Title of Invention

PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF RHEUMATOIDARTHRITIS

Abstract The present invention provides a pharmaceutical composition comprising a crude extract or extract of Plectranthus Amboinicus (Lour.) Spreng (PA) for the treatment of rheumatoid arthritis.
Full Text Background of the Invention
Field of the Invention

The present invention relates to Chinese herbal medicine extracts. In particular, the
present invention relates to a use of the crude extracts or extracts of Plectranthus
Amboinicus (Lour.) Spreng (PA) for treating rheumatoid arthritis (RA).
Description of the Prior Art
As a result of the use of various palliative medicaments and immunosuppressive
agents, the treatment of rheumatoid arthritis has made great progress. Most studies reveal
that when palliative medicaments or immunosuppressive agents are used as soon as the
early outbreak, the destruction rate of bones can be effectively reduced. At present, the
medicaments for treating rheumatoid arthritis are classified into the following three groups:
1. Non-steroidal anti-inflammatory drugs (NSAIDs), for example, aspirin,
indomethacin, or naproxen, which can effectively inhibit inflammation and alleviate pain
effect.
2. Anti-rheumatic drugs (ARDs), referred to as disease modifying anti-rheumatic
drugs (DMARDs), for example, gold preparation, hydroxychloroquine, methotrexate
(MTX) or penicillamine, which can inhibit conditions and improve immune abnormalities.
3. Steroids, referred to as corticosteroids, which are anti-inflammatory, and can be
used as immunosuppressive agents.
Additionally, clinically common palliative drugs include sulfasalazine, and
immunosuppressive agents include cyclosporine, azathioprine, or cyclophosphamide.
Furthermore, since some antibiotics, such as minocycline, can inhibit enzymes, inhibit bone


absorption, and inhibit production of inflammatory substances, they can be used for treating
rheumatoid arthritis as well. Many treatment models have been proposed for the use of
medicaments and treatment timing, such as Sawtooth therapy, Step-down bridge therapy,
staging therapy, and target therapy. The spirit of these treatment models undoubtedly is to
use various palliative medicaments or immunosuppressive agents alone or in combination in
the early stages.
However, the shortcoming of these medicaments is that side effects will be caused as
they work, especially steroids. Frequently, anti-inflammatory medicaments cause
abnormalities in the bowel tract, e.g., bleeding, and the like.
A protein antagonist, such as TNF-α, could be also used clinically to palliate
conditions rapidly, but it needs to be used in an invasive mode, which is inconvenient.
Additionally, as the herb Radix Tripterygii Wilfordii has anti-inflammatory, bactericidal,
and fever and pain relief effects, it is currently used for treating rheumatoid arthritis;
however, security considerations may be taken due to its toxic side effect.
According to statistics, 1% of the population suffers from rheumatoid arthritis across
the world. Accordingly, it is of great importance to develop convenient, safe, and efficient
medicaments for treating rheumatoid arthritis.
Plectranthus Amboinicus (Lour.) Spreng (PA) originates in Malaysia and India, and is
frequently planted by average families as an ornamental herb. The common herb PA is the
epigeal portion of the Plectranthus Amboinicus (Lour.) Spreng plant, the aliases of which
are Lysimachia capillipes Hemsl, Spearmint, Agastache ragosus, Patchouly, Indian
peppermint, or pogostemon cablin. East Indians used this as a cloth-perfuming agent, and
the English found PA's charming scent after they introduced shawl cloth from India in 1820.
If the leaves of PA were placed into clothes directly, not only did the clothes acquire a scent,
but also, the clothes were prevented from being moth-eaten. The PA is considered as having


bactericidal, exciting, or insect repelling functions in Southeast Asia. Also, PA can cure
bites from vipers or mosquitoes and insects, and also can relieve headaches, flatulence,
vomiting, diarrhea, and fever, and the like. Plectranthus Amboinicus (Lour.) Spreng
essential oil is the most popular flavor in Asia. In aromatherapy, PA is used to facilitate
epithelial cell regeneration, and the treatments of acne, eczema, Hong Kong foot, and dry
cracking of skin. Even more, PA is an excellent anti-depressant and aphrodisiac that
functions to relieve anxiety and enhance libido.
In the present invention, it is unexpectedly found that crude extracts or extracts of PA
have the efficacy of treating RA.
Summary of the Invention
One object of the invention is to provide a pharmaceutical composition for treating
rheumatoid arthritis, comprising a therapeutically effective amount of a crude extract or
extract of Plectranthus Amboinicus (Lour.) Spreng (PA).
Another object of the invention is to provide a use of the crude extract or extract of PA
for the manufacture of medicaments for treating rheumatoid arthritis.
Yet another object of the invention is to provide a method for manufacturing the crude
extract or extract of PA.
The invention will be illustrated in detail below. Other features, objects, and
advantages of the invention will be apparent from the specification and the claims.
Brief Description of the Accompanying Drawings
FIG. 1 is an HPLC pattern of the PA crude extract of Embodiment 1;


FIG. 2 illustrates a scheme of the animal test of Embodiment 2;
FIG. 3 illustrates the effect of feeding on PA crude extracts on the body weight of CIA
rats;
FIG. 4 illustrates the effect of feeding on PA crude extracts on the arthritic indices of
CIA rats;
FIG. 5 illustrates the effect of feeding on PA crude extracts on the arthritic swelling
degree of CIA rats;
FIG. 6 illustrates the effect of feeding on PA crude extracts on the serum RF of CIA
rats;
FIG. 7 illustrates the effect of feeding on PA crude extracts on the serum CRP of CIA
rats;
FIG. 8 illustrates the effect of feeding on PA crude extracts on cytokine TNF-α and IL-
1β in the abdominal cavity of CIA rats;
FIG. 9 illustrates the effect of feeding on PA crude extracts on cytokine IL-6 in the
abdominal cavity of CIA rats;
FIG. 10 is a HPLC pattern of the PA extract PA-F2 of Embodiment 3;
FIG. 11 illustrates a scheme of the animal test of Embodiment 4;
FIG. 12 illustrates the effect of feeding on PA extracts on the body weight of CIA rats;
FIG. 13 illustrates the effect of feeding on PA extracts on the arthritic indices of CIA
rats;
FIG. 14 illustrates the effect of feeding on PA extracts on the arthritic swelling degree
of CIA rats;
FIG. 15 illustrates the effect of feeding on PA extracts on the serum RF of CIA rats;
FIG. 16 illustrates the effect of feeding on PA extracts on cytokine IL-6 in the
abdominal cavity of CIA rats; and
FIG. 17 illustrates the effect of feeding on PA extracts on cytokine IL-1β in the
abdominal cavity of CIA rats.


Detailed Description
The term "treatment" or "treating," as used herein, refers to improving conditions.
The term "patient," as used herein, refers to animals, especially mammals. In a
preferred embodiment, the patient is human.
The term "therapeutically effective amount," as used herein, refers to the amount of
components of the pharmaceutical composition of the invention alone or in combination
with other medicaments that could provide therapeutical benefits in treatment.
The term "carrier" or "pharmaceutically acceptable carrier," as used herein, refers to
diluents, excipients, acceptable agents or the like that are wellknown by those of ordinary
skill in the art and can be used in the preparation of pharmaceutical composition.
The term "PA crude extracts" or "PA extracts," as used herein, refers to those obtained
by direct juice pressing or an extracting process used on the epigeal portion of the PA plant.
The term "high-polarity solvent," as used herein, refers to a solvent that has the
highest polarity in the solvents used in the preparation process. The high-polarity solvent
includes, but are not limited to water, methanol, ethanol, or a mixture of two or more of the
preceding solvents.
The term "low-polarity solvent," as used herein, refers to a solvent that has the lowest
polarity in the solvents used in the preparation process. The low-polarity solvent includes,
but are not limited to chloroform, isopropanol, acetone, ethyl acetate, a mixture of two or
more of the preceding solvents, or a mixture of one or more of the preceding solvents with
one or more solvent that have higher polarity in a ratio (v:v) from about 70:30 to about
50:50.


The term "sub-high-polarity solvent," as used herein, refers to a solvent that has a
polarity lower than that of the high-polarity solvent used in the preparation process but
higher than that of the medium-polarity solvent used in the preparation process. The sub-
high-polarity solvent can be obtained by mixing a high-polarity solvent with a solvent
having lower polarity in a ratio (v:v) from about 70:30 to about 30:70, preferably from
about 60:40 to about 40:60.
The term "medium-polarity solvent," as used herein, refers to a solvent that has a
polarity lower than that of the sub-high-polarity solvent used in the preparation process but
higher than that of the low-polarity solvent used in the preparation process. The medium-
polarity solvent can be obtained by mixing a high-polarity solvent with a solvent having
lower polarity in a ratio (v:v) from about 30:70 to about 5:95, preferably from about 15:85
to about 5:95.
The invention is characterized by the use of PA crude extracts or PA extracts for
treating RA. Thus, the invention provides a pharmaceutical composition for treating RA,
comprising a therapeutically effective amount of a PA crude extract or PA extract.
The most suitable route and dosage for treatment will be easily determined by those
skilled in the art. According to the invention, the preferred route is oral administration, for
example, but not limited to, capsule, tablet, powder, ointment, liquor, or spray, etc. Dosage
will depend on the nature and states of the symptoms being treated, ages and general
physical conditions of the patient being treated, administration route and any therapies
practiced previously. It should be understood by those skilled in the art that the dosage will
vary with patients, depending on age, size, health condition, and related factors.
Furthermore, if desired, the composition could be sterilized, or mixed with any
pharmaceutically acceptable carrier or excipient. The preparation of the pharmaceutical
composition of the invention can be performed by those skilled in the art according to
conventional methods.


In preferred embodiments of the invention, the preparation of PA crude extracts and
PA extracts is shown as follows.
Preparation of PA crude extract
A fresh PA plant was taken, washed with clean water, and then pressed by a juice
extractor to obtain juice. The PA juice then was freeze-dried, to provide a dry powder,
which was taken up in an appropriate solvent, such as chloroform or methanol, to give the
PA crude extract.
Preparation of PA extract
A certain amount of dry PA was soaked in a suitable amount of a high-polarity
solvent, filtrated, and then soaked again in a suitable amount of a high-polarity solvent.
After this, the PA extract liquor was condensed to 2-3% of its original volume under
reduced pressure by a rotary concentrator, diluted in a solvent, and then separated in a
column. Optionally, four segments of different solvents from high polarity to low polarity
(referred to as high-polarity solvent, sub-high-polarity solvent, medium-polarity solvent,
and low-polarity solvent) could be used for elution continuously. The high-polarity solvent,
sub-high-polarity solvent, medium-polarity solvent, and low-polarity solvent are as defined
above. Preferably, the column separation method uses a DIAION column that has already
been treated by methanol. For example, the same amount of DIAION as dry PA was
weighed out, soaked in methanol, and then filled into a column. After the filling, the
DIAION was washed with 1-2 times volume of methanol, followed by 5-6 times volume of
deionized and distilled water. Once the washing was finished, the filling was complete.


Ingredient analysis of the extracts of PA medicinal material
Instrument and Apparatus
High performance liquid chromatograph
Pump: Spectra-Physics P4000
Detector: UV/VIS Spectra-Physics Spectra System UV600OLP
Automated sampler: Thermo Separation Pruducts AS3500
Software: Thermo Separation Pruducts Chrom Quest
System control: Thermo Separation Pruducts SN4000
Conditions of liquid chromatography
Chromatograph column: COSMOSIL, 4.6x250mm, 5C18-MS
Flow rate: 1.0 ml/min Pressure Limite : 250 kgf/cm2
Sample injection: 10 µl
PDA condition:
Sampling: 0.64 sec
Wavelength range: 190-370 nm
uv wavelength: 254 nm

The invention is described in detail with reference to the following non-limiting
examples. The following procedures could be carried out to verify the effect of PA crude

extracts or extracts on treating RA. Any modifications and changes that can be easily
achieved by those skilled in the art are included in the scope of the disclosure of the
specification and appended claims.
EMBODIMENT
Embodiment 1: PA crude extracts obtained by using direct juice pressing
1.25 kg fresh PA was weighed, washed with clean water, and then pressed by a juice
extractor to obtain juice. A volumetric cylinder was used to measure the volume of the
juice, from which 1050 ml was taken out, and was freeze-dried, to obtain 19 g dry powder
(yield of 1.5%). The HPLC pattern is shown in FIG. 1.
Embodiment 2: animal test on treating RA in animals by PA crude extracts
[Test animall
The test animals, Lewis rats, 8 weeks old and about 155-165 g in weight, were all
purchased from National Laboratory Animal Center. They were raised in an animal house
with a 12-hour light/12-hour dark cycle, a room temperature of 23±1°C, moderate moisture,
and good air conditioning, wherein water and feed were provided ad libitum. In addition,
during the operation, all the test animals conformed to the criteria of the International
Committee on Laboratory Animals' standard regulation.
[Medicaments]
1. Collagen Type II (Sigma C-1188), obtained from bovine tracheal cartilage
2. Complete Freund's adjuvant, CFA (BD BBL™ 231131)
3. Incomplete Freund's adjuvant, IFA (BD BBL™ 263910)


4. ELISA kit of tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and interleukin-
lp(IL-1β) of rat (R&D, Duoset)
5. C-reactive protein (CRP) of rat, ELISA kit (BD™ Pharmingen 557825)
6. Indomethacin (commercially available from Johnson Chemical Pharmacy
Corporation, Sanchung City, Taipei County)
7. Fresh Chinese herb PA was pressed directly to obtain juice, concentrated under
reduced pressure to form a PA concentrated solution, and then diluted into a high
concentration of 22.5 g crude medicine / kg (PA-H) and a low concentration of 4.5 g crude
medicine / kg (PA-L), respectively. Finally, the solutions were directly administered orally
to the rats based on respective actual body weights.
[Apparatus]
1. Syringe: 1 ml, 3 ml, and 5 ml (Terumo)
2. Balance
3. Vernier Caliper (Mitutoyo Corporation)
4. Three-way piston tube
5. Oscillator (Vortex)
6. ELISA reader (Dynex, Thermo Labsystems)
[Protocol]
1. Antigen formulation and immune injection
The animal test scheme is shown as FIG. 2. Bovine collagen Type II (Bovine C II)
was dissolved in 0.1 M acetic acid solution, stirred to dissolve thoroughly, and formulated
into solutions with concentrations of 1.5 and 3 mg/ml, which were stored in 4°C for later
use. For the first immune injection, 100 µl of C II solution was emulsified with an equal
amount of CFA, and injected subcutaneously (200 µl/rat) in the root portions of tails of the
rats after the emulsification was complete. After the first immunization, the body weights
of the rats were recorded every three days, and they were observed in order to discover


whether or not swelling occurred in limbs. After about 15 days, the second immunization
was done. 100 µl of C II solution was emulsified with an equal amount of IFA, and injected
subcutaneously (200 µl/rat) in the root portions of tails of the rats after the emulsification
was complete. Approximately from Day 20, the symptoms of arthritis were observed (CIA
rats), and PA and indomethacin were fed until Day 45.
2. Grouping and treating of animals

[Test items and indicesl
1. Body weight observation: shown by weighing every 3 days
2. Evaluation of RA examination: evaluated in maximum arthritic index (MAI)
3. Joint swelling rate: measured by Vernier Caliper (Day 15-28 arthritis development)
4. Rheumatoid factor (RF)
5. Acute inflammatory C-reactive protein (CRP)
6. Cytokine: TNF-α, IL-1β, and IL-6 (inflammatory cytokine)
-Evaluation of RA examination
After immunization, the rats were observed three times a week. The changes of
redness and swelling or the like in limbs were recorded, and photographs were taken and
filed for comparison. The scoring of the examinations was based on 5 grades as follows:

0: No symptom of arthritis occurs.
1: Sole and tarsus show red and swell slightly.
2: Tarsus and ankle are red and swell moderately.
3: Tarsus and ankle are red and swell severely.
4: Joints are stiff, and bones deform.
Maximum arthritic index, average MAI for each group was calculated as follows:
Average MAI = Total of MAI recorded in limbs of each rat (0: no CIA occurs, 16:
highest score)/4 /total rat number of each group
- Measurement of arthritic swelling degree
The thickness changes of soles of rats were measured by a Vernier Caliper (twice per
week), and there were eight measurement sites in total in each rat, including one site at the
center of each sole of the two front feet respectively, and three sites in each of the two back
feet (ankle joint, sole, and root portion of toe) respectively.
-Serum RF analysis
1. Collagen was formulated in a coating buffer to a concentration of 40 µg/ml, 0.1 ml
of which was added into a 96-well micro plate respectively, and was kept in 4°C overnight.
2. After washing with Tris buffer three times, 0.2 ml of blocking buffer containing 1%
BSA was added into each well. The reaction lasted for 2 hours at room temperature, and
then was washed by Tris buffer three times again.
3. After the serum sample was diluted appropriately with Tris buffer containing 0.05%
Tween 20 (1/20 or 1/40), 0.1 ml serum sample was added into each well of the 96-well
micro plate. The reaction lasted for 2 hours at room temperature, and then was washed by
Tris buffer three times again.


4. Coated anti-rat immunoglobulin M (IgM) was combined with horseradish peroxide
(HRP), appropriately diluted (1/12000), and then added into the 96-well micro plate. The
reaction lasted 2 hours at room temperature, and then was washed by Tris buffer three times
again.
5. 0.1 ml of tetramethyl benzidine (TMB) was directly added into each well for color
reaction, and then a stopping solution was added to stop the reaction. Finally, absorption
(O.D) value was read out at the wavelength of 450 nm.
-Serum C-reactive protein (CRP) analysis
1. CRP ELISA kit was a pre-coated microplate, into which 0.1 ml of appropriately
diluted serum sample could be added directly. After being reacted for 1 hour at room
temperature, it was washed by the washing buffer four times.
2. Rabbit anti-rat C-reactive protein (CRP) was combined with HRP Ab, diluted with
the washing buffer 100 times, and then 0.1 ml was added into each well. After being
reacted for 1 hour at room temperature, it was washed by the washing buffer four times.
3. 0.1 ml of TMB was added into each well for color reaction, and after about 5 to 10
minutes, a stopping solution was added to stop the reaction. Finally, O.D value was read
out at the wavelength of 450 nm.
(The above analyzing methods were all carried out according to the examination
regulations appended to the kit)


- Abdominal cavity cell culture and cytokine analysis
1. After carbon dioxide euthanasia, the outer furs of the rat abdominal cavity were cut
out by scissors, to expose the whole abdominal cavity.

2. HBSS buffer was injected into the abdominal cavity batch-wise by a 10 ml syringe
to make a total volume of about 20 ml/rat.
3. The abdominal cavity of the rat was opened after the abdomen was gently kneaded.
An incision of about 2 cm was made in the abdominal cavity by using scissors. Peritoneal
exudation cell (PEC) liquid (about 10 to 15 ml of cellular fluid could be collected) was
drawn with a syringe, and placed into a 50 ml centrifuge tube.
4. The supernatant was removed after the liquid was centrifuged at 1500 rpm for 5
minutes. 10 ml of HBSS buffer was added to wash, and then centrifuged, after which the
supernatant was removed.
5. The cell concentration was adjusted to 2 x 106 cells/ml with a fresh culture medium
(containing antibiotics).
6. The cell suspension was divided into a 48-well plate at 0.5 ml/well.
7. An additional 0.5 ml of lipopolysaccharides (LPS) (20 ng/ml) was added separately,
making a final concentration of 10 |ig/ml.
8. Finally, it was placed into a 37°C incubator for 24 hours. The supernatant was
collected, and stored in -20°C. The concentrations of cytokine TNF-α, IL-6, and IL-1β
were analyzed by using ELISA kit.


[Results and discussion]
I. Changes in the body weight of rats
In the animal test, both applying drugs and applying other outer forces affect the
changes of body weight directly or indirectly. Therefore, the observation of body weight
directly is the most important index in appearance. The results of measurement of body
weight show that the arthritis symptoms induced by collagen occur after about Day 20, and
compared to normal rats, their body weights are reduced significantly, as shown in FIG. 3;
however, weight reduction is efficiently avoided in the groups of rats fed with PA-H and
indomethacin, with the same growth curve as the normal group. In addition, the weight
reduction phenomenon could not be efficiently inhibited in the group fed with PA-L.
II. Maximum arthritic index
As described in the section of Material and Method, maximum arthritic index (MAI),
with a 5-stage difference as the criterion for examination scoring, is one of the appearance
indices. As shown in FIG. 4, the arthritis of rats induced by collagen reaches a peak at about
Day 35, while the arthritic indices could be efficiently reduced in the groups of rats fed with
PA and indomethacin, wherein the results of PA-H and indomethacin are most preferred.
III. Joint swelling degree
Arthritis symptoms occurred successively at about Day 20 after the second antigen
injection of rats, and the joint sites of limbs were actually measured with a Vernier Caliper.
Joint swelling rate (the average obtained from 8 measurement sites) increased from 20% to
the highest, 61%, in Day 39, and as shown in FIG. 5, it has a significant difference
compared to the normal group (P indomethacin, both PA-H and PA-L can efficiently inhibit the joint swelling, wherein the


effects of PA-H and indomethacin are still most preferred, suggesting that PA-H may have a
similar anti-inflammatory effect to indomethacin.
IV. Effect of feeding on PA on serum RF
During RA development, there are several autoantibodies that occur. However, in
clinical diagnosis, the presence or absence of an autoantibody in patient serum is a major
criterion in determining whether it is RA or not, wherein the autoantibody of RF is most
important. Therefore, the animal model of CIA in the present test also takes RF as an
important biochemistry index. It has been pointed out in past studies that the RF in human
or rat serum could be analyzed using ELISA method (Vittecoq et al., 2001; Jonsson et al.,
1986). In the present study, we made some improvements on the basis of past analysis
methods, and rebuilt a technological platform for rat serum RF analysis. The analysis
results show that at Day 20 after two antigen injections, the serum RF titers of rats all reach
a peak, as shown in FIG. 6; however, compared with Day 20, at Day 35, the serum RF
values of the groups fed with PA and indomethacin reduced by 33% and 47% respectively,
and at Day 45, further reduced by 39% and 51% respectively. In addition, the present study
found that the serum RF value of the negative control group has the tendency of gradual
reduction from Day 20, suggesting that after arthritis symptoms are induced by collagen in
rats, the symptoms tend to be relieved and even recover over time; as a result, it is assumed
that the "golden time" for disease development is Day 20 to Day 45.
V. Effect of feeding on PA on serum CRP
Serum CRP is mainly produced by the liver, and is an index of causing systemic
inflammatory response; if is the most important reactant presented in serum during acute
inflammation. It is noted in some references that the CRP level in the serum of RA patients,
and the production of IL-1 and TNF-α are closely related to arthritis disorder development
(Nakamu, rheumatoid arthritis, 2000). The study results show that after two antigen


immune injections, CRP concentration in rat serum is significantly increased, and reaches a
peak at Day 35, as shown in FIG. 7, and it has a significant difference compared with the
normal group (P indomethacin, the serum CRP concentration could be efficiently inhibited in CIA rats, and
the same effects still exist at Day 45, suggesting that feeding on PA-H has the same
treatment effect of clinical anti-inflammatory administration as indomethacin. Feeding on
PA-L has no significant inflammatory inhibiting effect.
VI. Change of PEC cytokine
Besides the several important biochemistry indices above evaluating whether PA has
an anti-swelling or anti-inflammatory effect or not, the study also further explores the effect
of the secretion of cytokine on inflammatory response, in order to more easily understand
the change in states of rats before and after the occurrence of RA symptoms, wherein such
inflammatory cytokines as TNF-α, IL-6, and IL-1β are the most important indices, i.e., the
amount of these cytokines in the body is closely related to inflammatory response. The
study results demonstrate that feeding PA-H to CIA rats can significantly inhibit PEC from
secreting TNF-α and IL-ip (as shown in FIG. 8), as well as IL-6 (as shown in FIG. 9).
Additionally, in the feeding with indomethacin, there was no effect of inhibiting PEC from
secreting inflammatory cytokine, assuming that the effect mechanism of indomethacin was
not directed to this; therefore, the application value of PA in anti-swelling or anti-
inflammatory use is further emphasized.
It is known from the above that in the comparison of the drug of the present invention
and indomethacin of the control group, the drug efficacy of highly concentrated PA crude
extracts is equal to that of 2.5 mg/kg of indomethacin. In addition, indomethacin is an
inhibitor of COX, and its pharmaceutical effect may be different from that of PA, based on
the exhibited inhibiting phenomenon of cytokine.


Embodiment 3: PA extracts (PA-EtOH, PA-F1, PA-F2, PA-F3, and PA-F4) obtained by
using column separation purification
2 kg of PA dry material was taken, soaked in 10 times highly concentrated ethanol for
24 hours, filtrated, and then soaked in 10 times highly concentrated ethanol for 24 hours
again. After this, PA extract liquor is condensed to 2-3% of its origin volume under
reduced pressure by a rotary concentrator, dried into a powder and named PA-EtOH, 30g of
weight, 1.5% of yield.
After being diluted with a solvent, it was filled into a DIAION column that was
pretreated. It was washed with a high-polarity solvent with about 10 times volume of dry
herb, and then the elution was collected, named PA-F1, 8.5 g of weight, 0.43% of yield.
The column was washed with a sub-high-polarity solvent with about 5 to 10 times
volume of dry herb, and the elution was collected, named PA-F2, 12 g of weight, 0.6% of
yield. The HPLC pattern is shown in FIG. 10.
The column was washed again with a medium-polarity solvent with about 5 to 10
times volume of dry herb, and the elution was collected, named PA-F3, 15 g of weight,
0.75% of yield.
The column was washed again with a low-polarity solvent which is about 5 to 10
times volume of dry herb, and the elution was collected, named PA-F4, 12g of weight, 0.6%
of yield.
Embodiment 4: animal test of PA extracts for treating RA animals
In the present animal test, the test animals and apparatus used are the same as those
disclosed in embodiment 2.


[Medicamentsl
1. Collagen Type II (Sigma C-1188), obtained from bovine tracheal cartilage
2. Complete Freund's adjuvant, CFA (BD BBL™ 231131)
3. Incomplete Freund's adjuvant, IFA (BD BBL™ 263910)
4. ELISA kit of interleukin-6 (IL-6) and interleukin-1β(IL-1β) (R&D, Duoset)
5. Ceelebrex (CBX)
6. PA extracts (PA-EtOH, PA-F1, PA-F, PA-F3, and PA-F4) in Embodiment 3, directly
administered orally to the rats based on respective actual body weights.
[Protocol]
1. Antigen formulation and immune injection
The animal test scheme is shown as FIG. 11. Bovine collagen type II was dissolved in
0.1 M acetic acid solution, stirred to dissolve thoroughly, and formulated into solutions of
concentrations of 1.5 and 3 mg/ml, which were stored at 4°C for later use. For the first
immune injection, 100 µl of C II solution was emulsified with an equal amount of CFA, and
injected subcutaneously (200 µl/rat) in the root portions of tails of the rats after the
emulsification was complete. After the first immunization, the body weights of the rats
were recorded every three days, and they were observed to discover whether or not swelling
occurred in limbs. After about 15 days, the second immunization was done. 100 µl of C II
solution was emulsified with an equal amount of IFA, and injected subcutaneously (200
µl/rat) in the root portions of tails of the rats after the emulsification was complete.
Approximately from Day 18, the symptoms of arthritis were observed (CIA rats), and PA
and CBX were fed from Day 19 until Day 38.


2. Grouping and treating of animals

[Results and discussionl
I. Changes in the body weight of rats
As shown in FIG. 12, there was no obvious difference between the body weights of
normal rats and CIA rats in each group. In comparison with the control group that was fed
distilled water, the rats fed with PA-EtOH, PA-F2, and PA-F4 have a heavier weight.
II. Maximum arthritic index
As shown in FIG. 13, the arthritis index of rats induced by collagen reaches a peak at
about Day 31, while the arthritic indices could be efficiently reduced in the groups of rats
fed with PA-EtOH, PA-F1, PA-F2, PA-F3, PA-F4, and CBX, wherein the results of PA-F1
and CBX are most preferred.
III. Joint swelling degree
Arthritis symptoms occurred successively at about Day 20 after the second immune
injection of rats, and the joint sites of limbs were actually measured with a Vernier Caliper.
Joint swelling rate continuously increased, and reached the highest, 64%, at Day 27. As
shown in FIG. 14, it has a significant difference compared to the normal group. However,
the rats fed with PA-EtOH, PA-F1, PA-F2, PA-F3, PA-F4, and CBX all exhibited the
inhibiting effect of joint swelling, wherein the effect of PA-F1 and CBX is still most
preferred, showing that PA-F1 may have a similar anti-inflammatory drug efficacy to that of
CBX.

IV. Influence of feeding on PA on serum RF
The analysis results show that after two immune injections, at Day 18, the serum RF
titer of rats all reach the peak, as shown in FIG. 15. However, in the group feeding on PA-
F2 and CBX, PA-F2 reduced RF value by about 34% at Day 30, and about 44% at Day 37,
while CBX treatment reduced RF value by about 52% at Day 30, and about 66% at Day 37;
and when compared to Day 18, serum RF value reduced significantly respectively.
V. Change of PEC cytokine
The study results demonstrate that feeding PA-F2, PA-F3, and PA-F4 to CIA rats
significantly inhibits PEC from secreting IL-6 (as shown in FIG. 16), and feeding PA-F1,
PA-F2, and CBX to CIA rats significantly inhibits PEC from secreting IL-1β (as shown in
FIG. 17).
[Conclusion]
The analysis results of the activities of the above animals are summarized in Table I:


Embodiment 5: in vitro cell model analysis of rat macrophage
I. Determination method of concentrations of TNF-α and IL-1β
[Objective]
The concentrations of TNF-α and IL-1β in microphage RAW264.7 cell culture of rats
were measured, and thereby the active ingredients capable of inhibiting LPS-induced-a
synthesis were screened.
[Apparatus and Material]
1. Instrument
(l)ELISA reader
(2) Centrifuger
2. Assay kit DY410: Rat TNF-α/TNFSF1A (ELISA kit)(R&D, Duoset)

(1) Carrier protein-free goat anti-rat TNF-α or IL-ip antibody: 0.8 µg/ml in phosphate
buffered saline (PBS)
(2) Biotinlated goat anti-rat TNF-α or IL-1β antibody: 150 ng/ml in diluent agent
(3) Recombinant rat TNF-α or IL-1β in diluent agent: 2000 pg/ml
(4) Streptavidin-HRP
3. Solutions required
(1) PBS: 137 mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM disodium
hydrogen phosphate (Na2HPO4), 1.5 mM potassium dihydrogen phosphate (KH2PO4), pH
7.2-7.4
(2) Washing buffer: 0.05% Tween 20 in PBS
(3) Blocking buffer: 1% BSA, 5% sucrose, 0.05% sodium azide (NaN3) in PBS
(4) Diluent agent: 1% BSA in PBS


(5) Substrate solution: Mixture of 1:1 Color Reagent A and Color Reagent B (R&D
system #DY999)
(6) Stopping solution: 2 N sulfuric acid
4. Medicaments
(1) Griess reagent: 1% sulfanilamide and 0.1% N-(l-naphthyl)-ethylene diamine in
5% phosphoric acid
(2) Standard: sodium nitrite
(3) Immune stimulator: Lipopolysaccharide (LPS)
(4) Activity control agent: nitro-L-arginine methyl ester (L-NAME), indomethacin
(5) Culture medium: 10% fetal calf serum (FCS) in DMEM
(6) PBS
(7) Trypsin
5. Cell: RAW 264.7
[Method]
1. Cell culture and plating:
(1) The old culture medium in T-75 was taken out, washed with PBS 1 to 2 times, 3
ml trypsin was added, and reacted in 37°C for 3 minutes, after which 7 ml medium (DMEM
added with 10% FCS added) was added to stop trypsin action.
(2) It was centrifuged at 4°C for 10 min by using a centrifuger at 1000 rpm, to remove
trypsin-containing medium. 10 ml of medium was added, thoroughly mixed, and cells were
counted.
(3) RAW 264.7 cells were inoculated to a 24-well plate at a cell density of 5 x 105
cells/well, and were incubated at 37°C, in 5% carbon dioxide.
2. LPS stimulation and drug treatment
(1) L-LAME and indomethacin (activity control agent), as well as samples were
added into a phenol-free culture medium containing 1 µg/ml LPS.


(2) The old culture medium was taken out, and changed to a fresh medium containing
LPS and activity control agent or samples. This was repeated three times. They were
incubated at 37°C, in 5% carbon dioxide for 18 to 24 hrs.
3. Formulation of sandwich ELISAs
(1) 100A, of capture antibody (diluted to 0.8 µg/ml in PBS) was added to each well of a
96-well plate, and sealed at room temperature to incubate overnight.
(2) After the removal of free capture antibody, it was washed by a washing buffer
three times. 30λ. of a blocking buffer was added, and incubated at room temperature for at
least 1 hr to decrease non-specific binding.
(3) Cell suspension was collected, and centrifuged at 10 krpm with a centrifuger at
4°C for 10 min, and then stored at -20°C.
4. ELISAs analysis
(1) After the removal of the blocking buffer, it was washed by the washing buffer
three times. 100λ, of appropriately diluted cell culture or standard (the highest concentration
was 2000 pg/ml) was added, and incubated at room temperature for 2 hrs.
(2) After the removal of the cell culture or standard, it was washed by the washing
buffer three times. 100λ of detecting antibody (diluted to 100 ng/ml in a solvent) was
added, and incubated at room temperature for 2 hrs.
(3) After the removal of the free detecting antibody, it was washed by the washing
buffer three times. 100λ. of working diluted solution of Streptavidin-HRP was added, and
incubated at room temperature for 20 min, avoiding light.
(4) After the removal of the free Streptavidin-HRP, it was washed by the washing
buffer three times. 100λ, of substrate solution was added, and incubated at room
temperature for 20 min, avoiding light.
(5) 100λ, of stopping solution was added and shaken gently to make it thoroughly
mixed.


(6) O.D value was read out at a wavelength of 450 nm, and 540 nm or 570 nm
calibration was recommended, or the read value at 570 nm (or 540 nm) was detracted
directly from the read value at 450 nm.
II. NO determination
[Objective]
The concentration of nitrite (NO) in microphage RAW264.7 cell culture of rats was
measured, and thereby the active ingredients capable of inhibiting LPS-induced-NO
synthesis were screened.
[Apparatus and Materiall
1. Instrument
(1)ELISA reader
(2) Centrifuger
2. Medicaments

(1) Griess reagent: 1% sulfanilamide and 0.1% N-(1-naphthyl)-ethylene diamine in
5% phosphoric acid
(2) Standard: sodium nitrite
(3) Immune stimulator: Lipopolysaccharide (LPS)
(4) Activity control agent: nitro-L-arginine methyl ester (L-NAME), indomethacin
(5) Culture medium: 10% fetal calf serum (FCS) in DMEM
(6) PBS
(7) Trypsin
3. Cell: RAW 264.7
[Method]
1. Cell culture and plating:
(1) The old culture medium in T-75 was taken out, washed with PBS 1 to 2 times, 3
ml trypsin was added, and reacted in 37°C for 3 minutes, after which 7 ml medium (DMEM
added with 10% FCS) was added to stop trypsin action.


(2) It was centrifuged at 4°C for 10 min by using a centrifuger at 1000 rpm, to remove
trypsin-containing medium. 10 ml of medium was added, thoroughly mixed, and cells were
counted.
(3) RAW 264.7 cells were inoculated to a 24-well plate at a cell density of 5 x 105
cells/well, and were incubated at 37°C, in 5% carbon dioxide.
2. LPS stimulation and drug treatment
(1) L-NAME and indomethacin (activity control agent), as well as samples were
added into a phenol-free culture medium containing 1 µg/ml LPS.
(2) The old culture medium was taken out, and changed to a fresh medium containing
LPS and activity control agent or samples. This was repeated three times. They were
incubated at 37°C, in 5% carbon dioxide for 18 to 24 hrs.
(3) Cell suspension was collected, and centrifuged at 10 krpm with a centrifuger at
4°C for 10 min, and then stored in -20°C.
3. Measurement of NO concentration
(1) Formulation of standard: 100 µM/mL sodium nitrite (dissolved in culture medium)
was formulated, and diluted 2x to obtain 7 standards in total with concentrations of 50, 25,
12.5, 6.25, 3.13, and 1.56 µM/mL respectively.
(2) The standard or the supernatant of cell culture was mixed with Griess reagent in
1:1, and incubated for 15 min under room temperature, avoiding light.
(3) O.D value was read out at a wavelength of 550 nm.
III. Determination of prostaglandin E2 (PGE2)
RAW 264.7 cells were inoculated to a 24-well plate at a cell density of 105 cells/well,
and were incubated overnight (16 to 24 hrs). An activity control agent and test samples
were added to the phenol-free culture medium containing 1 jig/ml of LPS respectively.
After the removal of the old medium, 1 ml of fresh medium containing test sample and LPS


was added to co-incubate. After 24 hrs, they were centrifuged at 1000 rpm with a
centrifuger for 10 min. Supernatant was sucked out, and stored at -20°C, or PGE2 content
in the supernatant was directly quantified by using PGE2 Correlate-EIA kit (Amersham
RPN222).
[Conclusion]
The analysis results of the above cell models are summarized in Table II:

PA extracts can inhibit rat macrophage inflammation induced by LPS. PA-F1 can
significantly inhibit TNF-α produced by reddish swelling inflammatory cells induced from
LPS, and PA-F2 can inhibit the effects generated by IL-1β, whereas PA-F3 can inhibit
PGE2, which is a product of COX.
As described above, the PA crude extract or extract used in the invention is obtained
by direct juice pressing or by column separation, and it is a very safe herb that could be used
externally or orally. In addition, it is demonstrated in the invention by RA rat model that
when orally administrated, the PA crude extract or extract can effectively inhibit animal
disorders of autoimmune diseases and related biochemical caused by collagen plus immune
adjuvant, and therefore, the PA crude extract or extract has a potential anti-swelling or anti-
inflammatory efficiency, and could be effectively used to treat RA.

We Claim;
1. A pharmaceutical composition for treating rheumatoid arthritis, which comprises a
therapeutically effective amount of a crude extract or extract of Plectranthus Amboinicus
(Lour.) Spreng (PA), and a pharmaceutically acceptable carrier.
2. The pharmaceutical composition as claimed in claim 1, which is in the form of a
capsule, tablet, powder, ointment, liquor, or spray.
3. The pharmaceutical composition as claimed in claim 1, wherein the PA crude
extract is a crude extract obtained by direct juice pressing of PA.
4. The pharmaceutical composition as claimed in claim 1, wherein the PA extract is an
extract obtained by column separation after soaking PA with a polar solvent and condensing.
5. The pharmaceutical composition as claimed in claim 4, wherein the extract
obtained by soaking PA with the polar solvent has the HPLC peaks with following retention
times:



wherein the HPLC is performed under the following conditions:
flow rate: 1.0 ml/min pressure limite: 250 kgf/cm2
sample injection: 10 µl
PDA conditions:
sampling: 0.64 sec
wavelength range: 190-370 nm
uv wavelength: 254 nm
elution pattern:

6. The pharmaceutical composition as claimed in claim 4, wherein the extract
undergoes the column separation by:
- washing with a high-polarity solvent and collecting the elution buffer;
- washing with a sub-high-polarity solvent having an about five- to ten-fold volume
of the dry material volume and collecting the elution buffer;
- washing with a medium-polarity solvent having an about five- to ten-fold volume
of the dry material volume and collecting the elution buffer; and
- washing with a low-polarity solvent having an about five- to ten-fold volume of the
dry material volume and collecting the elution buffer.
7. The pharmaceutical composition as claimed in claim 6, wherein the PA extract that
is washed with a sub-high-polarity solvent having an about five- to ten-fold volume of the
dry material volume has the HPLC peaks with the following retention times:




wherein the HPLC is performed under the following conditions:
flow rate: 1.0 ml/min pressure limite: 250 kgf/cm2
sample injection: 10 µl
PDA conditions:
sampling: 0.64 sec
uv wavelength: 254 nm
elution pattern:


The present invention provides a pharmaceutical composition comprising a crude
extract or extract of Plectranthus Amboinicus (Lour.) Spreng (PA) for the
treatment of rheumatoid arthritis.

Documents:

00115-kol-2008-abstract.pdf

00115-kol-2008-claims.pdf

00115-kol-2008-correspondence others.pdf

00115-kol-2008-description complete.pdf

00115-kol-2008-drawings.pdf

00115-kol-2008-form 1.pdf

00115-kol-2008-form 2.pdf

00115-kol-2008-form 3.pdf

00115-kol-2008-form 5.pdf

0115-KOL-2008-CORRESPONDENCE OTHERS 1.2.pdf

0115-KOL-2008-FORM 1-1.1.pdf

0115-KOL-2008-OTHERS.pdf

115-KOL-2008-ABSTRACT.pdf

115-KOL-2008-CLAIMS.pdf

115-KOL-2008-CORRESPONDENCE 1.3.pdf

115-KOL-2008-CORRESPONDENCE OTHERS 1.1.pdf

115-KOL-2008-CORRESPONDENCE-1.2.pdf

115-kol-2008-correspondence-1.3.pdf

115-KOL-2008-CORRESPONDENCE.pdf

115-KOL-2008-DESCRIPTION (COMPLETE).pdf

115-KOL-2008-DRAWINGS.pdf

115-KOL-2008-EXAMINATION REPORT REPLY RECIEVED.pdf

115-kol-2008-examination report.pdf

115-KOL-2008-FORM 1.pdf

115-kol-2008-form 18.pdf

115-KOL-2008-FORM 2.pdf

115-kol-2008-form 3-1.1.pdf

115-KOL-2008-FORM 3.pdf

115-kol-2008-form 5.pdf

115-KOL-2008-FORM-27.pdf

115-kol-2008-granted-abstract.pdf

115-kol-2008-granted-claims.pdf

115-kol-2008-granted-description (complete).pdf

115-kol-2008-granted-drawings.pdf

115-kol-2008-granted-form 1.pdf

115-kol-2008-granted-form 2.pdf

115-kol-2008-granted-specification.pdf

115-KOL-2008-INTERNATIONAL SEARCH REPORT.pdf

115-KOL-2008-OTHERS-1.1.pdf

115-KOL-2008-OTHERS.pdf

115-kol-2008-pa-1.1.pdf

115-KOL-2008-PA.pdf

115-KOL-2008-PETITION UNDER RULE 137.pdf

115-kol-2008-reply to examination report.pdf


Patent Number 246818
Indian Patent Application Number 115/KOL/2008
PG Journal Number 11/2011
Publication Date 18-Mar-2011
Grant Date 16-Mar-2011
Date of Filing 16-Jan-2008
Name of Patentee DEVELOPMENT CENTER FOR BIOTECHNOLOGY
Applicant Address NO. 101, LANE 169, KANGNING ST., XIZHI CITY, TAIPEI COUNTY 221, TAIWAN R.O.C.
Inventors:
# Inventor's Name Inventor's Address
1 CHUNG, YUH-SHAN NO. 101, LANE 169, KANGNING ST., XIZHI CITY, TAIPEI COUNTY 221, TAIWAN R.O.C.
2 WU, REY-YUH NO. 101, LANE 169, KANGNING ST., XIZHI CITY, TAIPEI COUNTY 221, TAIWAN R.O.C.
3 CHENG, CHUN-MING NO. 101, LANE 169, KANGNING ST., XIZHI CITY, TAIPEI COUNTY 221, TAIWAN R.O.C.
4 CHANG, JIA-MING NO. 101, LANE 169, KANGNING ST., XIZHI CITY, TAIPEI COUNTY 221, TAIWAN R.O.C.
PCT International Classification Number A61K36/53; A61P19/02; A61K36/185
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA