High-performance liquid chromatography analysis of plant saponins: An update 2005-2010.

Saponins are widely distributed in plant kingdom. In view of their wide range of biological activities and occurrence as complex mixtures, saponins have been purified and separated by high-performance liquid chromatography using reverse-phase columns at lower wavelength. Mostly, saponins are not detected by ultraviolet detector due to lack of chromophores. Electrospray ionization mass spectrometry, diode array detector , evaporative light scattering detection, and charged aerosols have been used for overcoming the detection problem of saponins.

INTRODUCTION

Saponins are plant glycosides which are recognized by their ability to produce a soapy lather when shaken with water. They are widely distributed in the nature and have been reported to be present in at least 500 genera of plants. All saponins are polar in nature, thus they are freely soluble in water but insoluble in nonpolar solvents. They are glycosides of triterpenoid or steroid aglycones with a varying number of sugar side chains. A wide variety of plants belonging to family Liliacae are major source of saponins. The saponins isolated from Asparagus species, referred as Shatavarins such as shatavarin I, II, III, and IV, derived from steroidal aglycones, have been identified and characterized by spectroscopic method.[1] Saponins on hydrolysis yield an aglycone known as “sapogenin.” The applications of saponins stretch over several areas such as additives in food and cosmetics, as wetting agents for the agriculture and photographic industry, and as adjuvants in the pharmaceutical industry.[2]

The structural complexity of saponins results in a number of physical, chemical, and biological properties. Saponins are usually amorphous substances having high molecular weight. Due to the presence of a lipid-soluble aglycone and water-soluble sugar chain in their structure (amphiphilic nature), saponins are surface active compounds with detergent, wetting, emulsifying, and foaming properties. In aqueous solutions, surfactants form micelles above a critical concentration called critical micelle concentration (CMC). Saponins, including soybean saponins, saponins from Saponaria officinalis and Quillaja saponaria, form micelles in aqueous solutions, the size and structure of which are dependent on the type of saponin.[3] The micelle-forming properties such as CMC and the aggregation number (number of monomers in a micelle) of quillaja saponins were affected by temperature, salt concentration, and pH of the aqueous phase.[4]

Saponins possess a variety of biological activities, viz. antioxidant, immunostimulant, antihepatotoxic, antibacterial, anticarcinogenic, antidiarrheal, antiulcerogenic, antioxytocic, hypocholesterolemic, anticoagulant, hepatoprotective, hypoglycemic, neuroprotective, anti-inflammatory activity, useful in diabetic retinopathy, inhibition of dental caries, and platelet aggregation.[56] Many saponins are known to be antimicrobial to inhibit mould and to protect plants from insects. They may be considered forming the defense system and have been included in a large group of protective molecules found in plants named phytoanticipins or phytoprotectants. Saponin-rich plant has been found to improve growth, feed efficiency, and health in ruminants.[7] This article briefly reviews the high-performance liquid chromatography (HPLC) methods, columns, detectors, mobile and stationary phases used for saponins and sapogenin.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF SAPONINS

The normal- and reverse-phase HPLCs are commonly used for separation, identification, and purification of saponins. But for the best separation of saponins, RP-HPLC is normally used. HPLC is increasingly used in the separation of various compounds including saponins. This technique is rapid, selective, and highly sensitive. Separation of saponins can be affected by HPLC using variety of stationary and mobile phases. The separation of major saponin components in Liquorice, Bupleuri radix on a column of octadecylsilylated silica (ODS) gel LS-40 with a mixture of methanol, water, acetic acid, and triethylamine as mobile phase have been achieved using HPLC.[8] Saponins of Platycodi radix have been separated and quantitated by HPLC with light-scattering detector (ELSD) using C18 column and gradient aqueous acetonitrile mobile phase.[9] Most studies are concerned with triterpene saponins and have generally adopted the chromatographic conditions preferred for HPLC- ultraviolet (UV), i.e., a C18 column and mobile phase gradient of aqueous acetonitrile.[10] The chromatographic behaviors of steroidal saponins of Anemarrhena asphodeloides, Asparagus officinalis, Convallaria majalis, Digitalis purpurea, and Ruscus aculeatus were studied by HPLC-MS on C18 reverse-phase column and aqueous acetonitrile or aqueous methanol as mobile phase in gradient manner with or without the addition of 1% acetic acid.[11] The hydrolyzed saponins of Ilex paraguariensis were separated and quantified by HPLC method with UV detection. The total concentrations of saponins in aqueous extract were found to be 352 μg/ml.[12] HPLC analyses on octadecylsilyl porous glass were investigated for acidic saponins in ginseng, bupleurum root, and senega. These acidic saponins as well as neutral saponins were separated rapidly with aqueous acetonitrile containing K2HPO4 mobile phase at room temperature.[13] Only conjugated soyasaponins which have UV absorption maximum at 295 nm, glycyrrhetinic acid glycosides, and cucurbitacins could be successfully detected with UV-Vis detector. Saponins have also been successfully separated on Spherisorb ODS, C18, and C8 column using MeCN-H2O. Similar validated method using ELSD was developed for saponin determination in Flos lonicerae, a herb used in traditional Chinese medicine.[14]

The benzoate derivatives of the major sapogenins (hecogenin, dehydrohecogenin, and tigogenin) occurring in the Agave species have been analyzed by reversed phase HPLC on LiChrosorb RP-8 column using MeOH-H2O (80 : 20) mobile phase. Different stationary phases and mobile phases used in the separation of saponins are listed in Table 1.

Table 1

Separation of saponins by HPLC using different stationary and mobile phases

CONCLUSION

Most of the naturally occurring saponins are separated by C18 and ODS column using MeOH-H2O and CH3CN-H2O with or without addition of acids as mobile phase. HPLC-ELSD (evaporative light scattering detection), HPLC-MS (mass spectrometry), and HPLC-RI (refractive index) methods have been developed for overcoming the detection problem of saponins by UV detector.