Search

This study focused on the evaluation of Quillaja saponin extracts with the additional quality designation DAB-which means the abbreviation of the German Pharmacopoeia (Deutsches Arzneibuch). This label suggests that Quillaja saponin extracts marked in this way are of pharmacopoeial quality and thus stand out from other Quillaja saponin extracts. The DAB ninth edition listed Quillaia saponin as a reagent. According to DAB, its quality must be checked by thin-layer chromatography (TLC), and three closely spaced zones in a defined retention factor (Rf) interval specify the saponin reagent. All the Quillaja saponin extracts obtained from different manufacturers and labeled as DAB quality complied with the TLC test. However, the analysis with high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (HPLC-Q-ToF-MS) clearly showed additionally an intense peak pattern of Madhuca saponins in all measured samples. The TLC test for Mahua seed cake, which is the press residue from Madhuca longifolia, surprisingly showed the same three closely spaced zones in the defined Rf interval. The three zones could be identified as Mi-saponins from Madhuca after scraping and extracting them from the stationary phase of the TLC plate and subsequent measurement by HPLC-Q-ToF-MS. Therefore, the specification of the saponin reagent in DAB characterizes erroneously Madhuca saponins that are not listed as a saponin plant source for the saponin reagent.

Turmeric (Curcuma longa L.) extracts have a long history of use worldwide, but a major limitation of these extracts is their extremely low oral bioavailability, caused by low absorption, rapid metabolism and rapid excretion following ingestion. Thus, a new highly bioavailable turmeric extract formulation (comprising turmeric extract, acacia gum, sunflower oil and quillaia extract) has been developed and is intended for use as a food ingredient. Safety of this novel extract was evaluated using the standard Tier 1 battery of in vitro genotoxicity tests (bacterial reverse mutation test and an in vitro mammalian cell micronucleus test) followed by repeated-dose 28- and 90-day oral toxicity studies in rats. In the 90-day study, male and female Sprague-Dawley rats were dosed once daily, by oral gavage, either with the vehicle or the test item at 500, 1500 or 3000 mg/kg body weight/day. Clinical examinations were conducted regularly, and body weights and food consumption were recorded weekly throughout the study. At the end of the study, blood samples were analyzed for clinical pathology parameters, before a macroscopic necropsy was conducted and a full list of tissues were examined histopathologically. There was no evidence of genotoxicity in vitro. No test item-related adverse effects were observed in the 28- or 90-day studies; therefore, 3000 mg/kg body weight/day (the maximum feasible dose and highest dose tested in rats) was established as the no-observed-adverse-effect level.

The EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific opinion on Quillaia extract (E 999) when used as a food additive and the evaluation of the safety of its proposed extension of use as a food additive in flavourings. The Scientific Committee for Food (SCF) in 1978 established an acceptable daily intake (ADI) of 0–5 mg spray‐dried extract/kg body weight (bw) per day for E 999. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) established in its latest evaluation a group ADI of 0–1 mg/kg bw per day, expressed as quillaia saponins, for Quillaia extract for Type 1 and Type 2. The Panel considered it likely that intact Quillaia extract saponins are absorbed to a low extent, are hydrolysed in the gastrointestinal (GI) tract and that the aglycone is absorbed only to a limited extent. The Panel considered that the genotoxicity data available did not indicate a concern for genotoxicity. Taking into account the available toxicological database, various no observed adverse effect levels (NOAELs) relevant for the derivation of an ADI were identified. The Panel considered that the 2‐year study in rats was the most robust and that the NOAEL of 1,500 mg Quillaia extract/kg bw per day could be used to derive the ADI for E 999. Considering that the adverse effects reported were due to the presence of saponins in the extract, that saponins were present in Quillaia extract Type 1 (around 20%) and using an uncertainty factor of 100, the Panel derived a ADI of 3 mg saponins/kg bw per day for E 999. None of the exposure estimates for the different population groups of the refined brand‐loyal scenario exceeded the ADI of 3 mg saponins/kg bw per day. The proposed extension of use also would not result in an exceedance of this ADI for the refined scenario. The Panel proposed some recommendations for the European Commission to consider, in particular revising the EU specifications for E 999 in order to differentiate the extracts of Quillaia according to the saponins content and to include other parameters to better characterise the food additive.

The study aimed at determining the antioxidant effect of natural extracts on the oxidative quality and color variation of rainbow trout fillets during storage at 4 °C. The fillets were marinated and samples from the belly flap area and Norwegian quality cuts were used for lipid oxidation determination while the dorsal region was used for color measurements. The fillets were marinated with the different treatments: 470 mg l-1 of oregano extract, 6.84 ml l-1 of quillaia extract, 7.2 ml l-1 of rosemary extract and 2 ml l-1 of a synthetic antioxidant. Maximum TBARS (Thiobar-bituric acid reactive substances) values of belly flap and Norwegian quality cuts occurred at five and six days of storage, respectively. The susceptibility of treatments to lipid oxidation in decreasing order was: control>quillaia >oregano>rosemary>synthetic antioxidant. An increase in lightness (L*) and redness (a*) were observed for rosemary and quillaia extracts when compared to control samples. Marinates with natural antioxidants may be an al-ternative for extending shelf-life of trout fillets at least during the first six days of storage at 4 °C.

Ryokucha saponin (RSP), which is one of the ingredients of green tea, was administered orally to rats for 3 months at dose levels of 50, 150 and 500 mg/kg/day to assess any toxic effects. As positive control, 1200 mg/kg/day of quillaia saponin (QSP), which contains saponins equivalent in amount to those in 500 mg RSP/kg/day, was administered. The no-effect level of RSP was 50 mg/kg/day and the lowest-observed-effect level of RSP was 150 mg/kg/day for both sexes, so that the true no-effect level of RSP was estimated to be between these dose levels. In addition, it was confirmed that the toxicity of RSP at 500 mg/kg/day was less than that of QSP at 1200 mg/kg/day and RSP was demonstrated to be safer than QSP, which is a permitted food additive.

Saponins are chemically a heterogeneous group of sterol glycosides and triterpene glycosides which are common constituents of plants. One source of triterpenoid saponins obtained from the bark of Quillaia saponaria Molina (the soap bark tree) have been known to cause substantial enhancement of immune responses since the 1920s. Despite their use in animal vaccines, the development of saponin-based formulations for human vaccines has been impeded by their complexity and concerns about toxicity. This review briefly covers the use of saponins for animal vaccines but focuses mainly on the development of these adjuvants for use in man. Important aspects include preparation of purified or highly defined saponin fractions, improved understanding of the relationships between adjuvant activity, toxicity and structure of saponins and formulation of saponins into structures with reduced toxicity such as ISCOMs. Recent developments in the understanding of cellular interactions, cytokine induction and the in vivo localisation of saponin containing formulations will also be reviewed.

Saponins are a highly heterogenous group of glycosides which are common in plants and have been known to have adjuvant properties since the 1920s. The immunostimulating complex or ISCOM is a particulate adjuvant/antigen delivery system. ISCOMs are open cage-like complexes typically with a diameter of about 40 nm which are built up by cholesterol, lipid, immunogen and saponins from the bark of Quillaia saponaria Molina (soap bark tree). ISCOMs and saponins are used as adjuvants in some commercial veterinary vaccines and have been examined as adjuvants in a large number of human experimental vaccines. This review describes the current status and potential of saponin and ISCOMs as adjuvants for orally-administered vaccines with special reference to the induction of local and systemic immune responses and interactions with the intestinal epithelium. The structure and composition of saponins and ISCOMs will also be reviewed.

Aluminum salts are currently the only widely used adjuvant for human vaccines. Over the past 10–15 years, a large research effort has attempted to find novel adjuvants with ability to induce abroad range of immune responses, including cell-mediated immunity. The immunostimulating complex or ISCOM is one adjuvant with multiple adjuvant properties. ISCOMs are open cage-like complexes typically with a diameter of about 40 nm that are built up by cholesterol, lipid, immunogen, and saponins from the bark of the tree Quillaia saponaria Molina. ISCOMs have been demonstrated to promote antibody responses and induce T helper cell as well as cytotoxic T lymphocyte responses in a variety of experimental animal models, and have now progressed to phase I and II human trials. This review describes recent developments in the understanding of the structure, composition, and preparation of ISCOMs and will cover important aspects of the understanding of the adjuvant functions of ISCOMs and how they act on the immune system. J. Leukoc. Biol. 64: 713–723; 1998.

Detergent-disrupted influenza virus vaccines, formulated as Iscoms™, or oil-in-water (o/w) emulsions, were administered parenterally to mice and evaluated for immunogenicity and protective efficacy. Both formulations enhanced both primary and secondary serum antibody responses. The magnitude of these responses with o/w emulsions was further enhanced by the addition of the non-ionic block copolymer L121 in the emulsion. Four weeks after primary immunization, mice were challenged by exposure to an aerosol containing infectious virus. Resistance to challenge in terms of survival rate and weight change correlated well with serum antibody titre for all formulations. Two major differences were observed between the adjuvant formulations. Iscom™ vaccines, formulated with Quil-A or the less toxic Quillaia saponin preparation Iscoprep™ 703, induced specific cytotoxic T-lymphocyte responses, whereas the o/w-based vaccines did not. In addition, dose-site reactivity studies in sheep showed that Iscom™ vaccines were less reactive than o/w-based vaccines, the degree of reactivity of the latter increasing sharply with increasing L121 concentration. On the basis of these studies, Iscoms™ were chosen for development as a potential adjuvant for human influenza vaccines.

Adjuvant activity and immunostimulating complex (ISCOM) formation by a series of saponins and glycoalkaloids differing in the structures of their aglycones and sugar chains were examined. The only two saponins apart from Quillaia that were adjuvant-active were Gypsophila and Saponaria, which resemble Quillaia in that they contain saponins with branched sugar chains attached to positions 3 and 28 of the aglycone. Glycoalkaloids with a branched sugar chain lacked adjuvant activity. Saponaria saponins formed irregular ISCOM-like structures, and Gypsophila produced a sheet of joined pore-like structures. The alfalfa hederagenin saponin and Quinoa also formed pore-sheets, despite lacking adjuvanticity.

Groups of 48 male and 48 female mice were fed quillaia extract in the diet at levels of 0 (control), 0·1, 0·5 or 1·5% for 84 wk. The material had no adverse effect on the death rate or the incidence of histopathological findings, including tumours. However, there was a lower rate of body-weight gain at the 1·5% dietary level, and there were isolated statistically significant differences between the treated and control animals, mainly at the 1·5% dietary level, in the haematological examinations and in some absolute and relative organ weights of both sexes. It is concluded that, in mice, quillaia extract fed at levels up to 1·5% in the diet (approximately 2·2g/kg/day) did not exert a carcinogenic effect. The no-untoward-effect level from this study is considered to be 0·5% in the diet, giving an intake of approximately 0·7 g quillaia extract/kg/day.

1. Saponins are steroid or triterpene glycosides which occur in a number of important food plants, including such staples as soya beans (Glycine max) and chickpeas (Cicer arietinurn). They are known to be hypocholesterolaemic.2. Some saponins form an insoluble complex with cholesterol which prevents its absorption from the small intestine. Others cause an increase in the faecal excretion of bile acids, an indirect route for elimination of cholesterol.3. We have investigated the effects of different saponins on absorption of the bile salt sodium cholate from perfused loops of small intestine, in vivo, in the rat. Purified saponins from soapwort (Suponaria Officinalis), soya beans and quillaia (Quillata suponaria) reduced the rate of absorption of the bile salt; soya-bean and soapwort saponins substantially so but quillaia saponin to a much lesser extent.4. These results were explained by the formation of large mixed micelles by bile acid and saponin molecules in aqueous solution. These aggregates can have molecular weights in excess of 106daltons, consequently the bile acid molecules incorporated in them are not available for absorption.5. Control of plasma cholesterol and nutrient absorption through dietary saponins could provide substantial health and nutritional benefits in humans.

Groups of 48 male and 48 female rats were fed quillaia extract in the diet at levels of 0 (controls), 0.3, 1.0 and 3.0% for 2 yr. The material had no adverse effects on death rate, water consumption, serum chemistry or haematological parameters or on the incidence of histopathological findings, including tumours. In the males given the 3% dietary level, the death rate, total leucocyte count at wk 108, incidence of kidney lesions and weights of the kidneys, heart and thyroid were all below the control values. These differences were explicable, however, in terms of a lowered body weight consequent on a decreased food intake. It is concluded that, in rats, quillaia extract fed at levels up to 3.0% in the diet did not have any carcinogenic effect. The no-untoward-effect level established in this was 3.0% in the diet, approximately equivalent to an intake of 1.5 g/kg/day.

Saponins are very widely distributed in plants (Price et al, 1987), but the one that is used as an adjuvant is extracted from the bark of the South American tree Quillaia saponaria (Dalsgaard, 1978). It is a triterpene glycoside, consisting of the hydrophobic triterpene polycyclic ring, to which is attached two chains of sugars (Fig. 1), which are hydrophilic. This gives the molecule surface-active properties, and saponin is used as a foaming agent in soft drinks. Its potential as an adjuvant was discovered more than 50 years ago (Thibault and Richou, 1936), and it has since become widely used in veterinary vaccines, particularly those against foot-and-mouth disease virus (Dalsgaard, 1978). Recently new impetus has been given to research on saponin by the discovery that it can form small ordered structures, ISCOMS or immunostimulating complexes, with phospholipid, cholesterol, and proteins with a suitable hydrophobic tail such as those extracted from viral envelopes (Morein, 1988 and chapter by Morein in this volume). This chapter reviews what is known of the adjuvant properties of saponin and its mechanism of action.

Quillaia extract was fed to groups of 15 male and 15 female rats for 13 wk at dietary concentrations of 0 (control), 0·6, 2·0 or 4·0%. There was a transitory reduction in the rate of body-weight gain, associated with a reduced intake of food and water, but by the end of the study the weights of the treated rats did not differ significantly from those of the controls. The feeding of quillaia saponin did not affect the results of haematological examinations (including erythrocyte fragility tests in hypotonic saline), serum and urine analyses, renal concentrating ability or the urinary cell excretion. The relative liver weight was reduced in males given 2·0 or 4·0% quillaia and the relative stomach weight was increased in both sexes at the same levels. No histopathological effects attributable to treatment were found. The no-untoward-effect level in this study was 0·6% of the diet, equivalent to an intake of approximately 400 mg/kg/day.

Saponins occur in at least 400 plant species belonging to 60 different families. Some of these plants such as spinach, beetroot and asparagus are components of the human diet whilst others are animal foods. Because of their foaming properties saponins are used in the manufacture of foods, beverages, toilet preparations and pharmaceuticals. Several plant extracts used as flavouring agents in food contain active saponins. There is no legislation in the UK or USA controlling the use of foaming agents as a class in the manufacture of foods. The use of saponins as foaming agents is specifically prohibited in some countries, particularly in Central and Southern America. Flavouring legislation has yet to be formulated in the UK; in the USA several plant extracts containing saponins including quillaia (from Quillaja saponaria Molina), are permitted as flavourings, whilst in Federal Germany quillaja bark is specifically prohibited. There is some variation in the toxicity of saponins from different sources. Most toxicity tests have been with single doses administered orally or by intravenous injection. The lethal oral dose is 3–1000 times as great as the lethal intravenous dose. The majority of saponins are powerful haemolytics in vitro but large doses are needed to produce haemolysis on intravenous injection. Several authors have studied the effect of saponins on growth but no references have been found to experimental work on the chronic effects of those saponins likely to be consumed by man in the diet. However, a guide to the safety of some saponins is provided by experience based on common use as food additives or natural components of foods.

n/a

Light scattering, viscometry , fluorescence spectroscopy, electron microscopy and equilibrium dialysis have been used to study the aggregates formed by the interactions of saponins and bile acids in aqueous solution. Purified saponins from three plant sources were used: commercial 'saponin white' (from Saponaria officinalis), quillaia saponin (from Quillaia saponaria), and saponin from soya beans. Alone, the saponins formed small micelles, dimers in the case of saponin white and soya saponin , and larger aggregates of about 50 molecules for quillaia saponin . With bile acids, the saponins formed large mixed micelles. The size and structure of these depended on the chemical structure of the saponin . Saponin white and quillaia saponin gave filamentous structures with the hydrophobic triterpene groups of the saponin forming elongated stacks interleaved with bile acid anions. Soya saponin formed very different micelles with bile acids. These had a loose, open structure with considerable interpenetration of water. These results help to explain the increased faecal excretion of bile acids which has been observed in response to foods rich in saponins.

The solution of coal tar is prepared as follows: After mixing the coal tar with 700 cc. of alcohol, add the quillaia and macerate the mixture during seven days in a closed vessel, with occasional agitation. Then filter, and wash the contents of the filter with a quantity of alcohol sufficient to make the product measure 1,000 Gm. or ec. The procedure described is an unpleasant, time-consuming and labor-consuming process which yields variable results. The color of the product differs according to the origin of the tar as well as in the same tar. In spite of care, small particles of pitch frequently pass through the filter and are objectionable to the patient, especially when the compound is used on light-colored hair. For example, blondes often object to the brown color of scalp lotions containing coal tar solution. Because ethyl alcohol is used in the extraction, the cost of the