U.S. patent application number 11/652360 was filed with the patent office on 2007-05-17 for extraction and purification method of active constituents from stem of lonicera japonica thunb., its usage for anti-inflammatory and analgesic drug.
Invention is credited to Yong-Baik Cho, Chang-Kyun Han, Wie-Jong Kwak, Hae In Rhee, Keun Ho Ryu, Hee Jae Shin, Hunseung Yoo.
Application Number | 20070111955 11/652360 |
Document ID | / |
Family ID | 36165452 |
Filed Date | 2007-05-17 |
United States Patent
Application |
20070111955 |
Kind Code |
A1 |
Kwak; Wie-Jong ; et
al. |
May 17, 2007 |
Extraction and purification method of active constituents from stem
of Lonicera japonica Thunb., its usage for anti-inflammatory and
analgesic drug
Abstract
Disclosed is a method for extracting and purifying active
constituents from honeysuckle (Lonicera japonica Thunb.) and its
use. More particularly, this invention relates to an extraction and
purification method of active constituents including sweroside from
for honeysuckle stem (stem of honeysuckle where leaves are removed)
by removing tannins, hardly soluble flavonoids, saponins, and the
like. Thus obtained active constituents have better
anti-inflammatory and analgesic effect, are safer and more stable
than the conventional active constituents obtained from honeysuckle
flower or honeysuckle leaves, and include sweroside which is an
effective active ingredient of anti-inflammatory and analgesic
drug.
Inventors: |
Kwak; Wie-Jong; (Seoul,
KR) ; Cho; Yong-Baik; (Anyang-shi, KR) ; Han;
Chang-Kyun; (Seoul, KR) ; Shin; Hee Jae;
(Suwon-shi, KR) ; Ryu; Keun Ho; (Seoul, KR)
; Yoo; Hunseung; (Seoul, KR) ; Rhee; Hae In;
(Seoul, KR) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
36165452 |
Appl. No.: |
11/652360 |
Filed: |
January 11, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10527139 |
Mar 8, 2005 |
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PCT/KR03/01851 |
Sep 8, 2003 |
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11652360 |
Jan 11, 2007 |
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Current U.S.
Class: |
514/27 ;
536/17.1 |
Current CPC
Class: |
A61K 36/355 20130101;
A61P 29/00 20180101 |
Class at
Publication: |
514/027 ;
536/017.1 |
International
Class: |
A61K 31/7048 20060101
A61K031/7048 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 11, 2002 |
KR |
10-2002-0055106 |
Sep 26, 2002 |
KR |
10-2002-0058494 |
Claims
1. An anti-inflammatory and analgesic drug which comprises
sweroside represented by the following Chemical Formula 1: ##STR3##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a division of U.S. patent application Ser. No.
10/527,139 filed Mar. 8, 2005 entitled "Extraction and Purification
Method of Active Constituents from Stem of Lonicera Japonica
Thunb., its Usage for Anti-Inflammatory and Analgesic Drug" which
is a 371 of PCT/KR2003/001851 filed on Sep. 8, 2003, published on
Mar. 25, 2004 under publication number WO 2004/024172 A1 which
claims priority benefits from South Korean Patent Application
Number 10-2002-0055106 filed Sep. 11, 2002 and South Korean Patent
Application Number 10-2002-0058494 filed Sep. 26, 2002.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to an extraction and
purification method of active constituents from honeysuckle
(Lonicera japonica Thunb.) and its use, more particularly to an
extraction and purification method of active constituents including
sweroside from honeysuckle stem (stem of honeysuckle where leaves
are removed) by removing tannins, hardly soluble flavonoids,
saponins, and the like. Thus obtained active constituents have
better anti-inflammatory and analgesic effect, are safer and more
stable than the conventional active constituents obtained from
honeysuckle flower or honeysuckle leaves, and include sweroside
which is an effective active ingredient of anti-inflammatory and
analgesic drug.
[0004] The honeysuckle (Lonicera japonica Thunb.) is a
semi-evergreen viny shrub that grows naturally at the feet of
mountains or levees in the range of an altitude of 50 to 600 meters
in Japan, China and Korea. Its flower bud (honeysuckle flower) and
stem (honeysuckle stem) are used as herbal medicines for promotion
of urination, detoxification, hemostasis, blood purification,
treatment of tumor, edema treatment, cold, diarrhea, emesis, and
the like [Illustrated Book of Korean Flora, Chang-Bok Lee, 709,
1989, Hangmun Publishing Co., Seoul; Standard for Herbs (Herb
Medicines) Not Covered by Korean Pharmacopoeia, Hyung-Joon Chi,
Sang-In Lee, 87, 305, 1988, Korea Medical Index Co., Seoul;
Resource Plants in Korea, Tae-Kyung Kim, vol. 4, 148-149, 1996, SNU
Press, Seoul]. Also, many traditional Chinese medicinal books
including Sasang Constitutional Medicine and Gwangje Bigeup teach
that it is good for treating a variety of inflammatory abscesses
inside and outside human bodies [Sasang Constitutional Medicine for
Chosun People, Yenben Chosun Medical Institute, 276, 1991, Yeogang
Publishing Co., Seoul; Gwangje Bigeup, Kyung-Hwa Lee, 349-351,
1991, Yeogang Publishing Co., Seoul]. It has long been used as a
folk remedy for treatment of upper respiratory infections such as
cold, tonsillitis and neuralgia for its anti-inflammatory and
analgesic activity. Recently, the inflammatory and analgesic
activity of honeysuckle has been proved through a variety of
experimental animal models, and its effective physiologically
active constituents have been isolated and reported to the academic
circle [Development of the vegetable anti-inflammatory medicine:
Comparison of anti-inflammatory and analgesic actions of the
honeysuckle extract, Song-Jin Lee et al., Korean Journal of
Pharmacognosy, 363-367, 25, 1994; Flavonoids from the aerial parts
of Lonicera japonica, Son et al., Arch. Pharm. Res., 365-370, 15,
1992; Antiinflammatory activity of Lonicera japonica, Lee et al.,
Phytother. Res., 445-447, 12, 1998; Triterpenoid saponins from the
aerial parts of Lonicera japonica, Son et al., Phytochem.,
1005-1008, 35, 1994; Anti-inflammatory activity of the major
constituents of Lonicera japonica, Lee et al., Arch. Pharm. Res.,
133-135, 18, 1995].
[0005] 2. Description of the Related Art
[0006] To date, hydrolyzable tannins such as caffeoylquinic acid,
methyl caffeate, chlorogenic acid and iso-chlorogenic acid, and
iridoid glycosides such as loganin, sweroside, vogeloside and
epi-vogeloside are known as effective active constituents contained
in honeysuckle stem. Most conventional researches have been
centered on honeysuckle flower and honeysuckle leaves. It should be
noted that honeysuckle stem has a different distribution of
constituents from honeysuckle leaves or honeysuckle flower. That
is, unlike honeysuckle stem, major constituents of honeysuckle
leaves or honeysuckle flower are flavonoids, such as lonicerin,
rhoifolin and ochnaflavon, triterpene saponins having hederagenin
or oleanolic acid as non-sugar constituent, and various
hydrolyzable tannins.
[0007] These constituents are hard to be prepared into an
injection. If the injection is rich in polymer tannins, they may
bind to other constituents thus resulting in coprecipitation, and
may bind to serum proteins in the blood to form hardly soluble
precipitates, which can be a cause of angiostenosis. In addition,
since flavonoids included in honeysuckle in general are insoluble
to water, a fairly large amount of organic solvent or other agents
which mediate dissolution are bnecessary to dissolve them to the
level higher than the effective concentration. Also, active
constituents rich in flavonoids are extremely insoluble in
physiological salt solution for injection and may become unstable
if stored for a long time in an alkaline buffer solution. Lastly,
saponins obtained from honeysuckle, particularly monodesmosides,
are known to have strong hemolysis properties. Therefore, they
cannot be injected directly into the vein without purification
[Studies on the saponins of Lonicera japonica Thunb., Kawai et al.,
Chem. Pharm. Bull., 4769-4775, 36(12), 1988]. Honeysuckle leaves
and honeysuckle flower are greater in tannins and hardly soluble
flavonoids than honeysuckle stem. In the acute toxicity test for
injections, they confer more toxicity than honeysuckle stem even at
low contents, and provide poor analgesic and anti-inflammatory
effect.
[0008] Lonicera japonica, Swertia japonica, Gentiana scabra,
Gentiana triflora, Gentiana manshurica, Gentiana rigescens, and
Gentiana rigescens French var. stictantha Marquand have been used
for alleviation of fever or detoxification for many years. However,
it has not been clearly understood which constituents offer such
effects, and most of the researches have been concentrated on
identifying the activities of loganin, the major medicinal
ingredient of honeysuckle stem [J. Nat. Prod., 54(4),
1102.about.1104 , 1991: Planta Med., 60, 232.about.234, 1994:
Phytotherapy Res., 12, 405-408, 1998].
[0009] Further, sweroside has been known as effective only in liver
protection and inhibition of bacterial activity [J.
Ethnopharmacol., 42, 183-191, 1994: Chem. Pharm. Bull., 45(11),
1823-1827, 1997: Yakugaku Zasshi, 102(8), 755-759, 1982], and its
anti-inflammatory and analgesic effect has not been reported so
far.
SUMMARY OF THE INVENTION
[0010] The present inventors have identified that active
constituents of honeysuckle stem (stem of honeysuckle where leaves
are removed), from which tannins, hardly soluble flavonoids,
saponins, and the like have been removed, and sweroside, the
effective active ingredient of the active constituents, have
superior anti-inflammatory and analgesic effect.
[0011] Accordingly, it is an object of the present invention to
provide a preparation method for preparing active constituents from
honeysuckle stem, which have superior anti-inflammatory and
analgesic activity, safety and stability.
[0012] It is another object of the present invention to provide an
anti-inflammatory and analgesic drug comprising the active
constituents.
[0013] It is still another object of the present invention to
provide an anti-inflammatory and analgesic drug comprising
sweroside.
BRIEF DESCRIPTION OF THE DRAWING
[0014] FIG. 1 is a graph that shows medicinal effects of the active
constituents depending on their contents.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] The present invention provides a method for preparing active
constituents from honeysuckle stem, which have superior
anti-inflammatory and analgesic activity, safety and stability.
[0016] The present invention also provides an anti-inflammatory and
analgesic drug comprising the active constituents.
[0017] The present invention also provides an anti-inflammatory and
analgesic drug comprising sweroside.
[0018] Hereunder is given a more detailed description of the
present invention.
[0019] The present invention relates to a method extracting and
purifying active constituents and sweroside from honeysuckle by
removing tannins, hardly soluble flavonoids, saponins, and the
like. Thus obtained active constituents have significantly better
anti-inflammatory and analgesic effect, are safer and more stable
than the conventional active constituents obtained from honeysuckle
flower or honeysuckle leaves, and also include sweroside which is
an effective active ingredient of anti-inflammatory and analgesic
drug.
[0020] Active constituents and sweroside are extracted from
honeysuckle stem and purified by the following method.
[0021] A honeysuckle stem sample is reflux-extracted with about 7
to 10 volumes of distilled water for 2 to 3 hours, and then
filtered. The residue is collected and reflux-extracted with about
5 to 7 volumes of distilled water for 2 to 3 hours. Thus obtained
liquid is filtered and combined with the above filtrate,
concentrated under reduced pressure, and filtered again, so that
its volume becomes about 1 to 3 times (v/w) with reference to the
herb weight. In extracting with distilled water, if distilled water
is used too little, stirring becomes difficult and the extraction
efficiency decreases because the solubility of the extract becomes
poor. In contrast, if distilled water is used excessively, it
requires more time and cost. Then, equivalent water-saturated low
grade alcohol is added and stirring is carried out for about 10 to
20 minutes at 30 to 50 rpm. After the layers are separated, the
water-saturated low grade alcohol layer is filtered and
concentrated under reduced pressure to obtain primary active
constituents. Here, the water-saturated low grade alcohol to be
used is prepared by adding distilled water to low grade alcohol,
such as propyl alcohol and butyl alcohol, and stirring followed by
sedimentation. The process of layer separation is carried out for 2
to 3 times. In obtaining the low grade alcohol solvent fraction, if
low grade alcohol is used too little, the purification efficiency
decreases, thus the extraction yield and the effective ingredient
content decrease. In contrast, if low grade alcohol is used too
much, it results in increase in cost. Accordingly, it is
recommended to use 1 to 3 volumes (v/w) of low grade alcohol with
reference to the herb weight.
[0022] A column chromatography is carried out for the primary
active constituents using polyamide resin, polyvinylpyrrolidone
resin, and the like in order to remove unwanted materials and
detect effective ingredient. The filler is used in 1 to 10 volumes
(w/w) of the water-saturated low grade alcohol layer. Two three
volumes of 50% (v/v) methanol and methanol to the filler volume are
eluted, and then the distilled water solvent is eluted by
step-gradient method. The secondary active constituents obtained by
eluting the active constituents with distilled water had much less
aromatic organic acids, tannins and flavonoids, and showed better
medicinal effect, significantly reduced toxicity, increased
solubility and improved blood stability. A column chromatography is
carried out again for the secondary active constituents using ODS
(octadecylsilane) resin. Starting from a 10% (v/v) methanol, 2 to 3
volumes of solvent to the resin volume is eluted by the
step-gradient method while increasing the methanol content by 10%
(v/v). The polyamide resin or polyvinylpyrrolidone resin is used in
20 to 50 volumes for the weight of the purified primary active
constituents. The active constituents obtained by eluting 20 to 30%
(v/v) methanol showed the best anti-inflammatory and analgesic
effect. Analysis of the active constituents revealed that iridoid
substances, such as sweroside and loganin are major active
ingredients. The sweroside content was 15.1 to 72.1 wt %, and the
loganin content was 13.9 to 41.4 wt %.
[0023] The active constituents obtained by eluting 20 to 30% (v/v)
methanol has the highest sweroside content. Another column
chromatography was carried out for these active constituents to
separate sweroside represented by the following Chemical Formula 1:
##STR1##
[0024] For thus obtained active constituents and sweroside, an
arachidonic acid induced ear edema test and a croton oil induced
ear edema test were carried out to determine the anti-inflammatory
effect. And, an acetic acid induced writhing test was carried out
to measure the analgesic effect. As a result, they were found to
have much superior inflammatory and analgesic activities than the
conventional active constituents obtained from honeysuckle flower
or honeysuckle leaves.
[0025] The sweroside obtained by the present invention can be
prepared into treatment drugs by the methods known in the
pharmaceutical circle. And, it can be administered orally or
parenterally alone or along with a pharmaceutically. acceptable
carrier, a forming agent, a diluent, etc. Particularly, it can be
prepared in the form of powder, granule, tablet, capsule, syrup,
skin ointment or injection drug.
[0026] The human dosage of the active constituents or sweroside of
the present invention can be selected considering the absorptivity
of the active ingredient in the body, inactivation rate and
excretion rate, age, sex and physical status of the subject,
severity of the disease to be treated, and so forth. Preferably,
the active constituents or sweroside are administered from 1 to 200
mg a day for an adult. The drug may be administered according to
specialized method, if required by the experts' opinion. The drug
may be administered several times a day, preferably 1 to 3 times a
day, at regular intervals. The drug composition may be administered
orally or non-orally. When the drug is to be administered
parenterally, it can be administered through veins, muscles, rectum
or skin.
[0027] Because the active constituents of honeysuckle stem have
superior anti-inflammatory and analgesic effect and desirable
solubility, acute toxicity and blood stability, they are well
suited for the injection drug.
[0028] Hereinafter, the present invention is described in more
detail through the following examples. However, the following
Examples are only for the understanding of the present invention,
and they shall not be construed as limiting the scope of the
present invention.
EXAMPLES
Example 1
Medicinal Effects of Each Part of Honeysuckle
[0029] Samples of the whole body of honeysuckle (honeysuckle with
stem and leaves), honeysuckle leaves and honeysuckle stem were
taken at Yeongcheon, Gyeongsangbuk-do, Korea in July 1999. The
samples were dried in the shade and reflux-extracted with 7 volumes
of distilled water for 2.5 hours, and then filtered. The residue
was then collected and reflux-extracted with 7 volumes of distilled
water for 2.5 hours. Thus obtained liquid was filtered and combined
with the above filtrate, concentrated under reduced pressure, and
filtered again, so that its volume became about 2 times (v/w) with
reference to the herb weight. Then, equal volume of water-saturated
n-butyl alcohol was added and the mixture was stirred for 15
minutes at about 30 rpm. After the layers were separated, the
alcohol layer was filtered and concentrated under reduced pressure
to obtain primary active constituents. Then, a column
chromatography was carried out for obtaining the purified fraction
using polyamide resin (CAS NO. 63428-83-1). The resin amount was 5
volumes of the sample. Two volumes of 50% (v/v) methanol and
methanol were eluted, and then the distilled water solvent was
eluted by step-gradient method to obtain secondary active
constituents. A croton oil induced ear edema test was carried out
by administering the secondary active constituents of whole body of
honeysuckle, honeysuckle stem and honeysuckle leaves into the tail
veins of 6-week-old ICR mice (body weight: 20 to 30 g, n=6, SLC,
Japan), which had been fasted for 4 hours. 15 minutes later,
inflammation was induced with 2.5% croton oil. 4 hours later,
thicknesses of left and right ears of the mice were measured using
a dial thickness gauge. The rate of inflammation was calculated by
the following Equation 1 and the result is shown in Table 1. Rate
of inflammation (%)=[Thickness of inflamed (right) ear-Thickness of
normal (left) ear]/[Thickness of normal ear].times.100 Equation
1
[0030] TABLE-US-00001 TABLE 1 Classification Concentration (mg/kg)
Rate of Inhibition (%) Whole body of 1 27.6 honeysuckle 3 29.7 10
34.8 25 33.6 50 32.2 100 30.4 Honeysuckle stem 3 30.2 30 39.0
Honeysuckle leaves 3 19.6 30 21.9
[0031] As shown in Table 1, the active constituents of honeysuckle
stem showed best anti-inflammatory and analgesic activity.
Example 2
Comparison of Medicinal Effect of Active Constituents Obtained from
Honeysuckle Stem
[0032] A croton oil induced ear edema test was carried out as in
Example 1.
[0033] Further, an arachidonic acid induced ear edema test was
carried out by administering drugs (marobiven, primary constituents
of honeysuckle stem, and secondary active constituents of
honeysuckle stem) into the tail veins of 6-week-old ICR mice (body
weight: 20 to 30 g, n=6, SLC, Japan), which had been fasted for 4
hours. Fifteen minutes later, inflammation was induced with 0.05%
arachidonic acid. About 1 hour later, thicknesses of left and right
ears of the mice were measured, and the rate of inhibition was
calculated by Equation 1 and the result is shown in Table 2.
TABLE-US-00002 TABLE 2 Rate of Inhibition (%) Classification Dose
(mg/kg) CO* AA** Control group 50 21.0 NT (marobiven) 100 25.8 NT
200 30.1 NT Primary active 50 35.6 NT constituents 100 36.2 NT 200
38.0 NT Secondary active 0.1 19.8 11.7 constituents 0.3 35.2 16.5 3
38.0 26.3 10 36.6 32.4 30 37.1 48.7 *Croton oil induced ear edema
test result **Test result of arachidonic acid induced ear edema NT:
Not-tested
[0034] As shown in Table 2, the secondary active constituents
obtained from honeysuckle stem included no compounds like aromatic
organic acids, tannins and flavonoids, and had a greater content of
active ingredient than the primary active constituents.
Example 3
Comparison of Medicinal Effects of Active Constituents Obtained
from Honeysuckle Stem
[0035] The procedure of Example 1 was carried out by replacing
polyamide resin with polyvinylpyrrolidone resin (CAS NO.
25249-54-1). TABLE-US-00003 TABLE 3 Rate of Inhibition (%)
Classification Dose (mg/kg) CO* AA** Control group 50 20.2 NT
(marobiven) 100 24.1 NT 200 31.2 NT Primary active 50 33.2 NT
constituents 100 35.1 NT 200 37.5 NT Secondary active 0.1 20.1 10.3
constituents 0.3 34.1 16.7 3 39.7 24.9 10 35.6 33.3 30 36.4 49.6
*Croton oil induced ear edema test result **Test result of
arachidonic acid induced ear edema NT: Not-tested
Example 4
Preparation of Final Active Constituents and Medicinal Effect
Test
[0036] The secondary active constituents of honeysuckle stem
prepared in Example 1 were concentrated under reduced pressure to
obtain a powder. Another column chromatography was carried out for
the powder using ODS resin (YMC*GEL ODS-A 12 nm, S-150 m or ODS-AM
12 nm, S-50 m or ODS-AQ 12 nm, S-50 m). Three volumes of resin was
used and 20% (v/v) methanol was eluted to obtain the final active
constituents.
[0037] A croton oil induced ear edema test was carried out as in
Example 1.
[0038] Further, an acetic acid induced writhing test was carried
out by administering drugs (marobiven and the final active
constituents of honeysuckle stem) into the tail veins of ICR mice
(body weight: 20 to 30 g, n=8, SLC, Japan), which had been fasted
for a day. Twenty minutes later, 0.7% acetic acid was injected
intraperioneally. Fifteen minutes later, numbers of writhing for 10
minutes were counted to calculate the rate of inhibiting
inflammation. The result is shown in Table 4. TABLE-US-00004 TABLE
4 Rate of Inhibition (%) Classification Dose (mg/kg) CO* AA**
Control group 1 6.6 37.9 (marobiven) 10 24.0 48.8 100 29.8 56.5
Final active 0.1 29.6 62.1 constituents 1 34.8 68.4 10 45.8 76.9
*Croton oil induced ear edema test result **Test result of acetic
acid induced writhing NT: Not-tested
Example 5
Contents of Active Ingredients and Medicinal Effect Test
[0039] From high performance liquid chromatography (HPLC) for the
final active constituents prepared in Examples 1 and 4, the
contents of active ingredient were identified: sweroside=15.1 to
72.1 wt % and loganin=13.9 to 41.4 wt %. The sweroside and loganin
contents of each sample are given in Table 5. The medicinal effect
of the final active constituents depending on habitat and gathering
time are given in Table 6. TABLE-US-00005 TABLE 5 Contents of
sweroside and loganin depending on habitat and time of collection
Yeongcheon, Andong, Taishan, Xinxiang, Month Gyeongsangbuk-do (A)
Gyeongsangbuk-do (B) China China January Lo: 34.6 Lo: 38.3 Lo: 38.2
Lo: 31.4 Sw: 57.7 Sw: 55.4 Sw: 48.6 Sw: 59.2 Total: 92.3 Total:
93.7 Total: 86.8 Total: 90.6 February Lo: 30.2 Lo: 32.3 Lo: 33.3
Lo: 28.6 Sw: 64.1 Sw: 62.1 Sw: 54.2 Sw: 63.3 Total: 94.3 Total:
94.4 Total: 87.5 Total: 91.9 March Lo: 22.7 Lo: 26.1 Lo: 25.8 Lo:
20.5 Sw: 70.2 Sw: 67.6 Sw: 60.3 Sw: 72.1 Total: 92.9 Total: 93.7
Total: 86.1 Total: 92.6 April Lo: 13.9 Lo: 15.9 Lo: 19.1 Lo: 14.2
Sw: 63.1 Sw: 60.1 Sw: 65.3 Sw: 69.7 Total: 77.0 Total: 76.0 Total:
84.4 Total: 83.9 May Lo: 17.1 Lo: 14.2 Lo: 18.7 Lo: 14.9 Sw: 52.3
Sw: 55.7 Sw: 66.2 Sw: 55.2 Total: 69.4 Total: 69.9 Total: 84.9
Total: 70.1 June Lo: 19.4 Lo: 16.2 Lo: 23.5 Lo: 17.4 Sw: 33.4 Sw:
38.2 Sw: 43.5 Sw: 42.6 Total: 52.8 Total: 54.4 Total: 67.0 Total:
60.0 July Lo: 21.7 Lo: 25.3 Lo: 28.9 Lo: 22.4 Sw: 15.1 Sw: 17.5 Sw:
29.8 Sw: 35.2 Total: 36.8 Total: 42.8 Total: 58.7 Total: 57.6
August Lo: 23.5 Lo: 27.0 Lo: 30.1 Lo: 32.1 Sw: 29.4 Sw: 32.7 Sw:
35.9 Sw: 36.4 Total: 52.9 Total: 59.7 Total: 66.0 Total: 68.5
September Lo: 25.3 Lo: 30.1 Lo: 33.6 Lo: 39.2 Sw: 47.6 Sw: 45.3 Sw:
42.5 Sw: 40.2 Total: 72.9 Total: 75.4 Total: 76.1 Total: 79.4
October Lo: 28.7 Lo: 32.2 Lo: 33.8 Lo: 37..4 Sw: 52.2 Sw: 50.5 Sw:
48.2 Sw: 43.3 Total: 80.9 Total: 82.7 Total: 82.0 Total: 80.7
November Lo: 39.6 Lo: 37.3 Lo: 38.0 Lo: 41.4 Sw: 54.3 Sw: 58.3 Sw:
54.6 Sw: 45.6 Total: 93.9 Total: 95.6 Total: 92.6 Total: 87.0
December Lo: 36.2 Lo: 33.7 Lo: 35.2 Lo: 40.2 Sw: 56.8 Sw: 60.2 Sw:
58.3 Sw: 52.6 Total: 93.0 Total: 93.9 Total: 93.5 Total: 92.8 Lo:
Loganin, Sw: Sweroside
[0040] TABLE-US-00006 TABLE 6 Rate of Inhibition (%) Classification
Contents (mg/kg) CO* AA** Control group 10 25.6 46.9 (marobiven)
100 28.8 56.8 Yeongcheon, July 1 37.2 70.2 Lo: 21.7, Sw: 15.1 10
46.3 75.1 Total: 36.8 Andong, July 1 40.3 72.2 Lo: 25.3, Sw: 17.5
10 51.2 84.3 Total: 42.8 Xinxiang, July 1 49.8 78.8 Lo: 22.4, Sw:
35.2 10 52.6 85.6 Total: 57.6 Taishan, September 1 52.1 82.9 Lo:
33.6, Sw: 42.5 10 52.4 85.5 Total: 76.1 Andong, November 1 53.4
84.6 Lo: 37.3, Sw: 58.3 10 53.2 87.7 Total: 95.6 *Croton oil
induced ear edema test result **Test result of acetic acid induced
writhing
Example 6
Hemolysis Test for Final Active Constituents
[0041] Twenty mL of blood was collected from a rabbit's heart using
a syringe treated with heparin. The blood was centrifuged for about
10 minutes. The supernatant was discarded and the residue was
diluted with 10 volumes of physiological salt solution for
injection. After mixing by gentle shaking, 0.5 mL of the diluted
blood and 0.5 mL of each drug of Example 1 (final active
constituents of honeysuckle stem and primary active constituents of
honeysuckle stem, honeysuckle leaves and whole body of honeysuckle)
and the final active constituents of Example 4 were put in a test
tube (Physiological salt solution and distilled water (100%
hemolysis) control groups were also prepared). The test tube was
incubated in a bath kept at 37.degree. C. for 15 minutes, and then
placed at room temperature for 45 minutes. Lastly, after centrifuge
at 2500 rpm for 2 minutes, the upper layer was analyzed at 540 nm.
TABLE-US-00007 TABLE 7 Standard Classification Hemolysis (%)
deviation Control group (physiological salt solution 0.73 0.17 for
injection) Final active constituents, 5 .times. 10.sup.-4 g/mL 0.69
0.10 Final active constituents, 1.5 .times. 10.sup.-3 g/mL 0.73
0.11 Final active constituents, 5 .times. 10.sup.-3 g/mL 0.74 0.88
Honeysuckle stem, 5 .times. 10.sup.-3 g/mL 15.2 0.20 Honeysuckle
leaves, 5 .times. 10.sup.-3 g/mL 35.1 0.15 Whole body of
honeysuckle, 5 .times. 10.sup.-3 g/mL 23.0 0.45
Example 7
Preparation of Sweroside
[0042] A column chromatography was carried out for the final active
constituents prepared in Example 4 using octadecylsilane resin to
separate sweroside represented by the following Chemical Formula 1:
##STR2##
Example 8
Determination of Anti-Inflammatory Effect of Sweroside
[0043] A croton oil induced ear edema test was carried out by
administering sweroside into the tail veins of 6-week-old ICR mice
(body weight: 20 to 30 g, n=6, SLC, Japan), which had been fasted
for 4 hours. Fifteen minutes later, inflammation was induced with
2.5% croton oil. Four hours later, thicknesses of left and right
ears of the mice were measured using a dial thickness gauge. The
rate of inflammation was calculated by Equation 1 and the result is
shown in Table 8. The medicinal effect for oral administration is
given in Table 9. TABLE-US-00008 TABLE 8 Classification Dose
(mg/kg) Rate of Inhibition (%) Sweroside 0.1 49.8 1 56.2 10
65.7
[0044] TABLE-US-00009 TABLE 9 Classification Dose (mg/kg) Rate of
Inhibition (%) Sweroside 1 31.9 10 43.1 100 57.8
[0045] Further, an arachidonic acid induced ear edema test was
carried out by administering sweroside into the tail veins of
6-week-old ICR mice (body weight: 20 to 30 g, n=6, SLC, Japan),
which had been fasted for 4 hours. Fifteen minutes later,
inflammation was induced with 0.05% arachidonic acid. One hour
later, thickness of left and right ears of the mice was measured,
and the Rate of Inhibition was calculated by the Equation 1 and the
result is shown in Table 10. The medicinal effect for oral
administration is given in Table 9. TABLE-US-00010 TABLE 10
Classification Dose (mg/kg) Rate of Inhibition (%) Sweroside 0.1
48.3 1 55.1 10 69.1
[0046] TABLE-US-00011 TABLE 11 Classification Dose (mg/kg) Rate of
Inhibition (%) Sweroside 1 29.0 10 34.7 100 45.7
Example 9
Determination of Analgesic Effect of Sweroside
[0047] An acetic acid induced writhing test was carried out by
administering sweroside into the tail veins of ICR mice (body
weight: 20 to 30 g, n=8, SLC, Japan), which had been fasted for a
day. Twenty minutes later, 0.7% acetic acid was injected
intraperioneally. Fifteen minutes later, the number of writhing for
10 minutes were counted to calculate the rate of inhibiting
inflammation and the result is shown in Table 12. The medicinal
effect for oral administration is given in Table 13. TABLE-US-00012
TABLE 12 Classification Dose (mg/kg) Rate of Inhibition (%)
Sweroside 0.1 74.6 1 86.4 10 89.7
[0048] TABLE-US-00013 TABLE 13 Classification Dose (mg/kg) Rate of
Inhibition (%) Sweroside 1 65.9 10 79.1 100 87.9
Example 10
Toxicity Test
[0049] The active constituents of honeysuckle stem and sweroside,
in the amount of 1.0 g/kg, 1.5 g/kg and 2.0 g/kg, were administered
through the tail veins of SD rats (body weight: 120 to 170 g, 5
male and female rats for each administration dose, SLC, Japan),
respectively, which had been fasted for 4 hours. The rats were
observed for 30 minutes at first, and then observed at 30 minutes
intervals with naked eyes. Death rate, general symptoms and weight
change were observed for 2 weeks after administration. An autopsy
was conducted to identify presence of any abnormalities of
organs.
[0050] The lethal dose of the active constituents of honeysuckle
stem and sweroside was both over 5.0 g/kg for oral administration
(no dead rats observed), and over 2.0 g/kg for intravenous
injection (no dead rats observed). When 2.0 g/kg was intravenously
injected, the rats showed increased respiration counts and
decreased activity for about 10 minutes, which were restored soon.
No other symptoms were observed, and there was no change in weight
due to administration. The autopsy result showed no abnormalities
as is the case with the control groups.
[0051] A local toxicity test at the dose of 50, 100 and 150 mg/kg,
respectively, showed no difference from the group administered with
physiological salt solution. No toxicity, such as tissue necrosis
or inflammation, was observed.
Preparation Example 1
Preparation of Tablet
[0052] The active constituents of honeysuckle stem or sweroside was
prepared into a tablet with the following composition:
TABLE-US-00014 Active ingredient 160 mg Light anhydrous silicic
acid 20 mg Corn starch 87 mg Crystalline cellulose 72 mg Sodium
starch glyconate 60 mg Magnesium stearate 6 mg Total 672 mg
Preparation Example 2
Preparation of Syrup
[0053] The active constituents of honeysuckle stem or sweroside was
prepared into syrup with the following composition: TABLE-US-00015
Active ingredient 4,000 mg Methyl p-oxybenzoate (5% ethanol
solution) 60 mg Propyl p-oxybenzoate (5% ethanol solution) 40 mg
Sodium benzoate (5% solution) 100 mg Banana powder (10% solution)
600 mg D-Sorbitol 140,000 mg Distilled water 196 mL
Preparation Example 3
Preparation of Injection
[0054] The active constituents of honeysuckle stem or sweroside was
prepared into an injection with the following composition:
TABLE-US-00016 Injection ampule: Active ingredient 20 mg Mannitol
60 mg Corresponding solvent sample: Physiological salt solution
2000 mg for injection Total 2080 mg
Preparation Example 4
Preparation of Injection
[0055] The active constituents of honeysuckle stem or sweroside was
prepared into an injection with the following composition:
TABLE-US-00017 Injection ampule: Active ingredient 50 mg
KH.sub.2(PO.sub.4) 8.5 mg Physiological salt solution for injection
3000 mg Total 3058.5 mg
Preparation Example 5
Preparation of Injection
[0056] The active constituents of honeysuckle stem or sweroside was
prepared into an injection with the following composition:
TABLE-US-00018 Injection ampule: Active ingredient 100 mg Mannitol
300 mg KH.sub.2(PO.sub.4) 17 mg Physiological salt solution for
injection 3000 mg Total 3417 mg
Preparation Example 6
Preparation of Ointment Drug
[0057] The active constituents of honeysuckle stem or sweroside was
prepared into an ointment drug the following composition:
TABLE-US-00019 Active ingredient 5 g Liquid paraffin 10 g Hard lead
9 g Ethanol 8 g Sorbitan monooleate 2 g Polysorbate 4 g Propyl
p-oxybenzoate 0.05 g Methyl p-oxybenzoate 0.1 g Concentrated
glycerine 10 g Purified water Adequate
[0058] As described above, the active constituents of the present
invention obtained from honeysuckle stem have significantly
increased solubility and blood stability as compared to those of
the conventional active constituents. Further, they are shown to
have superior analgesic and anti-inflammatory effect, safety and
stability. Also, sweroside of the present invention obtained from
honeysuckle stem has very superior medicinal effect and shows
little toxicity, thus being very suitable for an anti-inflammatory
and analgesic drug.
[0059] While the present invention has been described in detail
with reference to the preferred embodiments, those skilled in the
art will appreciate that various modifications and substitutions
can be made thereto without departing from the spirit and scope of
the present invention as set forth in the appended claims.
* * * * *