Identification of saponins

Summary

Quantitative determination of saponins can be carried out by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), colorimetric methods and high-performance liquid chromatography-ultraviolet absorption (HPLC-UV).

Operation method

colorimetric method

Principle

Saponin is also known as alkali saponin; saponin; saponin; saponin; or saponin. Saponins are a class of glycosides whose glycosides are triterpenoids or helical steroidal compounds, mainly distributed in higher plants on land, but also present in small quantities in marine organisms such as starfish and sea cucumbers. The main active ingredients of many Chinese herbs such as ginseng, Yuanzhi, Platycodonopsis, Glycyrrhiza glabra, Zhimai and Chaihu contain saponins. Some saponins also have antibacterial activity or valuable biological activities such as antipyretic, sedative and anticancer.

Materials and Instruments

Saponin
Distilled water Acetic anhydride Concentrated sulfuric acid Chloroform
Test tubes Beakers Water baths Dispensing funnels Conical flasks Filter paper Slides Alcohol lamps Evaporators Cuvettes Color plates

Move

1. Preparation of test solution:

(1) take 1g of saponin fragments in a large test tube (or a small beaker), add 15ml of distilled water, in a 30-90 ° water bath on the impregnation for 15 minutes and then filtered, the filtrate for identification.

(2) Take 0.5g of Dioscorea crumbles plus the same method to prepare Dioscorea sativa.

(3) Take 0.5g of Dioscorea crumbles in a large test tube or small conical flask with 95% ethanol 10ml in a water bath for 15 minutes, filtrate for identification.

2. Identification test:

(1) hemolytic test: take a small piece of filter paper, in a careful place to add a drop of saponin infusion, to be dry in the same place and then add a drop, such as repeated operations to the drop to add a number of drops, after drying the spray-free hemocyte test solution (take bovine blood, sheep's blood, or rabbit blood a copy of the blood, with a glass rod or swab stirring, to remove agglutination of blood proteins, plus pH 7.4 phosphate buffer a dilution of the same), a few minutes later, observe the back of the bottom of whether the red After a few minutes, observe whether there is a yellow (or transparent) spot without red color in the red back ground (the origin of the saponification solution in the center). (This reaction can also be carried out in a test tube, blood cell test solution in the herbal infusion in the saponin dissolution, blood cell solution from turbid to clear. (In addition can also be carried out on slides and observed under the microscope before and after the rupture and dissolution of blood cells).

(2) Foam test: Dioscorea sativa infusion, saponin infusion 2ml each, respectively, placed in a test tube. Shake vigorously for one minute and then leave it, in 10 minutes to observe whether both tubes have persistent foam production?

(3) Acetic anhydride and concentrated sulfuric acid test (Liebermann-Burchard reaction), 5 ml of saponaria extract, evaporate in a dish on a water bath, add 1 ml of acetic anhydride to make it dissolve, drop it in a dry cuvette and add 1 drop of concentrated sulfuric acid slowly from the edge to observe the color change. Also take 5 ml of Dioscorea sativa infusion, put it in an evaporating dish, evaporate it on a water bath, add 1 ml of acetic anhydride solution and pour it into a cuvette, (test tube) add a few drops of concentrated sulphuric acid along the wall of the tube, and observe whether there is a purplish-red ring between the interfaces to be produced?

(4) chloroform - concentrated sulfuric acid test (Salkowski reaction): take 2 ml of Dioscorea alba extract, evaporate on a water bath, 1 ml of chloroform dissolved, transferred to a dry small test tube, along the wall, carefully add 1 ml of concentrated sulfuric acid, the chloroform layer shows red or blue, sulfuric acid layer has a green fluorescence, showing that it contains steroidal saponins.

Caveat

When using colorimetric method to determine the total saponin content, the standard can be selected two or more than two kinds of results for comparison, take the average of the two as the final determination, so that the results are more meaningful. When determining the recovery, two or more standards can also be selected for comparison.

Common Problems

The operation of the test was carried out by colorimetric method, and the enzyme marker was selected to process the data, which was easy and fast to operate, with good linear relationship, and reduced a large amount of time when operating with the UV spectrophotometer method, and reduced the effect of time on the results of the color development, and the stability, precision, reproducibility and recovery reached the standard requirements.

Sourced from "Comparative study of different ginsenosides as standards for the determination of saponin content" Food Industry.

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