PHARMACEUTICAL COMPOSITION FOR TREATING DIABETES AND PREPARATION METHOD THEREOF

COMPOSITION PHARMACEUTIQUE SERVANT AU TRAITEMENT DU DIABETE ET METHODE DE PREPARATION ASSOCIEE

English Abstract

The present invention declares a pharmaceutical composition for treating
diabetes,
said pharmaceutical composition mainly comprises Feculae Bombycis and Radix
Glycyrrhizae. The present invention declares also declares preparation method
and
quality control method of said pharmaceutical composition. Said pharmaceutical

composition can reduce blood sugar as well as relieve intestinal tympanites.

French Abstract

L'invention porte sur une composition pharmaceutique pour traiter le diabète et sur ses procédés de préparation, ses procédés de contrôle qualité et ses utilisations ; ladite composition pharmaceutique est préparée à l'aide d'excréments de ver à soie et de réglisse sauvage en tant que matières premières ; non seulement elle peut réduire le sucre dans le sang, mais encore elle peut améliorer la flatulence.

Claims

CLAIMS
1. A pharmaceutical composition consisting of:
6000-8000 parts by weight of a material of Feculae Bombycis; and
100-200 parts by weight of a material of Radix Glycyrrhizae;
wherein said composition is prepared by the following method:
taking Feculae Bombycis, adding 50-70% ethanol at 2-6 fold the amount of
Feculae
Bombycis, soaking overnight, heating up slowly to boil, refluxing for 1-3
times, each
time 0.5-1.5 hours, filtering, combining the filtrates, removing ethanol,
after the
ethanol is removed, heating the filtrates up to concentrate or vacuum
concentrating
the filtrates at a temperature below 80°C until the relative density of
0.95-1.10,
measured at the temperature 55°C, then obtaining a concentrated
solution, adding
water and heating to boiling point, cooling, leave to stand overnight, then
taking a
supernatant A, heating up to concentrate the supernatant, or vacuum
concentrating the
supernatant at 80°C to obtain a thick paste A with the relative density
of 1.00-1.15,
measured at the temperature 55°C, let the thick paste A stand; taking
Radix
Glycyrrhizae, adding 8-15 fold the amount of Radix Glycyrrhizae with water,
decocting 1-3 times, each time 1-3 hours, combining the decoctions, leave to
stand
overnight to precipitate, concentrating a supernatant B to a thick paste B
with the
relative density of 1.00-1.15, measured at a temperature 55°C, let the
thick paste B
stand; combining the thick paste A and the thick paste B thereby producing
said
composition.
2. The pharmaceutical composition of claim 1, said pharmaceutical composition
consisting of:
7150 parts by weight of the material of Feculae Bombycis; and
137.5 parts by weight of the material of Radix Glycyrrhizae.
3. The pharmaceutical composition of claim 1, said pharmaceutical composition
consisting of:
6150 parts by weight of the material of Feculae Bombycis; and
187.5 parts by weight of the material of Radix Glycyrrhizae.
26

4. The pharmaceutical composition of claim 1, said pharmaceutical composition
consisting of:
7850 parts by weight of the material of Feculae Bombycis
117.5 parts by weight of the material of Radix Glycyrrhizae.
5. The pharmaceutical composition of any one of claims 1 to 4, wherein said
method
is:
taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae
Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times,
each
time 1 hour, filtering, combining the filtrates, removing ethanol, after the
ethanol is
removed, heating the filtrates up to concentrate or vacuum concentrating the
filtrates
at a temperature below 80°C until the relative density of 1.01-1.06
measured at the
temperature 55°C, then obtaining concentrated solution, adding same
amount water
and heating to boiling, cooling, leaving to stand overnight, then taking
supernatant A,
heating up to concentrate the supernatant, or vacuum concentrating at
80°C to obtain
the thick paste A with the relative density of 1.06-1.15, measured at the
temperature
55°C; taking Radix Glycyrrhizae, adding 10 folds amount of Radix
Glycyrrhizae with
water, decocting 2 times, each time 2 hours, combining the decoctions, leaving
to
stand overnight to allow precipitation, concentrating the supernatant B to the
thick
paste B with the relative density of 1.04-1.15, measured at a temperature
55°C;
combining the thick paste A and the thick paste B.
6. The pharmaceutical composition of any one of claims 1 to 5, further
comprising a
general adjuvant.
7. The pharmaceutical composition of claim 6 formulated in the form of a
tablet,
capsule, powder, soft capsule, drop pill, honey pill, pill, granule, honey
refilled
extract, sustained-release preparation, rapid-release preparation, controlled
release
preparation, oral liquid preparation or injection preparation.
8. A method for controlling quality of the pharmaceutical composition of any
one of
claims 1 to 5, wherein said method includes the following identification and
content
determination steps
ldentification:
27

A Adding 0.4-0.6 g of the pharmaceutical composition in 4-6 mL anhydrous
ethanol
to produce a solution, refluxing the solution at 40-80°C in a water
bath for 20-40
minutes and then letting the solution standing. taking a supernatant from the
solution
and evaporating the supernatant above a water bath until 0.5-1.5 mL is left.
to provide
a test sample solution;
furthermore, taking .beta.-sitosterol as a reference substance which is added
to anhydrous
ethanol to provide a reference substance solution having a concentration of
0.0005-
0.001 5 g/mL;
performing a thin layer chromatography test by sucking 0.001-0.003 mL of said
test
sample solution and 0.001-0.003 mL of said reference substance solution,
spotting
them on a same TLC (thin layer chromatography) silica gel plate and developing

them by using a developing solvent composed of chloroform and acetone at a
weight
ratio of 18-21 : 0.3-0.7, then drying the silica gel plate and spraying it
with 5% -15%
phosphor-molybdic acid solution. followed by heating the silica gel plate at
100-
110°C until a chromatogram is visualized; the chromatogram produced by
the test
sample solution shows the same color spots as that displayed by the reference
substance solution;
B identification by high-performance liquid chromatography, using
octadecylsilanized silica gel as a packing; using a mixture of methanol,
ammonium
acetate of 0.2mol/I. and acetic acid at a weight ratio of 50-70 : 30-50 : 0.5-
1.5 as a
mobile phase; detection wavelength is 250nm; theoretical plate number counted
according to peak ammonium glycyrrhizinate is not less than 2000;
weighing 0.005-0.015 g mono-ammonium glycyrrhizinate accurately, dissolving it

with said mobile phase in a volumetric flask to 50 mL and shaking up. to
provide a
reference substance solution containing mono-ammonium glycyrrhizinate at
0.0001-
0.0003 g/mL, which is equivalent to 0.00019-0.00020 g/mL of glycyrrhizic acid;
taking 1.0-3.0 g of said pharmaceutical composition, putting it into a
volumetric flask
of 25 mL, adding 18-22 mL of said mobile phase into the flask to produce a
mixture,
exposing the mixture to ultrasound treatment for 20-40 minutes with power 250W

and frequency of 50KHz, then cooling the mixture, adding the mobile phase to
the
mixture until the volume reaches 25mL, shaking up the mixture, followed by
28

centrifuging the mixture, and then taking a supernatant from the centrifuged
mixture
for using as a test sample solution,
accurately sucking 0.005-0.015 ml, of the reference substance solution and
0.005-
0.015 mL of the test sample solution and injecting them into a liquid
chromatogram
instrument. ensuring that the test sample solution should show a
chromatographic
peak having a same retention time as that of the reference substance solution;
Content determination:
An applicability test of chromatogram condition and system
using octadecylsilanized silica gel as a packing; using a mixture of
acetonitrile,
dimethyl formamide and sodium acetate of 0.025mol/L at a weight ratio of 15-
25:
0.1-1: 75-85 as a mobile phase;
detection wavelength is 390nm, and the number of theoretical plates is not
less than
2500 counted according to the peak of pipecolic acid;
accurately weighing 0.004-0.006 g of pipecolic acid as reference compound,
putting
it into a first volumetric flask, adding water to dilute to 50 mL and shaking
up to
produce a first solution, measuring 0.5-1.5 mL of the first solution
accurately and
putting it into a first plugged test tube with a volume of 10 mL, adding 0.5-
1.5 mL
water, 1-3 mL 2,4-acetonitrile dinitrofluorobenzene solution having a
concentration
of 0.8% by weight and 1-3 mL sodium bicarbonate solution having a
concentration
of 0.5mol/L to the tube to produce a second solution, heating the second
solution at
60 C in a water bath for 0.5-1.5 hours, taking out the second solution in the
tube from
the water bath, cooling the second solution, and transferring the second
solution from
the tube into a second volumetric flask of 10mL. washing the tube with a
phosphate
hurler solution having a concentration of 0.2mol/L and a pH value of 7.0 for
several
times, collecting the phosphate buffer solution after each of said washing and

combining it into the second volumetric flask, adding a further amount of the
phosphate buffer solution to the second volumetric flask until the volume
reaches
10mL, and then shaking the second volumetric flask to provide a reference
compound
solution;
weighing 0.3-0.5 g of the pharmaceutical composition accurately, placing it in
a
second plugged test tube with a volume of 10 mL, adding 5 mL water in the tube
and

shaking it up fully to produce a third solution, heating up the tube
containing the third
solution at 50-70°C in a water bath for 20-40 minutes, taking out the
tube and cooling
the third solution, and transferring the third solution from the tube into a
third
volumetric flask of 10 mL, washing the tube with water for several times,
collecting
the water after each of said washing and combining it into the third
volumetric flask,
adding a further amount of water to the third volumetric flask until the
volume
reaches 10mL, and then shaking up the third volumetric flask to produce a
fourth
solution; centrifuging the fourth solution at 12000 rpm for 5-15 minutes to
produce a
centrifuged fourth solution, collecting a supernatant from the centrifuged
fourth
solution, measuring 1-3 mL of the supernatant accurately and placing it into a
third
plugged test tube of 10 mL, adding 1-3 mL 2,4-acetonitrile
dinitrofluorobenzene
solution having a concentration of 0.8% by weight and 1-3 mL sodium
bicarbonate
solution having a concentration of 0.5mol/L into the third tube to produce a
fifth
solution, heating up the fifth solution at 50-70°C in a water bath for
0.5-1.5 hours,
taking out the fifth solution from the water bath and cooling it, and
transferring it
from the third tube into a fifth volumetric flask of 10 mL, washing the third
tube with
a phosphate buffer solution having a concentration of 0.2mol/L and a pH of 7.0
for
several times, collecting the phosphate buffer solution after each of said
washing and
combining it into the fifth volumetric flask, adding a further amount of the
phosphate
buffer solution into the fifth volumetric flask until the volume reaches 10mL,
shaking
up, to provide a test sample solution;
accurately sucking 0.004-0.006 mL of the reference compound solution and 0.005-

0.015 mL of the test sample solution, respectively injecting them into a
liquid
chromatogram instrument for determination, and determining Feculae Bombycis
content in the pharmaceutical composition, counted according to pipecolic acid

(C6H11NO2), whereby said pharmaceutical composition has a positive quality
control
when said Feculae Bombycis content is not less than 0.0001g.
9. The method of claim 8, wherein said step of identification is:
A Adding 0.5 g of the pharmaceutical composition in 5 mL anhydrous ethanol to
produce a solution, refluxing the solution at 60°C in a water bath for
30 minutes and
then letting the solution standing, taking a supernatant from the solution and

evaporating the supernatant above a water bath until 1 mL is left, to provide
a test
sample solution;

furthermore, taking .beta.-sitosterol as a reference substance which is added
with
anhydrous ethanol to provide a reference substance solution to provide a
reference
substance solution having a concentration of-0.001 g/mL;
performing a thin layer chromatography test by sucking 0.002 mL of said test
sample
solution and 0.002 mL of said reference substance solution, spotting them on a
same
thin layer chromatography (TLC) silica gel plate and developing them by using
a
developing solvent composed of chloroform and acetone at a weight ratio of
19.5:0.5,
then drying the silica gel plate, and spraying it with 10% phosphor-molybdic
acid
solution, followed by heating the silica gel plate at 105°C until a
chromatogram is
visualized; the chromatogram produced by the test sample solution shows the
same
color spots as that displayed by the reference substance solution;
B ldentification by high-performance liquid chromatography, using
octadecylsilanized silica gel as a packing; using a mixture of methanol,
ammonium
acetate of 0.2mol/L and acetic acid at a weight ratio of 60:40:1 by weight as
a mobile
phase; detection wavelength is 250nm; theoretical plate number counted
according to
peak ammonium glycyrrhizinate is not less than 2000;
weighing 0.01 g mono-ammonium glycyrrhizinate accurately, dissolving it with
said
mobile phase in a volumetric flask to 50 mL and shaking up to provide a
reference
substance solution containing mono-ammonium glycyrrhizinate at 0.0002 g/mL,
which is equivalent to 0.0001959 g/mL of glycyrrhizic acid;
taking 2 g of said pharmaceutical composition, putting it into a volumetric
flask of 25
mL, adding 20 mL of said mobile phase, exposing the mixture to ultrasound
treatment
for 30 minutes with power 250W and frequency of 50KHz, then cooling the
mixture,
adding the mobile phase to the mixture until the volume reaches 25 mL, shaking
up
the mixture, followed by centrifuging the mixture, taking a supernatant from
the
centrifuged mixture for using as a test sample solution;
accurately sucking 0.01 mL of the reference substance solution and 0.01 mL of
the
test sample solution and injecting them into liquid chromatogram instrument,
ensuring that the test sample solution should show a chromatographic peak
having a
same retention time as that of the reference substance solution; and
said step of content determination is :
31

An applicability test of chromatogram condition and system
using octadecylsilanized silica gel as a packing;
using a mixture of acetonitrile, dimethyl formamide and sodium acetate of
0.025mol/L at a weight ratio of 21:0.5:79 as a mobile phase;
detection wavelength is 390nm, the number of theoretical plates is not less
than 2500
counted according to peak of pipecolic acid;
accurately weighing 0.01 g of pipecolic acid as reference compound, putting it
into a
first volumetric flask, adding water to dilute to 100 mL and shaking up to
produce a
first solution, measuring 1 mL of the first solution accurately and putting it
into a first
plugged test tube with a volume of 10 mL, adding 1 mL water, adding 2 mL 2,4-
acetonitrile dinitrofluorobenzene solution of 0.8% by weight and 2 mL sodium
bicarbonate solution having a concentration of 0.5mol/L to the tube to produce
a
second solution, heating the second solution at 60°C in a water bath
for 1 hour, taking
out the second solution in the tube from the water bath, cooling the second
solution,
and transferring the second solution into a second volumetric flask of 10 mL,
washing
the tube with phosphate buffer solution having a concentration of 0.2mol/L and
a pH
value of 7.0 for 3 times, collecting the phosphate buffer solution after each
of said
washing and combining it into the second volumetric flask, adding a further
amount
of the phosphate buffer solution to the second volumetric flask until the
volume
reaches 10mL, and then shaking the second volumetric flask to provide a
reference
compound solution;
weighing 0.4 g of the pharmaceutical composition accurately, placing it in a
second
plugged test tube with a volume of 10 mL, adding 5 mL water in the tube and
shaking
it up fully to produce a third solution, heating up the tube containing the
third solution
at 60°C in a water bath for 30 minutes, taking out the tube and cooling
the third
solution, and transferring the third solution from the tube into a third
volumetric flask
of 10 mL. washing the tube with water for 3 times, collecting the water after
each of'
said washing and combining it into the third volumetric flask and adding a
further
amount of water to the third volumetric flask until the volume reaches 10mL,
and
then shaking up the third volumetric flask to produce a fourth solution;
centrifuging
the fourth solution at 12000 rpm for 10 minutes to produce a centrifuged
fourth
solution, collecting a supernatant from the centrifuged fourth solution,
measuring 2
32

mL of the supernatant accurately and placing it into a third plugged test tube
of 10
mL, adding 2 mL of 2,4-acetonitrile dinitrofluorobenzene solution having a
concentration of 0.8% by weight and 2 mL of sodium bicarbonate solution having
a
concentration of 0.5mol/L into the third tube to produce a fifth solution,
heating up
the fifth solution at 60°C in a water bath for 1 hour, taking out the
fifth solution from
the water bath and cooling it, and transferring it from the third plugged tube
into a
fifth volumetric flask of 10 mL, washing the third plugged tube with phosphate
buffer
solution having a concentration of 0.2mol/L and a pH of 7.0 for 3 times,
collecting
the phosphate buffer solution after each of said washing and combining it into
the
fifth volumetric flask, adding a further amount of the phosphate buffer
solution into
the fifth volumetric flask until the volume reaches 10mL, shaking up to
provide a test
sample solution;
accurately sucking 0.005 mL reference compound solution and 0.01 mL test
sample
solution, respectively injecting them into a liquid chromatogram instrument
for
determination, and determining Feculae Bombycis content in the pharmaceutical
composition, counted according to pipecolic acid (C6H11NO2), whereby said
pharmaceutical composition has a positive quality control when said Feculae
Bombycis content is not less than 0.0002g.
10. Use of the pharmaceutical composition of any one of claims 1 to 5 in the
preparation of a medicine for treating diabetes.
11. The use of claim 10, wherein said treating diabetes is to improve on the
symptoms
of polydipsia or polyphagia compared to a control group.
12. The use of claim 10, wherein said treating diabetes is to decrease blood
glucose
and serum fructosamine compared to a control group.
13. The use of claim 10, wherein said treating diabetes is to decrease serum
cholesterol and triglyceride levels compared to a control group.
14. The use of claim 10, wherein said treating diabetes is to decrease serum
NAG-ase
activity and improve microvascular complications compared to a control group.
15. The use of claim 10, wherein said treating diabetes is to decrease
sorbitol content
in sciatic nerve and improve chronic neuropathy of diabetes compared to a
control
group.
33

16. The use of claim 10, wherein said treating diabetes is to increase GSH
content in
red blood cell, and enhance body's antioxidant capacity compared to a control
group.
17. The use of claim 10, wherein said treating diabetes is to lower kidney
weight,
body mass index, reduce serum creatinine levels or relieve glycogen deposition
in
renal tubular epithelial cells compared to a control group.
18. Use of said pharmaceutical composition of any one of claim 1 to 5 for
treating
diabetes.
19. The use of claim 18, wherein said treating diabetes is to improve on the
symptoms
of polydipsia or polyphagia compared to a control group.
20. The use of claim 18, wherein said treating diabetes is to decrease blood
glucose
and serum fructosamine compared to a control group.
21. The use of claim 18, wherein said treating diabetes is to decrease serum
cholesterol and triglyceride levels compared to a control group.
22. The use of claim 18, wherein said treating diabetes is to decrease serum
NAG-ase
activity and improve microvascular complications compared to a control group.
23. The use of claim 18, wherein said treating diabetes is to decrease
sorbitol content
in sciatic nerve and improve chronic neuropathy of diabetes compared to a
control
group.
24. The use of claim 18, wherein said treating diabetes is to increase GSH
content in
red blood cell, and enhance body's antioxidant capacity compared to a control
group.
25. The use of claim 18, wherein said treating diabetes is to lower kidney
weight,
body mass index, reduce serum creatinine levels or relieve glycogen deposition
in
renal tubular epithelial cells compared to a control group.
26. The use of claim 18, wherein said treating diabetes is to inhibit .alpha.-
glucosidase
activity.
34

Description

CA 02710862 2013-05-24
PHARMACEUTICAL COMPOSITION FOR TREATING DIABETES AND
PREPARATION METHOD THEREOF
FIELD OF THE INVENTION
The present invention relates to a pharmaceutical composition and its
preparation
method, especially relates to a pharmaceutical composition for treating
diabetes, and
its preparation method and quality control method.
BACKGROUD OF THE INVENTION
Incidence of Diabetes (diabetes) becomes increasingly in china and abroad,
which
seriously harms people's health and even their lives. At present, it is
understood that
there are more than 200 million diabetics in the worldwide, and 85% of them
are elder
diabetics. There are 30 million diabetics in china, only second to the United
States,
but increasing at an annual rate of 750,000 new patients. Therefore, diabetes
has
belonged to one of major difficult diseases in national "95" projects, but
efficacy of
anti-diabetic traditional Chinese medicine is presently not satisfactory, and
Acarbose
and other drugs commonly used for treating diabetes can cause flatulence,
borborygmus and other side effects. Consequently, it is necessary to carry out
research
on prevention and treatment of diabetes.
SUMMARY OF INVENTION
One purpose of present invention is to provide a pharmaceutical composition
for
treating diabetes; another purpose of the present invention is to provide
preparation
method and quality control method of this composition.
The purposes of present invention can be implemented through the following
technical schemes:
Raw materials of this invention pharmaceutical composition consist of:
Feculae Bombycis (silkworm excrement) 6000-8000 parts by weight
Radix Glycyrrhizae (licorice) 100-200 parts by weight.
Preferably, raw materials of this invention pharmaceutical composition consist
of:
Feculae Bombycis 7150 parts by weight
Radix Glycyrrhizae 137.5 parts by weight.
Preferably, raw materials of this invention pharmaceutical composition consist
of:
Feculae Bombycis 6150parts by weight
Radix Glycyrrhizae 187.5parts by weight.
Preferably, raw materials of this invention pharmaceutical composition consist
of:
Feculae Bombycis 7850 parts by weight
Radix Glycyrrhizae 117.5 parts by weight.
1

CA 02710862 2010-06-25
According to usual process by adding general adjuvant, this invention
pharmaceutical composition is produced to tablet, capsule, powder, soft
capsule, drop
pill, honey pill, pill, granule, honey refined extract, sustained-release
preparation,
rapid-release preparation, controlled-release preparation, oral liquid or
injection.
The preparation method of pharmaceutical composition of this invention can
also
comprise:
Taking Feculae Bombycis, adding 50-70% ethanol at 2-6 folds amount of Feculae
.. Bombycis, soaking overnight, heating up slowly to boil, refluxing for 1-3
times, each
time 0.5-1.5 hours, filtering, combining the filtrates, recover ethanol, after
the ethanol
is removed, heating the filtrates up to concentrate or vacuum concentrated at
a
temperature below 80 C until the relative density of 0.95-1.10 (measured at
the
temperature 55 C), then obtaining concentrated solution, adding water and
heating to
boiling, cooling, placing overnight, then taking supernatant A, heating up to
concentrate the supernatant, or vacuum concentrating the supernatant at 80 C
to
obtain the thick paste A with the relative density of 1.00-1.15 (measured at
the
temperature 55 C), stand it by service; taking Radix Glycyrrhizae, adding 8-15
folds
amount of Radix Glycyrrhizae with water, decocting 1-3 times, each time 1-3
hours,
combining the decoctions, placing overnight to precipitate, concentrating the
supernatant B to the thick paste B with the relative density of 1.00-1.15
(measured at a
temperature 5500), stand it by service; combining the thick paste A and the
thick paste
B, according to usual process by adding general adjuvant, then they are
produced to
tablet, capsule, powder, soft capsule, drop pill, honey pill, pill, granule,
honey refined
extract, sustained-release preparation, rapid-release preparation, controlled-
release
preparation, oral liquid or injection. or
Taking Feculae Bombycis, adding 50-70% ethanol at 2-6 folds amount of
Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for
1-3
.. times, each time 0.5-1.5 hours, filtering, combining the filtrates, recover
ethanol, after
the ethanol is removed, heating the filtrates up to concentrate or vacuum
concentrated
at a temperature below 80 C until the relative density of 0.95-1.10 (measured
at the
temperature 55 C), then obtaining concentrated solution, adding water and
heating to
boiling, cooling, placing overnight, then taking supernatant A, heating up to
concentrate the supernatant, or vacuum concentrating the supernatant at 80 C
to
obtain the thick paste A with the relative density of 1.00-1.15 (measured at
the
temperature 55 C), stand it by service; taking Radix Glycyrrhizae, adding 8-15
folds
amount of Radix Glycyrrhizae with water, decocting 1-3 times, each time 1-3
hours,
combining the decoctions, placing overnight to precipitate, concentrating the
supernatant B to the thick paste B with the relative density of 1.00-1.15
(measured at a
temperature 55 C), stand it by service; combining the thick paste A and the
thick paste
B, adding an appropriate amount of (3-cyclodextrin or micro silica, spraying
drying,
adding an appropriate amount of hydroxypropyl cellulose (L-HPC) or other
general
adjuvant, blending, granulating, according to usual process by adding general
2

_
. CA 02710862 2010-06-25
..
_
adjuvant, then they are produced to tablet, capsule, powder, soft capsule,
drop pill,
honey pill, pill, granule, honey refined extract, sustained-release
preparation,
rapid-release preparation, controlled-release preparation, oral liquid or
injection.
Preferably, the preparation method of pharmaceutical composition of this
invention
is:
Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae
Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times,
each
time 1 hour, filtering, combining the filtrates, recover ethanol, after the
ethanol is
removed, heating the filtrates up to concentrate or vacuum concentrated at a
temperature below 80 C until the relative density of 1.01-1.06 (measured at
the
temperature 55 C), then obtaining concentrated solution, adding same amount
water
and heating to boiling, cooling, placing overnight, then taking supernatant A,
heating
up to concentrate the supernatant, or vacuum concentrating the supernatant at
80 C to
obtain the thick paste A with the relative density of 1.06-1.15 (measured at
the
temperature 55 C), stand it by service; taking Radix Glycyrrhizae, adding 10
folds
amount of Radix Glycyrrhizae with water, decocting 2 times, each time 2 hours,

combining the decoctions, placing overnight to precipitate, concentrating the
supernatant B to the thick paste B with the relative density of 1.04-1.15
(measured at a
temperature 55 C), stand it by service; combining the thick paste A and the
thick paste
B, adding 13-cyclodextrin or micro silica with amount of 20% Feculae Bombycis
dry
paste , spraying drying, adding hydroxypropyl cellulose (L-HPC) in amount of
1%
drug powder, blending, according to usual process by adding general adjuvant,
then
they are produced to tablet, capsule, powder, soft capsule, drop pill, honey
pill, pill,
granule, honey refmed extract, sustained-release preparation, rapid-release
preparation,
controlled-release preparation, oral liquid or injection.
Quality control method of this invention pharmaceutical composition includes
one
or more of following identification and/or content determination:
Identification:
A. Taking 0.4-0.6g pharmaceutical composition of this invention, adding 4-6 ml
anhydrous ethanol, refluxing in 40-80 C water bath for 20-40 minutes, placing
and
taking supernatant, evaporating it until 0.5-1.5 ml above water bath, that is
test sample
solution; furthermore, taking 0-sitosterol as reference substance, adding
anhydrous
ethanol for 0.0005-0.0015g/m1 solution, that is reference substance solution;
according to thin layer chromatography test, sucking above two solutions 0.001-

0.003m1 respectively, pointing them on the same TLC plate of silica gel G
respectively, developing, taking out, drying, 18-21:0.3-0.7 of chloroform:
acetone as
mobile phase, and spraying with 5% -15% phosphor-molybdic acid solution,
heating
in 100-110 C until visualize the chromatogram; the chromatogram produced by
the
test sample solution shows the same color spots as that displayed by the
reference
substance solution in their respectively corresponding areas;
B. Identification by high-performance liquid chromatography, Using
3

CA 02710862 2010-06-25
. -
octadecylsilanized silica gel as packing; using methanol-(0.2mo1 / L) ammonium

acetate - acetic acid = 50-70:30-50:0.5-1.5 as mobile phase; detection
wavelength
250nm; theoretical plate number counted according to peak ammonium
glycyrrhizinate is not less than 2000; weighing 0.005-0.015g mono-ammonium
glycyrrhizinate reference compound accurately, putting it into 50m1 volumetric
flask,
dissolving by above mobile phase to the volume, shaking up, that is, 0.0001-
0.0003g
mono-ammonium glycyrrhizinate per ml, which is equivalent to 0.00019-0.00020g
glycyrrhizic acid, that is reference substance solution;
taking pharmaceutical compositions of this invention 1.0-3.0g, put it into 25
ml
volumetric flask, adding 18-22m1 above mobile phase, deal with ultrasound for
20-40
minutes with power 250W, frequency of 50KHz, taking out and cooling, adding
mobile phase to the volume, shaking up, centrifuging, taking the supernatant,
that is
test sample solution;
accurately sucking reference substance solution and test sample solution
0.005-0.015m1 respectively, injecting into liquid chromatogram instrument,
test
sample should be presented the same retention time peaks with the reference
substance.
Content determination:
An applicability test of chromatogram condition and system
Using octadecylsilanized silica gel as packing; using a solution with
acetonitrile-dimethyl formamide-sodium acetate(0.025mol/L)= 15-25: 0.1-1: 75-
85 as
mobile phase; detection wavelength is 390nm, theoretical plate number counted
according to peak Pipecolic acid is not less than 2500;
Weighing 0.004-0.006g Pipecolic acid reference compound accurately, put it
into
50m1 volumetric flask, adding water to dilute to the volume, shaking up,
measuring
0.5-1.5m1 the said solution accurately, putting it into 10m1 test tube with
plug, adding
0.5-1.5m1 water, adding 1-3m1 2,4-acetonitrile dinitrofluorobenzene
solution(0.8%)
and 1-3m1 sodium bicarbonate solution(0.5mol/L), heating up in 60 C water bath
for 0.5-1.5 hours, taking out, cooling, and transferring into 10m1 volumetric
flask,
washing container with phosphate buffer solution (0.2moUL, pH7.0) by several
times,
combining the washing solution into volumetric flask, adding phosphate buffer
solution to the volume, shaking up, that is reference compound solution;
weighing 0.3-0.5g the pharmaceutical composition of present invention
accurately,
placing it in 10m1 test tube with plug, adding water, shaking up fully,
heating up in
50-70 C water bath for 20-40 minutes, taking out, cooling, and transferring
into 10m1
volumetric flask, washing container with water by several times, combining the

washing solution into volumetric flask and adding water to volume, and shaking
up;
centrifuging 12000 mm for 5-15 minutes, measuring 1-3m1 the supernatant
accurately,
placing it into 10m1 test tube with plug, adding 1-3m1 2,4-acetonitrile
dinitrofluorobenzene solution(0.8%) and 1-3m1 sodium bicarbonate
solution(0.5moUL), heating up in 50-70 C water bath for 0.5-1.5 hours, taking
out,
cooling, and transferring into 10m1 volumetric flask, washing container with
4

CA 02710862 2010-06-25
phosphate buffer solution (0.2mol/L, pH7.0) by several times, combining the
washing
solution into volumetric flask, adding phosphate buffer solution to the
volume,
shaking up, that is test sample solution, accurately sucking 0.004-0.006m1
reference
substance solution and 0.005-0.015m1 test sample solution respectively,
injecting into
liquid chromatogram instrument, determining, that is; Feculae Bombycis content
in
the pharmaceutical composition of this invention in dosage per day, counted
according to Pipecolic acid (C6H1 IN02), is not less than 0.0001-0.0003g.
Preferably, Quality control method of this invention pharmaceutical
composition
includes one or more of following identification and/or content determination:

Identification:
A. Taking 0.5g pharmaceutical composition of this invention, adding 5 ml
anhydrous ethanol, refluxing in 60 C water bath for 30 minutes, placing and
taking
supernatant, evaporating it until 1 ml above water bath, that is test sample
solution;
furthermore, taking 13-sitosterol as reference substance, adding anhydrous
ethanol for
0.001g/m1 solution, that is reference substance solution; according to thin
layer
chromatography test, sucking above two solutions 0.002m1 respectively,
pointing
them on the same TLC plate of silica gel G respectively, developing, taking
out,
drying, 19.5:0.5 of chloroform: acetone as mobile phase, and spraying with 10%
phosphor-molybdic acid solution, heating in 105 C until visualize the
chromatogram;
the chromatogram produced by the test sample solution shows the same color
spots as
that displayed by the reference substance solution in their respectively
corresponding
areas;
B. Identification by high-performance liquid chromatography, Using
octadecylsilanized silica gel as packing; using methanol-(0.2mo1 / L) ammonium

acetate - acetic acid = 60:40:1 as mobile phase; detection wavelength 250nm;
theoretical plate number counted according to peak ammonium glycyrrhizinate is
not
less than 2000; weighing 0.01g mono-ammonium glycyrrhizinate reference
compound
accurately, putting it into 50m1 volumetric flask, dissolving by above mobile
phase to
the volume, shaking up, that is, 0.0002g mono-ammonium glycyrrhizinate per ml,

which is equivalent to 0.0001959g glycyrrhizic acid, that is reference
substance
solution;
taking pharmaceutical compositions of this invention 2g, putting it into 25 ml

volumetric flask, adding 20m1 above mobile phase, deal with ultrasound for 30
minutes with power 250W, frequency of 50KHz, taking out and cooling, adding
mobile phase to the volume, shaking up, centrifuging, taking the supernatant,
that is
test sample solution;
accurately sucking reference substance solution and test sample solution
0.01m1
respectively, injecting into liquid chromatogram instrument, test sample
should be
presented the same retention time peaks with the reference substance.
5

CA 02710862 2010-06-25
Content determination:
An applicability test of chromatogram condition and system
Using octadecylsilanized silica gel as packing; using a solution with
acetonitrile-dimethyl formamide-sodium acetate(0.025mol/L)= 21:0.5:79 as
mobile
phase; detection wavelength is 390nm, theoretical plate number counted
according
to peak Pipecolic acid is not less than 2500;
Weighing 0.01g Pipecolic acid reference compound accurately, putting it into
100m1 volumetric flask, adding water to dilute to the volume, shaking up,
measuring
1m1 the said solution accurately, putting it into 10m1 test tube with plug,
adding lml
water, adding 2m1 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 2m1
sodium bicarbonate solution(0.5mol/L), heating up in 60 C water bath for 1
hour,
taking out, cooling, and transferring into 10m1 volumetric flask, washing
container
with phosphate buffer solution (0.2mol/L, pH7.0) by 3 times, combining the
washing
solution into volumetric flask, adding phosphate buffer solution to the
volume,
shaking up, that is reference compound solution;
weighing 0.4g the pharmaceutical composition of the present invention
accurately,
placing it in 10m1 test tube with plug, adding 5m1 water, shaking up fully,
heating up
in 60 C water bath for 30 minutes, taking out, cooling, and transferring into
10m1
volumetric flask, washing container with water by 3 times, combining the
washing
solution into volumetric flask and adding water to volume, and shaking up;
centrifuging 12000 rpm for 10 minutes, measuring 2m1 the supernatant
accurately,
placing it into 10m1 test tube with plug, adding 2m1 2,4-acetonitrile
dinitrofluorobenzene solution(0.8%) and 2m1 sodium bicarbonate
solution(0.5mol/L),
heating up in 60 C water bath for 1 hour, taking out, cooling, and
transferring into
10m1 volumetric flask, washing container with phosphate buffer solution
(0.2mol/L,
pH7.0) by 3 times, combining the washing solution into volumetric flask,
adding
phosphate buffer solution to the volume, shaking up, that is test sample
solution,
Accurately sucking 0.005m1 reference substance solution and 0.01m1 test sample

solution respectively, injecting into liquid chromatogram instrument,
determining, that
is; Feculae Bombycis content in the pharmaceutical composition of this
invention in
dosage per day, counted according to Pipecolic acid (C61111NO2), is not less
than
0.0002g.
In this invention, said relationship between weight parts and volume parts is
g / ml.
Quality control methods of pharmaceutical compositions of this invention can
be
applied to a variety of dosage forms of compositions, such as tablets,
capsules,
powders, soft capsules, dipping pills, honey pills, granules, honey refining
pastes,
slow-release preparations, rapid-release preparations, controlled-release
preparations,
oral liquid preparations, or injection preparations, and other clinically
acceptable
dosage forms. Because different dosage forms of preparations contain same
considerable amount of crude drug, in quality control of various dosage forms,

selected samples can be converted into relatively uniform dose of crude drug
congruously. Preparation per unit is corresponding to amount of crude drug 65g
(daily
dose) in the quality control method, and it can also be preparation per
tablet, per piece,
per injection, per pill, and so on.
6

= CA 02710862 2010-06-25
,
DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph depicting influence of MC for blood glucose of normal mice
after
sucrose feeding;
Figure 2 is a graph depicting influence of MC for blood glucose of normal mice
after
starch feeding;
Figure 3 is a graph depicting influence of MC for blood glucose of alloxan-
induced
hyperglycemic mice after sucrose feeding;
Figure 4 is a graph depicting influence of MC for blood glucose of alloxan-
induced
hyperglycemic mice after starch feeding;
Figure 5 is a graph depicting influence of MC for blood glucose of healthy
volunteers
after steamed bread feeding;
Figure 6 is a chart depicting influence of MC for urine glucose of alloxan-
induced
hyperglycemic rats;
Figure 7 is a chart depicting influence of MC for kidney weight/body mass
index of
alloxan-induced hyperglycemic rats;
Figure 8 is a chart depicting kidney, glomerulus and renal tubules of normal
rats show
no lesion X 50;
Figure 9 is a chart depicting that renal tubules epithelial cells of some
alloxan-induced
hyperglycemic rats group became swell with vacuolar appearance X 50;
Figure 10 is a chart depicting renal tubules of MC rat group rats show no
significant
lesion X50.
Experiments prove that pharmaceutical composition of this invention is better
than
raw material drugs used respectively. In addition to a hypoglycemic effect,
two drugs
combined have a synergistic effect, which is to improve on intestinal
tympanites.
After administrating this pharmaceutical composition for 4 weeks chronically,
it can
significantly improve the alloxan-induced hyperglycemic rats on common
symptoms
of polydipsia or polyphagia; decrease blood glucose and serum fructosamine
significantly; decrease serum cholesterol markedly and also triglyceride
levels;
decrease serum NAG-ase activity markedly, and explain to improve on
microvascular
complications; decrease sorbitol content in sciatic nerve markedly, and clue
to
improve on chronic neuropathy of diabetes; increase GSH content in red blood
cell,
and enhance body's antioxidant capacity; lower kidney weight and body mass
index
markedly, reduce serum creatinine levels, relief glycogen deposition in renal
tubular
epithelial cells; in addition, inhibit a-glucosidase activity of the
pharmaceutical
compositions of this invention in vitro is stronger, while free for a-amylase.
It explain
the pharmaceutical compositions of this invention can reduce blood sugar
elevation
induced by disaccharide hydrolysis absorb into blood, while the incidence of
gastrointestinal side effects is lower than similar products.
7

CA 02710862 2010-06-25
=
The following experiments and examples are for further explanation, but not
limiting the present invention.
The following experiments 1-8 use all following test medicines and
experimental animals.
Test medicines:
1. Extract powder of the said pharmaceutical composition (obtain in experiment
1),
abbreviated as MC, offered by Institute of Materia Medica Chinese Academy of
Medical Sciences, batch number: 96102, per gram of extract powder is
equivalent to
16.7g crude drug; MC extract powder dissolved with water; 2. Acarbose:
produced
by Bayer pharmaceutical, Germany, batch number: 264086D.
Animals:
Kunming mice, Beijing animal quality control file number (1994), No. 029;
Wistar
rats, Beijing animal quality control file number (1994), No. 030, were both
purchased
from breeding plants of Chinese Academy of Medical Sciences Institute, mice 22-
25g,
rat 180-250g, Sex: Male, animal number in each group: 10.
Alloxan-induced hyperglycemic mouse and rat models: normal animals are
injected
alloxan intravenously (mice 90-100 mg / kg, rats 45-50 mg / kg), predicted
blood
glucose (glucose oxidase method) after administering 72 hours, animals which
are
their blood glucose values over 11.1 mmol / L are selected for experiments.
EXPERIMENT 1: influence of MC for blood glucose elevation of normal mice
after sucrose feeding
Normal mice are divided into 5 groups, 10 mice each group, are fasted
overnight
before the experiment, a group of oral sucrose solution 4.0 g / kg as a
control group, a
group of oral sucrose and Acarbose 10 mg / kg as the positive control group.
The
remaining three groups respectively, with oral sucrose and different doses of
MC 0.45
g / kg, 0.9 g / kg, 1.8 g / kg. Determine blood glucose values at 0 min and 30
min, 60
min, 120min after the administration, results are shown in Figure 1, Table 1:
Table 1. Influence of MC for Peak blood glucose, Peak time and AUC of normal
mice after sucrose feeding
(mean SD, n=10)
Group Peak blood glucose Peak time ( min. ) AUC (
mmol/L .hr )
( mmol/L )
Control 9.0 1.6 30 12.9 2.8
MC 0.45 7.6 0.6 60 12.2 1.9
MC O. 9 6. 8 + 1. 9 * 60 10. 6 3. 0
MC 1. 8 5. 4+ 1. 3 ** 60 9. 2 2. 2 **
Acarbose 5.0 1.6 *** 120 9.2 + 2.7 **
8

CA 02710862 2010-06-25
AUC is area under blood glucose curve; compared with control group *
P<0.05,** P<0.01, *** p<0.001; MC dose is g/kg.
Experimental results show that, MC can reduce blood glucose AUC (area under
curve) of normal mice after sucrose feeding, furthermore the peak blood
glucose
decreased significantly and shift back, which basically identical for the role
of
Acarbose, and show a certain dose-effect relationship.
EXPERIMENT 2: influence of MC for blood glucose elevation of normal mice
after starch feeding
50 normal mice are divided into five groups, 10 mice each group, are fasted
overnight, a group of soluble starch 3.0 g / kg by gavage as a control group,
a group of
oral sucrose and Acarbose 10 mg / kg as the positive control group. The
remaining
three groups respectively, with oral sucrose and different doses of MC (0.45 g
/ kg,
0.9 g / kg, 1.8 g / kg). Determine blood glucose values at 0 min and 30 min,
60 mm,
120min after the administration, results are shown in Figure 2, Table 2:
Table 2. Influence of MC for Peak blood glucose, Peak time and AUC of normal
mice after starch feeding
(mean SD, n=10)
Group Peak blood glucose Peak time( mm.) AUC( mmol/L .hr )
( mmol/L )
Control 9. 4 2. 1 30 12. 0 2. 6
MC 0.45 6.8 1.4 ** 60 10.6 1.6
MC O. 9 6. 2 + O. 6 ** 60 10. 3 1. 6
MC 1.8 4.4 0.4 *** 120 8.3 + 1.4 ***
Acarbose 4. 2 0. 5 *** 30 8. 1 + 1. 1 ***
AUC is area under blood glucose curve; compared with control group *
13<0.05,** P<0.01, *** p<0.001; MC dose is g/kg.
Results show that, MC can make the peak blood glucose decreased significantly
and shift back after of normal mice after starch feeding, and reduce blood
glucose
AUC markedly, and show a certain dose-effect relationship.
EXPERIMENT 3: influence of MC for blood glucose elevation of normal mice
after glucose feeding
9

- CA 02710862 2010-06-25
. .
Normal mice are divided into 4 groups, 10 mice each group, are fasted
overnight, a
group of glucose 4.0 g / kg by gavage as a control group, a group of glucose
and
Acarbose 10 mg / kg by gavage as the positive control group. The remaining two
groups respectively, with glucose and different doses of MC (0.45 g / kg, 1.8
g / kg)
by gavage. Determine blood glucose values at 0 min and 30 min, 60 min, 120min
after the administration, results are shown in Table 3:
Table 3. Influence of MC for Peak blood glucose, Peak time and AUC of normal
mice after glucose feeding
(mean SD, n=10)
Group Peak blood glucose Peak time ( min. ) AUC(
mmol/L . hr)
( mmol/L )
Control 10. 2 1. 9 30 14. 6 2. 1
MC 0.45 9.7 1.3 30 14.2 1.8
MC 1. 8 9. 4 1. 9 30 14. 8 1. 7
Acarbose 10.7 2.4 30 15.1 2.6
AUC is area under blood glucose curve; MC dose is g/kg.
Results show that, MC is identical for Acarboset, has no significant influence
for
blood glucose elevation of normal mice after glucose feeding. It explains that
this
composition does not directly affect intestinal absorption for glucose.
EXPERIMENT 4: Influence of MC for blood glucose elevation of
alloxan-induced hyperglycemic mice after sucrose feeding
Alloxan-induced hyperglycemic mice are divided into 5 groups, 10 mice each
group, are fasted overnight, a group of sucrose 4.0 g / kg by gavage as a
control group,
a group of sucrose and Acarbose 10 mg / kg by gavage as the positive control
group.
The remaining three groups respectively, with sucrose and different doses of
MC (0.6
g / kg, 1.2 g / kg, 1.8 g / kg) by gavage. Determine blood glucose values at 0
min and
30 min, 60 min, 120min after the administration, results are shown in Figure
3, Table
4:
Table 4. Influence of MC for Peak blood glucose, Peak time and AUC of
alloxan-induced hyperglycemic mice after sucrose feeding
(mean SD, n=10)

. CA 02710862 2010-06-25
. .
Group Peak blood glucose Peak time(min.)
AUC(mmol/L.hr )
( mmol/L )
Control 22.6 1.7 30 39.0 4.2
MC 0.6 19.3 2.7 ** 60 34.2 + 4.8 *
MC 1.2 17.1+1.3 *** 60 30. 8 2. 8 ***
MC 1.8 14.8 1.8 *** 60 28.2+ 4.8 ***
Acarbose 17.3 2.6 *** 60 30.7+ 4.3 ***
AUC is area under blood glucose curve; compared with control group *
P<0.05,** P<0.01, *** p<0.001; MC dose is g/kg.
Experimental results show that, different doses of MC can significantly reduce
blood glucose elevation of alloxan-induced hyperglycemic mice after sucrose
feeding,
reduce blood glucose AUC significantly, make peak blood glucose value shift
back,
MC is identical for Acarbose at effect.
EXPERIMENT 5: Influence of MC for blood glucose elevation of
alloxan-induced hyperglycemic mice after starch feeding
Alloxan-induced hyperglycemic mice are divided into 5 groups, 10 mice each
group, are fasted overnight, a group of starch 3.0 g / kg by gavage as a
control group,
a group of starch and Acarbose 10 mg / kg as the positive control group. The
remaining three groups respectively, with starch and different doses of MC
(0.6 g / kg,
1.2 g / kg, 1.8 g / kg). Determine blood glucose values at 0 min and 30 min,
60 min,
120min after the administration, results are shown in Figure 4, Table 5:
Table 5. Influence of MC for Peak blood glucose, Peak time and AUC of
alloxan-induced hyperglycemic mice after starch feeding
(mean SD, n=10)
Group Peak blood glucose Peak time(min.)
( mmol/L ) AUC( mmol/L
.hr )
-
Group 24.4 1.3 30 40.6 + 4.4
Control 23.0 2.9 60 36.6 5.9
MC 0.6 21.3 + 3.1 ** 60 35.9 4.6 *
MC 1.2 19.3+2.2 *** 60 32.9 3.9 ***
MC 1.8 17.0+2.9 *** 60 31.3+5.9 ***

AUC is area under blood glucose curve; compared with control group * P<0.05,**

P<0.01, *** p<0.001; MC dose is g/kg.
Experimental results show that, different doses of MC can inhibit blood
glucose
elevation of alloxan-induced hyperglycemic mice after starch feeding, reduce
blood
glucose AUC, make peak blood glucose value shift back.
11

CA 02710862 2010-06-25
EXPERIMENT 6: Influence of MC for blood glucose of healthy volunteers after
steamed bread feeding
7 volunteers (the Institute staff), 2 males and 5 females, aged 25-62 years.
In first
test, after fasting, each person ate 100 g steamed bread (offered by canteen
of the
Institute) as self-control; in second test, randomly, 4 volunteers take MC 1.5
g / person,
3 persons take MC 3.0 g / person, and ate 100 g steamed bread at one time,
Determine
blood glucose values at fasting (0 min) and 30 min, 60 min, 120 min after ate
steamed
.. bread. Interval of two experiments is 1 week, and the results shown in
Figure 5 and
Table 6.
Table 6. Influence of MC for Peak blood glucose, Peak time and AUC of healthy
volunteers after steamed bread feeding
(mean SD )
High dose (3. 0 g/person) Low dose( 1.5 g/ person)
Before After Before After
administration administration administration administration
3 3 4 4
Peak blood glucose 6.8 1.4 4. 6 0. 5 * 6.5 1.5 5.4 0.9 *
( mmol/L )
Peak time (mm.) 30 60 30 60
AUC(mmol/L. hr) 12.2 1.9 8.8 0.54 11.5 3.1 10.0 1.2
AUC is area under blood glucose curve; Compared after and before with themself
*
P<0.05, iT. P ===-, 0.05
The preliminary results show, MC can reduce peak blood glucose value OF
healthy
volunteers after steamed bread feeding, reduce blood glucose AUC, make blood
glucose peak value shift back, and show dose-effect relationship.
EXPERIMENT 7: influence of MC for urine glucose of alloxan-induced
hyperglycemic rats
Three groups of alloxan-induced hyperglycemic rats, 10 rats each group. A
group
ate high-sucrose diet as hyperglycemic control, the second and third groups
ate
high-sucrose diet with different doses of MC (equivalent to a dose of 0.6 g /
kg, 1.2 g /
kg) respectively. The fourth group is 10 normal rats as control group, ate
normal diet.
After two weeks, collect urine by metabolic cages, measure urine sugar levels
of 6
hours, simultaneously, collect urine of the fourth group, normal rat, measure
it as a
12

CA 02710862 2010-06-25
comparison, the results are shown in Figure 6.
The results show that: normal rats were not detected glucose in urine, MC can
significantly reduce urine sugar levels in alloxan-induced hyperglycemic rats,
and it
shows a certain dose-effect relationship.
EXPERIMENT 8: Influence of MC for general conditions, blood glucose, blood
lipids, fructosamine, serum N-acetyl-13-D glucosaminidase (short for NAG-ase)
activity, sorbitol content in organization, red GSH content in blood cell,
urea
nitrogen and creatinine levels in serum, as well as influences of renal
pathological
changes, and so on of alloxan-induced hyperglycemic rats
Three groups of alloxan-induced hyperglycemic rats, 10 rats each group. A
group
ate high-sucrose diet as hyperglycemic control, the second and third group ate

high-sucrose diet with different doses of MC (equivalent to a dose of 0.6 g /
kg, 1.2 g /
kg) respectively. The fourth group is 10 normal rats as control group, eat
normal diet.
Observe the general condition of animals every day, and record food and water
intake.
After 4 weeks, the animals were killed to take blood, crystalline lens,
sciatic nerve and
other organizations, measure blood glucose, blood lipids, fructosamine, serum
NAG
activity, sorbitol content in organization, GSH content in red blood cell,
urea nitrogen
and creatinine levels in serum, and the other biochemical indicators, and
observe renal
pathological changes, the results are as follow:
1. Changes of food and water intake, Table 7:
Table 7. Influence of MC for food and water intake of alloxan-induced
hyperglycemic
rats;
(mean SD )
Group Food intake( g/rat/day ) Water intake( ml/ rat/day )
Normal rats 22.6 3.3 19.5 3.4
Hyperglycemic rats 125.3 15.1 118.6 19.6
MC 0.6 99.0 + 6.0 ** 61.2 + 11.2 ***
MC 1.2 179.8+ 12.1 *** 39.4 + 4.9 ***
Compared with hyperglycemic rats, ** P<0.01, *** P<0.001; MC dose is g/kg.
2. Influence of MC for blood glucose, fructosamine of alloxan-induced
hyperglycemic rats, Table 8;
Fructosamine is an aldehyde-ketone condensation compound format through
molecular rearrangements in the process of nonenzymatic glycosylation between
plasma protein (albumin mainly) and glucose molecules. Since the stability of
albumin concentration in the body, so serum fructosamine level is also stable
relatively. It can reflect the blood glucose levels within 1-2 weeks.
13

CA 02710862 2010-06-25
Table 8. Influence of MC for blood glucose, fructosamine of alloxan-induced
hyperglycemic rats
(mean SD , n=10)
Group blood glucose ( mmol/L )
Fructosamine
no fasting fasting ( mmol/L )
Normal 5. 4 0. 2 3. 4 O. 5 1. 60 0. 12
25. 9 3. 6 16. 5 2. 1 2. 64 0. 16
Hyperglycemic
rats
MC 0. 6 19. 1+3. 4 *** 10. 4+3. 5 *** 2. 20 + O. 24
***
MC 1. 2 10. 9 4. 4 *** 8. 3 + 3. 7 *** 2. 00 + O. 16 ***
Compared with hyperglycemic rats, *** P<0.001; MC dose is g/kg.
3. Influence of MC for blood lipids, Table 9:
Animals, which have high blood sugar, often have high blood lipids. Reducing
blood glucose can improve high blood lipids. Blood lipids are determined by
Enzyme
method, kit purchased from Beijing Biosino Bio-technology & science INC.
Table 9. Influence of MC for blood lipids of alloxan-induced hyperglycemic
rats;
(mean SD, n=10 )
Group Blood lipids( mmol/L )
Triglycerides Total cholesterol
Normal rats 0.98 0.21 2.54 0.21
Hyperglycemic rats 1.83 1.00 2.78 0.24
MC 0.6 1.70 0.58 2.31 0.17 *
MC 1. 2 1. 16 0. 30 4 2. 44 O. 28 *
Compared with hyperglycemic rats, * P<0.05, 4 P==0.05 MC dose is g/kg
4. Influence of MC for serum NAG-ase activity, Table 10:
NAG enzyme is a lysosomal enzyme which present in renal parenchyma widely,
and has closely relation with urinary albumin excretion and retinal
microangiopathy
degree, and it is a sensitive indicator of microangiopathy. With the
microvascular
disease increases, NAG activity increases.
Table 10. Influence of MC for serum NAG-ase activity of alloxan-induced
hyperglycemic rats
(mean SD, n=10)
Group Serum NAG-ase ( iu )
14

= CA 02710862 2010-06-25
. .
Normal rats 48.3 5.9
Hyperglycemic rats 88. 0 28. 1
MC 0.6 67.5 17.0
MC 1.2 52.9 + 13.7 **
Compared with hyperglycemic rats, ** P<0.01; MC dose is g/kg
5. Influence of MC for sorbitol content in crystalline lens and sciatic nerve
of
alloxan-induced hyperglycemic rats, Table 11:
Sorbitol pathway is closely related with occurrence and development of chronic
complications of diabetes. Increased level of tissue sorbitol content can
directly reflect
the activity metabolism level of sorbitol channel. Determine it with sodium
arsenite-chromotropic acid method.
Table 11. Influence of MC for sorbitol content in crystalline lens and sciatic
nerve of
alloxan-induced hyperglycemic rats
(mean SD, n=10 )
Group Sorbitol( mol/g tissue)
Crystalline lens sciatic nerve
Normal rats 1. 35 0. 12 2. 44 +
0. 23
Hyperglycemic rats 3.56 0.78 9.54 1.93
MC 0.6 3.47 0.55 5.71 + 1.02
***
MC 1.2 3.39 0.82 4.15 + 1.11
***
Compared with hyperglycemic rats, *** P<0.001; MC dose is g/kg
6. Influence of MC for reduced glutathione (GSH) content in red blood cell on
alloxan-induced hyperglycemic rats, Table 12:
Free radical theory is one of major theories about occurrence and development
of
some diabetic chronic complications explained by molecular biology level. In
high
blood sugar state, GSH levels of red blood cell often low, it can reflect the
body's
ability to withstand free radical damage.
Table 12. Influence of MC for GSH in red blood cell of alloxan-induced
hyperglycemic rats
(mean SD, n=10)
Group GSH( mmol/L ) P value
Normal rats 0.95 + 0.16
Hyperglycemic 0. 61 1. 16
MC 0.6 1.02 0.19 <0.01
MC 1.2 1.322 0.32 <0.01
P value is compared with hyperglycemic rats; MC dose is g/kg

CA 02710862 2010-06-25
. .
7. Influence of MC for kidney weight/body mass index of alloxan-induced
hyperglycemic rats, Figure 7:
Diabetic kidney hypertrophy and hyperfiltration phenomenon are the earliest
changes appearing in renal pathophysiology. Whether or not control diabetic
kidney
hypertrophy is often one of symbols that effectiveness of treatment of this
disease.
8. Influence of MC for kidney serum urea nitrogen and creatinine levels of
alloxan-induced hyperglycemic rats (Table 13):
Determine serum urea nitrogen and creatinine levels can speculate changes of
renal
function. Serum urea nitrogen and creatinine levels rise, then it reflect
renal
dysfunction. Method uses reagent kit of Beijing Biosino Bio-technology &
science
INC.
Table 13. Influence of MC for kidney serum urea nitrogen and creatinine levels
of
alloxan-induced hyperglycemic rats
(mean SD, n10)
_____ Group Urea nitrogen(mmol/L) Creatinine ( mol/L )
Normal rats 6.64 1.36 47.74 6.19
Hyperglycemic 5. 39 0. 71 84. 86 14. 14
rats
MC 0.6 5.75 0.75 76.91 23.87
MC 1.2 5.82 1.39 53. 04 13.26 *
Compared with hyperglycemic rats * P<0.01; MCdose is g/kg
9. Influence of MC for renal pathological changes of alloxan-induced
hyperglycemic
rats
Experimental rat kidney in each group is 10% fixed with formalin, regular
slices,
stained with hematoxylin eosin, observed by light microscope. Kidney structure
of
normal rat is normal (see Figure 8), not seen in any lesions. Part of renal
epithelial
cells of kidney tubules of hyperglycemic rats are swollen and vacuolar, and
after their
intracellular glycogen has been dissolved during production process, membrane
is
visible clearly (see Figure 9); kidney tubules of MC rats isn't observed such
changes
(see Figure 10). Glycogen nephrosis causes by increasing blood glucose
absorption of
glomerular epithelial cells when through renal tubules. After reduction of
blood
glucose and urine glucose, other disorders can be reduced. Experiment shows
that,
MC can improve lesions of kidney tubules, while reduce blood and urine
glucose.
The experimental results show that, after long-term (4 weeks) administration,
MC can significantly improve on polydipsia or polyphagia and other general
conditions of alloxan-induced hyperglycemic rats; decrease significantly blood

glucose and serum fructosamine; decrease serum cholesterol levels
significantly,
reduce serum triglyceride levels; also decrease serum NAG activity
significantly,
which indicates that it has an improvement for microvascular complications;
reduce
16

CA 02710862 2010-06-25
=
. .
sorbitol content in sciatic nerve significantly, which suggests that it can
improve
chronic neurological disease of diabetes; increase GSH content in red blood
cell,
enhance the body's antioxidant ability; also reduce kidney weight/body mass
index
significantly, reduce serum creatinine levels, relieve glycogen deposition of
renal
tubular epithelial cells.
Experimental 9: Experiment in vitro of pharmaceutical composition
prepared in example 1
1. Test drugs: test sample: pharmaceutical composition prepared in example 1,
short for MC; positive control drug: Acarbose, Orlistat.
2. Experimental animals: normal wistar rats, 250-300 g, used to extract
intestinal
a-glucosidase.
3. Methods and Results:
(1) Inhibit effect of MC for a-glucosidase
With sucrose or maltose as substrate and Acarbose as a positive control,
measure
inhibition effect of MC for a-sucrose and maltose enzyme activity in rat
intestinal,
calculate IC50; with starch as substrate, Acarbose as a positive control,
measure
inhibition effect of MC for a-amylase activity, the results are shown in Table
14:
Table 14. inhibition in vitro IC50 ( pig/m1) of MC for a-glucosidase
Sample a-sucrase a-maltase a-amylase
MC 2.53 4.05 >100
Acarbose 0. 255 0. 076 0. 80

The results show that: MC has strong inhibitory activity on a-glucosidase in
vitro,
IC50 of a- sucrase and maltase are 2.53 lig / ml and 4.05 ttg / ml; and it has
no
inhibitory effect on a-amylase activity.
(2) Inhibit effect of MC for lipase activity
With triolein as substrate and Orlistat as a positive control, test samples MC

response to porcine pancreatic lipase and lipoprotein lipase respectively,
determine
inhibitory activity of sample to lipase, by release rate of oleic acid
released from
triolein. The results are shown in Table 15:
Table 15. Inhibitory rate of MC for lipase activity
Terminal concentration
Sample ( P g/ml ) Pancreatic lipase
Lipoprotein lipase
MC 10 0 0
Orlistat 10 100% 100%
The results show that MC almost no inhibitory effect on lipase.
4. Experimental result:
17

= CA 02710862 2010-06-25
Experimental results show that, samples of MC have a very strong inhibitory
effect
on a-sucrose and a-maltase activity, and IC50 are 2.53 itg / ml and 4.05 p.g /
ml
respectively; do not inhibit a-amylase. It shows that MC only inhibit
disaccharide
hydrolase without inhibiting a-amylase activity, thus it greatly reduces
amount of
starch and oligosaccharides without fully hydrolyzed into intestinal bottom
end, only
part remnant disaccharide into intestinal bottom end, reduce hydrolyzed
disaccharide absorbed into blood, make blood glucose elevation, their
incidence of
gastrointestinal side effects is lower than Acarbose. MC has no inhibitory
effect on
lipase, it shows that MC can not directly affect fat absorption from
intestine.
The following examples can entirely achieve efficacies in above experiments.
EMBODIMENTS
EXAMPLE 1: PREPARATION OF CAPSULE
Feculae Bombycis 7.15kg Radix Glycyrrhizae 0.1375kg
Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae
Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times,
each
time 1 hours, filtering, combining the filtrates, recovering ethanol,
concentrating at a
temperature 55 C, gaining concentrated solution of the relative density of
1.01-1.06,
adding same amount water and heating to boiling, cooling, placing overnight,
then
taking supernatant A, heating the supernatant to obtain the thick paste A with
the
relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by
service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with
water,
decocting 2 times, each time 2 hours, combining the decoctions, placing
overnight to
precipitate, concentrating the supernatant B to the thick paste B with the
relative
density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining
the thick paste A and the thick paste B, adding 0-cyclodextrin with amount of
20%
Feculae Bombycis dry paste, spraying drying, add hydroxypropyl cellulose (L-
HPC)
in amount of 1% drug powder, blending, drying, granulating, whole grain,
putting
into capsules, obtaining 1000 capsules, 2-4 tablets each time, three times
daily.
EXAMPLE 2: PREPARATION OF TABLET
Feculae Bombycis 7.15kg Radix Glycyrrhizae 0.1375kg
, taking above two crude drugs, according to usual process, adding general
adjuvant,
producing tablet.
EXAMPLE 3: PREPARATION OF GRANULE
Feculae Bombycis 6.15kg Radix Glycyrrhizae 0.1875kg
, taking above two crude drugs, according to usual process, adding general
adjuvant,
producing granule.
18

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CA 02710862 2010-06-25
EXAMPLE 4: PREPARATION OF PILL
Feculae Bombycis 7.85kg Radix Glycyrrhizae 0.1175kg
, taking above two crude drugs, according to usual process, adding general
adjuvant,
producing pill.
EXAMPLE 5: PREPARATION OF HONEY REFINING PASTE
Feculae Bombycis 7.15kg Radix Glycyrrhizae 0.1375kg
, taking above two crude drugs, according to usual process, adding general
adjuvant,
producing honey refining paste.
EXAMPLE 6: PREPARATION OF ORAL LIQUID PREPARATION
Feculae Bombycis 6.15kg Radix Glycyrrhizae 0.1875kg
, taking above two crude drugs, according to usual process, adding general
adjuvant,
producing oral liquid preparation.
EXAMPLE 7: PREPARATION OF INJECTION
Feculae Bombycis 7.85kg Radix Glycyrrhizae 0.1175kg
, taking above two crude drugs, according to usual process, adding general
adjuvant,
producing injection.
EXAMPLE 8: Identification and content determination methods of the
pharmaceutical composition capsule
Identification:
A. Taking 0.5g capsule content of this invention pharmaceutical composition
prepared in Example 1, adding 5 ml anhydrous ethanol, refluxing in 60 C water
bath
for 30 minutes, placing and taking supernatant, evaporating it until 1 ml
above water
bath, that is test sample solution; furthermore, taking 0-sitosterol as
reference
substance, adding anhydrous ethanol for 0.001g/m1 solution, that is reference
substance solution; according to thin layer chromatography test, sucking above
two
solutions 0.002m1 respectively, pointing them on the same TLC plate of silica
gel G
respectively, developing, taking out, drying, 19.5:0.5 of chloroform: acetone
as
mobile phase, and spraying with 10% phosphor-molybdic acid solution, heating
in
105 C until visualize the chromatogram; the chromatogram produced by the test
sample solution shows the same color spots as that displayed by the reference
substance solution in their respectively corresponding areas;
B. Identification by high-performance liquid chromatography, Using
octadecylsilanized silica gel as packing; using methanol-(0.2m01 / L) ammonium
acetate - acetic acid = 60:40:1 as mobile phase; detection wavelength 250nm;
theoretical plate number counted according to peak ammonium glycyrrhizinate is
not
less than 2000; weighing 0.01g mono-ammonium glycyrrhizinate reference
compound
19

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= CA 02710862 2010-06-25
.
accurately, putting it into 50m1 volumetric flask, dissolving by above mobile
phase to
the volume, shaking up, that is, 0.0002g mono-ammonium glycyrrhizinate per ml,

which is equivalent to 0.0001959g glycyrrhizic acid, that is reference
substance
solution;
taking 2g capsule content of this invention pharmaceutical compositions
prepared
in Example 1, putting it into 25 ml volumetric flask, adding 20m1 above mobile
phase,
deal with ultrasound for 30 minutes with power 250W, frequency of 50KHz,
taking
out and cooling, adding mobile phase to the volume, shaking up, centrifuging,
taking
the supernatant, that is test sample solution;
Accurately sucking reference substance solution and test sample solution
0.005ml
respectively, injecting into liquid chromatogram instrument, test sample
should be
presented the same retention time peaks with the reference substance.
Content determination:
An applicability test of chromatogram condition and system
Using octadecylsilanized silica gel as packing; using a solution with
acetonitrile-dimethyl formamide-sodium acetate(0.025mol/L)= 21:0.5:79 as
mobile
phase; detection wavelength is 390nm, theoretical plate number counted
according to
peak Pipecolic acid is not less than 2500;
Weighing 0.01g Pipecolic acid reference compound accurately, putting it into
100m1 volumetric flask, adding water to dilute to the volume, shaking up,
measuring
1m1 the said solution accurately, putting it into 10m1 test tube with plug,
adding 1m1
water, adding 2m1 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 2m1

sodium bicarbonate solution(0.5mol/L), heating up in 60 C water bath for 1
hour,
taking out, cooling, and transferring into 10m1 volumetric flask, washing
container
with phosphate buffer solution (0.2mol/L, pH7.0) by 3 times, combining the
washing
solution into volumetric flask, adding phosphate buffer solution to the
volume,
shaking up, that is reference compound solution;
accurately weighing 0.4g capsule content of this invention pharmaceutical
composition prepared in Example 1, placing it in 10m1 test tube with plug,
adding 5m1
water, shaking up fully, heating up in 60 C water bath for 30 minutes, taking
out,
cooling, and transferring into 10m1 volumetric flask, washing container with
water by
3 times, combining the washing solution into volumetric flask and adding water
to
volume, and shaking up; centrifuging 12000 rpm for 10 minutes, measuring 2m1
the
supernatant accurately, placing it into 10m1 test tube with plug, adding 2m1
2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 2m1 sodium
bicarbonate
solution(0.5mol/L), heating up in 60 C water bath for 1 hour, taking out,
cooling,
and transferring into 10m1 volumetric flask, washing container with phosphate
buffer
solution (0.2mol/L, pH7.0) by 3 times, combining the washing solution into
volumetric flask, adding phosphate buffer solution to the volume, shaking up,
that is
test sample solution, Accurately sucking 0.005m1 reference substance solution
and
0.01m1 test sample solution respectively, injecting into liquid chromatogram
instrument, determining, that is; dosage per day for the pharmaceutical
composition of

CA 02710862 2010-06-25
this invention is 0.5g, then Feculae Bombycis content counted according to
Pipecolic
acid (C6H11NO2), is not less than 0.0018g.
EXAMPLE 9: Identification method of the pharmaceutical composition powder
A. Taking 0.5g pharmaceutical composition powder of this invention, adding 6
ml
anhydrous ethanol, refluxing in 40 C water bath for 40 minutes, placing and
taking
supernatant, evaporating it until 0.5 ml above water bath, that is test sample
solution;
furthermore, taking 0-sitosterol as reference substance, adding anhydrous
ethanol for
0.0015g/m1 solution, that is reference substance solution; according to thin
layer
chromatography test, sucking above two solutions 0.00 1 ml respectively,
pointing
them on the same TLC plate of silica gel G respectively, developing, taking
out,
drying, 21:0.3 of chloroform: acetone as mobile phase, and spraying with 15%
phosphor-molybdic acid solution, heating in 100 C until visualize the
chromatogram;
the chromatogram produced by the test sample solution shows the same color
spots as
that displayed by the reference substance solution in their respectively
corresponding
areas;
B. Identification by high-performance liquid chromatography, Using
octadecylsilanized silica gel as packing; using methanol-(0.2mo1 / L) ammonium
acetate - acetic acid = 50:50:0.5 as mobile phase; detection wavelength 250nm;

theoretical plate number counted according to peak ammonium glycyrrhizinate is
not
less than 2000; weighing 0.005g mono-ammonium glycyrrhizinate reference
compound accurately, putting it into 50m1 volumetric flask, dissolving by
above
mobile phase to the volume, shaking up, that is, 0.0003g mono-ammonium
glycyrrhizinate per ml, which is equivalent to 0.00019g glycyrrhizic acid,
that is
reference substance solution;
taking 3.0g pharmaceutical composition powder of this invention, putting it
into 25
ml volumetric flask, adding 18m1 above mobile phase, deal with ultrasound for
20
minutes with power 250W, frequency of 50KHz, taking out and cooling, adding
mobile phase to the volume, shaking up, centrifuging, taking the supernatant,
that is
test sample solution;
accurately sucking reference substance solution and test sample solution
0.015m1
respectively, injecting into liquid chromatogram instrument, test sample
should be
presented the same retention time peaks with the reference substance.
EXAMPLE 10: Content determination method of the pharmaceutical
composition granule
An applicability test of chromatogram condition and system
Using octadecylsilanized silica gel as packing; using a solution with
acetonitrile-dimethyl formamide-sodium acetate(0.025mo1/L)= 15:1:75 as mobile
21

= CA 02710862 2010-06-25
phase; detection wavelength is 390nm, theoretical plate number counted
according
to peak Pipecolic acid is not less than 2500;
Weighing 0.006g Pipecolic acid reference compound accurately, putting it into
50m1 volumetric flask, adding water to dilute to the volume, shaking up,
measuring
0.5m1 the said solution accurately, putting it into 10m1 test tube with plug,
adding
1.5m1 water, adding lml 2,4-acetonitrile dinitrofluorobenzene solution(0.8%)
and
3m1 sodium bicarbonate solution(0.5mol/L), heating up in 60 C water bath for
0.5
hour, taking out, cooling, and transferring into 10m1 volumetric flask,
washing
container with phosphate buffer solution (0.2mo1/L, pH7.0) by several times,
combining the washing solution into volumetric flask, adding phosphate buffer
solution to the volume, shaking up, that is reference compound solution;
accurately weighing 0.5g granule of this invention pharmaceutical composition,

placing it in 10m1 test tube with plug, adding proper amount water, shaking up
fully,
heating up in 50 C water bath for 40 minutes, taking out, cooling, and
transferring
into 10m1 volumetric flask, washing container with water by several times,
combining
the washing solution into volumetric flask and adding water to volume, and
shaking
up; centrifuging 12000 rpm for 5 minutes, measuring 3m1 the supernatant
accurately,
placing it into 10m1 test tube with plug, adding lml 2,4-acetonitrile
dinitrofluorobenzene solution(0.8%) and 3m1 sodium bicarbonate
solution(0.5mol/L),
heating up in 50 C water bath for 1.5 hour, taking out, cooling, and
transferring into
10m1 volumetric flask, washing container with phosphate buffer solution
(0.2mol/L,
pH7.0) by several times, combining the washing solution into volumetric flask,

adding phosphate buffer solution to the volume, shaking up, that is test
sample
solution, Accurately sucking 0.004m1 reference substance solution and 0.015m1
test
sample solution respectively, injecting into liquid chromatogram instrument,
determining, that is; dosage per day for the pharmaceutical composition of
this
invention is 0.5g, then Feculae Bombycis content counted according to
Pipecolic acid
(C6H111\102), is not less than 0.0018g.
EXAMPLE 11: Identification and content determination methods of the
pharmaceutical composition tablet
Identification:
A. Taking 0.5g tablet of this invention pharmaceutical composition prepared in
Example 2, adding 5g anhydrous ethanol, refluxing in 60 C water bath for 30
minutes, placing and taking supernatant, evaporating it until 1 ml above water
bath,
that is test sample solution; furthermore, taking 0-sitosterol as reference
substance,
adding anhydrous ethanol for 0.001g/m1 solution, that is reference substance
solution;
according to thin layer chromatography test, sucking above two solutions
0.002m1
respectively, pointing them on the same TLC plate of silica gel G
respectively,
developing, taking out, drying, 19.5:0.5 of chloroform: acetone as mobile
phase, and
spraying with 10% phosphor-molybdic acid solution, heating in 105 C until
visualize
22

. .
CA 02710862 2010-06-25
. the chromatogram; the chromatogram produced by the test sample
solution shows the
same color spots as that displayed by the reference substance solution in
their
respectively corresponding areas;
Content determination:
An applicability test of chromatogram condition and system
Using octadecylsilanized silica gel as packing; using a solution with
acetonitrile-dimethyl formamide-sodium acetate(0.025mo1/L)= 21:0.5:79 as
mobile
phase; detection wavelength is 390nm, theoretical plate number counted
according
to peak Pipecolic acid is not less than 2500;
Weighing 0.005g Pipecolic acid reference compound accurately, putting it into
50m1 volumetric flask, adding water to dilute to the volume, shaking up,
measuring
1m1 the said solution accurately, putting it into 10m1 test tube with plug,
adding lml
water, adding 2m1 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 2
parts
by volume of sodium bicarbonate solution(0.5mo1/L), heating up in 60 C water
bath
for 1 hour, taking out, cooling, and transferring into 10m1 volumetric flask,
washing
container with phosphate buffer solution (0.2m01/L, pH7.0) by several times,
combining the washing solution into volumetric flask, adding phosphate buffer
solution to the volume, shaking up, that is reference compound solution;
accurately weighing 0.4g tablet of this invention pharmaceutical composition
prepared in Example 2, placing it in 10m1 test tube with plug, adding proper
amount
water, shaking up fully, heating up in 60 C water bath for 30 minutes, taking
out,
cooling, and transferring into 10m1 volumetric flask, washing container with
water by
several times, combining the washing solution into volumetric flask and adding
water
to volume, and shaking up; centrifuging 12000 rpm for 10 minutes, measuring
2m1 the
supernatant accurately, placing it into 10m1 test tube with plug, adding 2m1
2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 2m1 sodium
bicarbonate
solution(0.5mol/L), heating up in 60 C water bath for 1 hour, taking out,
cooling,
and transferring into 10m1 volumetric flask, washing container with phosphate
buffer
solution (0.2mol/L, pH7.0) by several times, combining the washing solution
into
volumetric flask, adding phosphate buffer solution to the volume, shaking up,
that is
test sample solution, accurately sucking 0.005m1 reference substance solution
and
0.01m1 test sample solution respectively, injecting into liquid chromatogram
instrument, determining, that is; dosage per day for the pharmaceutical
composition of
this inventionis 0.5g, then Feculae Bombycis content counted according to
Pipecolic
acid (C6H1 INN, is not less than 0.0018g.
EXAMPLE 12: PREPARATION OF TABLET
Feculae Bombycis 7.15kg Radix Glycyrrhizae 0.1375kg
Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae
Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times,
each
time 1 hour; filtering, combining the filtrates, recovering ethanol,
concentrating at a
temperature 55 C, gaining concentrated solution of the relative density of
1.01-1.06,
adding same amount water and heating to boiling, cooling, placing overnight,
then
taking supernatant A, heating the supernatant to obtain the thick paste A with
the
23

CA 02710862 2010-06-25
relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by
service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with
water,
decocting 2 times, each time 2 hours, combining the decoctions, placing
overnight to
precipitate, concentrating the supernatant B to the thick paste B with the
relative
density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining
the thick paste A and the thick paste B, according to usual process, adding
general
adjuvant, producing tablet.
EXAMPLE 13: PREPARATION OF GRANULE
Feculae Bombycis 6.15kg Radix Glycyrrhizae 0.1875kg
Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae
Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times,
each
time 1 hour, filtering, combining the filtrates, recovering ethanol,
concentrating at a
temperature 55 C, gaining concentrated solution of the relative density of
1.01-1.06,
adding same amount water and heating to boiling, cooling, placing overnight,
then
taking supernatant A, heating the supernatant to obtain the thick paste A with
the
relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by
service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with
water,
decocting 2 times, each time 2 hours, combining the decoctions, placing
overnight to
.. precipitate, concentrating the supernatant B to the thick paste B with the
relative
density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining
the thick paste A and the thick paste B, according to usual process, adding
general
adjuvant, producing granule.
EXAMPLE 14: PREPARATION OF PILL
Feculae Bombycis 7.85kg Radix Glycyrrhizae 0.1175kg
Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae
Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times,
each
time 1 hour, filtering, combining the filtrates, recovering ethanol,
concentrating at a
temperature 55 C, gaining concentrated solution of the relative density of
1.01-1.06,
adding same amount water and heating to boiling, cooling, placing overnight,
then
taking supernatant A, heating the supernatant to obtain the thick paste A with
the
relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by
service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with
water,
decocting 2 times, each time 2 hours, combining the decoctions, placing
overnight to
precipitate, concentrating the supernatant B to the thick paste B with the
relative
density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining
the thick paste A and the thick paste B, according to usual process, adding
general
adjuvant, producing pill.
EXAMPLE 15: PREPARATION OF HONEY REFINING PASTE
Feculae Bombycis 7.15kg Radix Glycyrrhizae 0.1375kg
Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae
Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times,
each
24

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= CA 02710862 2010-06-25
. .
time 1 hour, filtering, combining the filtrates, recovering ethanol,
concentrating at a
temperature 55 C, gaining concentrated solution of the relative density of
1.01-1.06,
adding same amount water and heating to boiling, cooling, placing overnight,
then
taking supernatant A, heating the supernatant to obtain the thick paste A with
the
relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by
service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with
water,
decocting 2 times, each time 2 hours, combining the decoctions, placing
overnight to
precipitate, concentrating the supernatant B to the thick paste B with the
relative
density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining
the thick paste A and the thick paste B, according to usual process, adding
general
adjuvant, producing honey refining paste.
EXAMPLE 16: PREPARATION OF ORAL LIQUID PREPARATION
Feculae Bombycis 6.15kg Radix Glycyrrhizae 0.1875kg
Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae
Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times,
each
time 1 hour, filtering, combining the filtrates, recovering ethanol,
concentrating at a
temperature 55 C, gaining concentrated solution of the relative density of
1.01-1.06,
adding same amount water and heating to boiling, cooling, placing overnight,
then
taking supernatant A, heating the supernatant to obtain the thick paste A with
the
relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by
service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with
water,
decocting 2 times, each time 2 hours, combining the decoctions, placing
overnight to
precipitate, concentrating the supernatant B to the thick paste B with the
relative
density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining
the thick paste A and the thick paste B, according to usual process, adding
general
adjuvant, producing oral liquid preparation.
EXAMPLE 17: PREPARATION OF INJECTION
Feculae Bombycis 7.85kg Radix Glycyrrhizae 0.1175kg
Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae
Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times,
each
time 1 hour, filtering, combining the filtrates, recovering ethanol,
concentrating at a
temperature 55 C, gaining concentrated solution of the relative density of
1.01-1.06,
adding same amount water and heating to boiling, cooling, placing overnight,
then
taking supernatant A, heating the supernatant to obtain the thick paste A with
the
relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by
service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with
water,
decocting 2 times, each time 2 hours, combining the decoctions, placing
overnight to
precipitate, concentrating the supernatant B to the thick paste B with the
relative
density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining
the thick paste A and the thick paste B, according to usual process, adding
general
adjuvant, producing injection.