .uparw. 0.01 13.9283 .+-. 1.7330 130.35
.uparw. AA 1 17.3164 .+-. 1.6150 162.06 .uparw. 0.1 18.2169 .+-.
1.8700 170.48 .uparw. 0.01 21.3347 .+-. 1.7800 199.66 .uparw.
IsoAS1 1 14.4734 .+-. 1.1350 135.41 .uparw. 0.1 21.3107 .+-. 1.5130
199.44 .uparw. 0.01 14.4776 .+-. 0.5519 135.49 .uparw. IsoAS2 1
12.6518 .+-. 0.3680 118.40 .uparw. 0.1 14.1059 .+-. 0.1815 132.01
.uparw. 0.01 14.7577 .+-. 0.2837 138.11 .uparw.
[0044] It is concluded that the absorption of arginine can be
enhanced with the administration of astragalosides isolated from
Astragalus membranaceus var. mongholicus, including AS1 of Formula
I, AS2 of Formula II, AS3 of Formula III, AS4 of Formula IV, AS6 of
Formula VI, AA of Formula VIII, IsoAS1 of Formula V or IsoAS2 of
Formula VII.
Example 3
Regulatory Effects of Isolated Astragalosides on Tryptophan
Absorption
Trytophan Absorption Assay
[0045] The experimental procedures similar to those in Example 2
were used for measuring the uptake of tryptophan molecules across
the Caco-2 membrane, except using a tryptophan incubation buffer
consisting of 137 mM choline chloride, 10 mM Hepes, 0.6 mM
KH.sub.2PO.sub.4, 5.4 mM KCl, 2.8 mM CaCl.sub.2, 1 mM MgSO.sub.4,
and 10 mM glucose, and having its pH adjusted to 7.4. Results were
expressed as the nanomoles of tryptophan transport across the
Caco-2 cell monolayers with respect to time in minutes
(nmol/min).
[0046] From the tryptophan absorption assay results shown in Table
4, it was found that isolated astragalosides, such as AS1 of
Formula I, AS2 of Formula II, AS3 of Formula III, AS4 of Formula
IV, AS6 of Formula VI, AA of Formula VIII, IsoAS1 of Formula V and
IsoAS2 of Formula VII had regulatory effects on the tryptophan
transport across the Caco2 cell monolayer. As shown in FIG. 3 and
Table 4, the tryptophan transport rate was increased when the Caco2
cell monolayer was treated with AS2 of Formula II, AS3 of Formula
III, AS4 of Formula IV, AS6 of Formula VI, AA of Formula VIII,
IsoAS1 of Formula V or IsoAS2 of Formula VII respectively at a
concentration from 0.01 .mu.M to 1 mM, and with AS1 of Formula I at
a concentration from 0.01 .mu.M to 0.1 mM. The regulatory effects
of the isolated astragalosides on the tryptophan transport in Caco2
cells are listed in Table 4 below, wherein the arrows that point up
represent the enhancing effect on the tryptophan transport.
TABLE-US-00004 TABLE 4 Regulatory effects of isolated
astragalosides on Tryptophan transport Compound (.mu.M) Transport
rate (nmol/min) Percentage (%) * Control 8.9420 .+-. 0.3670 100 --
AS1 1 16.000 .+-. 1.3190 178.93 .uparw. 0.1 23.130 .+-. 1.3120
258.67 .uparw. 0.01 22.220 .+-. 0.8695 248.49 .uparw. AS2 1 -- --
-- 0.1 11.650 .+-. 0.5789 130.28 .uparw. 0.01 10.290 .+-. 0.4115
115.07 .uparw. AS3 1 24.200 .+-. 1.0260 270.63 .uparw. 0.1 13.590
.+-. 1.0080 151.98 .uparw. 0.01 14.290 .+-. 1.3910 159.81 .uparw.
AS4 1 9.6640 .+-. 0.2770 108.07 .uparw. 0.1 12.730 .+-. 0.4470
142.36 .uparw. 0.01 10.130 .+-. 0.7025 113.29 .uparw. AS6 1 15.490
.+-. 0.2161 173.23 .uparw. 0.1 13.850 .+-. 0.6567 151.87 .uparw.
0.01 15.510 .+-. 0.3688 173.45 .uparw. AA 1 12.100 .+-. 0.5197
135.32 .uparw. 0.1 14.000 .+-. 0.6445 156.56 .uparw. 0.01 11.900
.+-. 0.6231 133.08 .uparw. IsoAS1 1 10.600 .+-. 0.8058 118.54
.uparw. 0.1 13.370 .+-. 0.3301 149.52 .uparw. 0.01 10.370 .+-.
0.8808 115.97 .uparw. IsoAS2 1 20.070 .+-. 0.1931 224.45 .uparw.
0.1 13.060 .+-. 0.5530 146.05 .uparw. 0.01 13.000 .+-. 0.5547
145.38 .uparw.
[0047] It is concluded that the absorption of tryptophan may be
enhanced with the administration of the astragaloside isolated from
Astragalus membranaceus var. mongholicus, including AS1 of Formula
I, AS2 of Formula II, AS3 of Formula III, AS4 of Formula IV, AS6 of
Formula VI, AA of Formula VIII, IsoAS1 of Formula V or IsoAS2 of
Formula VII.
Example 4
Regulatory Effects of Isolated Astragalosides on Folate Uptake
Folate Uptake Assay
[0048] The Caco2 cells were subjected to folate uptake test in a
manner similar to that described in the glucose uptake assay in
Example 1 above. In the folate uptake test, the Caco2 cells were
pretreated with the culture medium containing 5% FBS and isolated
astragalosides at a concentration of 0.1 .mu.M for 2 days before
the cells were cultured in a folate uptake buffer (Hank's balanced
salt solution, supplemented with 0.14 g/L CaCl.sub.2, 0.1 g/L
MgCl.sub.2, and 0.1 g/L MgSO.sub.4, pH 6.0) for 1 hour. The buffer
was then aspirated, and uptake was initiated by adding 0.2 mL of
fresh folate uptake buffer containing 2 .mu.Ci/mL radioactive
folate (3,5,7,9-.sup.3H-folic acid, 25 mCi/mmol, ARC) and cold,
unlabeled folate giving a final folate concentration of 5 .mu.M.
The folate uptake was terminated by removing the uptake buffer at
designated time intervals. The cells were then washed three times
with ice-cold PBS and lysed by the addition of 0.2 mL of 0.2N NaOH,
followed by incubation at 65.degree. C. for 20 min. Intracellular
uptake of .sup.3H-folate was determined by transferring 20 .mu.L of
the cell lysate to the filter-bottomed UniFilter plates
(Perkin-Elmer) and counting as described previously in Example 1.
The amount of folate accumulated in the cells was calculated and
normalized to protein concentration, and uptake rate was expressed
as picomoles of folate per minutes per milligram of cell protein
(pmol/min/mg). Protein concentration was determined by a standard
Bicinchoninic acid (BCA) protein assay as described above.
[0049] Referring to FIG. 4, Caco2 cells treated with AS1 of Formula
I at the concentration of 0.1 .mu.M was found to exhibit an
increased folate uptake from the control group having non-treated
Caco2 cells. The regulatory effects of the isolated astragalosides
on the folate uptake in Caco2 cells are listed in Table 5 below,
wherein the arrows that point up represent the enhancing effect on
folate uptake.
TABLE-US-00005 TABLE 5 Regulatory effects of isolated
astragalosides on folate uptake Uptake rate (pmol/mg/min)
Percentage ( % ) * Control 53.140 .+-. 3.5540 -- 0.1 .mu.M AS1
79.710 .+-. 3.0410 150.00 .uparw. Control 54.220 .+-. 3.1730 -- 0.1
.mu.M AS2 69.970 .+-. 3.7720 129.05 .uparw. Control 55.280 .+-.
0.8527 -- 0.1 .mu.M AS3 80.380 .+-. 6.2170 145.41 .uparw. Control
56.030 .+-. 0.9678 -- 0.1 .mu.M AS4 75.710 .+-. 5.2390 135.12
.uparw. Control 60.240 .+-. 6.6510 -- 0.1 .mu.M AS6 84.560 .+-.
4.7200 140.37 .uparw. Control 53.010 .+-. 6.3290 -- 0.1 .mu.M AA
84.030 .+-. 4.9410 158.52 .uparw. Control 50.720 .+-. 3.7550 -- 0.1
.mu.M 73.460 .+-. 3.6060 144.83 .uparw. IsoAS1 Control 53.19 .+-.
1.98 -- 0.1 .mu.M 86.63 .+-. 2.82 162.87 .uparw. IsoAS2
[0050] It is concluded that the uptake of folate can be enhanced
with the administration of the astragaloside isolated from
Astragalus membranaceus var. mongholicus, including AS1 of Formula
I, AS2 of Formula II, AS3 of Formula III, AS4 of Formula IV, AS6 of
Formula VI, AA of Formula VIII, IsoAS1 of Formula V or IsoAS2 of
Formula VII.
[0051] Although the above examples described regulating nutrient
absorption of the colon cancer cells, it should be noted that the
present invention is not limited as such. The gut cells and cells
of gastrointestinal system should also be expected to benefit from
the regulatory effect of the astragaloside compounds proposed in
the present invention as long as these cells have similar nutrient
transporting mechanisms. Besides a regulatory role in glucose,
arginine, tryptophan and folate absorption, the astragaloside
compounds described in the present invention may equivalently apply
to regulate absorption of nutrients which include vitamins, amino
acids, hormones, growth factors, and other elements important for
cell metabolism. Moreover, the nutrient absorption test and
nutrient uptake test described in the embodiments may be
implemented interchangeably for assessing and evaluating the
regulatory effect of the isolated astragaloside on the nutrient
absorption of the individual according to the present
invention.
[0052] It will be appreciated by those skilled in the art that
changes could be made to the embodiments described above without
departing from the broad inventive concept thereof. It is
understood, therefore, that this invention is not limited to the
particular embodiments disclosed, but it is intended to cover
modifications within the spirit and scope of the present invention
as defined by the appended claims.
* * * * *