Abstract
The colorimetric assay used for saponin quantification in plant extracts is subject to interference by common solvents used to extract the saponins from plant materials. Therefore, the degree of interference of ten common solvents was investigated. It was found that the presence of acetone, methanol and n-butanol in the reaction solution caused an intense darkening of the reaction solution in the absence of saponins, which likely could lead to erroneous saponin content values. Using aescin to construct standard curves with different solvents—such as water, ethanol, and methanol— also showed significant differences in the standard curves obtained, which led to different values when they were applied to quantify the saponin content of an ethanol extract from dried and powdered Gac (Momordica cochinchinensis Spreng) seed kernels. To improve the method, a solvent evaporation step was added prior to the colorisation reaction to prevent undesired solvent interference during the reaction step. Using this modified protocol for the aescin standard curve and the Gac seed kernel extract eliminated any solvent interference. Thus, this improved protocol is recommended for the quantification of the saponin content of plant extracts irrespective of which extraction solvent is used.
Highlights
Saponins are a diverse group of compounds widely distributed in the plant kingdom, which are characterised by their structure containing a triterpenoid (C30) or steroid (C27) aglycone, and one or more carbohydrate chains [1]
The method is known as the vanillin-sulfuric acid assay because the basic principle of the method is the reaction of sulphuric acid-oxidised triterpene saponins with vanillin, which gives a distinctive red-purple colour measured at wavelengths ranging from 473 to 560 nm [4] using a spectrophotometer [10]
The vanillin-sulphuric acid assay for determining the total saponin content (TSC) of plant materials [12] is usually done [11,20] by incubating 0.25 mL of plant sample extracts, standards or reagent blank (Table 1) with
Summary
Saponins are a diverse group of compounds widely distributed in the plant kingdom, which are characterised by their structure containing a triterpenoid (C30) or steroid (C27) aglycone, and one or more carbohydrate chains [1]. Their physicochemical (e.g., surfactant) properties [1], coupled with mounting evidence on their biological activities (e.g., anticancer and anti-cholesterol activity) [2], have led to the emergence of saponins as significant compounds with expanding applications in the food, cosmetics and pharmaceutical sectors [3]. The TSC of a plant sample is determined from a calibration curve with a standard saponin (e.g., aescin, oleanolic acid, diosgenin, quillaja saponin) [4,11] and it is expressed in terms of the standard’s equivalence (e.g., mg standard equivalents per g sample; or g standard equivalents per 100 g sample)
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