US2004247701A1PendingUtilityA1
Fagopyrum cymosum (Trev.) meisn composition, method to prepare and analyze the same and uses thereof
Priority: Sep 29, 1997Filed: Aug 9, 2004Published: Dec 9, 2004
Est. expirySep 29, 2017(expired)· nominal 20-yr term from priority
A61P 35/00A61K 31/70G01N 33/50G01N 2333/415
38
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Claims
Abstract
This invention provides different compositions extracted from Fagopyrum cymosum (Trev.) Meisn. Said compositions comprise active components for therapeutic applications. This invention also provides a method of preparation of the compositions and a method of identification and determination of individual components of said compositions. Finally, this invention provides various uses of the compositions.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A method for identifying by chromatography a composition of Fagopyrum cymosum (Trev.) Meisn capable of treating cancer comprising the steps of:
a) preparing the assay comprising the steps of:
I. ultrasonically dissolving 0.5 g of Fagopyrum cymosum (Trev.) Meisn composition with 30 ml. of distilled water for 30 minutes to form a solution;
II. removing the solution to the separator funnel; extracting the solution from the water with chloroform (30 ml. each time for 3 times) in order to degrease;
III. continuously extracting the water layer from said solution with acetic ether (30 ml. each time for 5 times) to form a fraction;
IV. collecting and blending the acetic ether fraction to form a blend;
V. drying the blend with anhydrous calcium chloride;
VI. filtering the blend to form a filtrate;
VII. drying the filtrate;
VIII. mixing said filtrate with water in constant volume of 10 ml. to form a suspension;
IX. injecting 1 ml. of the suspension into C 18 ods cartridge and eluting it with 5 ml. of water, 5 ml. of 50% methanol and 5 ml. of methanol respectively;
X. making the eluent of 50% methanol the constant volume of 5 ml.; and
XI. filtering the eluent with 0.45 μ filtration membrane;
b). using (−) epicatechin as the standard; and c). performing HPLC assay under following conditions:
I. Gradient mobile phase:
Time(minutes) Water(PH = 3.00) Acetonitrile 0 90 10 40 74 26 50 20 80
II. column temperature: room temperature; flow rate: 0.8 ml/min;
III. injection volume: 10 μl;
IV. wavelength: 282 nm; and
V. calculating according to the following formula: (−) epicatechin=As×Cst/Ast×Ws×100% wherein As=peak area of sample, Cst=concentration of standard, Ast=peak area of standard, Ws=weight of sample.
2 . A method for identifying a composition of Fagopyrum cymosum (Trev.) Meisn capable of treating cancer comprising the steps of:
a) preparing the assay comprising the steps of:
I. dissolving a suitable amount of Fagopyrum cymosum (Trev.) Meisn composition in distilled water to form a solution;
II. extracting said solution from the water with chloroform to degrease;
III. continuously extracting the water layer from said degreased solution with acetic ether;
IV. collecting said acetic ether fractions to form a blend;
V. drying said blend with anhydrous calcium chloride;
VI. filtering said dried blend to form a filtrate;
VII. drying said filtrate;
VIII. mixing said filtrate with water in constant volume of 10 ml. to form a suspension;
IX. injecting 1 ml. of said suspension into a C 18 ods cartridge and eluting it with 5 ml. of water, 5 ml. of 50% methanol and 5 ml. of methanol respectively;
X. making the eluent of 50% methanol the constant volume of 5 ml.; and
XI. filtering the eluent with a 0.45 μ filtration membrane;
b). using (−) epicatechin as the standard; c). performing HPLC assay under the following conditions:
I. Gradient mobile phase:
Time(minutes) Water(PH = 3.00) Acetonitrile 0 90 10 40 74 26 50 20 80
II. column temperature: room temperature; flow rate: 0.8 ml/min;
III. injection volume: 10 μl;
IV. wavelength: 282 nm; and
d). calculating according to the following formula: (−) epicatechin=As×Cst/Ast×Ws×100% wherein As=peak area of sample, Cst=concentration of standard, Ast=peak area of standard, Ws=weight of sample.
3 . A composition comprising the product that when subjected to method of claim 1 produces 6 peaks as shown in FIG. 1.
4 . A composition comprising the product that when subjected to the method of claim 1 produces a fingerprint as tabulated herein:
Relative
Area
Peak
Retention
Retention
Apearance
Area
Ratio
#
Time
Time
Probability
Area
Ratio
Range
6
14.57
0.76
80-100
705156
0.69
0.07-3.45
8
16.81
0.88
80-100
1833159
1.80
0.18-9.0
9
19.07
1.0
100
1018514
1.00
10
21.45
1.12
80-100
208454
0.20
0.02-3.0
14
29.70
1.56
80-100
51306
0.05
0.01-2.0
15
30.91
1.62
80-100
145424
0.14
0.03-3.0
5 . A composition as in claim 3 , wherein:
the occurrence frequency of the 6 peaks are 80-100%; the retention time ratios of the 6 peaks in comparison with (−) epicatechin are 0.76, 0.88, 1.0, 1.12, 1.56, and 1.62 respectively; the area ratios of the 6 peaks in comparison with (−epicatechin are 0.69, 1.80, 1.00, 0.20, 0.05 and 0.14 respectively; and the ranges of the area ratios of the 6 peaks in comparison with (−) epicatechin are 0.07-3.45, 0.18-9.0, 1.0, 0.02-3.0, 0.01-2.0 and 0.03-3.0 respectively.
6 . A composition comprising:
a). 30-70% tannin content; and b). 0.2-1.0% (−) epicatechin.
7 . The composition of claim 6 extracted from Fagopyrum cymosum (Trev.) Meisn rhizome.
8 . The composition of claim 7 wherein the Fagopyrum cymosum (Trev.) Meisn rhizome is obtained from cultivated plants.
9 . A method for obtaining a Fagopyrum cymosum (Trev.) Meisn composition comprising steps of:
a). obtaining, washing and cutting rhizome of fresh Fagopyrum cymosum (Trev.) Meisn; b). drying the rhizome; c). crushing the rhizome into particles; d). putting the particles through a process of hot reflux in a solution selected from water, methanol, ethanol, acetone, water-methanol mixture, water-ethanol mixture, water-acetone mixture and ethyl acetate several times to produce extracts; e). mixing the extracts; f). separating the extract from precipitate and solid materials; g). concentrating the separated extract; h). diluting the extract with water; i). passing the suspension through pretreated macroporous resin column; j). eluting the chromatographic column; k). concentrating the eluant at reduced pressure; and l). spray-drying the extract into amorphous red-brown powder, thereby producing an extract containing the active constituents of Fagopyrum cymosum (Trev.) Meisn.
10 . A method for obtaining a Fagopyrum cymosum (Trev.) Meisn composition as in claim 9 further comprising the steps of:
I. in step (b) drying the rhizome to less than 10% moisture;
II. In step (c) crushing the dried rhizome into particles smaller than 5 mm. in diameter while less than 10% of the particles are smaller than 0.1 mm. in diameter;
III. in step (d) putting the particle through a process of hot reflux in the solution of 10-90% ethanol with a ratio of 1:10 (w/v) twice (1-3 hours and 1-2 hours respectively) to produce extracts at the temperature of 50-70° C.;
IV. in step (g) concentrating the separated extract at reduced pressure under the temperature of 70° C. at a density of 1.3 and a temperature of 50° C.;
V. in step (h) diluting the extract with 5-10 times the amount of water;
VI. in step (i) passing the suspension through pretreated macroporous resin column (model D101) and other suitable polystyrene resin, saturated with water pre-column;
VII. in step (j) eluting the chromatographic column with distilled water and 80-90% ethanol repeatedly until the effluent does not react positively with ferric chloride reagent; and
VIII. in step (k) concentrating the eluant at reduced is pressure under 50-70° C. to the density of 1.10-1.13.
11 . The method of claim 10 , wherein the particle of Fagopyrum cymosum (Trev.) Meisn rhizome is put through a process of hot reflux in the solvent of 10-90% ethanol twice (for 1-3 hours and 1-2 hours respectively with the volume of solvent being 3-8 times and 2-7 times the medicinal material).
12 . The method of claim 10 , wherein the macroporous resins are packed in columns.
13 . The method of claim 10 , wherein the chromatographic material is selected from porous polymer, silicon gel, aluminum oxide, polyamide, activated charcoal, cellulose or sephadex in addtion to macroporous resin (model D101).
14 . The method of claim 10 , wherein the chromatographic column eluate is concentrated at reduced pressure under 60° C. to a relative density of 1.10-1.13.
15 . The composition comprising the product produced by the method of claims 9 , 10 , 11 , 12 , 13 or 14 .
16 . A method for determination of total tannin content in a Fagopyrum cymosum (Trev.) Meisn composition comprising the steps of:
a). preparing the assay comprising the steps of:
I. dissolving a suitable amount of Fagopyrum cymosum (Trev.) Meisn composition with 20% ethanol to form a solution;
II. separating the solution from the residue by filtration;
III. discarding the first filtrate;
IV. drawing 75 ml. of subsequent filtrate;
V. evaporating the filtrate to the absence of ethanol on a water bath;
VI. cooling the filtrate;
VII. transferring the filtrate into a 250 ml. volumetric flask; and
VIII. diluting the filtrate;
b). determining the total water soluble portion by evaporating 25 ml. of the assay to dryness and drying the precipitate at 105° C. for 3 hours (T 1 ); c). determining the water soluble portion not bound with crude powder comprising the steps of:
I. adding 6 g. of crude powder of Fagopyrum cymosum (Trev.) Meisn composition to 100 ml. of the assay and shaking for 15 minutes;
II. separating the solution from the residue by filtration; and evaporating 25 ml. of the filtrate to dryness; and
III. drying the precipitate at 105° C. for 3 hours (T 2 ); and
d). determining the water soluble portion of crude powder comprising the steps of:
I. dissolving 6 g. of crude powder of Fagopyrum cymosum (Trev.) Meisn composition with 100 ml. of water;
II. shaking the solution for 15 minutes; separating the solution from residue by filtration;
III. evaporating 25 ml. of the filtrate to dryness; and
IV. drying the precipitate at 105° C. for 3 hours (T 0 ); and The total tannin content in percentage is calculated according to the following formula: Total tannin content %=(T 1 −T 2 +T 0 )×10/W×100% wherein W=quantity of sample (dried substance).
17 . A method for determination of the amount of (−) epicatechin in a Fagopyrum cymosum (Trev.) Meisn composition comprising the steps of:
a). performing adaptability test of the system under following conditions:
I. using octadecyl silicomethane-linked silica gel as the packing;
II. using water (pH 3.0)/acetonitrile for mobile phase gradient elution; the proportions being 0 minutes: 90/10→40 minutes: 74/26→50 minutes: 20/80→110 minutes: stop; and making detection at the wavelength 282 nm; and
III. the theoretical number of plate calculated with regard to (−) epicatechin should be no less than 20,000;
b). preparing the standard comprising steps of:
I. dissolving 10 mg. (−) epicatechin with 20 ml. of mobile phase solution by ultrasonic treatment in a 25 ml. volumetric flask (power not lower than 150 W, frequency not lower than 25 Hz) for 10 minutes;
II. removing the solution after complete dissolution; cooling the solution to room temperature;
III. diluting the solution with mobile phase solution to the mark; and
IV. shaking the solution to homogeneity, 40 μg/ml (−) epicatechin is obtained;
c). preparing the assay comprising the steps of:
I. dissolving 0.5 g. of the crude powder of Fagopyrum cymosum (Trev.) Meisn composition with 30 ml. of distilled water by ultrasonic treatment for 30 minutes;
II. transferring the solution to a separating funnel; extracting and defatting the solution by chloroform for 3 times;
III. extracting the water layer with 30 ml. of ethyl acetate for 5 times;
IV. pooling the ethyl acetate portions;
V. dehydrating the pooled ethyl acetate portions with anhydrous calcium chloride;
VI. separating the residue by filtration;
VII. volatilizing the filtrate to dryness;
VIII. making the precipitate into suspension with water with a constant volume of 10.0 ml.;
IX. passing the assay from step (I) through C 18 ODS cartridge and performing solid phase extraction;
X. eluting the assay with 5 ml. of water, 50% methanol and methanol respectively, the 50% methanol portion having a constant volume of 5 ml.; and
XI. passing the 50% methanol portion through a filter membrane as the assay; and
d). performing the assay comprising the steps of:
I. pipetting the standard and the assay, 10 μl. each;
II. transferring the pipetted standard and assay into a liquid chromatograph; and
III. performing the calculation and obtaining the results.
18 . The method of claim 16 , wherein (−) epicatechin is used as the standard to determine the amount of (−) epicatechin in a Fagopyrum cymosum (Trev.) Meisn composition.
19 . A pharmaceutical composition comprising an effective amount of the composition of claims 3 , 5 , 6 , 7 or 8 and a pharmaceutically acceptable carrier.
20 . The formulation of claim 19 , wherein the formulation is a pill, capsule, granule, tablet, suspension, injection, syrup, or tincture.
21 . A method for treating lung cancer in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
22 . A method for alleviating clinical symptoms in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
23 . A method for alleviating the symptom of cough in a subject suffering from common cold, bronchitis, pneumonia, pulmonary tuberculosis, pulmonary abscess, lung cancer, and upper respiratory track infection comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
24 . A method for alleviating the symptom of expectoration in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
25 . A method for alleviating the symptom of chest pain in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
26 . A method for alleviating the symptom of bloody sputum in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
27 . A method for alleviating the symptom of fever in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
28 . A method for improving blood picture in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
29 . A method for increasing hemoglobin count in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
30 . A method for increasing leucocyte count in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
31 . A method for increasing platelet count in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
32 . A method for improving clinical signs in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
33 . A method for increasing body weight in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
34 . A method for increasing daily food consumption in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
35 . A method for decreasing nausea occurrence in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
36 . A method for decreasing vomiting occurrence in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
37 . A method for decreasing diarrhea occurrence in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
38 . A method for decreasing debility occurrence in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
39 . A method for decreasing blood sedimentation rate in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
40 . A method for decreasing blood urea nitrogen in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
41 . A method for improving Karnofsky performance scores in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 .
42 . A method for treating lung cancer in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 combined with chemotherapy.
43 . A method for treating lung cancer in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 combined with radiotherapy.
44 . A method for increasing Complement C3 level in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 combined with radiotherapy.
45 . A method for increasing E Rosette level in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 combined with radiotherapy.
46 . A method for increasing lymphocyte transforming factor level in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 combined with radiotherapy.
47 . A method for increasing IgA level in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 combined with radiotherapy.
48 . A method for increasing IgG level in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 combined with radiotherapy.
49 . A method for increasing IgM level in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 combined with radiotherapy.
50 . A method for increasing macrophage phagocytic rate in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 combined with radiotherapy.
51 . A method for increasing macrophage phagocytic index in a subject suffering from lung cancer comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 combined with radiotherapy.
52 . A method for treating gastric cancer, cervical cancer, sarcoma and other neoplasms, relieving inflammation, and alleviating toxic and adverse effects associated with chemotherapy and radiotherapy in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19.Join the waitlist — get patent alerts
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