Liquid chromatography to detect the content of mallow fruit

Liquid chromatography to detect the content of mallow fruit

(1) The surface view of this product: the epidermis stellate hair consists of 2 to 8 (more than 4 to 8) cells, with a single cell length of 50 to 1140 μm, a diameter of about 75 μm, and a slightly thicker wall; Oval, 5-7 cells, 25 to 38 μm in diameter. The upper epithelial non-glandular hairs are slender, curved or straight, about 1190 μm long, and the walls are thin or slightly thick. The upper stomata of the lower epidermis are inequalities. The parenchyma parenchyma cells contain calcium oxalate cluster crystals with a diameter of 6 to 25 μm and sharp edges.

The cross section of the peel of the product: the outer peel is a layer of rectangular epidermal cells, the wall is slightly thick, and the outer layer is cuticle. The mesocarp consists of 2 to 3 layers of round parenchyma cells and a layer of cells containing calcium oxalate prisms, with large mucous cells scattered in the parenchyma. The crystal-containing cells are round, thick and woody. Large mucous cells are scattered in the parenchyma. There are more than 10 bundles of fiber bundles between the mesocarp and the endocarp, which are arranged in a ring shape. The endocarp is a series of radially elongated stone cells, which are fence-like, with thick sidewalls and inner walls, and woody.

The powder of this product is light brownish yellow; the surface of epidermal cells of exocarp is elongated, and the pericarp cells contain many calcium oxalate crystals. Pigment cells are round and contain reddish-brown material. The grid-like cell fragments are colorless and are 1 column of long cylindrical cells with extremely thick walls and a cell cavity in the middle.

(2) Take 2g of this product powder, add 20ml of water, shake for 15 minutes, filter, add 1g of activated carbon to the filtrate, heat on the water bath for 15 minutes, filter, take 2ml of filtrate, add 4 drops of alkaline tartrate test solution, set Heat on the water bath for 5 minutes to form a brown-red precipitate; another 2 ml of the filtrate, add 3 drops of 10%-naphthol ethanol solution, shake well, add 0.5 ml of sulfuric acid along the tube wall, and a purple-red ring at the junction of the two liquids.

(3 ) Take 1g of this product powder , place it in a round bottom flask, add 70% ethanol and heat to reflux for 2 hours, filter it, and evaporate the filtrate, add 10 ml of methanol to dissolve the residue , take 2 ml of the supernatant and pass the C ₁₈ column. 5 ml of distilled water was eluted, and distilled water eluate was collected as a test solution. Another caffeic acid reference substance was added, and methanol was added to make a solution containing 1 mg per 1 ml as a reference solution. According to the thin layer chromatography ( Appendix VI B) test, draw 20 μl of the test solution and 4 μl of the reference solution , respectively, on the same polyamide film, and use methanol- water- glacial acetic acid (3 :2 :0.1) as the developing solvent. , unroll, remove, dry, and set under UV light (365nm ). In the chromatogram of the test sample, fluorescent spots of the same color are displayed at positions corresponding to the chromatogram of the reference substance .

[Check] Moisture must not exceed 10.0% (Appendix IX H first method).

The total ash should not exceed 11.0% (Appendix IX K ).

[Content determination] Preparation of the reference solution accurately weigh the appropriate amount of caffeic acid reference substance, add anhydrous methanol to make a solution containing 0.03mg per 1ml , that is.

Preparation of standard curve Precision extraction of 0.25ml , 0.5ml , 1.0ml , 1.5ml , 2.0ml , 2.5ml , 3.0ml and 4.0ml of caffeic acid reference solution , placed in a 25ml volumetric flask, add absolute ethanol to 5ml , add 0.3 % sodium dodecyl sulfate and 2.0ml of 0.6% -0.9% potassium ferricyanide, ferric chloride (1: 0.9) mixed solution 1.0ml, mixing, placing in the dark for 5min, add 0.1mol / L hydrochloric acid solution was added to The scale was placed in the dark for 20 min , and the corresponding reagent was used as a blank. The absorbance was measured by ultraviolet- visible spectrophotometry (Appendix VA ) at a wavelength of 700 nm, and the absorbance was plotted on the ordinate and the concentration was plotted on the abscissa.

Assay   Weigh accurately 2.5g of this product powder , place it in a round bottom flask, add 50ml of 70% ethanol , extract by heating and reflux for 2 hours, filter it, wash the container twice with 20ml of 20 % ethanol , and wash the solution into the same round bottom flask. The solvent was recovered under reduced pressure at 40 ° C to near dryness, dissolved in an appropriate amount of anhydrous methanol, and transferred to a 25 ml volumetric flask, and diluted to the mark with anhydrous methanol. Precisely measure 5.0ml into a 10ml volumetric flask, dilute to the mark with anhydrous methanol, shake well, and keep it away from light. Precisely measure 0.5ml and place it in a 25ml volumetric flask. According to the method under the standard curve preparation, the absorbance is determined according to the law from the "addition of absolute ethanol to 5ml", and the caffeic acid in the test solution is read from the standard curve. The amount, the calculation, that is.

This product is calculated as dry product, and the total phenolic acid is not less than 0.15% based on caffeic acid (C ₉ H ₈ O ₄ ) .