Patent Application
20090104293
The present invention is related to a new use of a potent product extracted from rhizomes of Zingiber officinale in treating a disease associated with Helicobacter pylori such as gastritis, gastric ulcer or duodenal ulcer in a patient.
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. Both gingerol and shogaol are mutagenic, whereas zinger and zingerone have been found to exhibit antimutagenic 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].
U.S. Pat. No. 6,855,347 discloses a composition for treating gastric ulcer and a process for preparing the same, wherein the composition comprising an extract obtained from the plant parts of Aegle marmetos and Withania somnifra and from the plant parts of at least one member selected from the group consisting of eight plants, wherein one of them is Zingiber officinale. The extract of the composition is preferably an aqueous extract.
Japanese patent publication No. 2004-115536 discloses anti-Helicobacter pylori potent product useful for prophylaxis and treatment of gastritis and gastric and duodenal ulcer, which comprises using one or more galenicals selected from Sophorae radix, Anisi stellati Frutus, Myristica fragrans, Isodon japonicus Hara, Swertia japonica, Florence fennel, Zingiber siccatum, Atractylodes rhizome, ginger, Saussureae radix and gayangae rhizoma.
It is concluded in the study of an article published in Anticancer Research 23(5A): 3699-3702, 2003, that ginger root extracts containing the gingerols inhibit the growth of H. pylori CagA+ strains.
An objective of the present invention is to provide a potent product in treating a disease associated with Helicobacter pylori which is prepared from rhizomes of Zingiber officinale. Preferably, the potent product prepared in the present invention is significantly more effective in comparison with a crude aqueous extract or a crude organic solvent extract from rhizomes of Zingiber officinale.
It is disclosed in the present invention that an aqueous eluate obtained by subjecting the crude organic solvent extract to an elution treatment with a reverse phase chromatography column has the weakest potency in treating said disease, and an eluate of an eluent having a relatively lower polarity has an improved potency. It is surprised to find that an eluate of an eluent having a polarity sufficiently low so that the eluate is free of both 6-gingerol and 6-shogaol has the strongest potency. This finding is advantageous because 6-gingerol and 6-shogaol are pungent components and are relatively less stable chemicals.
Preferred embodiments of the invention include (but not limited to) the following:
1. A method of treating a disease associated with Helicobacter pylori in a patient comprising administering to the patient a therapeutically effective amount of a potent product prepared from rhizomes of Zingiber officinale, said potent product being prepared by a process comprising the following steps:
a) preparing a crude extract from rhizomes of Zingiber officinale, said crude extract comprising 6-gingerol and 6-shogaol;
b) introducing the crude extract to a reverse phase chromatography column or a normal phase chromatography column, and eluting the column with a first eluent and a second eluent in sequence, i) said first eluent having a polarity lower than water and said second eluent having a polarity lower than that of the first eluent when the reverse phase chromatography column is used, or ii) said second eluent having a polarity lower than that of a mixture of ethyl acetate (EA) and methanol in a weight ratio of 1:1 and said first eluent having a polarity lower than that of the second eluent when the normal phase chromatography column is used, 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; and
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 or 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;
wherein step a) comprises steps i) to iv), or comprises step 1), 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 extract;
said step 1) is:
I) extracting dried rhizomes of Zingiber officinale with an organic solvent which is the same as said second organic solvent, recovering the resulting extraction solution of the organic solvent, and evaporating the organic solvent from the extraction solution to obtain the crude extract;
said step I′) is:
I′) steam distilling dried rhizomes of Zingiber officinale, and concentrating the resulting distillate by evaporation to obtain the crude extract; and
said step I″) is:
I″) extracting powder of dried rhizomes of Zingiber officinale with supercritical CO2, recovering the resulting extraction solution of the supercritical CO2, and evaporating CO2 from the extraction solution to obtain the crude extract.
2. The method according to Item 1, wherein said disease is gastric ulcer.
3. The method according to Item 1, wherein said crude extract is prepared from the method comprising step i) to iv).
4. The method according to Item 3, wherein said first organic solvent is ethyl ether, and said second organic solvent is acetone, methanol, ethanol, a mixture of methanol and water, a mixture of ethanol and water, or a combination of them
5. The method according to Item 4, wherein said second organic solvent is acetone.
6. The method according to Item 1, wherein said crude extract is prepared from the method comprising step 1).
7. The method according to Item 6, wherein said organic solvent is acetone, methanol, ethanol, a mixture of methanol and water, a mixture of ethanol and water, or a combination of them.
8. The method according to Item 7, wherein said organic solvent is acetone, ethanol or a mixture of ethanol and water.
9. The method according to Item 1, wherein the reverse phase chromatography column is used, and the first eluent has a polarity lower than that of a mixture of ethanol and water having 40% of ethanol by weight.
10. The method according to Item 9, wherein the first eluent is methanol, a mixture of methanol and water, ethanol, a mixture of ethanol and water, a mixture of acetone and water, or a combination of them.
11. The method according to Item 10, wherein the first eluent is a mixture of ethanol and water.
12. The method according to Item 11, wherein the reverse phase chromatography column is eluted with water prior to the first eluent.
13. The method according to Item 9, wherein said potent product is said first concentrated eluate, and said first concentrated eluate comprises 6-gingerol and 6-shogaol.
14. The method according to Item 1, wherein the reverse phase chromatography column is used, said potent product is said second concentrated eluate, and the first eluent has a polarity lower than that of a mixture of ethanol and water having 40% of ethanol by weight.
15. The method according to Item 14, wherein said second eluent is acetone, a mixture of acetone and water, a mixture of acetone and methanol, a mixture of acetone and ethanol, a mixture of acetone and C4-C6 alkane, C4-C6 alkane, a mixture of C4-C6 alkane and methanol, a mixture of C4-C6 alkane and ethanol, or a combination of them.
16. The method according to Item 15, wherein said second eluent is acetone.
17. The method according to Item 14, wherein said second concentrated eluate is substantially free of 6-gingerol or 6-shogaol.
18. The method according to Item 14, wherein said second concentrated eluate is substantially free of both 6-gingerol and 6-shogaol.
19. The method according to Item 1, wherein the normal phase chromatography column is used, said potent product is said first concentrated eluate, and the first eluent has a polarity lower than that of a mixture of n-hexane and ethyl acetate in a weight ratio of 6:4.
20. The method according to Item 19, wherein the first eluent has a polarity close to that of a mixture of n-hexane and ethyl acetate in a weight ratio of 9:1.
21. The method according to Item 20, wherein the first eluent is a mixture of n-hexane and ethyl acetate in a weight ratio of 9:1.
22. The method according to Item 19, wherein said first concentrated eluate is substantially free of 6-gingerol or 6-shogaol.
23. The method according to Item 19, wherein said first concentrated eluate is substantially free of both 6-gingerol and 6-shogaol.
24. The method according to Item 1, wherein the normal phase chromatography column is used, said potent product is said second concentrated eluate, the second eluent has a polarity close to that of a mixture of n-hexane and ethyl acetate in a weight ratio of 6:4, and said second concentrated eluate comprises 6-gingerol and 6-shogaol.
25. The method according to Item 24, wherein said second eluent is a mixture of n-hexane and ethyl acetate in a weight ratio of 6:4.
The potent product is preferably administered orally.
The applicant of this application in GB 2366565 discloses a method of preparing an extract from Zingiber officinale, which is potent in anti-inflammation, anti-platelet aggregation and anti-fungal activity, includes the following steps: preparing a crude liquid from rhizomes of ginger by extraction with an organic solvent or supercritical CO2 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 lower than that of the first eluent but higher 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. The details disclosed in GB 2366565 are incorporated herein by reference.
The invention is further described by means of example, but not in any limitative sense.
Percentages and other amounts referred to in this specification are by weight unless indicated otherwise. Percentages are selected from any ranges used to total 100%.
Sliced shade dried rhizomes of ginger were pulverized and screened with a sieve of mesh No. 10. One part by weight of the resulting powder was mixed with 8 parts by weigh of 95% ethanol, and the resulting mixture was boiled under refluxing for one hour and filtered to obtain a filtrate A and a residue. The residue was mixed with 95% ethanol in a weight ratio of 1:8, and the resulting mixture was boiled under refluxing for one hour and filtered to obtain a filtrate B. The filtrate A and filtrate B were combined, and it was concentrated in a water bath of 70° C. and in vacuo (Rotavapor R-220, BÜCHI, Switzerland) by evaporating the solvent therefrom. The resulting concentrate was introduced to a reverse phase chromatography column (7.1 cm×90 cm) packed with Diaion® HP-20 resin (Mitsubishi, Japan) in an amount of 20 times by dry weight of the concentrate of the combined filtrate, which was then eluted with 5 bed volumes of water, 4 bed volumes of 40% ethanol and 4 bed volumes of acetone in sequence. The eluate from the elution of 40% ethanol, and the eluate from the elution of acetone were collected separately. The eluate from 40% ethanol was concentrated in vacuo (Rotavapor R-220, BÜCHI, Switzerland) to dry, and then concentrated again in a water bath of 80° C. and in vacuo (40 mbar) for one hour to obtain Sample 1. The eluate from acetone was concentrated in vacuo (Rotavapor R-220, BÜCHI, Switzerland) to dry, and then concentrated again in a water bath of 80° C. and in vacuo (40 mbar) for one hour to obtain Sample 2.
The procedures of Example 1 were repeated except that the eluent of 40% ethanol was replaced by 70% ethanol. Sample 3 was obtained from the elution of 70% ethanol, and Sample 4 was obtained from the elution of acetone.
The procedures of Example 1 were repeated to obtain Sample 5, except that the eluent of 40% ethanol was replaced by 95% ethanol.
Sliced shade dried rhizomes of ginger were pulverized and screened with a sieve of mesh No. 10. One part by weight of the resulting powder was mixed with 8 parts by weigh of 95% ethanol, and the resulting mixture was boiled under refluxing for one hour and filtered to obtain a filtrate A and a residue. The residue was mixed with 95% ethanol in a weight ratio of 1:8, and the resulting mixture was boiled under refluxing for one hour and filtered to obtain a filtrate B. The filtrate A and filtrate B were combined, and it was concentrated in a water bath of 70° C. and in vacuo (Rotavapor R-220, BÜCHI, Switzerland) to ⅕ of its original weight by evaporating the solvent therefrom. To the resulting concentrate 4 times by weight of water was added to resume its original weight, and the resulting liquid was introduced to a reverse phase chromatography column (7.1 cm×90 cm) packed with Diaion® HP-20 resin (Mitsubishi, Japan) in an amount of 20 times by dry weight of the concentrate of the combined filtrate, which was then eluted with 1 bed volume of 20% ethanol and 4 bed volumes of 70% ethanol in sequence. The eluate from the elution of 70% ethanol was collected and concentrated in vacuo (Rotavapor R-220, BÜCHI, Switzerland) to dry, and then concentrated again in a water bath of 80° C. and in vacuo (40 mbar) for one hour to obtain Sample 6.
The procedures of Example 4 were repeated to obtain Sample 7, except that the eluent of 70% ethanol was replaced by 95% ethanol.
Sliced shade dried rhizomes of ginger were pulverized and screened with a sieve of mesh No. 10. One part by weight of the resulting powder was mixed with 8 parts by weigh of 95% ethanol, and the resulting mixture was stirred in a homogenizer at 2000 rpm (Type X 50/10, Ystral, Germany) for one hour and filtered to obtain a filtrate A and a residue. The residue was mixed with 95% ethanol in a weight ratio of 1:8, and the resulting mixture was stirred in a homogenizer at 2000 rpm for one hour and filtered to obtain a filtrate B. The filtrate A and filtrate B were combined, and it was concentrated in a water bath of 70° C. and in vacuo (Rotavapor R-220, BÜCHI, Switzerland) to ⅕ of its original weight by evaporating the solvent therefrom. To the resulting concentrate 4 times by weight of water was added to resume its original weight, and the resulting liquid was introduced to a reverse phase chromatography column (7.1 cm×90 cm) packed with Diaion® HP-20 resin (Mitsubishi, Japan) in an amount of 20 times by dry weight of the concentrate of the combined filtrate, which was then eluted with 1 bed volume of 20% ethanol and 4 bed volumes of 70% ethanol in sequence. The eluate from the elution of 70% ethanol was collected and concentrated in vacuo (Rotavapor R-220, BÜCHI, Switzerland) to dry, and then concentrated again in a water bath of 80° C. and in vacuo (40 mbar) for one hour to obtain Sample 8.
The procedures of Example 6 were repeated to obtain Sample 9, except that the eluent of 70% ethanol was replaced by 95% ethanol.
Sliced shade dried rhizomes of ginger were pulverized and screened with a sieve of mesh No. 10. One part by weight of the resulting powder was mixed with 8 parts by weigh of acetone, and the resulting mixture was boiled under refluxing for one hour and filtered to obtain a filtrate A and a residue. The residue was mixed with acetone in a weight ratio of 1:8, and the resulting mixture was boiled under refluxing for one hour and filtered to obtain a filtrate B. The filtrate A and filtrate B were combined, and it was concentrated in a water bath of 70° C. and in vacuo (Rotavapor R-220, BÜCHI, Switzerland) by evaporating the solvent therefrom. The resulting concentrate was introduced to a reverse phase chromatography column (7.1 cm×90 cm) packed with Diaion® HP-20 resin (Mitsubishi, Japan) in an amount of 20 times by dry weight of the concentrate of the combined filtrate, which was then eluted with 5 bed volumes of water, 4 bed volumes of 40% ethanol and 2 bed volumes of acetone in sequence. The eluate from the elution of acetone were collected and concentrated in vacuo (Rotavapor R-220, BÜCHI, Switzerland) to dry, and then concentrated again in a water bath of 80° C. and in vacuo (40 mbar) for one hour to obtain Sample 10.
The procedures of Example 8 were repeated to obtain Sample 11, except that the eluent of 40% ethanol was replaced by 70% ethanol. The eluate from the elution of 70% ethanol were collected and concentrated in vacuo (Rotavapor R-220, BÜCHI, Switzerland) to dry, and then concentrated again in a water bath of 80° C. and in vacuo (40 mbar) for one hour to obtain Sample 11.
The procedures of Example 8 were repeated to obtain Sample 12, except that the powder and the residue were boiled with ethyl acetate instead of acetone.
The procedures of Example 9 were repeated to obtain Sample 13, except that the powder and the residue were boiled with ethyl acetate instead of acetone.
Sliced shade dried rhizomes of ginger were pulverized and screened with a sieve of mesh No. 10. One part by weight of the resulting powder was mixed with 8 parts by weigh of 95% ethanol, and the resulting mixture was boiled under refluxing for one hour and filtered to obtain a filtrate A and a residue. The residue was mixed with 95% ethanol in a weight ratio of 1:8, and the resulting mixture was boiled under refluxing for one hour and filtered to obtain a filtrate B. The filtrate A and filtrate B were combined, and it was concentrated in a water bath of 70° C. and in vacuo (Rotavapor R-220, BÜCHI, Switzerland) by evaporating the solvent therefrom. The resulting concentrate was introduced to a normal phase chromatography column packed with 20 times by weight of silica gel 60 (Merck, Germany), which was then eluted with 4 bed volumes of a mixed eluent of n-hexane:ethyl acetate=9:1, 3 bed volumes of a mixed eluent of n-hexane:ethyl acetate=6:4 and 2 bed volumes of a mixed eluent of ethyl acetate:methanol=1:1 in sequence. The eluate from the elution of n-hexane:ethyl acetate=9:1 was collected and concentrated in vacuo (Rotavapor R-220, BÜCHI, Switzerland) to dry, and then concentrated again in a water bath of 80° C. and in vacuo (40 mbar) for one hour to obtain Sample 14. Samples 15 and 16 were obtained from the elution of n-hexane:ethyl acetate=6:4, and the elution of ethyl acetate methanol=1:1 respectively by the same procedures as used in obtaining Sample 14.
The procedures of Example 12 were repeated except that the 95% ethanol was replaced by acetone. Samples 17, 18 and 19 were obtained from the elution of n-hexane:ethyl acetate=9:1, n-hexane:ethyl acetate=6:4, and ethyl acetate:methanol=1:1 respectively.
Sliced shade dried rhizomes of ginger were pulverized and screened with a sieve of mesh No. 10. One part by weight of the resulting powder was mixed with 8 parts by weigh of 95% ethanol, and the resulting mixture was boiled under refluxing for one hour and filtered to obtain a filtrate A and a residue. The residue was mixed with 95% ethanol in a weight ratio of 1:8, and the resulting mixture was boiled under refluxing for one hour and filtered to obtain a filtrate B. The filtrate A and filtrate B were combined, and it was concentrated with a thin-film vacuum evaporator (CEP-L, Okawara Mfg. Co., Japan) to ⅕ of its original weight by evaporating the solvent therefrom. The resulting concentrate was introduced to a reverse phase chromatography column (34.5 cm×100 cm) packed with Diaion® HP-20 resin (Mitsubishi, Japan) in an amount of 20 times by dry weight of the concentrate, which was then eluted with 2 bed volumes of 95% ethanol and 2 bed volumes of acetone in sequence. The eluate from the elution of acetone was collected and concentrated in vacuo (Rotavapor R-220, BÜCHI, Switzerland) to dry, and then concentrated again in a water bath of 80° C. and in vacuo (40 mbar) for one hour to obtain Sample 20.
Accurately weigh sample into a sample bottle and dissolve with methanol to make a sample solution with concentration of 10 mg/ml. Filter the sample solution with 0.45 μm filter and analyze the filtrate with HPLC.
The results of the HPLC analysis of Sample 1 to Sample 20 prepared above are shown in
Samples 1 to 13 and 20 were prepared by using the reverse phase chromatography column. In comparison with the HPLC analysis fingerprints shown in
Minimum inhibitory concentration (MIC) of sample on Helicobacter pylon was determined by the agar dilution method [Malanosk G. J. et al. Effect of pH variation on the susceptibility of Helicobacter pylori to three marcolide antimicrobial agents and temafloxacin. Eur. J. Clin. Microbial Infect Dis. 12: pp. 131-133, 1993.]. The test substance was dissolved and serially diluted in DMSO to desired stock concentrations. For each concentration tested, a 0.01 ml aliquot is added to a 48-well plate containing 0.99 ml of Columbia agar base (Oxoid, England) supplemented 7% defibrinated rabbit blood. The inoculum of Helicobacter pylori (ATCC 43504) is prepared by suspending in brain heart infusion broth to a density of 5×105 CFU/ml. The final maximal concentration of DMSO is 1% and the initial test substance concentration is 300 μg/ml. The plates are incubated at 35° C. for 72 hours in the microaerophilic condition (Mixed gas N2 85%, CO2 10% and O2 5%) and then visually examined and scored positive (+) for inhibition of the colonies growth or negative (−) for no effect upon growth colonies. Vehicle-control and gentamicin are used as blank and positive controls, respectively. Each concentration is evaluated in duplicate. The results are shown in Table 2.
2. Helicobacter pylori-Induced Gastric Ulcer
Effects of samples on Helicobacter pylori-induced gastric ulcer were evaluated as described previously [Marchetti, M., Arico, B., Burroni, D., Figura, N., Rappuoli, R. and Ghiara, P. Development of a mouse model of Helicobacter pylori infection that mimics human disease. Science 267: 1655-1658, 1995.]. Groups of 5 male CD-1 (Crl.) derived mice weighing 24±2 g were fasted for 18 hours prior to intragastric inoculation of clinical isolated Helicobacter pylori in suspension at 3.2×109 colony formation units/0.4 ml/mouse. Test samples and vehicle (2% CMC, 10 ml/kg) were each administered orally to test animals, starting one hour after the Helicobacter pylori inoculation, dosing twice daily (10:00 am and 4:00 pm) for 7 consecutive days. Omeprazole (1 mg/kg) and clarithromycin (10 mg/kg), in combination, were used for the positive control agents and administered orally to test animal once daily for 7 consecutive days under the same schedule. Eight days after infection, all animals were fasted overnight, sacrificed and stomachs dissected along the greater curvature. Gastric ulceration scored at four levels according to the degree of hemorrhage and severity of ulcerative lesions:
0=normal appearance
1=mild red spots
2=moderate red spots and/or hemorrhage spots
3=marked hemorrhage spots
Percentage inhibition on Helicobacter pylori-induced gastric ulcer was calculated as follows:
(Scores of vehicle group−Scores of test group)/(Scores of vehicle group)×100%
The results are shown in Table 3.
pylori - induced gastric ulcer
Only Sample 1 is less effective than 6-gingerol in treating gastric ulcer induced by Helicobacter pylori among the samples prepared in the above examples. Sample 20 is the most effective, which has 83%, 75%, 67% and 50% inhibition on Helicobacter pylori-induced gastric ulcer at dosages of 100 mg/kg, 30 mg/kg, 10 mg/kg and 3 mg/kg, respectively.
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.
