Fagopyrum cymosum (Trev.) meisn composition, method to prepare and analyze the same and uses thereof

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 C18ods 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 0.45μ filtration membrane;
• b). using (−) epicatechin as the standard;
• c). performing HPLC assay under the following conditions:
• 63 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.

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.

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 (T1);
• 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 (T2);
• 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 (T0);
• and The total tannin content in percentage is calculated according to the following formula:
• Total tannin content %= (T1−T2+T0)×10/W×100% wherein W=quantity of sample (dried substance).

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 C18 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.