U.S. patent application number 09/887488 was filed with the patent office on 2002-04-18 for anti-fungal pharmaceutical compositions comprising an active ingredient prepared from zingiber officinal.
This patent application is currently assigned to Pharmaceutical Industry Technology and development Center. Invention is credited to Ko, Feng-Nien, Kuo, Sheng-Chu, Teng, Che-Ming, Wu, Tian-Shung.
Application Number | 20020044979 09/887488 |
Document ID | / |
Family ID | 29254736 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020044979 |
Kind Code |
A1 |
Wu, Tian-Shung ; et
al. |
April 18, 2002 |
Anti-fungal pharmaceutical compositions comprising an active
ingredient prepared from Zingiber officinal
Abstract
A method of preparing an extract which is potent in anti-fungal
activity from Zingiber officinale, includes the following steps:
preparing a crude liquid from rhizomes of ginger by extraction with
an organic solvent or supercritical CO.sub.2, or by distillation
with steam; introducing the crude liquid to a reverse phase
chromatography column, and eluting the column with water, a first
eluent and a second eluent having a polarity weaker than that of
the first eluent but stronger than that of chloroform, so that a
first eluate resulting from elution of the first eluent and a
second eluate resulting from elution of the second eluent are
obtained; removing the first eluent and the second eluent from the
first eluate and the second eluate by evaporation, respectively, so
that a first concentrated eluate and a second concentrated eluate
are obtained as the potent extract.
Inventors: |
Wu, Tian-Shung; (Tainan,
TW) ; Kuo, Sheng-Chu; (Taichung, TW) ; Teng,
Che-Ming; (Taipei, TW) ; Ko, Feng-Nien;
(Taipei, TW) |
Correspondence
Address: |
Jackson Walker, L.L.P.
Suite 600
2435 N. Central Expressway
Richardson
TX
75080
US
|
Assignee: |
Pharmaceutical Industry Technology
and development Center
5F, No. 101, Lane St., Hsi Chih Cheng
Taipei Hsien
TW
|
Family ID: |
29254736 |
Appl. No.: |
09/887488 |
Filed: |
June 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09887488 |
Jun 22, 2001 |
|
|
|
09648662 |
Aug 26, 2000 |
|
|
|
6274177 |
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Current U.S.
Class: |
424/756 ;
424/74 |
Current CPC
Class: |
A61K 36/9068 20130101;
Y10S 514/864 20130101; A61K 36/9066 20130101 |
Class at
Publication: |
424/756 ;
424/74 |
International
Class: |
A61K 035/78; A61K
007/06 |
Claims
1. An anti-fungal pharmaceutical composition comprising a
therapeutically effective amount of a product prepared from
rhizomes of Zingiber officinale, as an active ingredient, in
admixture with a pharmaceutically acceptable carrier or diluent for
the active ingredient, wherein said product is prepared by the
following steps: a) preparing a crude liquid from rhizomes of
Zingiber officinale; b) introducing the crude liquid to a reverse
phase chromatography column, and eluting the column with water, a
first eluent and a second eluent in sequence, said second eluent
having a polarity weaker than that of the first eluent but stronger
than that of chloroform, so that a first eluate resulting from
elution of the first eluent and a second eluate resulting from
elution of the second eluent are obtained; c) removing the first
eluent from the first eluate by evaporation, so that a first
concentrated eluate is obtained and is able to be used as the
product; and d) removing the second eluent from the second eluate
by evaporation, so that a second concentrated eluate is obtained
and is able to used as the product; wherein step a) comprises steps
i) to iv), or comprises step I), step I'), or step I"), wherein
said steps i) to iv) are: i) shedding fresh rhizomes of Zingiber
officinale and filtering the resulting mixture to obtain a filtrate
and a residue; ii) extracting the filtrate with a first organic
solvent, recovering the resulting extraction solution of the first
organic solvent, and evaporating the first organic solvent from the
extraction solution to obtain a first concentrated extraction
solution; iii) extracting the residue with a second organic
solvent, recovering the resulting extraction solution of the second
organic solvent, and evaporating the second organic solvent from
the extraction solution to obtain a second concentrated extraction
solution; and iv) combining the first concentrated extraction
solution and the second concentrated extraction solution to obtain
the crude liquid; said step I) is: I) extracting powder of dried
rhizomes of Zingiber officinale with the second organic solvent,
recovering the resulting extraction solution of the second organic
solvent, and evaporating the second organic solvent from the
extraction solution to obtain the crude liquid; said step I') is:
I') steam distilling powder of dried rhizomes of Zingiber
officinale, and concentrating the resulting distillate by
evaporation to obtain the crude liquid; and said step I") is: I")
extracting powder of dried rhizomes of Zingiber officinale with
supercritical CO.sub.2, recovering the resulting extraction
solution of the supercritical CO.sub.2, and evaporating CO.sub.2
from the extraction solution to obtain the crude liquid.
2. The pharmaceutical composition according to claim 1, wherein the
product as the active ingredient comprises 0-10 mg 6-shogaol per
gram of the product, 1-150 mg 6-gingerol per gram of the product,
and 0-40 mg 6-dehydrogingerdione per gram of the product.
3. The pharmaceutical composition according to claim 1, wherein
said first eluent is methanol, and said second eluent is
acetone.
4. The pharmaceutical composition according to claim 3, wherein
step a) comprises steps i) to iv).
5. The pharmaceutical composition according to claim 4, wherein
said first organic solvent is ethyl ether.
6. The pharmaceutical composition according to claim 4, wherein
said second organic solvent is acetone, methanol, ethanol or a
combination of them.
7. The pharmaceutical composition according to claim 6, wherein
said second organic solvent is acetone.
8. The pharmaceutical composition according to claim 3, wherein
step a) comprises step I).
9. The pharmaceutical composition according to claim 8, wherein
said second organic solvent is acetone, methanol, ethanol or a
combination of them.
10. The pharmaceutical composition according to claim 9, wherein
said second organic solvent is acetone.
11. The pharmaceutical composition according to claim 3, wherein
step a) comprises step I').
12. The pharmaceutical composition according to claim 3, wherein
step a) comprises step I").
13. The pharmaceutical composition according to claim 1, wherein
said reverse phase chromatography column is packed with a porous
resin.
14. An anti-fungal pharmaceutical composition comprising a
therapeutically effective amount of the crude liquid prepared
according to step a) in claim 1, as an active ingredient, in
admixture with a pharmaceutically acceptable carrier or diluent for
the active ingredient.
15. The pharmaceutical composition according to claim 14, wherein
step a) comprises steps i) to iv).
16. The pharmaceutical composition according to claim 15, wherein
said first organic solvent is ethyl ether.
17. The pharmaceutical composition according to claim 16, wherein
said second organic solvent is acetone, methanol, ethanol or a
combination of them.
18. The pharmaceutical composition according to claim 17, wherein
said second organic solvent is acetone.
19. The pharmaceutical composition according to claim 14, wherein
step a) comprises step I).
20. The pharmaceutical composition according to claim 19, wherein
said second organic solvent is acetone, methanol, ethanol or a
combination of them.
21. The pharmaceutical composition according to claim 20, wherein
said second organic solvent is acetone.
22. The pharmaceutical composition according to claim 14, wherein
step a) comprises step) I').
23. The pharmaceutical composition according to claim 14, wherein
step a) comprises step I").
24. The pharmaceutical composition according to claim 1, which is
used in the treatment of a disease associated with Trichophyton
mentagrophytes or Pityrosporum ovale.
25. The pharmaceutical composition according to claim 24, in which
said disease is selected from the group consisting of tinea pedis,
tinea capitis, tinea cruris, tinea glabrosa, onychomycosis,
pityriasis capitis, pityriasis vesicolor, pityrosporum
folliculitis, seborrheic dermatitis and dandruff.
26. The pharmaceutical composition according to claim 24, which is
in the form of a shampoo, a bath gel, soap, a body lotion, a body
cream or a detergent.
27. The pharmaceutical composition according to claim 26, which is
in the form of a shampoo for use in the treatment of dandruff.
28. The pharmaceutical composition according to claim 14, which is
used in the treatment of a disease associated with Trichophyton
mentagrophytes or Pityrosporum ovale.
29. The pharmaceutical composition according to claim 28, in which
said disease is selected from the group consisting of tinea pedis,
tinea capitis, tinea cruris, tinea glabrosa, onychomycosis,
pityriasis capitis, pityriasis vesicolor, pityrosporum
folliculitis, seborrheic dermatitis and dandruff.
30. The pharmaceutical composition according to claim 28, which is
in the form of a shampoo, a bath gel, soap, a body lotion, a body
cream or a detergent.
31. The pharmaceutical composition according to claim 30, which is
in the form of a shampoo for use in the treatment of dandruff.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation-in-part
application of U.S. patent application Ser. No. 09/648,662, filed
Aug. 26, 2000. The above-listed application of Ser. No. 09/648,662
is commonly assigned with the present invention and the entire
content of which application is incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention is related to a method of preparing an
extract potent in anti-fungal activity from Zingiber
officinale.
BACKGROUND OF THE INVENTION
[0003] Chinese crude drugs or spices eg. Zingiber officinale,
Eugenia caryophyllata, Allium sativum, have been used in medicine
and in flavoring foods. Crude ginger is used as an anti-emetic and
expectorant, an anti-tussive and accelerator of the digestive
organs. Semi-dried old crude ginger is also used for stomachache,
chest pain, low back pain, cough, common cold and as a cure for a
form of edema being called "stagnate of water". Zingerone is the
major component which accounts for the spicy character of ginger;
gingerol and shogaol are other pungent components in ginger.
Gingerol has cardio-tonic action, suppresses the contraction of
isolated portal veins in mice, and modulates the eicosanoid-induced
contraction of mouse and rat blood vessels. Shogaol exhibits
pressor response. Both gingerol and shogaol are mutagenic, whereas
zinger and zingerone have been found to exhibit anti-mutagenic
activity. Shogaol has inhibitory activity on the
carrageenin-induced paw edema and platelet aggregation [U.S. Pat.
No. 5,804,603, Background of the Invention].
[0004] Heretofore, many reports have shown that Zingiber officinale
exhibits various physiological activities. Typical examples include
a cancer metastasis suppressing agent disclosed in Japan patent
publication No. 7-258104; a synthesis promoter for neurotropic
factor, which is effective for nerve deteriorative diseases such as
Alzheimer's dementia or Parkinson's disease, disclosed in Japan
patent publication No. 7-25777; an anti-rheumatic agent disclosed
in Japan patent publication No. 6-293653, U.S. Pat. Nos. 5,494,668
and 5,683,698; an anti-microbial composition disclosed in Japan
patent publication No. 6-227931; and an analgesic composition
disclosed in Japan patent publication No. 6-107556. Ginger contains
1-4% essential oil (oleoresin). During the last 45 years many
chemical investigations have been carried out on the constituents
of the essential oil. Altogether more than 200 different volatiles
have been identified in essential oil wherein the pharmacological
activity is confined. The essential oil contains a mixture of
various terpenes as well as some other non-terpenoid compounds.
Although this is mostly speculative, the experimental data and
observations suggest that ginger inhibits both the cyclooxygenase
and lypoxygenase products, i.e. it can be a dual inhibitor of
eicosanoid synthesis. In all 56 patients (28 with rheumatoid
arthritis, 18 with osteoarthritis and 10 with muscular discomfort)
used powdered ginger against their afflictions. Amongst the
arthritis patients more than three-quarters experienced, to varying
degrees, relief in pain and swelling. All the patients with
muscular discomfort experienced relief in pain. None of the
patients reported adverse effects during the period of ginger
consumption which ranged from 3 months to 2.5 years. (Srivastava
and Mustafa; Medical Hypotheses; 1992; 39 342-348)
[0005] Non-steroidal anti-inflammatory drugs have three major
actions, all of which are related to inhibition of cyclo-oxygenase
resulting in decreased formation of prostanoids. Firstly, an
anti-inflammatory action achieved by reduced production of
vasodilator prostaglandins (PGE.sub.2, PGI.sub.2) which means less
vasodilation and, indirectly less edema. Secondly, an analgesic
effect achieved by reduced prostaglandin production (less
sensitization of nociceptic nerve endings to the inflammatory
mediators bradykinin and 5-hydroxytryptamine). Thirdly, an
antipyretic effect which is probably due to a decrease in the
mediator PGE.sub.2 generated in response to inflammatory pyrogens,
much as interleukin-1. Since ginger inhibits prostanoid synthesis
and also products of 5-lipoxygenase, its ameliorative effects in
arthritis and muscular discomforts could be related to reduced
formation of prostanoids and leukotrienes. Because of such a
possibility a decrease in the carageenan-induced edema formation in
the rat's paw after 3 g of ginger extract administration has been
demonstrated and the potency of the extract in the acute
inflammation test appears to be comparable to that exhibited by
acetyl salicylic acid reported in the same study (Mascolo N. et
al., Journal of Ethnopharmocology 1989, 27, 129-140).
[0006] Dermatophytes, especially Trichophyton rubrum and
Trichophyton mentagrophytes, are the usual pathogens of
onychomycosis and tinea pedis [Roberts D T., British Journal of
Dermatology. 141 Supple 56:1-4, 1999 Nov.; Roldan Y B. et al.,
Mycoses, 43(5):181-3, 2000]. Pityrosporum ovale (Malassezia furfur)
is the etiological agent of pityriasis vesicolor, Pityrosporum
folliculitis and Malassezia intertrigo. Several studies indicate a
strong association of Pityrosporum ovale with seborrheic dermatitis
and dandruff, a milder form of seborrheic dermatitis [Nenoff P. et
al., Dermatology. 191(4):311-4, 1995; Bulmer A C. et al.,
Mycopathologia, 147(2)63-5, 1999].
SUMMARY OF THE INVENTION
[0007] The present invention provides extracts from rhizomes of
ginger which show in vitro an antifungal activity against
Trichophyton mentagrophytes and Pityrosporum ovale. The extracts
are prepared by extracting rhizomes of ginger with an organic
solvent (such as ethyl ether, acetone, methanol and ethanol) or
supercritical CO.sub.2, or by steam distilling rhizomes of ginger
to obtain a crude liquid, and subjecting said crude liquid to a
reverse phase chromatography to obtain the extracts containing
shogaols, gingerols and/or dehydrogingerdione.
DETAILED DESCRIPTION OF THE INVENTION
[0008] As introduced in the Background of the Invention, ginger has
been used for anti-inflammation and pain relief.
[0009] The present invention is to provide an effective method of
preparing a product potent in antifungal activity from rhizomes of
ginger. The potent product prepared in accordance with the method
of the present invention has a substantially constant composition,
so that the pharmacological effects thereof are definite.
[0010] The effective method of preparing product potent in
antifungal activity from rhizomes of ginger according to the
present invention comprises the following steps:
[0011] a) preparing a crude liquid from rhizomes of ginger;
[0012] b) introducing the crude liquid to a reverse phase
chromatography column, and eluting the column with water, a first
eluent and a second eluent in sequence, said second eluent having a
polarity weaker than that of the first eluent but stronger than
that of chloroform, so that a first eluate resulting from elution
of the first eluent and a second eluate resulting from elution of
the second eluent are obtained;
[0013] c) removing the first eluent from the first eluate by
evaporation, so that a first concentrated eluate is obtained and is
able to be used as the potent product, and
[0014] d) removing the second eluent from the second eluate by
evaporation, so that a second concentrated eluate is obtained and
is able to be used as the potent product;
[0015] wherein step a) comprises steps i) to iv), or comprises step
I), step I'), or step I"), wherein said steps i) to iv) are:
[0016] i) shedding fresh rhizomes of ginger and filtering the
resulting mixture to obtain a filtrate and a residue;
[0017] ii) extracting the filtrate with a first organic solvent,
recovering the resulting extraction solution of the first organic
solvent, and evaporating the first organic solvent from the
extraction solution to obtain a first concentrated extraction
solution;
[0018] iii) extracting the residue with a second organic solvent,
recovering the resulting extraction solution of the second organic
solvent, and evaporating the second organic solvent from the
extraction solution to obtain a second concentrated extraction
solution; and
[0019] iv) combining the first concentrated extraction solution and
the second concentrated extraction solution to obtain the crude
liquid;
[0020] said step I) is:
[0021] I) extracting powder of dried rhizomes of ginger with the
second organic solvent, recovering the resulting extraction
solution of the second organic solvent, and evaporating the second
organic solvent from the extraction solution to obtain the crude
liquid;
[0022] said step I') is:
[0023] I') steam distilling powder of dried rhizomes of ginger, and
concentrating the resulting distillate by evaporation to obtain the
crude liquid; and
[0024] said step I") is:
[0025] I") extracting powder of dried rhizomes of ginger with
supercritical CO.sub.2, recovering the resulting extraction
solution of the supercritical CO.sub.2, and evaporating CO.sub.2
from the extraction solution to obtain the crude liquid.
[0026] The product potent in antifungal activity prepared according
to the method of the present invention preferably comprises 0-10 mg
6-shogaol per gram of the product, 1-150 mg 6-gingerol per gram of
the product, and 0-40 mg 6-dehydrogingerdione per gram of the
product.
[0027] The present invention also provides a pharmaceutical
composition potent in antifungal activity comprising a
therapeutically effective amount of said crude liquid prepared in
step a) of the method of the present invention, as an active
ingredient, in admixture with a pharmaceutically acceptable carrier
or diluent for the active ingredient.
[0028] The present invention also provides a pharmaceutical
composition potent in antifungal activity comprising a
therapeutically effective amount of said product prepared according
to the method of the present invention, as an active ingredient, in
admixture with a pharmaceutically acceptable carrier or diluent for
the active ingredient. Preferably, said product prepared according
to the method of the present invention is the first concentrated
eluate prepared in step c). Alternatively, said product prepared
according to the method of the present invention is the second
concentrated eluate prepared in step d).
[0029] Preferably, said first eluent is methanol, and said second
eluent is acetone.
[0030] Preferably, step a) of the method of the present invention
comprises steps i) to iv).
[0031] Preferably, said first organic solvent is ethyl ether.
[0032] Preferably, said second organic solvent is acetone,
methanol, ethanol or a combination thereof. More preferably, said
second organic solvent is acetone.
[0033] Preferably, step a) of the method of the present invention
comprises step I).
[0034] Preferably, step a) of the method of the present invention
comprises step I').
[0035] Preferably, step a) of the method of the present invention
comprises step I").
[0036] A suitable reverse phase chromatography column for use in
the method of the present invention includes (but not limited
thereto) a reverse phase chromatography column packed with a porous
resin, for examples Diaion HP-20 (Mitsubishi Co.), Sephadex LH-20
(Pharmicia Co.) and RP-18 (Nacalai tesque Co.).
[0037] The pharmaceutical composition Dotent in antifungal activity
of the present invention is preferably applied topically, for
examples as a shampoo, a bath gel, soap, a body lotion, a body
cream and a detergent. Preferably, the pharmaceutical composition
potent in antifungal activity of the present invention is used in
the treatment of diseases associated with Trichophyton
mentagrophytes or Pityrosporum ovale, including but are not limited
to tinea pedis, tinea capitis, tinea cruris, tinea glabrosa,
onychomycosis, pityriasis capitis, pityriasis vesicolor,
pityrosporum folliculitis, seborrheic dermatitis and dandruff. In
particular, the antifungal pharmaceutical composition of the
present invention is in the form of a shampoo for use in the
treatment of dandruff.
[0038] Without further elaboration, it is believed that the above
description has adequately enabled the present invention. The
following specific examples are, therefore, to be construed as
merely illustrative, and not limitations on the remainder of the
disclosure in any way whatsoever.
Determination of Active Ingredients
[0039] In the following examples, high performance liquid
chromatography (abbreviated as HPLC) was used to determine the
active ingredients of the products prepared therein. HPLC spectra
were recorded on a HPLC instrument (HPLC Shimadzu LC-10AT, Japan)
using a Cosmosil 5C-18 column (250 mm.times.4.6 mm, packed with
particles having 5 .mu.m diameter) by an elution method. An HPLC
sample was prepared by diluting an appropriate amount of a product
with a mobile phase solution (hydrogen cyanide:water=65:35, V/V) to
25 ml, and filtered with a 0.25 .mu.m membrane. The filtrate was
introduced into the HPLC column, and eluted with the mobile phase
solution. An UV detector (Shimadzu SPD-6AV, Japan) was used to
detect the absorption of the eluate at 230 nm.
EXAMPLE 1
[0040] 2100 g of fresh rhizomes of ginger were shredded and
filtered to obtain a filtrate and a residue. 500 ml of the filtrate
was extracted with 500 ml ethyl ether three times, the organic
phase layers were separated from the aqueous phase layers, and
combined. Ethyl ether was evaporated from the combined extraction
solution in vacuo to obtain a concentrated ethyl ether extraction
product (I-OE). The ginger residue was extract with 3000 ml acetone
three times, the extraction solutions were recovered by filtration,
and combined. Acetone was evaporated from the combined extraction
solution in vacuo to obtain a concentrated acetone extraction
product (I-O) (14.5 g). To a reverse phase chromatography column
300 mm.times.30 mm packed with 180 g Diaion HP-20 resin having a
diameter of 500 .mu.m-800 .mu.m, 7 g of a mixture of the
concentrate ethyl ether extraction product (I-OE) and the
concentrated acetone extraction product (I-O) was injected. 1500 ml
water, 2500 ml methanol, 2000 ml acetone and 2000 ml chloroform
were used to carry out elution. The water eluate, methanol eluate,
acetone eluate and chloroform eluate were collected separately, and
concentrated in vacuo to obtain 0.27 g concentrated water eluate
(I-OW), 1.45 g concentrated methanol eluate (I-OM), 2.68 g
concentrated acetone eluate (I-OA), and 0.83 g concentrated
chloroform eluate (I-OC). The amounts (mg) of 6-shogaol, 6-gingerol
and 6-dehydrogingerdione per gram of the I-O, I-OM and I-OA
determined by HPLC are listed in Table 1.
1TABLE 1 Content (mg/g) I-O I-OM I-OA 6-shogaol 1.10 .+-. 0.14 1.15
.+-. 0.0 -- 6-gingerol 59.98 .+-. 0.99 103.37 .+-. 8.57 2.51 .+-.
0.89 6-dehydrogingerdione 7.68 .+-. 0.42 8.94 .+-. 0.41 --
EXAMPLE 2
[0041] 500 g of shade dried rhizomes of ginger were pulverized and
the resulting powder was extracted with 30 L acetone trice (each
time with 10 L). The three extraction solutions were combined
together after filtration, and then concentrated in vacuo to obtain
24 g of concentrated acetone extraction product (II-O). To a
reverse phase chromatography column packed with 600 g Diaion HP-20
resin 20 g of the concentrated acetone extraction product (II-O)
was injected, which was then eluted with 4 L water, 6.5 L methanol,
15 L acetone and 5 L chloroform in sequence. The water eluate,
methanol eluate, acetone eluate and chloroform eluate were
collected separately, and concentrated in vacuo to obtain 2.5 g
concentrated water eluate (II-OW), 7.1 g concentrated methanol
eluate (II-OM), 6.9 g concentrated acetone eluate (II-OA), and 3.5
g concentrated chloroform eluate (II-OC). The amounts (mg) of
6-shogaol, 6-gingerol and 6-dehydrogingerdione per gram of the
II-O, II-OM and II-OA determined by HPLC are listed in Table 2.
2TABLE 2 Content (mg/g) II-O II-OM II-OA 6-shogaol 1.98 .+-. 0.00
4.96 .+-. 0.00 -- 6-gingerol 43.06 .+-. 0.84 70.87 .+-. 1.85 2.54
.+-. 0.00 6-dehydrogingerdione 9.33 .+-. 0.85 19.15 .+-. 4.57 2.35
.+-. 0.28
EXAMPLE 3
[0042] 10 Kg of shade dried rhizomes of ginger were pulverized and
the resulting powder was steam distilled for five hours. The
distillate was concentrated in vacuo to obtain 410 g of
concentrated distillate (III-O). To a reverse phase chromatography
column packed with 600 g Diaion HP-20 resin 20 g of the
concentrated distillate (III-O) was injected, which was then eluted
with 4.5 L water, 4.5 L methanol, 3 L acetone and 5 L chloroform in
sequence. The water eluate, methanol eluate, acetone eluate and
chloroform eluate were collected separately, and concentrated in
vacuo to obtain 0.03 g concentrated water eluate (III-OW), 14.5 g
concentrated methanol eluate (III-OM), 0.85 g concentrated acetone
eluate (III-OA), and 0.2 g concentrated chloroform eluate (III-OC).
The concentrated distillate (III-O) contains no 6-shogaol,
6-gingerol and 6-dehydrogingerdione determined by HPLC.
EXAMPLE 4
[0043] 10 g of powder of shade dried rhizomes of ginger was
extracted with 1000 ml acetone at 50.degree. C. for two hours. The
extraction solution was separated and concentrated in vacuo
(40.degree. C., 75 mmHg) to obtain a concentrated acetone
extraction product (IV-O). The color and viscosity of the product
(IV-O) together with its yield are listed in Table 3.
EXAMPLE 5
[0044] 10 g of powder of shade dried rhizomes of ginger was steam
distilled, and the oily distillate after being separated from the
aqueous distillate was freeze dried to obtain an oily extract
(V-O). The color and viscosity of the oily extract (V-O) together
with its yield are listed in Table 3
EXAMPLE 6
[0045] To 10 g of powder of shade dried rhizomes of ginger in a 250
ml extraction chamber CO.sub.2 was introduced at a flow rate of 45
L/min, wherein the chamber pressure was controlled at 2500 to 4000
psia with a high pressure pump (Model No. EK-1, LEWA Co., U.S.) and
the chamber temperature was maintained at 35-60.degree. C. with a
heat exchanger (Model No. H-2410, HOTEC Co., U.S.) and an exterior
circulation system. The extraction was stopped when the volume of
CO.sub.2 introduced reached 300 L, and a supercritical CO.sub.2
extraction product (VI-O) was obtained after evaporation of
CO.sub.2. The color and viscosity of the product (VI-O) together
with its yield are listed in Table 3. The contents of pungent
components determined by HPLC are listed in Table 4.
3 TABLE 3 IV-O V-O VI-O L* 87.6 80.4 96.3 A* -9.1 -0.1 -9.6 B* 31.1
9.6 22.0 Viscosity (cPs) 15.6 11.8 12.1 Yield (%) 3.8 2.2 3.9 *the
values of L, A, and B were determined by using a .SIGMA.90 color
measuring system, (Nippon Denshoku Inc, Co., Ltd., Japan), wherein
L represents lightness, A is the red/green difference and B is the
yellow/blue difference.
[0046]
4 TABLE 4 Content (mg/g) VI-O 6-shogaol 17.30 .+-. 0.00 6-gingerol
26.29 .+-. 0.00 6-dehydrogingerdione 19.20 .+-. 1.19
EXAMPLE 7
Evaluation of Inhibitory Activity on Trichophyton mentagrophytes or
Pityrosporum ovale
[0047] Anti-Trichophyton mentagrophytes assay
[0048] Minimum inhibitory concentration (MIC) of ginger extract was
determined and conducted according to the method previously
described by Edwards, J. R. et al. (Edwards, J. R. et al.,
Antimicrobial Agents Chemotherapy 33: 215-222, 1989). The test
substance was dissolved and serially diluted in solvent (100% DMSO)
to desired stock concentrations. For each concentration tested, a
0.01 ml aliquot was added to a 48-well plate containing 0.99 ml of
Potato Dextrose Broth (DIFCO, U.S.A.) with 10.sup.3-10.sup.4 CFU/ml
of Trichophyton mentagrophytes (ATCC 9533). The plates were
incubated at 28.degree. C. for 72 hours and then visually examined
and scored. Vehicle-control was used as blank control. Each
concentration was evaluated in duplicate. The results are shown in
Table 5.
Anti-Pityrosporum Ovale Assay
[0049] Minimum inhibitory concentration (MIC) of ginger extract was
determined and conducted by the method as mentioned above (Edwards,
J. R. et al., Antimicrobial Agents Chemotherapy 33: 215-222, 1989).
The test substance was dissolved and serially diluted in solvent
(100% DMSO) to desired stock concentrations. For each concentration
tested, a 0.01 ml aliquot was added to a 48-well plate containing
0.99 ml of Fluid Sabouraud Medium (DIFCO, U. S. A.) with
10.sup.3-10.sup.4 CFU/ml of Pityrosporum ovale (ATCC 38593). The
plates were incubated at 37.degree. C. for 48 hours and then
visually examined and scored. Vehicle-control was used as blank
control. Each concentration was evaluated in duplicate. The results
are shown in Table 5.
5TABLE 5 The inhibitory effects of ginger extracts on Pityrosporum
ovale (Po) and Trichophyton mentagrophytes (Tm) MIC (.mu.g/ml)
Ginger extract.sup.a) Po Tm I-O 100 30 I-OM 100 30 II-O 500 30
II-OC 100 30 II-OM 100 30 III-O 500 100 Blank control --.sup.b)
--.sup.b) .sup.a)Prepared in Examples 1, 2 and 3 .sup.b)no
inhibitory effect upon growth
[0050] Although the present invention has been described with
reference to specific details of certain embodiments thereof, it is
not intended that such details should be regarded as limitations
upon the scope of the invention except as and to the extent that
they are included in the accompanying claims. Many modifications
and variations are possible in light of the above disclosure.
* * * * *