Note: Descriptions are shown in the official language in which they were submitted.
USE OF PHARMACEUTICAL COMPOSITION IN PREPARING ANTIBACTERIAL
DRUG
[0001] This application claims the priority of Chinese Patent Application No.
202010153831.8,
filed with the China National Intellectual Property Administration on March 7,
2020, and titled
with "USE OF TRADITIONAL CHINESE MEDICINE COMPOSITION IN PREPARATION OF
ANTIBACTERIAL DRUG", which is hereby incorporated by reference in entirety.
FIELD
[0002] The present disclosure relates to the use of a pharmaceutical
composition in the
manufacture of antibacterial drugs, and belongs to the field of medicine.
BACKGROUND
[0003] Antibiotics refer to a class of secondary metabolic product, with anti-
pathogen and other
activities, from the life processes of microorganisms (including bacteria,
fungi and actinomycetes)
or higher animals and plants, and are chemical substances that can interfere
with the developmental
functions of other living cells. Commonly used antibiotics in clinics include
extracts from microbial
cultures and chemically synthesized or semi-synthetic compounds. The
bacteriostatic or
bactericidal effect of antibiotics mainly includes four major mechanisms,
namely: inhibiting
synthesis of bacterial cell wall, enhancing permeability of bacterial cell
membrane, interfering with
synthesis of bacterial protein, and inhibiting replication and transcription
of bacterial nucleic acids.
Antibiotics play a major role in the treatment of many bacterial infectious
diseases. However,
misuse of antibiotics has caused more and more bacteria to develop drug
resistance. The improper
uses antibiotics, including the abuse (which may causes long course of
treatment), arbitrary
replacement, combination and blind use of antibiotics, have caused problem
that many strains
develop multi-drug resistance and extensive drug resistance.
[0004] Therefore, effective antibacterial drugs, especially for those with
broad-spectrum
antibacterial properties or capable of effectively improving drug resistance,
are still desired.
- i -
CA 03167551 2022- 8- 8
[0005] The present disclosure is an improved invention based on the
pharmaceutical composition
disclosed in the patent CN101549060B, the contents of which are incorporated
herein in its entirety.
The above-mentioned patent does not disclose that the pharmaceutical
composition has
antibacterial effect.
SUMMARY
[0006] The inventors unexpectedly found in the research that the
pharmaceutical composition of
this disclosure has excellent antibacterial effect, and the pharmaceutical
composition is prepared
from the following materials in parts by weight:
ephedrae herba 52-86, gypsum fibrosum 194-324,
forsythiae fructus 194-324, scutellariae radix 78-130,
mori cortex 194-324, armeniacae semen amarum 78-
130,
peucedani radix 78-130, pinelliae rhizoma 78-130,
citri reticulatae pericarpium 78-130, fritillariae bulbus 78-130,
arctii fructus 78-130, lonicerae japonicae flos 78-
130,
rhei radix et rhizoma 39-65, platycodonis radix 46-76,
glycyrrhizae radix et rhizoma 39-65.
[0007] The pharmaceutical composition of this disclosure is preferably
prepared from the
following materials in parts by weight:
ephedrae herba 52, gypsum fibrosum 324,
forsythiae fructus 194, scutellariae radix 78,
mori cortex 194, armeniacae semen amarum 130,
peucedani radix 78, pinelliae rhizoma 130,
citri reticulatae pericarpium 78, fritillariae bulbus 78,
arctii fructus 130, lonicerae japonicae flos 130,
- 2 -
CA 03167551 2022- 8- 8
rhei radix et rhizoma 39, platycodonis radix 76,
glycyrrhizae radix et rhizoma 65.
[0008] The pharmaceutical composition of this disclosure is also preferably
prepared from the
following materials in parts by weight:
ephedrae herba 86, gypsum fibrosum 194,
forsythiae fructus 324, scutellariae radix 130,
mori cortex 324, armeniacae semen amarum 78,
peucedani radix 130, pinelliae rhizoma 78,
citri reticulatae pericarpium 130, fritillariae bulbus 130,
arctii fructus 78, lonicerae japonicae flos 78,
rhei radix et rhizoma 65, platycodonis radix 46,
glycyrrhizae radix et rhizoma 39.
[0009] The pharmaceutical composition of this disclosure is also preferably
prepared from the
following materials in parts by weight:
ephedrae herba 69, gypsum fibrosum 259,
forsythiae fructus 259, scutellariae radix 104,
mori cortex 259, armeniacae semen amarum 104,
peucedani radix 104, pinelliae rhizoma 104,
citri reticulatae pericarpium 104, fritillariae bulbus 104,
arctii fructus 104, lonicerae japonicae flos 104,
rhei radix et rhizoma 52, platycodonis radix 61;
glycyrrhizae radix et rhizoma 52.
[0010] The pharmaceutical composition of this disclosure is also preferably
prepared from the
following materials in parts by weight:
- 3 -
CA 03167551 2022- 8- 8
ephedrae herba 55, gypsum fibrosum 254,
forsythiae fructus 318, scutellariae radix 107,
mori cortex 203, armeniacae semen amarum 107,
peucedani radix 82, pinelliae rhizoma 105,
citri reticulatae pericarpium 84, fritillariae bulbus 125,
arctii fructus 122, lonicerae japonicae flos 113,
rhei radix et rhizoma 42, platycodonis radix 60,
glycyrrhizae radix et rhizoma 50.
[0011] Preferably, in the pharmaceutical composition of this disclosure, the
armeniacae semen
amarum is stir-baked armeniacae semen amarum, the fritillariae bulbus is
fritillariae thunbergii
bulbus, the lonicerae japonicae flos is lonicerae flos, and the pinelliae
rhizoma is pinelliae rhizoma
praeparatum cum alumine.
[0012] The pharmaceutical composition of this disclosure has been proved by
clinical
experiments that it can effectively kill various bacteria with a remarkable
effect.
[0013] The active ingredient of the pharmaceutical composition of this
disclosure can be
prepared by a method including:
[0014] step A. pulverizing fritillariae thunbergii bulbus weighed according to
formula ratio into
fine powder;
[0015] step B. to a mixture of ephedrae herba, forsythiae fructus, armeniacae
semen amarum,
pinelliae rhizoma, arctii fructus, and rhei radix et rhizoma weighed according
to formula ratio,
adding 40-70% ethanol in an amount of 8-10 times of the mixture for a first
extraction under reflux
for 1-4 h, adding 40-70% ethanol in an amount of 6-9 times of the mixture for
a second extraction
under reflux for 1-4 h, combining extracts, filtering, and concentrating
filtrate under reduced
pressure into a clear paste with a relative density of 1.14-1.16 measured at
60 C while recovering
ethanol;
[0016] step C. to a mixture of gypsum fibrosum, mori cortex, peucedani radix,
citri reticulatae
pericarpium, lonicerae japonicae flos, platycodonis radix, and glycyrrhizae
radix et rhizoma
- 4 -
CA 03167551 2022- 8- 8
weighted according to a formula ratio, adding water in an amount of 9-11 times
of the mixture for
a first boiling for 1-4 h, adding water in an amount of 7-9 times of the
mixture for a second boiling
for 1-4 h, combining decoctions, filtering, concentrating filtrate into a
clear paste with a relative
density of 1.14-1.16 measured at 60 C, and combining with the clear paste
obtained in the step B
to obtain a clear paste mixture; and
[0017] step D. mixing the fine powder obtained in the step A and the clear
paste mixture obtained
in the step C to obtain the active ingredients of the pharmaceutical
composition.
[0018] The dosage form of the medicament of this disclosure can be in a form
selected from the
group consisting of capsule, tablet, powder, granule, oral liquid, pill,
tincture, syrup, suppository,
gel, spray and injection.
[0019] To obtain the above dosage forms, it is preferable to add a
pharmaceutically acceptable
adjuvant during the preparation of these dosage forms, including fillers,
disintegrants, lubricants,
suspending agents, binders, sweeteners, flavoring agents, preservatives, bases
and the like. The
fillers include starch, pregelatinized starch, lactose, mannitol, chitin,
microcrystalline cellulose,
sucrose, and the like. The disintegrants include starch, pregelatinized
starch, microcrystalline
cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-
substituted
hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose and the
like. The lubricants
include magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide and
the like. The
suspending agents include polyvinylpyrrolidone, microcrystalline cellulose,
sucrose, agar,
hydroxypropyl methylcellulose and the like. The binders include starch syrup,
polyvinylpyrrolidone, hydroxypropyl methylcellulose and the like. The
sweeteners include sodium
saccharin, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid and the
like. The flavoring
agents include sweeteners and various flavors. The preservatives include
parabens, benzoic acid,
sodium benzoate, sorbic acid and salts thereof, benzalkonium bromide,
chlorhexidine acetate,
eucalyptus oil and the like. The bases include PEG6000, PEG4000, insect wax
and the like.
[0020] The tablet can be prepared by a process including:
[0021] step A. pulverizing fiitillaiiae bulbus weighed according to formula
ratio into fine powder;
[0022] step B. to a mixture of ephedrae herba, forsythiae fructus, armeniacae
semen amarum,
pinelliae rhizoma, arctii fructus, and rhei radix et rhizoma weighed according
to formula ratio,
- 5 -
CA 03167551 2022- 8- 8
adding 40-70% ethanol in an amount of 8-10 times of the mixture for a first
extraction under reflux
for 1-4 h, adding 40-70% ethanol in an amount of 6-9 times of the mixture for
a second extraction
under reflux for 1-4 h, combining extracts, filtering, and concentrating
filtrate under reduced
pressure into a clear paste with a relative density of 1.14-1.16 measured at
60 C while recovering
ethanol;
[0023] step C. to a mixture of gypsum fibrosum, mori cortex, peucedani radix,
citri reticulatae
pericarpium, lonicerae japonicae flos, platycodonis radix, and glycyrrhizae
radix et rhizoma
weighted according to a formula ratio, adding water in an amount of 9-11 times
of the mixture for
a first boiling for 1-4 h, adding water in an amount of 7-9 times of the
mixture for a second boiling
for 1-4 h, combining decoctions, filtering, concentrating filtrate into a
clear paste with a relative
density of 1.14-1.16 measured at 60 C, and combining with the clear paste
obtained in the step B
to obtain a clear paste mixture;
[0024] step D. spray-drying the clear paste mixture obtained in the step C and
collecting spray
powder; and
[0025] step E. preparing the spray powder obtained in the step D and the fine
powder obtained
in the step A into soft material by using ethanol as binder, sieving,
pelletizing, and tableting by
conventional methods of pharmacy.
[0026] Preferably, the process for preparing the tablet includes:
[0027] step A. pulverizing fritillariae bulbus weighed according to formula
ratio into fine powder;
[0028] step B. to a mixture of ephedrae herba, forsythiae fructus, armeniacae
semen amarum,
pinelliae rhizoma, arctii fructus, and rhei radix et rhizoma weighed according
to formula ratio,
adding 50% ethanol in an amount of 10 times of the mixture for a first
extraction under reflux for
3 h, adding 50% ethanol in an amount of 6 times of the mixture for a second
extraction under reflux
for 3 h, combining extracts, filtering, and concentrating filtrate under
reduced pressure into a clear
paste with a relative density of 1.15 measured at 60 C while recovering
ethanol;
[0029] step C. to a mixture of gypsum fibrosum, mori cortex, peucedani radix,
citri reticulatae
pericarpium, lonicerae japonicae flos, platycodonis radix, and glycyrrhizae
radix et rhizoma
weighted according to a formula ratio, adding water in an amount of 10 times
of the mixture for a
first boiling for 2 h, adding water in an amount of 7 times of the mixture for
a first boiling for 2 h,
- 6 -
CA 03167551 2022- 8- 8
combining decoctions, filtering, concentrating filtrate into a clear paste
with a relative density of
1.15 measured at 60 C, and combining with the clear paste obtained in the step
B to obtain a clear
paste mixture;
[0030] step D. spray-drying the clear paste mixture obtained in the step C and
collecting spray
powder; and
[0031] step E. preparing the spray powder obtained in the step D and the fine
powder obtained
in the step A into soft material by using ethanol as binder, sieving,
pelletizing, drying, granulating,
mixing well with sodium carboxymethyl starch, microcrystalline cellulose and
magnesium stearate,
and tableting.
[0032] Other dosage forms of the medicament according to this disclosure can
be prepared from
the materials weighed in proportion by conventional preparation methods. For
example, according
to the preparation process described in Fan Biting's "Pharmaceuticals of
Traditional Chinese
Medicine" (Shanghai Science Press, December 1997 1st edition),
pharmaceutically acceptable
conventional dosage forms can be made.
[0033] Experiments have demonstrated that the pharmaceutical composition of
this disclosure
has significant bacteriostatic effect on a-hemolytic streptococcus, 13-
hemolytic streptococcus,
staphylococcus aureus and streptococcus pneumoniae.
[0034] In another aspect of this disclosure, a method of inhibiting or killing
bacteria includes
administering the pharmaceutical composition of this disclosure to a subject
in need thereof In
another aspect of this disclosure, a method of preventing bacterial infection
or killing bacteria
includes administering the pharmaceutical composition of this disclosure to a
subject in need
thereof. Preferably, the bacteria are gram-positive bacteria; and more
preferably, the gram-positive
bacteria are selected from the group consisting of staphylococcus aureus, a-
hemolytic
streptococcus, 13-hemolytic streptococcus and streptococcus pneumoniae. In
another preferred
aspect, the bacteria are gram-negative bacteria; and more preferably, the gram-
negative bacteria
are selected from the group consisting of escherichia coli and shigella
dysenteriae.
BRIEF DESCRIPTION OF DRAWINGS
- 7 -
CA 03167551 2022- 8- 8
[0035] FIG. 1 shows the protective effect of the pharmaceutical composition of
this disclosure
on mice infected with staphylococcus aureus.
DETAILED DESCRIPTION
[0036] The following examples are intended to illustrate the preparation of
the pharmaceutical
compositions of this disclosure. It should be understood that they are
intended to limit the scope of
the present invention.
Examples
Example 1
[0037] Formula:
ephedrae herba 52 g, gypsum fibrosum 324 g,
forsythiae fructus 194 g, scutellariae radix 78 g,
mori cortex 194 g, armeniacae semen amarum 130
g,
peucedani radix 78 g, pinelliae rhizoma 130 g,
citri reticulatae pericarpium 78 g, fi-itillariae thunbergii
bulbus 78 g,
arctii fructus 130 g, lonicerae flos 130 g,
rhei radix et rhizoma 39 g, platycodonis radix 76 g,
glycyrrhizae radix et rhizoma 65 g.
[0038] Preparation:
[0039] A. Fritillaiiae thunbergii bulbus was weighed according to the formula
ratio and
pulverized into fine powder.
[0040] B. Ephedrae herba, forsythiae fructus, armeniacae semen amarum,
pinelliae rhizoma,
arctii fructus, and rhei radix et rhizoma were weighed according to the
formula ratio. To the mixture,
50% ethanol was added in an amount of 10 times of the mixture for a first
extraction under reflux
for 3 h, and 50% ethanol was added in an amount of 6 times of the mixture for
a second extraction
under reflux for 3 h. The extracts were combined, and filtered. The filtrate
was concentrated under
- 8 -
CA 03167551 2022- 8- 8
reduced pressure into a clear paste with a relative density of 1.15 measured
at 60 C, with ethanol
being recovered.
[0041] C. Gypsum ustum, mori cortex, peucedani radix, citri reticulatae
pericarpium, lonicerae
japonicae flos, platycodonis radix, and glycyrrhizae radix et rhizoma were
weighed according to
the formula ratio. To the mixture, water was added in an amount of 10 times of
the mixture for a
first boiling for 2 h, and water was added in an amount of 7 times of the
mixture for a first boiling
for 2 h. The decoctions was combined, and filtered. The filtrate was
concentrated into a clear paste
with a relative density of 1.15 measured at 60 C. The clear paste was combined
with the clear paste
obtained in the step B to obtain a clear paste mixture.
[0042] D. The clear paste mixture obtained in the step C was spray-dried to
collect spray powder.
[0043] E. The spray powder obtained in the step D and the fine powder obtained
in the step A
were prepared into soft material using 80% ethanol as binder. The soft
material was sieved,
pelletized, dryed at 60 C, and granulated. The granules was mixed well with
sodium
carboxymethyl starch, microcrystalline cellulose and magnesium stearate, and
prepared into tablets
by conventional preparation methods.
Example 2
[0044] Formula:
ephedrae herba 86 g, gypsum fibrosum 194 g,
forsythiae fructus 324 g, scutellariae radix 130 g,
mori cortex 324 g, stir-baked armeniacae semen
amarum 78 g,
peucedani radix 130 g, pinelliae rhizoma 78 g,
citri reticulatae pericarpium 130 g, fritillariae bulbus 130 g,
arctii fructus 78 g, lonicerae flos 78 g,
rhei radix et rhizoma 65 g, platycodonis radix 46 g,
glycyrrhizae radix et rhizoma 39 g.
[0045] Preparation:
- 9 -
CA 03167551 2022- 8- 8
[0046] A. Fritillariae bulbus was weighed according to the formula ratio and
pulverized into fine
powder.
[0047] B. Ephedrae herba, forsythiae fructus, stir-baked armeniacae semen
amarum, pinelliae
rhizoma, arctii fructus, and rhei radix et rhizoma were weighed according to
the formula ratio. To
the mixture, 40% ethanol was added in an amount of 8 times of the mixture for
a first extraction
under reflux for 4 h, and 40% ethanol was added in an amount of 9 times of the
mixture for a
second extraction under reflux for 4 h. The extracts was combined and
filtered. The filtrate was
concentrated under reduced pressure into a clear paste with a relative density
of 1.14 measured at
60 C, with ethanol being recovered.
[0048] C. Gypsum ustum, mori cortex, peucedani radix, citri reticulatae
pericaTium, lonicerae
japonicae flos, platycodonis radix, and glycyrrhizae radix et rhizoma were
weighted according to
the formula ratio. To the mixture, water was added in an amount of 9 times of
the mixture for a
first time boiling for 4 h, and water was added in an amount of 7 times of the
mixture for a second
boiling for 4 h. The decoctions were combined and filtered. The filtrate was
concentrated into a
clear paste with a relative density of 1.16 measured at 60 C. The clear paste
was combined with
the clear paste obtained in the step B to obtain a clear paste mixture.
[0049] D. The clear paste mixture obtained in the step C was spray-dried to
collect spray powder.
[0050] E. The spray powder obtained in the step D and the fine powder obtained
in the step A
were prepared into soft material using 80% ethanol as binder. The soft
material was sieved,
pelletized, dried at 60 C, and granulated. The granules was mixed well with
sodium carboxymethyl
starch, microcrystalline cellulose and magnesium stearate, and prepared into
tablets by
conventional preparation methods.
Example 3
[0051] Formula:
ephedrae herba 69 g, gypsum fibrosum 259 g,
forsythiae fructus 259 g, scutellariae radix 104 g,
mori cortex 259 g, stir-baked armeniacae semen amarum
104 g,
- 10 -
CA 03167551 2022- 8- 8
peucedani radix 104 g, pinelliae rhizoma praeparatum cum
alumine 104 g,
citri reticulatae pericarpium 104 g, fiitillariae thunbergii bulbus 104
g,
arctii fructus 104 g, lonicerae flos 104 g,
rhei radix et rhizoma 52 g, platycodonis radix 61 g,
glycyrrhizae radix et rhizoma 52 g.
[0052] Preparation:
[0053] A. Fritillaiiae thunbergii bulbus was weighed according to the formula
ratio and
pulverized into fine powder.
[0054] B. Ephedrae herba, forsythiae fructus, stir-baked armeniacae semen
amarum, pinelliae
rhizoma, arctii fructus, and rhei radix et rhizoma were weighed according to
the formula ratio. To
the mixture, 70% ethanol was added in an amount of 10 times of the mixture for
a first extraction
under reflux for 1 h, and 70% ethanol was added in an amount of 6 times of the
mixture for a
second extraction under reflux for 1 h. The extracts were combined and
filtered. The filtrate was
concentrated under reduced pressure into a clear paste with a relative density
of 1.16 measured at
60 C, with ethanol being recovered.
[0055] C. Gypsum ustum, mori cortex, peucedani radix, citri reticulatae
pericaTium, lonicerae
japonicae flos, platycodonis radix, and glycyrrhizae radix et rhizoma were
weighted according to
the formula ratio. To the mixture, water was added in an amount of 11 times of
the mixture for a
first boiling for 1 h, and water was added in an amount of 7 times of the
mixture for a second
boiling for 1 h. The decoctions were combined and filtered. The filtrate was
concentrated into a
clear paste with a relative density of 1.14 measured at 60 C. The clear paste
was combined with
the clear paste obtained in the step B to obtain a clear paste mixture.
[0056] D. The clear paste mixture obtained in the step C was spray-dried to
collect spray powder.
[0057] E. The spray powder obtained in the step D and the fine powder obtained
in the step A
were prepared into soft material using 80% ethanol as binder. The soft
material was sieved,
pelletized, dried at 60 C, and granulated. The granules was mixed well with
sodium carboxymethyl
starch, microcrystalline cellulose and magnesium stearate, and prepared into
tablets by
conventional preparation methods.
- 11 -
CA 03167551 2022- 8- 8
Example 4:
[0058] Formula of materials:
ephedrae herba 55 g, gypsum fibrosum 254 g,
forsythiae fructus 318 g, scutellariae radix 107 g,
mori cortex 203 g, stir-baked armeniacae semen
amarum 107 g,
peucedani radix 82 g, pinelliae rhizoma 105 g,
citri reticulatae pericarpium 84 g, fritillariae thunbergii bulbus
125 g,
arctii fructus 122 g, lonicerae flos 113 g,
rhei radix et rhizoma 42 g, platycodonis radix 60 g,
glycyrrhizae radix et rhizoma 50 g.
[0059] Preparation:
[0060] A. Fritillariae thunbergii bulbus was weighed according to the formula
ratio and
pulverized into fine powder.
[0061] B. Ephedrae herba, forsythiae fructus, stir-baked armeniacae semen
amarum, pinelliae
rhizoma, arctii fructus, and rhei radix et rhizoma were weighed according to
the formula ratio. To
the mixture, 60% ethanol was added in an amount of 9 times of the mixture for
a first time
extraction under reflux for 2 h, and 60% ethanol was added in an amount of 7
times of the mixture
for a second time extraction under reflux for 2 h. The extracts were combined
and filtered. The
filtrate was concentrated under reduced pressure into a clear paste with a
relative density of 1.15
measured at 60 C, with ethanol being recovered.
[0062] C. Gypsum ustum, mori cortex, peucedani radix, citri reticulatae
pericarpium, lonicerae
japonicae flos, platycodonis radix, and glycyrrhizae radix et rhizoma were
weighted according to
the formula ratio. To the mixture, water was added in an amount of 10 times of
the mixture for a
first boiling for 2.5 h, and water was added in an amount of 7 times of the
mixture for a second
boiling for 2.5 h. Decoctions were combined and filtered. The filtrate was
concentrated into a clear
paste with a relative density of 1.14 measured at 60 C. The clear paste was
combined with the clear
paste obtained in the step B to obtain a clear paste mixture.
- 12 -
CA 03167551 2022- 8- 8
[0063] D. The clear paste mixture obtained in the step C was spray-dried to
collect spray powder;
and
[0064] E. The spray powder obtained in the step D and the fine powder obtained
in the step A
were prepared into soft material using 80% ethanol as binder. The soft
material was sieved,
pelletized, dried at 60 C, and granulated. The granules was mixed well with
sodium carboxymethyl
starch, microcrystalline cellulose and magnesium stearate, and prepared into
tablets by
conventional preparation methods.
Example 5:
[0065] Capsules were prepared by conventional methods from the following
medical materials:
ephedrae herba 62 g, gypsum fibrosum 220 g, forsythiae fructus 256 g,
scutellariae radix 90 g, mori
cortex 300 g, armeniacae semen amarum 90 g, peucedani radix 90 g, pinelliae
rhizoma 90 g,
citri reticulatae pericamium 100 g, fritillariae bulbus 100 g, arctii fructus
100 g, lonicerae japonicae
flos 100 g, rhei radix et rhizoma 50 g, platycodonis radix 66 g, and
glycyrrhizae radix et rhizoma
50g.
Example 6:
[0066] Granules were prepared by conventional methods from the following
medical materials:
ephedrae herba 68 g, gypsum fibrosum 215 g, forsythiae fructus 215 g,
scutellariae radix 100 g,
mori cortex 220 g, armeniacae semen amarum 90 g, peucedani radix 90 g,
pinelliae rhizoma 90 g,
citri reticulatae pericarpium 90 g, fritillariae bulbus 90 g, arctii fructus
90 g, lonicerae japonicae
flos 90 g, rhei radix et rhizoma 50 g, platycodonis radix 50 g, and
glycyrrhizae radix et rhizoma 50
g.
Example 7:
[0067] Injections were prepared by conventional methods from the following
medical materials:
ephedrae herba 50 g, gypsum fibrosum 200 g, forsythiae fructus 300 g,
scutellariae radix 100 g,
mori cortex 250 g, armeniacae semen amarum 100 g, peucedani radix 100 g,
pinelliae rhizoma 100
g, citri reticulatae pericarpium 100 g, fritillariae bulbus 100 g, arctii
fructus 100 g, lonicerae
japonicae flos 100 g, rhei radix et rhizoma 50 g, platycodonis radix 50 g, and
glycyrrhizae radix et
rhizoma 50 g.
- 13 -
CA 03167551 2022- 8- 8
Example 8:
[0068] Pills were prepared by conventional methods from the following medical
materials:
ephedrae herba 60 g, gypsum fibrosum 200 g, forsythiae fructus 200 g,
scutellariae radix 95 g, mori
cortex 230 g, armeniacae semen amarum 95 g, peucedani radix 95 g, pinelliae
rhizoma 95 g, citri
reticulatae pericamium 95 g, fritillariae bulbus 95 g, arctii fructus 95 g,
lonicerae japonicae flos 95
g, rhei radix et rhizoma 50 g, platycodonis radix 50 g, and glycyrrhizae radix
et rhizoma 50 g.
Biological activity test examples:
[0069] In order to confirm that the pharmaceutical composition of this
disclosure has
antibacterial effects, the pharmaceutical composition prepared in Example 3 of
this disclosure, that
is, the granules after granulation but before being prepared into tablets in
Step E of Example 3
(hereinafter referred to as the drug of this disclosure), was studied for
pharmacology:
[0070] Study on the in vitro antibacterial effect of the pharmaceutical
composition of this
disclosure
[0071] Experimental Materials
[0072] 1. Drugs and reagents:
[0073] (1) The test drug was the pharmaceutical composition of this disclosure
(prepared in
Example 3), which was brown-yellow granules, and each gram of granules was
equivalent to 4.095
g of crude drugs. The drug was administered orally at a daily dosage of 22 g
of crude drugs/person,
which was equivalent to 0.367 g of the pharmaceutical composition/kg of body
weight based on
60 kg of human body weight.
[0074] (2) The positive control drug was Shuanghuanglian Oral Liquid, produced
by Harbin
Pharmaceutical Group Sanchine Pharmaceutical Co., Ltd., with the batch number
of 07101243.
[0075] 2. Bacterial strains:
[0076] (1) Standard strains: staphylococcus aureus (no. 26112), a-hemolytic
streptococcus (no.
32209), I3-hemolytic streptococcus (no. 32210), streptococcus pneumoniae (no.
31001),
escherichia coli (no. 44155), staphylococcus epidermidis (no. 26487),
micrococcus catarrhalis (no.
29103), pseudomonas aeruginosa (no. 10104), shigella dysenteriae (no. 51592),
and salmonella
typhi (no. 50071). They are all purchased from national institute for the
Control of Pharmaceutical
- 14 -
CA 03167551 2022- 8- 8
and Biological Products.
[0077] (2) Clinical strains: 5 strains each of staphylococcus aureus, a-
hemolytic streptococcus,
13-hemolytic streptococcus and streptococcus pneumonia. The 20 strains of
bacteria were clinically
isolated from the Dongfang Hospital Affiliated to Beijing University of
Traditional Chinese
Medicine.
[0078] 3. Culture medium:
[0079] Broth medium and 2% agar broth medium were used for passage and test of
staphylococcus aureus, escherichia coli, staphylococcus epidermidis,
pseudomonas aeruginosa,
shigella dysenteriae and salmonella typhi; 10% serum broth medium and 10%
serum agar broth
medium were used for passage and test of a-hemolytic streptococcus, 0-
hemolytic streptococcus
and streptococcus pneumoniae.
[0080] Methods and Results
[0081] 1. Preparation of the pharmaceutical composition of this disclosure:
[0082] 4.88 g granules of the pharmaceutical composition of this disclosure
was weighed and
dissolved in 40 ml of physiological saline. The resultant was equivalent to
500 mg crude drug/ml.
After being sterilized under high pressure at 4 C and refrigerated for 5 days,
the resultant was
subjected to centrifugation at 5000 rpm for 20 min to collect the supernatant
for in vitro
experiments.
[0083] 2. Bacteriostatic effect of the pharmaceutical composition of this
disclosure on standard
strains (steel ring method):
[0084] Each standard strain used in the bacteriostatic test was passaged for
two times with broth
medium and serum broth medium respectively. Then, the 18-hour culture of each
strain was diluted
10-2-fold with broth medium. 100 p.1 of the 10-2-fold diluted bacterial
solution was added dropwise
to a 2% agar broth plate and a 10% serum agar broth plate respectively, and
spread evenly on the
plate with a sterilized L-shaped glass rod. 5 sterile stainless steel rings
(diameter 7 mm) were placed
at certain intervals on the plate. Then sterile physiological saline (negative
control) and the test
drug solutions, namely the original solution of Shuanghuanglian, the original
solution of the
pharmaceutical composition of this disclosure (500 mg/ml), 1:1 dilution of the
pharmaceutical
- 15 -
CA 03167551 2022- 8- 8
composition of this disclosure (250mg/m1) and 1:2 dilution of the
pharmaceutical composition of
this disclosure (125 mg/ml), were added dropwise into each steel ring and
cultured in an incubator
at 37 C for 18 h, and then the diameter (mm) of the bacteriostatic ring was
measured. According
to the size of the bacteriostatic ring, the bacteriostatic effect of drugs was
determined. The results
are shown in Table 1 below.
[0085] Table 1 In vitro antibacterial effect of the pharmaceutical composition
of this disclosure
(diameter of bacteriostatic ring, mm)
Pharmaceutical Pharmaceutical
Pharmaceutical
composition of composition of
composition of
Strain Control Shuanghuanglian this disclosure
this disclosure this disclosure
500 mg/ml 250 mg/ml 125
mg/ml
Pseudomonas
aeruginosa
13-hemolytic
10.7 12.5 8.5
streptococcus
Escherichia coli ¨ 9.4 9.5 ¨ ¨
Staphylococcus
¨ 12.2 13.7
9.3 ¨
epidermidis
Staphylococcus
¨ 15.2 16.4
8.2 ¨
aureus
Micrococcus
¨ 8.0 10.5
8.4 ¨
catanhalis
Shigella
¨ 11.0 12.5 ¨
¨
dysenteriae
Streptococcus
¨ 14.2 14.7
9.8 ¨
pneumoniae
a-hemolytic
¨ 9.9 10.8
8.4 ¨
streptococcus
Salmonella
¨ 12.1 ¨ ¨
¨
typhi
Note: "¨" means no bacteriostatic ring
[0086] 2. In vitro inhibitory effect of the pharmaceutical composition of this
disclosure against
standard strains (test tube method):
[0087] The results of the above tests show that the pharmaceutical composition
of this disclosure
had a certain inhibitory effect against gram-positive bacteria. Therefore,
four standard strains of
gram-positive bacteria (streptococcus aureus, a-hemolytic streptococcus, 13-
hemolytic
streptococcus and streptococcus pneumoniae) and one strain of gram-negative
bacteria (escherichia
coli) were selected for this test.
- 16 -
CA 03167551 2022- 8- 8
[0088] (1) Drug dilution: 8 test tubes were numbered in sequence, and 4 ml of
serum-containing
broth medium was added by a pipette into each test tube except the first tube
according to the
aseptic operation procedure. To the first tube, instead of culture medium, 8
ml of the original
solution of the pharmaceutical composition of this disclosure (500 mg/ml) was
added. 4 ml of the
solution in the first tube was transferred into the second tube and mixed well
by shaking repeatedly.
Similarly, 4 ml of the solution in the second tube was transferred into the
third tube and mixed well
by shaking repeatedly. The above steps were repeated until the serial dilution
was performed in the
eighth tube. The titer of the drug dilutions were original solution, 1:1, 1:2,
1:4, 1:8, 1:16, 1:32 and
1:64, respectively, and the corresponding concentrations of the pharmaceutical
composition of this
disclosure were 500 mg/ml, 250 mg/ml, 125 mg/ml, 62.5 mg/ml, 31.3 mg/ml, 15.6
mg/ml, 7.8
mg/ml and 3.9 mg/ml. Shuanghuanglian Oral Liquid was diluted in the same way.
[0089] (2) Dilution of bacterial solution: 10 pi of the culture of
streptococcus pneumoniae, a-
hemolytic streptococcus, 13-hemolytic streptococcus, escherichia coli and
staphylococcus aureus
cultured for 18 h was diluted at 1:100 with 1 ml of broth medium.
[0090] (3) Test method: Into 40 test tubes, each tube was added with 1.8 ml of
broth medium or
serum-containing broth medium, followed by 0.2 ml of the pharmaceutical
composition of this
disclosure or Shuanghuanglian in different dilutions. The final concentrations
of the
pharmaceutical composition of this disclosure in each test tube were
respectively 50 mg/ml, 25
mg/ml, 12.5 mg/ml, 6.25 mg/ml, 3.13 mg/ml, 1.56 mg/ml, 0.78 mg/ml and 0.39
mg/ml. Note that
Shuanghuanglian Oral Liquid failed to be expressed by concentration because
its concentration
was not indicated. Bacterial inoculation: the diluted bacterial solutions of
streptococcus
pneumoniae, a-hemolytic streptococcus and 13-hemolytic streptococcus were
added respectively in
an amount of 20 pl to the serum-containing mediums, and the cultures of
escherichia coli and
staphylococcus aureus were added respectively in an amount of 10 pl to the
serum-free mediums.
These bacteria were cultured at 37 C for 24 h to observe their growth. The
lowest drug
concentration that results in no bacterial growth was taken as the minimum
inhibitory concentration
(MIC) of the drug against the strain. 10 pl of culture, from each tube without
visible bacterial
growth, was transferred to sterile medium, and cultured at 37 C for 24 h to
observe whether there
was bacterial growth. No bacterial growth observed in a tube means that the
concentration in the
tube was the minimum bactericidal concentration (MBC) of the drug. The results
are shown in
- 17 -
CA 03167551 2022- 8- 8
Tables 2 and 3.
[0091] Table 2: Minimum inhibitory concentration of the pharmaceutical
composition of this
disclosure against standard strains in vitro (test tube method)
Strains
Pharmaceutical composition of this disclosure Shuanghuanglian
Minimum inhibitory concentration (MIC) Drug dilution Drug
dilution
Escherichia coli ¨ ¨
¨
Staphylococcus aureus 12.5mg/m1 1:2 1:16
Streptococcus pneumoniae 6.25 mg/ml 1:4
1:16
a-hemolytic streptococcus 25 mg/ml 1:1
1:8
0-hemolytic streptococcus 12.5mg/m1 1:2
1:16
Table 3: Minimum bactericidal concentration of the pharmaceutical composition
of this
disclosure against standard strains in vitro (test tube method)
Pharmaceutical composition of this disclosure
Shuanghuanglian
Strains
Minimum bactericidal
Drug dilution
Drug dilution
concentration (MBC)
Escherichia coli ¨ ¨ ¨
Staphylococcus aureus 25mg/m1 1:1 1:32
Streptococcus pneumoniae 12.5mg/m1 1:2 1:32
a-hemolytic streptococcus 50mg/m1 Original solution 1:16
0-hemolytic streptococcus 25mg/m1 1:1 1:16
[0092] 3. In vitro inhibitory effect of the pharmaceutical composition of this
disclosure against
clinical strains (test tube method):
[0093] 20 strains of clinically isolated gram-positive bacteria (including 5
strains each of
staphylococcus aureus, a-hemolytic streptococcus, 13-hemolytic streptococcus
and Streptococcus
pneumoniae) were selected for the study on the inhibitory effect of the
pharmaceutical composition
of this disclosure against the clinical strains. The test method was the same
as above, and the results
- 18 -
CA 03167551 2022- 8- 8
are shown in Tables 4 and 5.
[0094] Table 4: Bacteriostatic effect of the pharmaceutical composition of
this disclosure on
clinical strains (test tube method)
Pharmaceutical composition of this disclosure Shuanghuanglian
Strains
Minimum inhibitory concentration
Drug (MIC) dilution
Drug dilution
Staphylococcus aureus 12.5 mg/ml 1:2
1:32
Streptococcus
12.5 mg/m1 1:2
1:32
pneumoniae
Original
a-hemolytic streptococcus 50 mg/ml
1:16
solution
I3-hemolytic streptococcus 12.5-25mg/m1 1:1-1:2
1:16-1:32
Table 5: Bactericidal effect of the pharmaceutical composition of this
disclosure on clinical
strains (test tube method)
Pharmaceutical composition of this disclosure Shuanghuanglian
Strains
Minimum bactericidal concentration
Drug dilution(MBC)
Drug dilution
Staphylococcus aureus 25 mg/ml 1:1
1:32
Streptococcus pneumoniae 25 mg/ml 1:1
1:32
a-hemolytic streptococcus ¨ ¨
1:16
I3-hemolytic streptococcus 50 mg/ml Original solution
1:16
[0095] Conclusions:
[0096] (1) The study on in vitro effect of the pharmaceutical composition of
this disclosure
against standard strains by using the steel ring method shows that the
pharmaceutical composition
of this disclosure had an in vitro inhibitory effect against the standard
strains including
staphylococcus aureus, a-hemolytic streptococcus, 13-hemolytic streptococcus,
streptococcus
pneumoniae, staphylococcus epidermidis, micrococcus catarrhalis, escherichia
coli and shigella
dysenteriae.
[0097] (2) The study on in vitro effect of the pharmaceutical composition of
this disclosure
- 19 -
CA 03167551 2022- 8- 8
against standard strains by using the test tube method shows that the
pharmaceutical composition
of this disclosure had a minimum inhibitory concentration of 25 mg/ml and a
minimum bactericidal
concentration of 50 mg/ml against a-hemolytic streptococcus, a minimum
inhibitory concentration
of 12.5 mg/ml and a minimum bactericidal concentration of 25 mg/ml against
staphylococcus
aureus or 13-hemolytic streptococcus, and a minimum inhibitory concentration
of 6.25 mg/m1 and
a minimum bactericidal concentration of 12.5 mg/ml against streptococcus
pneumonia.
[0098] (3) The study on in vitro effect of the pharmaceutical composition of
this disclosure
against clinically isolated strains by the test tube method shows that the
pharmaceutical
composition of this disclosure had a minimum inhibitory concentration of 12.5
mg/ml and a
minimum bactericidal concentration of 25 mg/ml against staphylococcus aureus
or streptococcus
pneumoniae, a minimum inhibitory concentration of 50 mg/ml against a-hemolytic
streptococcus,
and a minimum inhibitory concentration of 12.5-25 mg/ml and a minimum
bactericidal
concentration of 50 mg/ml against 13-hemolytic streptococcus.
[0099] It can be seen that the pharmaceutical composition of this disclosure
had an effective in
vitro antibacterial effect on gram-positive bacteria (a-hemolytic
streptococcus, 13-hemolytic
streptococcus, streptococcus pneumonia and staphylococcus aureus) common to
respiratory tract
infections.
[0100] II. Study on the in vivo antibacterial effect of the pharmaceutical
composition of this
disclosure
[0101] Experimental Materials
[0102] 1. Animals:
[0103] ICR mice, half male and half male, weighing 18-22 g, produced by
Beijing Charles River
Laboratory Animal Technology Co., Ltd., license number SCXK (Beijing 2005-
2006).
[0104] 2. Drugs and reagents:
[0105] (1) The test drug was the pharmaceutical composition of this
disclosure, which was the
same as that in the study I on the in vitro antibacterial effect. The test
drug was formulated with
0.5% CMC-Na to the required concentration before use.
[0106] (2) The positive control drug was Shuanghuanglian Oral Liquid, which
was the same as
- 20 -
CA 03167551 2022- 8- 8
that in the study I on the in vitro antibacterial effect.
[0107] (3) Gastrin was purchased from Beijing Yidelong Trading Company. 5 g of
gastrin was
ground in a mortar where 100 ml of physiological saline was added little by
little, and then sterilized
in an autoclaved at 10 pounds for 10 min.
[0108] 3. Strain:
[0109] Staphylococcus aureus (No. 26112) was purchased from the National
Institute for the
Control of Pharmaceutical and Biological Products.
[0110] 4. Instruments:
[0111] UV-120-02 UV-Vis spectrophotometer was a product of Shimadzu, Japan,
and 400R
high-speed refrigerated centrifuge was a product of Heraeus, Germany.
[0112] Methods and Results
[0113] 1. Preparation, injection and counting of staphylococcus aureus
bacterial solution:
[0114] Staphylococcus aureus was subcultured once. The resulting bacterial
culture cultured for
6 h was cultured with broth medium in a 37 C incubator for another 16 h and
then centrifuged at
3000 rpm for 10 min to obtain precipitation. The precipitation was diluted
with sterile physiological
saline, and then placed in an UV-Vis spectrophotometer to compare the color at
a wavelength of
640 nm, as the OD value of the bacterial solution was adjusted to 0.200. Part
of the bacterial
solution was taken out for counting the number of bacteria. The bacterial
solution was centrifuged
at 3000 rpm for 10 min to remove the supernatant and obtain bacteria which
were then restored to
the original volume with sterilized 5% gastrin. The resulting bacterial
solution was
intraperitoneally injected to each mouse at 0.4 ml which corresponded to about
1.6x108 bacteria.
[0115] 2. Animal grouping and treatment:
[0116] 92 ICR mice were randomly divided into 5 groups, namely, a control
group (gavage of
equal volume of 0.5% CMC-Na), a Shuanghuanglian group (gavage at 10 ml/kg
which is
equivalent to 10 times of the daily dosage for humans), and three
pharmaceutical composition
groups of this disclosure, administered by gavage, with doses of 1.9 g/kg
(equivalent to 5 times of
the daily dosage for humans), 3.7 g/kg (equivalent to 10 times of the daily
dosage for humans), and
7.4 g/kg (equivalent to 20 times of the daily dosage for humans),
respectively. The five groups were
-21 -
CA 03167551 2022- 8- 8
administered, 1 h before the injection of bacteria, at 0.4 m1/10 g of body
weight, twice a day, for 3
days. Observation was performed continuously for a week, every 3 hours on the
first day, and twice
a day after that. The results show that the pharmaceutical composition of this
disclosure at high and
medium doses had a significant protective effect on mice infected with the
lethal dose of
staphylococcus aureus, and delayed the death time of the mice. Statistical
analysis was performed
using SPSS software for chi-square test. The results are shown in Tables 6, 7
and FIG. 1.
[0117] Table 6: In vivo effect of the pharmaceutical composition of this
disclosure against
staphylococcus aureus infection
Number of mice surviving (after
Dosage Number of
administration)
Groups
(g/kg) animals
6h 9h 12h 24h 48h 96h 7days
Control group ¨ 18 18 18 12 6 2 1
1
Shuanghuanglian group 1 Oml/kg 18 18 18 15 13 10
9 9
Pharmaceutical composition
7.4 19 19 19 18 18 12 9
9
group of this disclosure
Pharmaceutical composition
3.7 19 19 19 17 16 10 8
8
group of this disclosure
Pharmaceutical composition
1.9 18
18 18 15 10 6 6 5
group of this disclosure
Table 7: In vivo protective effect of the pharmaceutical composition of this
disclosure against
staphylococcus aureus infection
Number Average
Dosage Number Number Mortality
Groups of
survival time
(g/kg) of animals of deaths rate (%)
survival (h)
(x sd)
Control group ¨ 18 1 17 94
37.3+39.0
Shuanghuanglian group 10m1/kg 18 9 9 50**
102.0+70.5
Pharmaceutical
composition group of 7.4 19 9 10 53**
110.5+59.4
this disclosure
Pharmaceutical
composition group of 3.7 19 8 11 58**
98.5+64.3
this disclosure
Pharmaceutical
composition group of 1.9 18 5 13 72
72.7+15.4
this disclosure
Note: *represents p<0.05, **represents p<0.01 compared with the control group.
- 22 -
CA 03167551 2022- 8- 8
[0118] Results and conclusions:
[0119] (1) The mortality rate of mice in the control group injected with
staphylococcus aureus
was 94% within 7 days.
[0120] (2) The mortality rate of mice orally administrated with
Shuanghuanglian decreased to
50%, which had significant difference (p<0.01) compared with the control
group, indicating that
Shuanghuanglian had a significant in vivo protective effect on mice injected
with staphylococcus
aureus.
[0121] (3) The mortality rates of mice in the high and medium dose groups of
the pharmaceutical
composition of this disclosure decreased to 53% and 58%, respectively, which
had significant
difference (p<0.01) compared with the control group. Although the low dose
group had no
statistical difference, the mortality rate of mice appeared a decreasing
trend, showing a dose-effect
relationship in general. The results show that the pharmaceutical composition
of this disclosure had
a significant in vivo protective effect on mice injected with staphylococcus
aureus, and can delay
their death time.
- 23 -
CA 03167551 2022- 8- 8
