U.S. patent application number 10/478048 was filed with the patent office on 2004-07-29 for herbal drug composition for cartilage protection.
Invention is credited to Cho, Yong-Baik, Han, Chang-Kyun, Joung, Ki Won, Jung, In Ho, Kim, Joo-Hyon, Kim, Taek Su, Kum, Do Seung, Kwak, Wie-Jong, Lee, So Yoon, Rhee, Hae In, Ryu, Keun Ho, Um, Key An, Yi, Jung Bum, Yoo, Hun Seung.
Application Number | 20040146593 10/478048 |
Document ID | / |
Family ID | 19709626 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146593 |
Kind Code |
A1 |
Han, Chang-Kyun ; et
al. |
July 29, 2004 |
Herbal drug composition for cartilage protection
Abstract
The present invention relates to a herbal drug composition for
cartilage protection comprising plant extracts of Clematis Radix,
Trichosanthis Radix, and Prunellae Spica and an optimal content of
rosmarinic acid to: (i) alleviate pains; (ii) inhibit the
acute/chronic inflammation, platelet/whole blood aggregation,
immunocyte (B-lymphcyte and T-lymphcyte) proliferation,
inflammation-inducing enzyme activities, and enzyme activities
associated with degradation of joint tissue; (iii) scavenge
activity of toxic active oxygen radicals; and (iv) further provide
excellent cartilage protection activity to be effectively used as
an anti-inflammatory agent with analgesic effects, blood
circulation enhancer, arthritis therapeutic agent and cartilage
protective.
Inventors: |
Han, Chang-Kyun; (Seoul,
KR) ; Kwak, Wie-Jong; (Seoul, KR) ; Joung, Ki
Won; (Kyunggi-do, KR) ; Yoo, Hun Seung;
(Seoul, KR) ; Kum, Do Seung; (Seoul, KR) ;
Cho, Yong-Baik; (Kyunggi-do, KR) ; Ryu, Keun Ho;
(Seoul, KR) ; Rhee, Hae In; (Seoul, KR) ;
Kim, Taek Su; (Kyunggi-do, KR) ; Jung, In Ho;
(Kyunggi-do, KR) ; Lee, So Yoon; (Seoul, KR)
; Yi, Jung Bum; (Kyunggi-do, KR) ; Kim,
Joo-Hyon; (Seoul, KR) ; Um, Key An;
(Kyunggi-do, KR) |
Correspondence
Address: |
Ronald R Santucci
Frommer Lawrence & Haug
745 Fifth Avenue
New York
NY
10151
US
|
Family ID: |
19709626 |
Appl. No.: |
10/478048 |
Filed: |
November 17, 2003 |
PCT Filed: |
January 4, 2002 |
PCT NO: |
PCT/KR02/00010 |
Current U.S.
Class: |
424/773 |
Current CPC
Class: |
A61P 37/02 20180101;
A61K 36/536 20130101; A61P 19/04 20180101; A61P 7/02 20180101; A61P
19/02 20180101; A61P 19/00 20180101; A61K 36/428 20130101; A61P
29/00 20180101; A61K 36/716 20130101; A61K 36/428 20130101; A61K
2300/00 20130101; A61K 36/536 20130101; A61K 2300/00 20130101; A61K
36/716 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/773 |
International
Class: |
A61K 035/78 |
Foreign Application Data
Date |
Code |
Application Number |
May 18, 2001 |
KR |
2001-0027231 |
Claims
What is claimed is:
1. A herbal drug composition for the cartilage protection
comprising Clematis Radix, Trichosanthis Radix, Prunellae Spica,
wherein more than 0.6 wt. % of rosmarinic acid is present in the
total composition.
2. The herbal drug composition for the cartilage protection
according to claim 1, wherein said rosmarinic acid is present in
the range of from 0.6 to 5.0 wt. % relative to the total
composition.
3. The herbal drug composition for the cartilage protection
according to claim 1, wherein said herbal drug composition
comprises oleanolic acid and 4-hydroxybenzoic acid in addition to
said rosmarinic acid.
4. The herbal drug composition for the cartilage protection
according to claim 1, wherein said oleanolic acid is present in the
range of from 2.0 to 6.0 wt. % to the total composition.
5. The herbal drug composition for the cartilage protection
according to claim 3, wherein said 4-hydroxybenzoic acid is present
in the range of from 0.01 to 0.04 wt. % to the total
composition.
6. A cartilage protective comprising the herbal drug composition
selected from the possible combinations consisting of said claims
1-5 as an active ingredient for cartilage protection and arthritis
therapeutic treatment.
7. The cartilage protective according to claim 6, wherein said
cartilage protective is formulated in the form of tablets or soft
gelatin capsules for oral administration, injection solutions,
ointments, or transdermals.
8. The cartilage protective according to claim 7, wherein the
dosage of said tablet or soft gelatin capsule for oral
administration is in the range of from 50 to 2400 mg daily.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a herbal drug composition
for cartilage protection and more particularly, to the drug
composition for cartilage protection comprising plant extracts of
Clematis Radix, Trichosanthis Radix, and Prunellae Spica and an
optimal content of rosmarinic acid to: (i) alleviate pains; (ii)
inhibit the acute/chronic inflammation, platelet/whole blood
aggregation, immunocyte (B-lymphocyte and T-lymphocyte)
proliferation, inflammation-inducing enzyme activities, and enzyme
activities associated with degradation of joint tissue; (iii)
scavenge activity of toxic active oxygen radicals; and (iv) further
provide excellent cartilage protection activity to be effectively
used as an anti-inflammatory agent with analgesic effects, blood
circulation enhancer, arthritis therapeutic agent and cartilage
protective.
BACKGROUND OF THE INVENTION
[0002] Clematis Radix, Trichosanthis Radix, and Prunellae Spica are
well known for medicinal plants. Each medicinal plant has long been
used for the treatment of general inflammations, such as various
swellings, wounds, bronchitis, mastitis, tonsillitis, and anal
fistula and also for the relief of various symptoms such as cold or
numb hands, painful knees, painful waist and shoulder, feeble in
health and pain in the skin, in the form of aqueous plant extract.
These symptoms are similar to general arthritis including chronic
rheumatism in terms of the modern pathological concept. Clematis
Radix, a root of Clematis mandshurica and the same genera in plant
taxonomy, is distributed in the shady forest throughout Asia. It is
collected in autumn, washed cleanly after removing cormophyte and
root hair, chopped finely and dried in the sun to be used as a
medicinal use. Clematis Radix, a non-toxic medicinal plant, has
long been used for the treatment of the following symptoms: pains
in the extremities; motor disturbance in knee joints; and paralysis
in the extremities. In particular, Clematis Radix has been
frequently used as a miraculous drug in those patients who feel
uncomfortable while standing due to the coldness in waist, knees
and feet. It is well known that Clematis Radix has various
constituents of flavanone glycosides such as clematin, etc. and
saponins such as clemontanoside A, clemontanoside B, clemontanoside
C, and clemontanoside S, glucoses, and sterols [Research Archives
of Useful Plants Resources in Korea, Korea Research Institute of
Chemical Technology, pp780-781(1988), 2. An Explanatory Diagram of
Korean Medicinal plants, Youngrim Pub., pp489-490(1990)].
[0003] Trichosanthis Radix, known as "multifarious medicine" or
"Karokon", is a non-toxic medicinal herb prepared by collecting
roots of Trichosnathes kirilowii and the same genera in plant
taxonomy, which are perennial liana plants, in autumn. The outer
shells of cleanly washed roots are removed and the rest of the
roots are cut appropriately and dried in the sun for medicinal use.
Trichosanthis Radix has been widely used for excretion of pus,
vanishing the boil, detoxification and antipyretic effect and also
effective for diseases symptomized by thirst, various swellings,
mastitis, and anal fistula. It has been investigated up to now that
Trichosanthis Radix contains trichosanthin as proteins, arginine
and citruline as amino acids, and palmitic acid and linoleic acid
as fatty acids. Recently Trichosanthis Radix is found to contain
bryonolic acid, 4-hydroxybenzoic acid, .alpha.-spinastero as
sterols [Research Archives of Useful Plants Resources in Korea,
Korea Research Institute of Chemical Technology, pp 354-357(1988),
2. An Explanatory Diagram of Korean Medicinal Plants, Youngrim
Pub., pp960-963(1990)].
[0004] Prunellae Spica, a flower or upper part of Prunella vulgaris
and the same genera in plant taxonomy, is a non-toxic medicinal
herb prepared by collecting the flower, when it is half withered
during summer, and drying in the sun. Prunellae Spica has been
widely used for the treatment of the following symptoms: chronic
swellings, smallpox, acute mastitis and lymphatic tuberculosis.
Prunellae Spica is also effective in the destructing lumps
(generated in a lower stomach owing to extravasated blood) or
others, while treating beriberi and numbness in the extremities. It
has been reported that Prunellae Spica contains saponins such as
oleanolic acid glycosides and ursolic acid glycosides, etc, and
also contains carotene, vitamin C, vitamin K, tannin, caffeic acid
and chlorogenic acid. Rosmarinic acid is also found in Prunellae
Spica [Research Archives of Useful Plant Resources in Korea, Korea
Research institute of Chemical Technology, pp 480-482(1988);
Chemical Research for Prunellae Spica, Lee Jak-pyung et al.,
Bulletin of Medical College in Beijing, 17(4), pp297-299(1985);
Pharm. Acta. Helv., 66, No. 7, pp185-188(1991)].
[0005] The conventional oriental herbal books (e.g.,
Dong-Eui-Bo-gam, Hyangyak Gibsung-bang and Kwangjee Beakub) or
related literatures refer to the medical efficacy of herbs and
processes of manufacturing aqueous herbal solutions. However, they
only described a single prescription of each of these medicinal
plants but not a formulation available for the manufacture of
aqueous herbal solution from appropriate combinations of sorted
medicinal plants by harvest place and harvest time to control the
content of active ingredients. Furthermore, these medicinal plants
were prepared by hot water extraction method, and any substances
extracted by above method showed no acquisition of detailed
knowledge on biologically active ingredients.
[0006] On the other hand, the inventors of the present invention
have disclosed a process of extraction and purified biologically
effective ingredients from an extract of Clematis Radix,
Trichosanthis Radix, and Prunellae Spica in a certain ratio, being
useful for alleviating acute/chronic inflammation; for inhibiting
platelet and whole blood aggregation, enzyme activities associated
with degradation of joint tissue, abnormally proliferated
immunocytes, and inflammation-inducing enzymes; for scavenging
activity of toxic active oxygen radical; and further for the
treatment of chronic rheumatoid arthritis (U.S. Pat. No.
5,910,307). U.S. Pat. No. 5,910,307 is characterized by mixing
Clematis Radix, Trichosanthis Radix, and Prunellae Spica in a
weight ratio of 1:0.5-2:0.5-1.5 and extracting the mixture with
water or aqueous alcoholic solution; partitioning with
water-saturated n-butanol and concentrating the alcohol layer under
reduced pressure; and concentrating the result with water under
constant boiling and lyophilizing to obtain an extract in powder
form.
[0007] In the continuous study, the inventors have realized that it
is difficult to standardize the extracts of Clematis Radix,
Trichosanthis Radix, and Prunellae Spica with simple weight ratio
since the content variation of each ingredient significantly varies
with harvest place and harvest time. This further makes it
difficult to merchandize such combined extracts since it is hard to
obtain the reproducibility of active ingredients having analgesic
and anti-inflammatory effects, blood circulation enhancing effect,
arthritis therapeutic effect and cartilage protection. Thereupon,
the inventors have made an extensive research to maximize the
pharmacological efficiency of herbal drug composition with the
reproducibility. As a result, we have developed a method to
optimize the efficiency of cartilage protection effect as well as
the analgesic and anti-inflammatory effect, blood circulation
enhancing effect, and arthritis therapeutic effect by controlling
the content of rosmarinic acid in the herbal drug composition, not
the extract weight ratio of Clematis Radix, Trichosanthis Radix,
and Prunellae Spica. In addition, it was possible to merchandize
medicinal herbs with the reproducibility.
[0008] The present invention, an improved invention of U.S. Pat.
No. 5,910,307, provides significant improvement in merchandizing by
optimizing the herbal extract composition of Clematis Radix,
Trichosanthis Radix, and Prunellae Spica with the proper content of
rosmarinic acid, maximizes pharmacological efficiencies over the
conventional herbal composition and further provides novel
therapeutic effects.
SUMMARY OF THE INVENTION
[0009] The present invention is to provide a herbal drug
composition comprising Clematis Radix, Trichosanthis Radix, and
Prunellae, wherein the ingredients extracted and purified are
standardized and merchandized based on the content of rosmarinic
acid for the purpose of reproducibility and useful for an
anti-inflammatory agent with analgesic effects, blood circulation
enhancer, arthritis therapeutic agent and cartilage protective,
etc. The herbal drug composition of the present invention has
similar or superior efficiency for analgesic and anti-inflammatory
effect and improving blood circulation to the conventional herbal
composition of U.S. Pat. No. 5,910,307. In addition to that, it has
excellent inhibitory activities against enzymes associated with
degradation of joint tissue and a protection activity toward
cartilage and thus, it is superiorly effective to general arthritis
including rheumatism.
[0010] Accordingly, the object of the present invention is to
provide a herbal drug composition having excellent analgesic and
anti-inflammatory effect, improving effect of peripheral blood
circulation, arthritis therapeutic effect and cartilage protection
effect by standardizing the extracts from mixed Clematis Radix,
Trichosanthis Radix, and Prunellae Spica with the proper content of
rosmarinic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 represents the inhibitory activity of the herbal drug
composition on edema induced by carrageenan.
[0012] FIG. 2 represents the anti-coagulant activity of the herbal
drug composition on platelet aggregation.
[0013] FIG. 3 represents the anti-coagulant activity of the herbal
drug composition on whole blood aggregation.
[0014] FIG. 4 represents the inhibitory activity of the herbal drug
composition on chronic inflammation.
[0015] FIG. 5 represents the inhibitory activity of the herbal drug
composition on B-lymphocyte proliferation.
[0016] FIG. 6 represents the inhibitory activity of the herbal drug
composition on T-lymphocyte proliferation.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention is characterized by a combined herbal
drug composition comprising Clematis Radix, Trichosanthis Radix,
Prunellae Spica and more than 0.6 wt. % of rosmarinic acid based on
the total composition.
[0018] The present invention is described in detail as set forth
hereunder.
[0019] The herbal drug composition of the present invention
provides significant biological effects such as analgesic and
anti-inflammations, improvement of blood circulation,
immunoregulation and inhibitions on enzymes associated with
degradation of joint tissue, and particularly, cartilage protection
and thus, being useful as arthritis therapeutic agent and cartilage
protective as well as an analgesic and anti-inflammatory agent and
blood circulation enhancer.
[0020] The content of active ingredients in herbal drugs varies
remarkably with harvest place, harvest time, storage period and
storage condition. Therefore, it is desirable to combine herbal
extracts or herbal plants in an appropriate ratio depending on the
harvest place as shown in Tables 1 to 3. That is, if harvest place,
harvest time, storage period and storage condition are different,
the content of active ingredients contained in the same weight of
herbal plant has different contents, thus the limitation with the
weight ratio of herbal extracts or herbal plants becomes
insignificant.
[0021] Therefore, the present invention is characterized by
selecting rosmarinic acid as a reference material to obtain the
herbal drug composition with optimal pharmacological efficiency.
Rosmarinic acid is known to have excellent activities such as: (i)
antioxidant activity by inhibiting lipid peroxidation and/or
biosynthesis of prostacyclin generated in the metabolism of
arachidonic acid, and by scavenging the active oxygen generated
from polymorphonuclear leukocytes; (ii) anti-inflammatory activity
such as inhibition against the generation of metabolites which are
carriers for inflammation reaction and immunoregulation to inhibit
allergic inflammation; (iii) blood circulation enhancing activity
by inhibition of platelet blood aggregation and fiber degradation
[Agent and Action, 17, pp375-376(1985); Pharm. Acta. Helv., 66, No.
7, pp185-188(1991); Biochem. Pharmae., 29, pp533-538(1980);
Yoa-Hsueh-Hseuh-Pao, 27, pp96-100(1992); Int. J. of Immunopharmae.,
10, No. 6, pp729-737(1988); J. of Natural Products, 50, No. 3,
pp392-399(1987); Yoa-Hsueh-Hseuh-Pao, 28, No. 4, pp241-245(1993)].
The variation of a rosmarinic acid content is broad depending on
harvest place, harvest time, storage period and storage condition
etc. and further the efficiency varies with the content of
rosmarinic acid. This is the reason why rosmarinic acid is selected
as a reference material in the present invention. Accordingly, the
herbal drug composition of the present invention can be prepared by
mixing the herbal extracts of Clematis Radix, Trichosanthis Radix,
Prunellae Spica or extracting the mixture of Clematis Radix,
Trichosanthis Radix, Prunellae Spica not depending on the weight
ratio of each herbal plant but depending on the content of
rosmarinic acid contained in the total herbal plants.
[0022] The herbal drug composition can obtain the desired
pharmaceutical efficiencies when the content of rosmarinic acid is
higher than 0.6 wt. % of rosmarinic acid, preferably 0.6-5.0 wt. %.
When the content of rosmarinic acid is higher than 0.6 wt. % in the
herbal drug composition comprising Clematis Radix, Trichosanthis
Radix, Prunellae Spica, it provides optimal effects such as
cartilage protection activity which has not been taught in the
conventional herbal drug compositions as well as alleviation of
pains, improvement of blood circulation, immunoregulation, and
inhibition of enzymes associated with degradation of joint tissue.
If the content of rosmarinic acid is lower than 0.6 wt. %, the
herbal drug composition provides alleviation of pains, improvement
of blood circulation, immunoregulation, and inhibition of enzymes
associated with degradation of joint tissue but very sluggish
cartilage protection activity. The present invention has no upper
limitation for the amount of the rosmarinic acid but when it is
higher than a certain level, the activities are not improved any
further, thus it is not desirable technically and economically to
increase the content of rosmarinic acid to higher than a certain
level.
[0023] According to the present invention, when the herbal drug
composition contains 2.0-6.0 wt. % of oleanolic acid and 0.01-0.04
wt. % of 4-hydroxybenzoic acid in addition to higher than 0.6 wt. %
of rosmarinic acid, it provides far more potent efficacy toward
cartilage protection activity as well as alleviation of pains,
improvement of blood circulation, immunoregulation, and inhibition
of enzymes associated with degradation of joint tissue.
[0024] Large quantity of oleanolic acid is present in the extract
of Clematis Radix. When the extract is hydrolyzed, sufficient
amounts of oleanolic acid are present as a sapogenin, a sugar-free
form of saponins. According to an analysis, it is found that
oleanolic acid is present as saponins bonded to various glycosides.
It has been reported that oleanolic acid has not only remarkable
anti-inflammatory and analgesic effects but also an excellent
effect for chronic rheumatoid arthritis induced by Mycobacterium
butyricum [J. of Pharm. Pharmacol., 44, No.5, pp456-458(1992);
Chung-Kuo-Li-Hsueh-Pao, 10, No.4, pp381-384(1984); Chem. Pharm,
Bull., 28, No.4, pp1183-1188(1980); Biochem. Int., 24, No.5,
pp981-990(1991)].
[0025] The extract of Trichosanthis Radix contains various organic
acids including 4-hydroxybenzoic acid. 4-hydroxybenzoic acid is
known to have excellent anti-oxidant activity, anti-inflammatory
activity and hormone-like effect in the uterus ablation
osteoporotic model and further is one of reference materials for
the present invention because it is maintained in a certain amount
regardless of harvest place, harvest time, storage period and
storage conditions [Free Radical Biol. & Medcine, 27, No.11/12,
pp1427-1436(1999); J. Ethnopharma., 53, 11-14(1996); Environ. Res.,
75, pp130-134(1997)].
[0026] Rosmarinic acid, oleanolic acid and 4-hydroxybenzoic acid
selected as reference materials are active ingredients of herbal
drug composition obtained from this invention. The efficacy is far
more potent due to remarkable synergic effect when they are
combined in a certain ratio based on the contents of active
ingredients. Further, in addition to such active ingredients of the
herbal drug composition from this invention, other different
ingredients cannot be ruled out in this invention.
[0027] To contain certain amounts of reference materials, the
herbal drug composition of the present invention is prepared by
either mixing each extract in powder form of Clematis Radix,
Trichosanthis Radix, and Prunellae or extracting the mixture of
Clematis Radix, Trichosanthis Radix, and Prunellae Spica based on
the chemical analysis of original herbal plants. The method for
preparing herbal drug composition makes a little difference in
cartilage protection activity. The method for preparing herbal drug
composition is described in detail as set forth hereunder.
[0028] Each Clematis Radix, Trichosanthis Radix, and Prunellae
Spica or a mixture thereof is extracted with 5-10 volumes of water
or aqueous alcoholic solution under reflux for 4 to 6 hours and
filtered. The filtrate is further extracted with 5-10 volumes of
water or aqueous alcoholic solution under reflux and filtered. Each
extract is combined and concentrated to dryness. Hence, if a small
amount of the solvent is used, the extraction efficiency is low due
to the lower solubility of extract and the difficulty of stirring
the extract. In case of using an excess of the solvent, however, a
larger amount of solvent saturated with lower alcohol in water has
to be evaporated and removed, therefore which is uneconomical and
difficult in handling. The present invention performs a series of
extraction steps, i.e., first and second extraction, because when
the extraction is on a large scale, significant losses are
anticipated due to high contents of water in medicinal plants, in
spite of effective filtration. So the second extraction is
responsible for preventing the reduced extraction efficiency rather
than the first extraction only. Further, it is revealed that about
85-95% of the total extract amount is obtained through two
extractions. It is also found that more than three steps of
extraction are not to be desirable in economical matter.
[0029] The extracts are filtered and concentrated, and the filtrate
is purified to remove some impurities such as proteins,
polysaccharides and fatty acids by extracting with the same amount
of lower alcohol saturated with 3-4 volumes of water. Examples of
lower alcohol used in the present invention include butyl alcohol
and propyl alcohol. If the amount of water-saturated lower alcohol
is less than that of the filtrate, a higher concentration of
impurities (e.g., polysaccharides, and proteins) having relatively
strong polarity causes lower concentration of active ingredients in
the extracts due to poor separation of layers.
[0030] After separating the layers, the obtained fractions
extracted with alcohol are concentrated under reduced pressure at
60-70.degree. C. to remove lower alcohol in the sample. Then, the
extract is further concentrated 2-3 times under constant boiling
with 25-50 volumes of water to the total extract amount and
followed with another same amount of water for homogeneous
suspension. The reason why the residue is concentrated under
constant boiling with water is to control the contents of remaining
lower alcohol so as to use the extracting solution as
pharmaceutical raw materials. The obtained extract is then
lyophilized to give an extract powder.
[0031] The obtained extract powder has significant pharmacological
effects, such as analgesic and anti-inflammatory agents, blood
circulation enhancers, rheumatoid arthritis therapeutic agents, and
cartilage protection agents.
[0032] Based on the general manufacturing method, the powdered
extract of this invention is formulated in oral or parenteral
administration such as tablets, soft gelatin capsules, injection
solutions, ointments, and transdermals.
[0033] For human use, the herbal drug composition of this invention
can be administered alone, but will generally be administered in
admixture with a pharmaceutical carrier selected with regard to the
intended route of administration and standard pharmaceutical
practice. For example, the herbal drug composition may be
administered orally, buccally or sublingually, in the form of
tablets containing excipients such as starch or lactose, or in
capsules or ovules either alone or in admixture with excipients, or
in the form of elixirs or suspensions containing flavoring or
coloring agents. Such liquid preparations may be prepared with
pharmaceutically acceptable additives such as suspending agent
(e.g., methylcellulose, a semi-synthetic glyceride such as witepsol
or mixtures of glycerides such as a mixture of apricot kernel oil
and PEG-6 esters or mixtures of PEG-8 and caprylic/capric
glycerides). A compound may also be injected parenterally, for
example intravenously, intramuscularly, subcutaneously or
intracoronarily. For parenteral administration, the compound is
best used in the form of a sterile aqueous solution that may
contain other substances, for example salts, or monosaccharides
such as mannitol or glucose, to make the solution isotonic with
blood.
[0034] For administration to man, the effective dose of the herbal
drug composition may vary with the age, weight, physical condition
and response of the particular patient and administration may be
made once a day or a few times a day according to the physician.
Preferred dosages for an average adult patient (70 Kg) are as
follows: in the range of from 50 to 2400 mg daily for oral
administration such as individual tablets or capsules; in the range
of from 50 to 400 mg daily for parenteral administration; in the
range of from 300 to 2400 mg daily for ointments; and in the range
of from 150 to 1200 mg daily for transdermal administration. The
above dosages are exemplary of the average case but there can be
individual instances in which higher or lower dosage ranges may be
merited, and such are within the scope of this invention. Further,
if the daily dosages of the herbal drug composition are less than
the above ranges, the desired effects are not obtained. On the
other hands, if they are higher than the above ranges, it is also
not preferable due to toxic side effects.
[0035] In particular, while the herbal drug composition of the
present invention was administered to human, the toxic side effect
is far less than other chemically synthesized drugs. As a matter of
fact, several toxicological tests reveal that the combined extract
of the present invention is not toxic to the human.
[0036] The present invention is explained in more detail with
reference to the following examples that should not be taken to
limit the scope of the invention.
REFERENCE EXAMPLE 1
[0037] 250 g of well air dried Clematis Radix where debris were
removed by washing with water was extracted from 2 l of 30% (v/v)
ethanol-containing water under reflux for 6 hrs while stirring.
After filtration, the residue was extracted again from 1.5 l of 30%
(v/v) ethanol-containing water under reflux for 6 hrs while
stirring. The filtrates were combined and concentrated to have 1 l
of the total volume. The filtrate was extracted with a same volume
of water-saturated n-butanol three times. The n-butanol layers were
combined and concentrated under reduced pressure at 60-70.degree.
C. to dryness. 0.3 l of water was added to the residue and
extracted under constant boiling and repeated the procedure twice.
The extract was well suspended in the same amount of distilled
water and lyophilized to give powdered extract.
[0038] Other Clematis Radix harvested in different places were
prepared by the above procedure and analyzed by high performance
liquid chromatography. The result was summarized in Table 1 with
the content (%) of oleanolic acid.
1 TABLE 1 Harvest Content of Yield place (China) oleanolic acid (%)
(%, w/w) Heilongjiang A 6.98 3.25 Heilongjiang B 13.59 3.95
Heilongjiang C 2.68 2.76 Jilin A 9.29 2.58 Jilin B 0.12 4.05 Jilin
C 8.96 2.94 Liaoning A 8.53 3.08 Liaoning B 6.75 2.83 Liaoning C
3.97 1.79 Sichuan A 0.15 3.64 Sichuan B 6.98 2.36 *A, B, C
represent different places
REFERENCE EXAMPLE 2
[0039] 250 g of well air dried Trichosanthis Radix where debris
were removed by washing with water was extracted from 2.5 l of 30%
(v/v) ethanol-containing water under reflux for 4 hrs while
stirring. After filtration, the residue was extracted again from
1.5 l of water under reflux for 3 hrs while stirring. The filtrates
were combined and concentrated to have 1 l of the total volume. The
filtrate was extracted with a same volume of water-saturated
n-butanol three times. The n-butanol layers were combined and
concentrated under reduced pressure at 60-70.degree. C. to dryness.
0.2 l of water was added to the residue and extracted under
constant boiling and repeated the procedure twice. The extract was
well suspended in the same amount of distilled water and
lyophilized to give powdered extract.
[0040] Other Trichosanthis Radix harvested in different places were
prepared by the above procedure and analyzed by high performance
liquid chromatography. The result was summarized in Table 2 with
the content (%) of rosmarinic acid.
2 TABLE 2 Harvest Content of Yield place (China) rosmarinic acid
(%) (%, w/w) Henan A 3.13 2.17 Henan B 12.05 2.28 Henan C 8.34 2.06
Hubei A 1.89 2.40 Hubei B 7.22 3.20 Hubei C 5.75 1.52 Hunan A 10.96
2.74 Hunan B 4.89 1.92 Hunan C 13.00 3.19 Sichuan A 6.14 1.32
Sichuan B 12.45 1.40 Sichuan C 1.59 2.82 *A, B, C represent
different places
REFERENCE EXAMPLE 3
[0041] 250 g of well air dried Prunellae Spica having a length of
2.0-4.0 cm where debris were removed by washing with water was
extracted from 2 l of water under reflux for 5 hrs while stirring.
After filtration; the residue was extracted again from 2 l of water
under reflux for 3 hrs while stirring. The filtrates were combined
and concentrated to have 1 l of the total volume. The filtrate was
extracted with a same volume of water-saturated n-butanol three
times. The n-butanol layers were combined and concentrated under
reduced pressure at 60-70.degree. C. to dryness. 0.1 l of water was
added to the residue and extracted under constant boiling and
repeated the procedure twice. The extract was well suspended in the
same amount of distilled water and lyophilized to give powdered
extract.
[0042] Other Trichosanthis Radix harvested in different places were
prepared by the above procedure and analyzed by high performance
liquid chromatography. The result was summarized in Table 3 with
the content (%) of 4-hydroxybenzoic acid.
3 TABLE 3 Harvest Content of place 4-hydroxybenzoic Yield (China)
acid (%) (%, w/w) Hebei A 0.018 0.95 Hebei B 0.024 0.76 Hebei C
0.037 0.82 Henan A 0.047 0.91 Henan B 0.058 1.05 Henan C 0.019
0.064 Anhui A 0.041 0.71 Anhui B 0.058 1.23 Anhui C 0.098 0.86
Zhejiang A 0.045 0.79 Zhejiang B 0.073 1.08 Zhejiang C 0.062 1.19
*A, B, C represent different places
EXAMPLE 1
Preparation of a Mixture of the Herbal Drug Extracts
[0043] Clematis Radix, Trichosanthis Radix, and Prunellae Spica
having 1.5% (w/w) of rosmarinic acid, 3.5% (w/w) of oleanolic acid,
and 0.02% (w/w) of 4-hydroxybenzoic acid were mixed and n-butanol
fractionation was performed three times, respectively. The extract
obtained each step was combined and well suspended in the same
amount of distilled water and lyophilized to give powdered
extract.
EXAMPLE 2
Preparation of a Mixture of the Herbal Drug Extracts
[0044] Clematis Radix, Trichosanthis Radix, and Prunellae Spica
having 0.7% (w/w) of rosmarinic acid, 5.0% (w/w) of oleanolic acid,
and 0.01% (w/w) of 4-hydroxybenzoic acid were mixed and n-butanol
fractionation was performed three times, respectively. The extract
obtained each step was combined and well suspended in the same
amount of distilled water and lyophilized to give powdered
extract.
EXAMPLE 3
Preparation of a Mixture of the Herbal Drug Extracts
[0045] Clematis Radix, Trichosanthis Radix, and Prunellae Spica
having 4.0% (w/w) of rosmarinic acid, 2.0% (w/w) of oleanolic acid,
and 0.04% (w/w) of 4-hydroxybenzoic acid were mixed and n-butanol
fractionation was performed three times, respectively. The extract
obtained each step was combined and well suspended in the same
amount of distilled water and lyophilized to give powdered
extract.
EXAMPLE 4
Preparation of a Herbal Extract of Mixed Plants
[0046] 200 g of well dried Clematis Radix harvested in Heilongjiang
A where debris were removed by washing with water, 450 g of
Trichosanthis Radix harvested in Henan A with a length of 2.0-4.0
cm, and 350 g of Prunellae Spica harvested in Henan B were mixed
and the mixture was extracted with 10 l of water under reflux for 6
hrs. After filtration, the residue was extracted again from 7 l of
water under reflux for 3 hrs while stirring. The filtrates were
combined and concentrated to give 5 l of the total volume. The
filtrate was extracted with the same volume of water-saturated
n-butanol three times. The n-butanol layers were combined and
concentrated under reduced pressure at 60-70.degree. C. to dryness.
One liter of water was added to the residue and extracted under
constant boiling and repeated the procedure twice. The extract was
well suspended in the same amount of distilled water and
lyophilized to give powdered extract.
[0047] The powdered extract was determined by HPLC analysis having
2.1% (w/w) of rosmarinic acid, 2.1% (w/w) of oleanolic acid, and
2.5% (w/w) of 4-hydroxybenzoic acid.
COMPARATIVE EXAMPLE 1
[0048] Well dried Clematis Radix harvested in Heilongjiang B where
debris were removed by washing with water, 500 g of Trichosanthis
Radix harvested in Anhui C with a length of 2.0-4.0 cm, and 250 g
of Prunellae Spica harvested in Hubei A were mixed and the mixture
was extracted with 15 l of water under reflux for 6 hrs. After
filtration, the residue was extracted again with 7 l of water under
reflux for 3 hrs while stirring. The filtrates were combined and
concentrated to give 5 l of the total volume. The filtrate was
fractionated with the same volume of water-saturated n-butanol
three times. The n-butanol layers were combined and concentrated
under reduced pressure at 60-70.degree. C. to dryness. One liter of
water was added to the residue and extracted under constant boiling
and the procedure was repeated twice. The extract was well
suspended in the same amount of distilled water and lyophilized to
give powdered extract.
[0049] The powdered extract was determined by HPLC analysis having
0.4% (w/w) of rosmarinic acid, 6.8% (w/w) of oleanolic acid, and
0.05% (w/w) of 4-hydroxybenzoic acid.
COMPARATIVE EXAMPLE 2
Preparation of a Mixture of Herbal Drug Extract
[0050] Each extract of Clematis Radix, Trichosanthis Radix, and
Prunellae Spica prepared in Reference Examples 1, 2, and 3 was
mixed and dissolved in aqueous alcoholic solution to have the
contents of 0.45% (w/w) of rosmarinic acid, 6.11% (w/w) of
oleanolic acid, and 0.02% (w/w) of 4-hydroxybenzoic acid. The
mixture was concentrated under reduced pressure. The extract was
well suspended in the same amount of distilled water and
lyophilized to give powdered extract.
EXPERIMENTAL EXAMPLE 1
Test for Analgesic Effects
[0051] To investigate the analgesic effects of various extracts
prepared by said Examples 1-4 and Comparative Examples 1-2,
writhing test was conducted as presented in the following
experimental method and the result was expressed as Table 4.
[0052] Experimental Method:
[0053] The herbal extracts, prepared by said Examples 1-4 and
Comparative Examples 1-2, were orally administered to ICR
(Institute of Cancer Research) mice at doses of 200 mg or 400 mg
per Kg of body weight.
[0054] One hour after administration, 0.6% (v/v) acetic acid was
intraperitoneally injected to the animals at a volume of 0.1 ml per
10 g of body weight and from 5 minutes after administration,
writhing frequency of each mice was observed for 10 minutes as a
pain threshold.
4 TABLE 4 Dose of herbal Avg. Rate of drug extract writhing
inhibition Category (mg/Kg) frequency (%) Control -- 19 -- Example
1 200 12 36.8 400 9 52.6 Example 2 200 11 42.1 400 8 57.9 Example 3
200 11 42.1 400 9 52.6 Example 4 200 10 47.4 400 7 63.2 Comparative
200 13 31.6 Example 1 400 10 47.4 Comparative 200 14 26.3 Example 2
400 11 42.1
[0055] According to the Table 4, it is revealed that the extract
prepared by the present invention has superior analgesic effects
from reduced writhing frequencies.
EXPERIMENTAL EXAMPLE 2
Test for Inhibitory Activity on Acute Inflammation
[0056] The inhibitory activity of the herbal extracts, prepared by
said Examples 1-4 and Comparative Examples 1-2, on acute
inflammation was investigated in rats. In comparison with the
control, the anti-inflammatory effect on hind paw in edema was
expressed as percent and the result is presented in the attached
FIG. 1.
[0057] Experimental Method:
[0058] The herbal extracts, prepared by said Examples 1-4 and
Comparative Examples 1-2, was orally administered to white SD
(Spraque-Dawley) rats. At one hour after administration, 0.1 ml of
1% carrageenan was intradermally injected to the left hind paw of
rats and edema at that site was measured at 1 hour interval for 5
hours.
[0059] As noted in the attached in FIG. 1, it is revealed that the
herbal extracts prepared by said Examples 1-4 of the present
invention significantly inhibited the carrageenan-induced
inflammation.
EXPERIMENTAL EXAMPLE 3
Test for the Anti-Aggregating Activity
[0060] The anti-coagulant activity of the herbal extracts, prepared
by said Examples 1-4 and Comparative Examples 1-2, was investigated
in rabbits plasma and the aggregation was induced by collagen and
the result is presented in the attached FIG. 2.
[0061] Experimental Method:
[0062] PRP (platelet rich plasma) was prepared from the blood
sample of rabbits and the number of platelet in blood was adjusted
at 2.times.10.sup.8/ml. The herbal extracts, prepared by said
Examples 1-4 and Comparative Examples 1-2, were added to the PRP
and adjusted on a cuvette of aggregometer at 37.degree. C. for 2
minutes. With the addition of collagen platelet aggregation was
measured with a dual aggregometer.
[0063] As noted in the attached FIG. 2, there was no increase in
platelet aggregation by the herbal.
EXPERIMENTAL EXAMPLE 4
Test for the Anti-Coagulant Activity on Whole Blood Coagulation
[0064] The anti-coagulant activity of the herbal extracts, prepared
by said Examples 1-4 and Comparative Examples 1-2, was investigated
using whole blood of rabbit and the result is presented in the
attached FIG. 3.
[0065] Experimental Method:
[0066] The same amount of saline solution was added to whole blood
of rabbit and mixed well prior to use in this experiment. The
herbal extracts, prepared by said Examples 1-4 and Comparative
Examples 1-2, were added to reconstituted whole blood and prewormed
for 2 minutes. And, the blood coagulation was induced by the
addition of collagen. The whole blood coagulation was measured by
an aggregometer. As noted in the attached FIG. 3, there was no
increase in whole blood coagulation when the herbal extracts
prepared by said Examples 1-4 of the present invention was
added.
EXPERIMENTAL EXAMPLE 5
Test for the Inhibitory Activity on Hyaluronidase, an Enzyme
Associated with Degradation of Joint Tissue
[0067] The inhibitory activity of the herbal extracts, prepared by
said Examples 1-4 and Comparative Examples 1-2, on hyaluronidase,
an enzyme associated with degradation of joint tissue, was
investigated and the result is presented in the following Table
5.
[0068] Experimental Method:
[0069] Hyaluronidase was prepared in the presence of acetate buffer
solution at 37.degree. C. for 20 minutes and activated. Then the
herbal extracts, prepared by said Examples 1-4 and Comparative
Examples 1-2, and potassium hyaluonate as a substrate were added to
the buffer solution and cultured for about 40 minutes. After
terminating the reaction with sodium hydroxide, potassium borate
was added to the cultures and heated at 100.degree. C. The
absorbance was measured by the development of DMBA
(dimethylbenzanthracene) and the rate of inhibition was calculated
in comparison with control.
5 TABLE 5 Test Rate of concentration inhibition Category (mg/ml)
(%) Example 1 1 82.3 Example 2 1 83.7 Example 3 1 89.6 Example 4 1
87.5 Comparative Example 1 1 80.5 Comparative Example 2 1 79.1
[0070] As shown in Table 5, the combined herbal extracts prepared
by the present invention significantly inhibited the activation of
the enzyme associated with degradation of joint tissue.
EXPERIMENTAL EXAMPLE 6
Test for the Inhibitory Activity on Chronic Inflammation
[0071] The anti-inflammatory activity of the herbal extracts,
prepared by said Examples 1-4 and Comparative Examples 1-2, on
chronic rheumatoid arthritis was investigated in Mycobacterium
butyricum injected rat models. The result is presented in the
attached FIG. 4.
[0072] Experimental Method:
[0073] To induce chronic edema, Mycobacterium butyricum suspended
in mineral oil and treated with heat was injected to the right hind
paw of white rats at each dose of 0.05 ml. Then, the herbal
extracts, prepared by said Examples 1-4 and Comparative Examples
1-2, were orally administered to the rats once daily for 16 days,
and the paw edema was measured with plethysmometer.
[0074] As shown in the attached FIG. 4, the combined herbal
extracts by the present invention significantly inhibited the
edema.
EXPERIMENTAL EXAMPLE 7
Test for the Inhibitory Activity on Leukotriene B.sub.4
[0075] The inhibitory activity of the herbal extracts, prepared by
said Examples 1-4 and Comparative Examples 1-2, on 5-lipoxygenase
was evaluated by the inhibition rate of leukotriene B.sub.4
(LTB.sub.4) biosynthesis induced by arachidonic acid and calcium
ionophore (A23187) and the result is presented in the Table 6.
[0076] Experimental Method:
[0077] The extracts, prepared by said Examples 1-4 and Comparative
Examples 1-2, were added to rat basophilic leukemia-1 (RBL-1) cells
adjusted at 37.degree. C. and reacted for 5 minutes. Then the
reacting mixture was treated with 20 .mu.g/ml A23187 and
arachidonic acid for 15 minutes so as to induce the generation of
LTB.sub.4. The generated LTB.sub.4 was extracted with ethyl acetate
and was subjected to HPLC.
6 TABLE 6 Test Rate of concentration inhibition Category (mg/ml)
(%) Example 1 0.5 87.3 Example 2 0.5 82.5 Example 3 0.5 79.1
Example 4 0.5 85.7 Comparative Example 1 0.5 71.5 Comparative
Example 2 0.5 70.6
[0078] As shown in the Table 6, the combined herbal extracts
prepared by the present invention significantly inhibited
5-lipoxygenase.
EXPERIMENTAL EXAMPLE 8
Test for the Inhibitory Activity on Cyclooxygenase-I
[0079] The inhibitory activity of the herbal extracts, prepared by
said Examples 1-4 and Comparative Examples 1-2 on cyclooxygenase-I
was evaluated by arachidonic acid and the result is presented in
the Table 7.
[0080] Experimental Method:
[0081] The extracts, prepared by said Examples 1-4 and Comparative
Examples 1-2, were added to cyclooxygenase-I adjusted at 37.degree.
C., and 100 .mu.M arachidonic acid was added and reacted for 2
minutes, trichloroacetic acid (TCA) was added to the reacting
mixture for terminating the reaction and absorbance was measured at
530 nm.
7 TABLE 7 Test Rate of concentration inhibition Category (mg/ml)
(%) Example 1 0.5 61.8 Example 2 0.5 62.5 Example 3 0.5 59.8
Example 4 0.5 67.3 Comparative Example 1 0.5 51.5 Comparative
Example 2 0.5 49.1
[0082] As shown in the Table 7, the combined herbal extracts
prepared by the present invention significantly inhibited
cyclooxygenase-I.
EXPERIMENTAL EXAMPLE 9
Test for the Inhibitory Activity on Cyclooxygenase-II
[0083] The inhibitory activity of the extracts, prepared by said
Examples 1-2 and 5 Comparative Examples 1-3, on cyclooxygenase-II
was evaluated and the result is presented in the Table 8.
[0084] Experimental Method:
[0085] The extracts prepared by said Examples 1-2 and Comparative
Examples 1-3, were added to cyclooxygenase-II and placed at a test
tube adjusted at 27.degree. C. After reaction with 500 mM
arachidonic acid for 90 seconds, trichloroacetic acid (TCA) was
added to the reaction mixture for terminating the reaction and
absorbance was measured at 532 nm.
8 TABLE 8 Test Rate of concentration inhibition Category (mg/ml)
(%) Example 1 0.5 72.5 Example 2 0.5 69.8 Example 3 0.5 65.7
Example 4 0.5 77.5 Comparative Example 1 0.5 61.5 Comparative
Example 2 0.5 58.8
[0086] As shown in the Table 8, it is noted that the combined plant
extracts prepared by this invention significantly inhibited
cyclooxygenase-II.
EXPERIMENTAL EXAMPLE 10
Test for the Inhibitory Activity on the Proliferation of
B-Lymphocyte
[0087] The inhibitory activity of the herbal extracts, prepared by
said Examples 1-4 and Comparative Examples 1-2 on the proliferation
of B-lymphocyte induced by lipopolysaccharide (LPS) was evaluated
and the result is presented in the attached FIG. 5.
[0088] Experimental Method:
[0089] Cultures were set up with 10.sup.6 B-lymphocyte/ml of medium
at 37.degree. C. The extracts, prepared by said Examples 1-4 and
Comparative Examples 1-2, were added to the cultures, and then the
cultures were treated with 10 .mu.g/ml of LPS for 24 hours. With
the addition of 2 .mu.Ci Thymidine-.sup.3H expressed by tritium as
radioactivity for 48 hours, cultures were quantified on liquid
scintillation counter (LSC).
[0090] As shown in the attached FIG. 5, it is noted that the
combined herbal extracts prepared by the present invention
significantly inhibited the proliferation of B-lymphocyte.
EXPERIMENTAL EXAMPLE 11
Test for the Inhibitory Activity on the Proliferation of
T-Lymphocyte
[0091] The inhibitory activity of the herbal extracts, prepared by
said Examples 1-4 and Comparative Examples 1-2, on the
proliferation of T-lymphocyte induced by concanavalin-A (Con-A)
were investigated and the result is presented in the attached FIG.
6.
[0092] Experimental Method:
[0093] Cultures were set up with 10.sup.6 T-lymphocyte/ml of medium
at 37.degree. C. The extracts, prepared by said Examples 1-4 and
Comparative Examples 1-2, were added to the cultures, which were
treated with 3 .mu.g/ml of concanavalin-A for 24 hours. With the
addition of 2 .mu.Ci Thymidine-.sup.3H expressed by tritium as
radioactivity for 48 hours, cultures were quantified on LSC.
[0094] As shown in the attached FIG. 6, it is noted that the
combined herbal extracts prepared by the present invention
significantly inhibited the proliferation of T-lymphocyte.
EXPERIMENTAL EXAMPLE 12
Test for the Scavenging Activity on Elimination of Superoxide
Radicals
[0095] The scavenging activity of the herbal extracts, prepared by
said Examples 1-4 and Comparative Examples 1-2, was assessed on the
elimination of superoxide radicals generated from xanthine-xanthine
oxidase and the result is presented in the Table 9.
[0096] Experimental Method:
[0097] Cytochrome-c (Cyt-c) and herbal extracts, prepared by
Examples 1-4 and Comparative Examples 1-2, were added to xanthine
oxidase adjusted at 37.degree. C. so as to induce the generation of
oxygen radicals by xanthine. The changes in color along with
oxidation of cytochrome-c (Cyt-c) was measured by spectrophotometer
at 540 nm and scavenging rate of oxygen radicals was also measured
as slope.
9 TABLE 9 Test Rate of concentration inhibition Category (mg/ml)
(%) Example 1 0.5 82.7 Example 2 0.5 89.5 Example 3 0.5 83.6
Example 4 0.5 92.1 Comparative Example 1 0.5 81.5 Comparative
Example 2 0.5 78.4
[0098] As shown in the Table 9, it is noted that the combined
herbal extracts prepared by the present invention significantly
scavenged active oxygen.
EXPERIMENTAL EXAMPLE 13
Test for the Inhibitory Activity on Proteoglycan Degradation
[0099] The inhibitory activity of the herbal extracts, prepared by
said Examples 1-4 and Comparative Examples 1-2, on the Proteoglycan
degradation which is an important element in cartilage tissue was
investigated and the result is presented in the Table 10.
[0100] Experimental Method:
[0101] Cartilage from rabbits was dissected from each joint and 50
mg of cartilage was placed in a culture medium adjusted at
37.degree. C. The herbal extract, prepared by said Examples 1-4 and
Comparative Examples 1-2, and 5 .mu.g of interleukin-1.alpha.
(IL-1.alpha.) per 50 mg of cartilage were added to the culture
medium and cultured for 72 hours. The production of
glucosaminoglycan (GAG), which is produced by proteoglycan
degradation, was measured at 525 nm by coloring with
1,9-dimethylmethylene blue dye.
10 TABLE 10 Test Rate of inhibition conc. on the proliferation of
Category (mg/ml) glucosaminoglycan (%) Example 1 1.0 115 0.3 83 0.1
68 Example 2 1.0 103 0.3 91 0.1 79 Example 3 1.0 99 0.3 81 0.1 73
Example 4 1.0 121 0.3 94 0.1 75 Comparative 1.0 84 Example 1 0.3 52
0.1 38 Comparative 1.0 81 Example 2 0.3 46 0.1 37
[0102] As shown in the Table 10, it is noted that the combined
herbal extracts prepared by the present invention significantly
inhibited the proteoglycan degradation activity.
EXPERIMENTAL EXAMPLE 14
Test for the Inhibitory Activity on Type II Collagen
Degradation
[0103] The inhibitory activity of the herbal extracts, prepared by
said Examples 1-4 and Comparative Examples 1-2, on the Type II
collagen degradation which is an important element in articular
cartilage tissue was investigated and the result is presented in
the Table 11.
[0104] Experimental Method:
[0105] Cartilage from rabbits was dissected from each joint and 50
mg of cartilage was placed in a culture medium adjusted at
37.degree. C. The herbal extract, prepared by said Examples 1-4 and
Comparative Examples 1-2, and 5 .mu.g of interleukin-l.alpha.
(IL-1.alpha.) per 50 mg of cartilage were added to the culture
medium and cultured for 21 days. The production of hydroxy proline,
which is produced by type II collagen degradation, was measured at
560 nm by coloring with chloramines-T and
dimethylaminobenzaldehyde.
11TABLE 11 Rate of inhibition on the proliferation Category Test
conc. (mg/ml) of hydroxy-Proline (%) Example 1 1.0 91 0.3 63 0.1 38
Example 2 1.0 89 0.3 59 0.1 39 Example 3 1.0 97 0.3 76 0.1 53
Example 4 1.0 93 0.3 65 0.1 46 Comparative 1.0 67 Example 1 0.3 36
0.1 19 Comparative 1.0 65 Example 2 0.3 36 0.1 20
[0106] As shown in the Table 11, it is noted that the combined
herbal extracts prepared by the present invention showed more
significant inhibitory activity on the Type II collagen degradation
than that of Comparative Example 1.
EXPERIMENTAL EXAMPLE 15
Test for the Therapeutic Efficacy Against Osteoarthritis
[0107] The activity on oateoarthritis of the herbal extracts,
prepared by said Examples 1-4 and Comparative Examples 1-2, on
cartilage protection was investigated by monitoring rabbit models
of which cartilage was injured by injecting collagenase to cavum
articular of rabbit to have similar symptoms to human
osteoarthritis. The result is presented in the Table 12.
[0108] Experimental Method:
[0109] 1 mg of collagenase per 1 Kg of body weight was injected at
the first and fourth days to the cavum articular of rabbit having
2.0-2.5 Kg of body weight to injure joint tissue and to have
collagenase-induced arthritis. The extracts, prepared by said
example 1-4 and comparative example 1-2, were given orally at a
dose of 200 mg/Kg for 4 weeks. After 4 weeks of administration, the
cartilage part was dissected and withdrawn from the rabbit and then
dyed with sapranin-O. The dyed cartilage part was divided into
cartilaginous tissue and synovial tissue and the degree of
arthritis severity was recorded with the integer scale of 0-4 to
quantify the levels: 0=normal; 1=slight; 2=moderate; 3=severe; and
4=maximum.
12 TABLE 12 Cartilaginous Category tissue.sup.1) Synovial
tissue.sup.2) Total.sup.3) Control 11.8 10.8 21.6 Example 1 6.0 6.5
12.5 Example 2 6.2 7.2 13.4 Example 3 6.3 7.1 13.4 Example 4 5.0
6.0 11.0 Comparative 8.6 8.2 16.8 Example 1 Comparative 8.7 8.0
16.7 Example 2 .sup.1)Cartilaginous tissue (score for complete
arthritis: 24): Loss of superficial layer, Erosion of cartilage,
Fibrillation and/or fissures, Disorganization of chondrocytes, Loss
of chondrocytes, Cluster formation .sup.2)Synovial tissue (score
for complete arthritis: 24): hyperplasia of synovial lining cell,
hypertrophy of synovial lining layer, infiltration of inflammatory
cells, proliferation of granulation tissue, vascularization
.sup.3)Total (score for complete arthritis: 48)
[0110] As shown in the Table 12, it is noted that the combined
herbal extracts prepared by the present invention showed more
significant alleviation activity on collagenase-induced arthritis
than that of Comparative Example 1.
MANUFACTURING EXAMPLE 1
[0111] The following chemical composition was employed for the
manufacture of oral tablets using the powdered extracts of the
present invention.
[0112] Chemical Composition: the herbal drug composition 200 mg;
hard anhydrous silicate 10 mg; Magnesium stearate 2 mg;
microcrystalline cellulose 50 mg; Sodium starch glycolate 25 mg;
corn starch 113 mg; and anhydrous ethanol in an appropriate
amount.
MANUFACTURING EXAMPLE 2
[0113] The following chemical composition was employed for the
manufacture of ointments using the powdered extracts of the present
invention.
[0114] Chemical Composition: the herbal drug composition 5 g; cetyl
palmitate 20 g; cetyl alcohol 40 g; stearyl alcohol 20 g; isopropyl
myristate 80 g; sorbitan monostearate 20 g; polysorbate 60 g;
propyl p-hydroxybenzoate 1 g; methyl p-hydroxybenzoate 1 g; and
phosphoric acid and distilled water in an appropriate amount.
MANUFACTURING EXAMPLE 3
[0115] The following chemical composition was employed for the
manufacture of injection solutions using the powdered extracts of
the present invention.
[0116] Chemical Composition: the herbal drug composition 100 mg;
mannitol 180 mg; Na.sub.2HPO.sub.4 25 mg; and water for injection
2,974 mg.
MANUFACTURING EXAMPLE 4
[0117] The following chemical composition was employed for the
manufacture of transdermal using the powdered extracts of the
present invention.
[0118] Chemical Composition 1: the herbal drug composition 0.4 g;
poly(acrylic acid, sodium salt) 1.3 g; glycerin 3.6 g; aluminum
hydroxide 0.04 g; methyl paraben 0.2 g; and water 14 g.
[0119] Chemical Composition 2: the herbal drug composition 0.8 g;
propylene glycol 1.6 g; fluid paraffin 0.8 g;
[0120] Several dosage forms (e.g., tablets, ointments, transdermal
and injection solutions) prepared by said manufacture 1-4 related
to combined herbal preparations using Clematis Radix, Trichosabthis
Radix and Prunellae Spica according to this invention. Said
preparations contain concentrations of oleanolic acid and
rosmarinic acid as reference materials, thus being effectively used
for anti-inflammatory agent with analgesic effects, chronic
rheumatoid arthritis drug and agent for improving peripheral blood
circulation.
* * * * *