U.S. patent application number 10/532687 was filed with the patent office on 2006-03-09 for rabbit skin comprising biological active substances and its use.
Invention is credited to Wing Sum Cheung.
Application Number | 20060051375 10/532687 |
Document ID | / |
Family ID | 32686811 |
Filed Date | 2006-03-09 |
United States Patent
Application |
20060051375 |
Kind Code |
A1 |
Cheung; Wing Sum |
March 9, 2006 |
Rabbit skin comprising biological active substances and its use
Abstract
A rabbit skin containing biologically active substances is
obtained by the process including vaccinating rabbit skin tissues
with vaccinia virus, feeding a rabbit vaccinated with vaccinia
virus, killing the rabbit when its skin tissues are sufficiently
inflamed, and skinning the rabbit. The rabbit skin of the present
invention can be used for preparing drugs and health foods.
Inventors: |
Cheung; Wing Sum; (Jiansu,
CN) |
Correspondence
Address: |
GREER, BURNS & CRAIN
300 S WACKER DR
25TH FLOOR
CHICAGO
IL
60606
US
|
Family ID: |
32686811 |
Appl. No.: |
10/532687 |
Filed: |
October 30, 2003 |
PCT Filed: |
October 30, 2003 |
PCT NO: |
PCT/CN03/00923 |
371 Date: |
April 26, 2005 |
Current U.S.
Class: |
424/232.1 ;
424/572 |
Current CPC
Class: |
A61K 39/12 20130101;
A61P 35/02 20180101; A61K 39/285 20130101; A61P 17/02 20180101;
A23J 1/10 20130101; A61P 29/00 20180101; A61P 37/08 20180101; A61K
35/36 20130101; A61P 25/04 20180101; A61K 35/12 20130101; A61P
25/20 20180101; A61K 2039/54 20130101 |
Class at
Publication: |
424/232.1 ;
424/572 |
International
Class: |
A61K 39/275 20060101
A61K039/275; A61K 35/12 20060101 A61K035/12; A61K 39/285 20060101
A61K039/285 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 31, 2002 |
CN |
02145975.4 |
Claims
1. A rabbit skin containing biologically active substances, said
skin is obtained by the following process comprising: vaccinating
rabbit skin tissues with vaccinia virus, feeding rabbit vaccinated
with vaccinia virus, killing the rabbit when its skin tissues is
sufficiently inflamed, and skinning the rabbit.
2. The rabbit skin according to claim 1, wherein the said vaccinia
virus is vaccinia virus Lister strain.
3. The rabbit skin according to claim 1, wherein the said vaccinia
virus is vaccinia virus Ikeda strain.
4. The rabbit skin according to claim 1, wherein the said vaccinia
virus is vaccinia virus Dairen strain.
5. The rabbit skin according to claim 1, wherein the said vaccinia
virus is vaccinia virus EM-63 strain.
6. The rabbit skin according to claim 1, wherein the said
vaccinating rabbit skin tissues with vaccinia virus is effected by
injecting subcutaneously 0.1.about.0.4 ml solution containing
10.sup.6.about.10.sup.9 viruses/ml each site, 100 to 250 sites per
rabbit weighing 1.5.about.3 Kg.
7. The rabbit skin according to claim 1, wherein the said rabbit is
a Japanese white rabbit.
8. The rabbit skin according to claim 1, wherein the said rabbit is
a New Zealand white rabbit.
9. The rabbit skin according to claim 1, wherein the said rabbit is
a Chinese rabbit.
10. The rabbit skin according to claim 1, wherein the said rabbit
is a Blue-violet rabbit.
11. The rabbit skin according to claim 1, wherein the said killing
of the rabbit when its skin tissues are sufficiently inflamed is
effected when the rabbit skin inflammatory tissue shows visible
blains accompanying with changing colour from redness to mauveness
and becomes thick, and its subcuticle and hip become swollen.
12. The rabbit skin according to claim 1, which possesses 0.5 iu/g
SART activity or more.
13. The rabbit skin according to claim 1, which possesses
kallikrein-protease inhibition activity.
14. Use of the rabbit skin according to claim 1, characterized in
that the rabbit skin is used for preparing at least one drug.
15. Use of the rabbit skin according to claim 1, characterized in
that the rabbit skin is used for preparing health food.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a rabbit skin containing
biologically active substances and its use.
BACKGROUND OF THE INVENTION
[0002] It was reported that the extracts from inflammatory rabbit
skin tissues vaccinated with vaccinia virus can be used for the
treatment of allergic disease and have the analgesic effect. There
has not been any method to prepare rabbit skin that contains
strongly active and high yield biologically active substances.
DETAILED DESCRIPTION OF THE INVENTION
Aims
[0003] The objective of the present invention is to provide a
rabbit skin which is rich in biologically highly active substances
that can be used for preparing drugs and health foods.
Project
[0004] As a result of many years of hard work, the inventors of the
present invention have reached this aim.
[0005] The rabbit skin of the present invention is obtained by the
process including vaccinating rabbit (Oryctolagus cuniculus) skin
tissues with vaccinia virus, feeding rabbit vaccinated with
vaccinia virus, killing the rabbit when its skin tissues is
inflamed enough, and peeling the rabbit.
[0006] Vaccinia virus has been used widely since the 20th century.
All kinds of vaccinia virus can be used to prepare the rabbit skin
of the present invention, such as Lister strain, Ikeda strain,
Dairen strain, EM-63 strain, Temple of Heaven strain, LMC strain,
Tashkent strain, Williamsport strain, and New York City Board of
Health strain. The preferred strains are Lister strain, Ikeda
strain, Dairen strain, EM-63 strain, the most preferred strain is
Lister strain. All these vaccinia virus strains can be purchased
from the market. The vaccinia virus strains used in the present
invention can be purchased strain or strain obtained from
subculture with rabbit.
[0007] The preferred vaccination method is subcutaneous
vaccination, the said vaccinating rabbit skin tissues with vaccinia
virus is effected by injecting subcutaneously 0.1.about.0.4 ml
solution containing 10.sup.6.about.10.sup.9 viruses/ml each site,
100 to 250 sites per rabbit weighing 1.5.about.3 Kg.
[0008] The rabbit used in preparing the rabbit skin of present
invention can be all kinds of rabbits, such as Japanese white
rabbit, New Zealand white rabbit, Chinese rabbit, Blue-violet
rabbit, Silver Fox rabbit, Viennese rabbit, Long hair rabbit,
Himalayan albio rabbit, Pex, Belgian Hare rabbit, Lop, California
rabbit, Chekered Giant, Denmark white rabbit, West Germany long
hair rabbit, the preferred rabbits are Japanese white rabbit, New
Zealand white Rabbit, Chinese rabbit, Blue-violet rabbit, the most
preferred rabbit is Japanese white rabbit.
[0009] The said killing the rabbit when its skin tissues is
inflamed enough is effected when the rabbit skin inflammatory
tissue shows visible blains accompanying with changing colour from
redness to mauveness and becomes thick, and its subcuticle and hip
become swollen. The preferred method to kill rabbit is cervical
vertebrae dislocation.
Effect
[0010] The rabbit skin of the present invention possesses 0.5 iu/g
SART activity or more, and which also possesses the
kallikrein-protease inhibition activity.
[0011] By extracting with organic solvent, processing with acid,
processing with alkali, absorbing, eluting and concentrating,
biologically active preparations being rich in amino acids and
nucleic acids can be prepared from the rabbit skin. The said amino
acids include glutamic acid, glycine, alanine, valine, isoleucine,
leucine, tyrosine, phenylalanine, lysine, histidine, aspartic acid,
threonine and serine. The said nucleic acids include urocanic acid,
uracil, hypoxanthine, xanthine and thymine.
[0012] Drug can be prepared by combining the biologically active
preparations of the present invention with pharmaceutical
acceptable adjuvants. This drug can be various form of preparations
used clinically, including injection and tablet, the preferred form
is the injection. For injections, the adjuvants can be for example
injectable distilled water, normal saline, injectable vegetable
oil, glucose injection, propylene glycol, polyethylene glycol, or
it may be all kinds of stabilizers and emulsifiers. For tablets,
capsules and granules, the adjuvants can be excipients such as
starch, lactose, mannitol; binders such as crystalline cellulose,
arabic gum, corn starch, glutin, polyethylene, polyvinyl alcohol,
polyvinyl-pyrrolidone; disintegrants such as carboxylmethyl
cellulose, poly-ethylene glycol, potato starch; lubricants such as
talcum powder, magnesium stearic acid; moistening agents such as
glycerol. For ointments, the adjuvants can be fat oil, paraffin,
wool fat, vaseline, glycol, glycerol.
[0013] The pharmacological and clinic experiments showed that the
drugs prepared from the rabbit skin of the present invention have
analgesic effect against all kinds of symptomatic neuralgia,
lambago, cholecystagia, angina, arterial embolism pains, acute
pains from wound, burn and scald, pains in surgery or post-surgery,
peptic ulcer pain, dysmenorrhea, labor pains posterior to
childbirth, headache, pains induced by various tumor and so on.
[0014] The study showed that the drugs prepared from the rabbit
skin of the present invention can effectively promote activation of
macrophage, significantly inhibit 48-hour homologous PCA reaction
induced by antibody of IgE in the model of type I allergic reaction
in mice, inhibit the activity of anti-complement in type II
allergic reaction. The effects have linear correlation with the
doses. So the drugs have effects on inhibiting inflammatory
reaction correlated with immunity and improving immunity
function.
[0015] Moreover, the drugs prepared from the rabbit skin of the
present invention have anti-allergic, anti-ulcer and sedative
effects and so on.
[0016] After a continuous 28-day intraperitoneally administration
of the drugs prepared from the rabbit skin of the present invention
in rats, no rats died and no changes induced by the drugs existed
in examinations of urine, eye, blood biochemistry, pathology and
anatomy. Therefore, the analgesic drugs of the present invention
have little toxic effects.
[0017] Health food can be prepared by combining the biologically
active preparations of the present invention with edible additives
and nutritious substances, the said edible additives and nutritious
substances include all kinds of vitamins and flavoring agents and
so on. These kinds of health food have effects on improving
immunity, alleviating pains, anti-allergy and anti-stress and so
on.
[0018] The method of determination of SART (Specific Alteration of
Rhythm in Environmental Temperature) activity is well known in the
art (Folia pharmacol. japon. 71:211.about.220, 1975).
[0019] The kallikrein-protease inhibition activity referred in this
description is determined as follows:
[0020] The rabbit skin was cut in pieces of 1 cm.sup.2, 4 times
(w/w) of 3% phenol aqueous solution was added, then placed the
mixture at 4.degree. C. for 72 h, centrifuged after liquid changed
into emulsion. The supernatant was filtrated to collect brown
solution A. The brown solution A was boiled for 40 min in a water
bath after pH was adjusted to 5.0 by 1M HCl, cooled to 28.degree.
C. promptly, centrifuged and filtrated to collect solution B. The
solution B was boiled for 40 min in a water bath after pH of
filtrate was adjusted to 9.2 by 1M NaOH, cooled to 28.degree. C.
promptly, and filtrated to collect solution C. pH of the solution C
was adjusted to 4.5 by 1M HCl, activated charcoal was added at
30.degree. C., stirred continuously for 4 h, stopped stirring, left
it for 30 min, removed the supernatant, filtrated under nitrogen
atmosphere. Then the activated charcoal was dipped in injectable
water and washed, filtrated and discarded filtrate to collect the
activated charcoal, reserved the activated charcoal. The activated
charcoal was then put into injectable water, adjusted pH to 11.0 by
1M NaOH, stirred continuously for 4 h, filtrated with a 0.45-.mu.m
Millipore filter under nitrogen atmosphere, washed the activated
charcoal by injectable water to collect solution D. After pH of the
solution D was adjusted to 6.0 by 1M HCl, the vessel was sealed,
heated up and kept the temperature at 121.degree. C. for 20 min,
and then cooled down to under 40.degree. C. to collect solution E.
The solution E was placed into a decompression distillator,
replaced the air with nitrogen in the decompression distillator,
distilled at 60.degree. C. under decompression, filtrated to
collect solution containing biologically active substances, whose
SART activity was determined. The SART activity of the solution was
adjusted to 1.2 iu/ml by evaporating, concentrating and diluting
with distilled water. 10 ml of this solution was desalted at 10
.mu.s/cm of final conductance, dried under decompression condition,
into which 1.5 ml of 0.25 M NaCl solution was added to obtain test
solution. 0.2 ml of 0.25M NaCl solution was regarded as the control
solution and treated with parallel process with the 0.2 ml test
solution. 0.5 ml human plasma were added respectively to both
solutions, placed at freezing point for 5 min, added 0.25 ml of
suspension of argilla, placed at freezing point for 20 min, and
filtrated. 0.1 ml of filtrate was mixed with 0.2 ml of 0.1 M
Tris-HCl buffer and 0.1 ml of basic solution. Effected reaction for
20 min at 30.degree. C., and stopped the reaction by adding 0.8 ml
of 1% citric acid. The absorbance A of test solution was determined
in 405 nm as the absorbance of control solution was initialized as
0.4. If A was less than 0.4, the rabbit skin from which the test
solution was prepared was regarded as possessing the
kallikrein-protease inhibition activity.
EXAMPLES
[0021] The present invention will be illustrated with following
non-limited examples.
Example 1
[0022] Preparation of Rabbit Skin
[0023] The dry variola vaccine of vaccinia virus Lister strain was
dissolved by PBS(-) (NaCl 80 g, KCl 2 g, NaH.sub.2PO.sub.4 11.5 g,
KH.sub.2PO.sub.4.2H.sub.2O 2 g, adding injectable H.sub.2O to 10 L)
and well shaken. 0.4 ml of this solution was injected in central
inner lamina of testicle of Japanese white rabbit. Dislocating its
cervical vertebra on the fourth day. Cut the scrotum, removed
connective tissue in the testicle. The testicle was placed in
vessel full of ice, preserved at -80.degree. C. refrigerator. The
testicle was taken from refrigerator, softened for 1 h, grinded at
4.degree. C., mixed in 1:1 with EAGLES' cultural medium (Eagle's
powder 9.4 g, 10% NaHCO.sub.3 12.5.about.22.0 ml, glutamine 10 ml,
injectable H.sub.2O 1 L), packed, frozen at -80.degree. C. in
refrigerator for 1 h, thawed at 37.degree. C. in water bath,
centrifuged with 3500 rpm at 4.degree. C. for 20 min, packed by 10
ml. The subculture antigen was preserved at -80.degree. C. in
refrigerator. The virus solution of subculture antigen was taken
from the -80.degree. C. refrigerator, thawed at 30.degree. C. in
incubator. 5 ml of the virus solution was added into 500 ml of
PBS(-) by a 10-ml syringe, well shaken and diluted to injection of
10.sup.9 virus/ml. A Japanese white rabbit weighing 3 kg with its
back shaved, was sterilized with 75% ethanol, injected
subcutaneously with the virus injection 0.4 ml per site in 200
sites with no leaking, no injecting without the virus injection and
no puncturing throughout the skin. The rabbit injected was fed for
4 days. When inflammatory tissue showed that skin surface had
visible blains accompanying with changing colour from redness to
mauveness and skin became thick, and subcuticle and hip became
swollen, killed the rabbit by cervical vertebra dislocation and
peeled in 15 min. The skin of rabbit was packed in plastic bag,
preserved at -18.degree. C. in refrigerator prior to use. The
rabbit skin weighed 349 g, its SART activity was 0.85 iu/g, and its
absorbance was 0.07. The results indicated that it possessed the
kallikrein-protease inhibition activity.
Example 2
[0024] Preparation of Rabbit Skin
[0025] The vaccinia virus Ikeda strain and New Zealand white rabbit
were used to prepare the subculture antigen according to example
1.
[0026] The virus solution of subculture antigen was taken from the
-80.degree. C. refrigerator, thawed at 30.degree. C. in incubator.
5 ml of the virus solution was added into 500 ml of PBS(-) by a
10-ml syringe, well shaken and diluted to injection of 10.sup.9
virus/ml. A New Zealand white rabbit weighing 2.75 kg with its back
shaved, was sterilized with 75% ethanol, injected subcutaneously
with the virus injection 0.3 ml per site in 250 sites with no
leaking, no injecting without the virus injection and no puncturing
throughout skin. The rabbit injected was fed for 3 days. When
inflammatory tissue showed that skin surface had visible blains
accompanying with changing colour from redness to mauveness and
skin became thick, and subcuticle and hip became swollen, killed
the rabbit by cervical vertebra dislocation and peeled in 15 min.
The skin of rabbit was packed in plastic bag, preserved at
-18.degree. C. in refrigerator prior to use. The skin of rabbit
weighed 302 g, its SART activity was 0.60 iu/g, and its absorbance
was 0.1. The results indicated that it possessed the
kallikrein-protease inhibition activity.
Example 3
[0027] Preparation of Rabbit Skin
[0028] The vaccinia virus Dairen strain and Chinese rabbit were
used to prepare the subculture antigen according to example 1.
[0029] The virus solution of subculture antigen was taken from the
-80.degree. C. refrigerator, thawed at 30.degree. C. in incubator.
5 ml of the virus solution was added into 500 ml of PBS(-) by a
10-ml syringe, well shaken and diluted to injection of 10.sup.6
virus/ml. A Chinese rabbit weighing 1.5 kg with its back shaved,
was sterilized with 75% ethanol, injected subcutaneously with the
virus injection 0.1 ml per site in 250 sites with no leaking, no
injecting without the virus injection and no puncturing throughout
skin. The rabbit injected was fed for 3 days. When inflammatory
tissue showed that skin surface had visible blains accompanying
with changing colour from redness to mauveness and skin became
thick, and subcuticle and hip became swollen, killed the rabbit by
cervical vertebra dislocation and peeled in 15 min. The skin of
rabbit was packed in plastic bag, preserved at -18.degree. C. in
refrigerator prior to use. The skin of rabbit weighed 176 g, its
SART activity was 0.50 iu/g, and its absorbance was 0.15. The
results indicated that it possessed the kallikrein-protease
inhibition activity.
Example 4
[0030] Preparation of Rabbit Skin
[0031] The vaccinia virus EM-63 strain and Blue-violet rabbit were
used to prepare the subculture antigen according to example 1.
[0032] The virus solution of subculture antigen was taken from the
-80.degree. C. refrigerator, thawed at 30.degree. C. in incubator.
5 ml of the virus solution was added into 500 ml of PBS(-) by a
10-ml syringe, well shaken and diluted to injection of 10.sup.7
virus/ml. A Blue-violet rabbit weighing 2 kg with its back shaved,
was sterilized with 75% ethanol, injected subcutaneously with the
virus injection 0.2 ml per site in 100 sites with no leaking, no
injecting without the virus injection and no puncturing throughout
skin. The rabbit injected was fed for 3 days. When inflammatory
tissue showed that skin surface had visible blains accompanying
with changing colour from redness to mauveness and skin became
thick, and subcuticle and hip became swollen, killed the rabbit by
cervical vertebra dislocation and peeled in 15 min. The skin of
rabbit was packed in plastic bag, preserved at -18.degree. C. in
refrigerator prior to use. The skin of rabbit weighed 230 g, its
SART activity was 0.55 iu/g, and its absorbance was 0.12. The
results indicated that it possessed the kallikrein-protease
inhibition activity.
Example 5
[0033] Preparation of Rabbit Skin
[0034] The vaccinia virus Lister strain and New Zealand white
rabbit were used to prepare the subculture antigen according to
example 1.
[0035] The virus solution of subculture antigen was taken from the
-80.degree. C. refrigerator, thawed at 30.degree. C. in incubator.
5 ml of the virus solution was added into 500 ml of PBS(-) by a
10-ml syringe, well shaken and diluted to injection of 10.sup.9
virus/ml. A New Zealand white rabbit weighing 2.75 kg with its back
shaved, was sterilized with 75% ethanol, injected subcutaneously
with the virus injection 0.3 ml per site in 200 sites with no
leaking, no injecting without the virus injection and no puncturing
throughout skin. The rabbit injected was fed for 3 days. When
inflammatory tissue showed that skin surface had visible blains
accompanying with changing colour from redness to mauveness and
skin became thick, and subcuticle and hip became swollen, the
rabbit was killed by cervical vertebra dislocation and peeled in 15
min. The skin of rabbit was packed in plastic bag, preserved at
-18.degree. C. in refrigerator prior to use. The skin of rabbit
weighed 310 g, its SART activity was 0.79 iu/g, and its absorbance
was 0.09. The results indicated that it possessed the
kallikrein-protease inhibition activity.
Example 6
[0036] Preparation of Rabbit Skin
[0037] The vaccinia virus Lister strain and Chinese rabbit were
used to prepare the subculture antigen according to example 1.
[0038] The virus solution of subculture antigen was taken from the
-80.degree. C. refrigerator, thawed at 30.degree. C. in incubator.
5 ml of the virus solution was added into 500 ml of PBS(-) by a
10-ml syringe, well shaken and diluted to injection of 10.sup.6
virus/ml. A Chinese rabbit weighing 1.5 kg with its back shaved,
was sterilized with 75% ethanol, injected subcutaneously with the
virus injection 0.1 ml per site in 250 sites with no leaking, no
injecting without the virus injection and no puncturing throughout
skin. The rabbit injected was fed for 3 days. When inflammatory
tissue showed that skin surface had visible blains accompanying
with changing colour from redness to mauveness and skin became
thick, and subcuticle and hip became swollen, killed the rabbit by
cervical vertebra dislocation and peeled in 15 min. The skin of
rabbit was packed in plastic bag, preserved at -18.degree. C. in
refrigerator prior to use. The skin of rabbit weighed 185 g, its
SART activity was 0.71 iu/g, and its absorbance was 0.11. The
results indicated that it possessed the kallikrein-protease
inhibition activity.
Example 7
[0039] Preparation of Rabbit Skin
[0040] The vaccinia virus Lister strain and Blue-violet rabbit were
used to prepare the subculture antigen according to example 1.
[0041] The virus solution of subculture antigen was taken from the
-80.degree. C. refrigerator, thawed at 30.degree. C. in incubator.
5 ml of the virus solution was added into 500 ml of PBS(-) by a
10-ml syringe, well shaken and diluted to injection of 107
virus/ml. A Blue-violet rabbit weighing 2 kg with its back shaved,
was sterilized with 75% ethanol, injected subcutaneously with the
virus injection 0.2 ml per site in 100 sites with no leaking, no
injecting without the virus injection and no puncturing throughout
skin. The rabbit injected was fed for 3 days. When inflammatory
tissue showed that skin surface had visible blains accompanying
with changing colour from redness to mauveness and skin became
thick, and subcuticle and hip became swollen, killed the rabbit by
cervical vertebra dislocation and peeled in 15 min. The skin of
rabbit was packed in plastic bag, preserved at -18.degree. C. in
refrigerator prior to use. The skin of rabbit weighed 235 g, its
SART activity was 0.74 iu/g, and its absorbance was 0.13. The
results indicated that it possessed the kallikrein-protease
inhibition activity.
Example 8
[0042] Preparation of Rabbit Skin
[0043] The vaccinia virus Ikeda strain and Japanese white rabbit
were used to prepare the subculture antigen according to example
1.
[0044] The virus solution of subculture antigen was taken from the
-80.degree. C. refrigerator, thawed at 30.degree. C. in incubator.
5 ml virus solution was added into 500 ml PBS (-) by a 10-ml
syringe, well shaken and diluted to injection of 10.sup.9 virus/ml.
A Japanese white rabbit weighing 3 kg with its back shaved, was
sterilized with 75% ethanol, injected subcutaneously with the virus
injection 0.3 ml per site in 200 sites with no leaking, no
injecting without the virus injection and no puncturing throughout
skin. The rabbit injected was fed for 3 days. When inflammatory
tissue showed that skin surface had visible blains accompanying
with changing colour from redness to mauveness and skin became
thick, and subcuticle and hip became swollen, the rabbit was killed
by cervical vertebra dislocation and peeled in 15 min. The skin of
rabbit was packed in plastic bag, preserved at -18.degree. C. in
refrigerator prior to use. The skin of rabbit weighed 335 g, its
SART activity was 0.70 iu/g, and its absorbance was 0.12. The
results indicated that it possessed the kallikrein-protease
inhibition activity.
Example 9
[0045] Preparation of Rabbit Skin
[0046] The vaccinia virus Dairen strain and Japanese white rabbit
were used to prepare for the subculture antigen according to
example 1.
[0047] The virus solution of subculture antigen was taken from the
-80.degree. C. refrigerator, thawed at 30.degree. C. in incubator.
5 ml of the virus solution was added into 500 ml of PBS (-) by a
10-ml syringe, well shaken and diluted to injection of 106
virus/ml. A Japanese white rabbit weighing 3 kg with its back
shaved, was sterilized with 75% ethanol, injected subcutaneously
with the virus injection 0.1 ml per site in 200 sites with no
leaking, no injecting without the virus injection and no puncturing
throughout skin. The rabbit injected was fed for 3 days. When
inflammatory tissue showed that skin surface had visible blains
accompanying with changing colour from redness to mauveness and
skin became thick, and subcuticle and hip became swollen, killed
the rabbit by cervical vertebra dislocation and peeled in 15 min.
The skin of rabbit was packed in plastic bag, preserved at
-18.degree. C. in refrigerator prior to use. The skin of rabbit
weighed 336 g, its SART activity was 0.61 iu/g, and its absorbance
was 0.14. The results indicated that it possessed the
kallikrein-protease inhibition activity.
Example 10
[0048] Preparation of Rabbit Skin
[0049] The vaccinia virus EM-63 strain and Japanese white rabbit
were used to prepare the subculture antigen according to example
1.
[0050] The virus solution of subculture antigen was taken from the
-80.degree. C. refrigerator, thawed at 30.degree. C. in incubator.
5 ml of the virus solution was added into 500 ml of PBS(-) by a
10-ml syringe, well shaken and diluted to injection of 10.sup.7
virus/ml. A Japanese white rabbit weighing 3 kg with its back
shaved, was sterilized with 75% ethanol, injected subcutaneously
with the virus injection 0.2 ml per site in 200 sites with no
leaking, no injecting without the virus injection and no puncturing
throughout skin. The rabbit injected was fed for 3 days. When
inflammatory tissue showed that skin surface had visible blains
accompanying with changing colour from redness to mauveness and
skin became thick, and subcuticle and hip became swollen, killed
the rabbit by cervical vertebra dislocation and peeled in 15 min.
The skin of rabbit was packed in plastic bag, preserved at
-18.degree. C. in refrigerator prior to use. The skin of rabbit
weighed 335 g, its SART activity was 0.66 iu/g, and its absorbance
was 0.12. The results indicated that it possessed the
kallikrein-protease inhibition activity.
Example 11
[0051] Extraction of Bioactive Substances.
[0052] 200 g each of the rabbit skins prepared according to example
1.about.10 were cut into pieces of 1 cm.sup.2, added into 4 times
(w/w) of 3% phenol solution, placed at 4.degree. C. for 72 h,
centrifuged after liquid changed into emulsion. The centrifuged
liquid was filtrated to collect brown solution A. After solution
A's pH was adjusted to 5.0 by 1M HCl, boiled solution A in a water
bath for 40 min and cooled down to 28.degree. C. immediately,
centrifuged, and filtrated to collect solution B. After pH of
filtrate was adjusted to 9.2 by 1M NaOH, the solution B was boiling
for 40 min in a water bath, cooled down to 28.degree. C.
immediately, and filtrated to collect solution C. After pH of
filtrate was adjusted to 4.5 by 1M HCl, 50 g of activated charcoal
was added to the solution C at 30.degree. C., stirred continuously
for 4 h, stopped stirring, left it for 30 min, removed the
supernatant, filtrated under nitrogen atmosphere. The activated
charcoal was dipped in injectable water and washed, filtrated and
removed filtrate to collect and reserve the activated charcoal. The
activated charcoal was then added into 400 ml injectable water in
which pH was adjusted to 11.0 by 1M NaOH, stirred continuously for
4 h, filtrated with a 0.45-.mu.m Millipore filter under nitrogen
atmosphere, washed by 40 ml of injectable water to collect solution
D. After pH of the solution D was adjusted to 6.0 by 1M HCl, the
vessel was sealed, heated up and kept the temperature at
121.degree. C. for 20 min and cooled down to under 40.degree. C. to
collect solution E. The solution E in decompression distillator
under nitrogen was decompressed and evaporated at 60.degree. C.
till the volume was 5 ml and filtrated to collect preparation of 5
ml of solution containing bioactive substances. The content of
amino acids and nucleic acids below were determined (.mu.g/ml):
TABLE-US-00001 Sample Exam. 1 Exam. 2 Exam. 3 Exam. 4 Exam. 5 Exam.
6 Exam. 7 Exam. 8 Exam. 9 Exam. 10 Glutamic acid 1.64 0.85 0.70
0.80 1.54 1.20 1.40 1.12 0.98 1.03 Glycine 0.92 0.51 0.33 0.49 0.86
0.73 0.80 0.70 0.55 0.61 Alanine 0.96 0.64 0.59 0.60 0.92 0.77 0.83
0.76 0.66 0.70 Valine 0.66 0.34 0.23 0.29 0.62 0.57 0.61 0.51 0.39
0.45 Isoleucine 0.42 0.17 0.10 0.14 0.40 0.32 0.38 0.30 0.26 0.28
Leucine 0.67 0.22 0.11 0.16 0.66 0.53 0.60 0.46 0.35 0.41 Tyrosine
0.83 0.30 0.25 0.20 0.77 0.61 0.69 0.52 0.36 0.44 Phenylalanine
0.55 0.26 0.24 0.25 0.53 0.42 0.48 0.34 0.30 0.33 Lysine 0.47 0.11
0.09 0.10 0.45 0.39 0.34 0.34 0.19 0.26 Histidine 0.64 0.24 0.18
0.21 0.57 0.43 0.53 0.41 0.31 0.35 Aspartic acid 0.68 0.44 0.39
0.40 0.61 0.57 0.58 0.49 0.45 0.46 Threonine 0.51 0.24 0.11 0.16
0.50 0.42 0.46 0.38 0.30 0.34 Serine 1.01 0.69 0.66 0.67 0.98 0.79
0.88 0.75 0.70 0.71 Urocanic acid 25.00 13.24 12.52 13.00 24.75
22.39 24.00 20.01 16.55 17.64 Uracil 16.12 6.66 5.51 6.16 14.31
10.46 13.19 10.00 7.12 8.54 Hypoxanthine 1.71 0.85 0.80 0.81 1.65
1.11 1.34 1.01 0.89 0.99 Xanthine 12.44 6.13 5.21 5.79 12.00 9.98
11.67 9.62 6.39 8.13 Thymine 3.38 1.99 1.15 1.54 3.30 2.77 3.19
2.49 2.04 2.44
Example 12
[0053] Preparation of Drug
[0054] The analgesic injection was prepared by formula below using
regular method. TABLE-US-00002 Preparation from rabbit skin
according to example 2 5 ml NaCl 2.6 g Injectable distilled water
300 ml
Example 13
[0055] Preparation of Tablet
[0056] The analgesic tablet was prepared by formula below using
regular method. TABLE-US-00003 Preparation from rabbit skin
according to example 1 50 ml Lactose 125 mg Crystalline cellulose
20 g Magnesium stearic acid 5 mg
Example 14
[0057] Preparation of Health Food
[0058] The health food was prepared by formula below using regular
method. TABLE-US-00004 Preparation from rabbit skin according to
example 1 50 ml Sucrose 125 mg Citric acid 20 mg Vitamin C 5 mg
Water 1000 ml
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