Cezary Pączkowski

Influence of environmental abiotic factors on the content of saponins in plants

Saponins occur constitutively in many plant species as part of their defense system. However, saponin content in plants seems to be dynamic, responding to many external factors including various biotic stimuli connected to herbivory attack and pathogenic infection, as well as involved in plant mutualistic symbioses with rhizobial bacteria and mycorrhizal fungi. Thus, not only saponins influence the living organisms interacting with plants, but in turn, all these interactions can impact the plant saponin content. According to their constitutive occurrence in plants, saponins are regarded mainly as phytoanticipins. Nevertheless, some presented data clearly point out to induced biosynthesis of saponins, especially in plant response to insect herbivory or inoculation with root symbionts, while the best studied examples of interactions between plants and their microbial pathogens show rather qualitative change of saponin composition based on chemical modifications of preformed, pre-infectional precursors. Simultaneously, despite evident inducibility of saponin production in plant cell cultures, the possible role of these compounds as phytoalexins synthesized in intact plants after pathogen infection is still not well documented. Some practical patterns and ecological consequences of biotic factors influencing saponin content in plants are briefly highlighted, with the special attention paid to microbial inoculants applied for optimisation of saponin synthesis in cultivated medicinal plants.

Fruit cuticular waxes as a source of biologically active triterpenoids

The health benefits associated with a diet rich in fruit and vegetables include reduction of the risk of chronic diseases such as cardiovascular disease, diabetes and cancer, that are becoming prevalent in the aging human population. Triterpenoids, polycyclic compounds derived from the linear hydrocarbon squalene, are widely distributed in edible and medicinal plants and are an integral part of the human diet. As an important group of phytochemicals that exert numerous biological effects and display various pharmacological activities, triterpenoids are being evaluated for use in new functional foods, drugs, cosmetics and healthcare products. Screening plant material in the search for triterpenoid-rich plant tissues has identified fruit peel and especially fruit cuticular waxes as promising and highly available sources. The chemical composition, abundance and biological activities of triterpenoids occurring in cuticular waxes of some economically important fruits, like apple, grape berry, olive, tomato and others, are described in this review. The need for environmentally valuable and potentially profitable technologies for the recovery, recycling and upgrading of residues from fruit processing is also discussed.

The Formation of Sugar Chains in Triterpenoid Saponins and Glycoalkaloids

Triterpenoid saponins and structurally related steroidal glycoalkaloids are a large and diverse family of plant glycosides. The importance of these compounds for chemical protection of plants against microbial pathogens and/or herbivores is now well-documented. Moreover, these compounds have a variety of commercial applications, e.g. as drugs or raw materials for pharmaceutical industry. Until recently there were only sparse data on the biosynthesis of saponins and glycoalkaloids, especially at the enzyme level. Substantial progress has recently been made, however, in our understanding of biosynthetic routes leading to the formation of the diverse array of aglycone skeletons found in these compounds as well as mechanisms of synthesis of their sugar moieties. This review highlights some of the advances made over past two decades in our understanding of the formation and modification of sugar moieties in triterpenoid saponins and glycoalkaloids.

Comparison of the Triterpenoid Content of Berries and Leaves of Lingonberry Vaccinium vitis-idaea from Finland and Poland

Triterpenoid compounds extracted from fruits and leaves of lingonberry ( Vaccinium vitis-idaea L.) collected in Finland and Poland were identified and quantitated by GC-MS/FID. The main lingonberry triterpenoid profile consisted of α-amyrin, β-amyrin, betulin, campesterol, cycloartanol, erythrodiol, fern-7-en-3β-ol, friedelin, lupeol, sitosterol, stigmasterol, stigmasta-3,5-dien-7-one, swert-9(11)-en-3β-ol, taraxasterol, urs-12-en-29-al, uvaol, oleanolic acid, and ursolic acid. To our knowledge, this is the first thorough description of triterpenoid compounds in this species. Ursolic acid was identified as a principal triterpene in lingonberry fruit. The influence of geographical origin on the level of individual triterpenoid compounds was examined, and considerable variations in triterpenoid profile between berries and leaves obtained from the two locations were observed. The most striking difference concerned the occurrence of fernenol and taraxasterol, which were found to be the major triterpenol in lingonberry leaves of Finnish and Polish origin, respectively.

Triterpenoid Content of Berries and Leaves of Bilberry Vaccinium myrtillus from Finland and Poland

Triterpenoid compounds found in free and ester forms in extracts of entire fruits and leaves and in fruit and leaf cuticular waxes of bilberry (Vaccinium myrtillus L.) collected in Finland and Poland were identified and quantitated by gas chromatography-mass spectrometry coupled to a flame ionization detector (GC-MS/FID). The main bilberry triterpenoid profile consisted of α- and β-amyrin, α- and β-amyrenone, campesterol, cholesterol, citrostadienol (in berries), cycloartanol, erythrodiol, lupeol, 24-methylenecycloartanol, sitosterol, sitostanol, stigmasterol, stigmasta-3,5-dien-7-one, uvaol, oleanolic and ursolic aldehydes, and oleanolic, ursolic, 2α-hydroxyoleanolic, and 2α-hydroxyursolic acids. Friedelin and D:A-friedooleanan-3β-ol were found only in Finnish plants, whereas D:C-friedours-7-en-3β-ol and taraxasterol were found only in Polish plants. To our knowledge, this is the first thorough description of triterpenoid compounds in this species. The presented results revealed that the triterpenoid profile of bilberry varied considerably between different organs of the plant, regardless of the plant origin, as well as between plant samples obtained from the two geographical locations.

Triterpenoid saponins affect the function of P-glycoprotein and reduce the survival of the free-living stages of Heligmosomoides bakeri

We studied the effect of triterpenoid saponins on the development of free-living stages of Heligmosomoides bakeri, a parasitic nematode of the mouse intestine. We evaluated the effectiveness of oleane-type glucuronides (GlcUAOA) isolated from Calendula officinalis and Beta vulgaris. The rhodamine 123 retention assay was used to detect dysfunctions of the major membrane transporter for xenobiotics, P-glycoprotein (Pgp). Both C. officinals and B. vulgaris GlcUAOA affect the development of the free living stages and function of Pgp in H. bakeri. The GlcUAOA inhibits egg hatching and moulting of larvae and also changes their morphology. These saponin fractions reversed the toxic effect of thiabendazole on the nematode; the function of Pgp was also inhibited.

Triterpenoid profile of flower and leaf cuticular waxes of heather Calluna vulgaris

Analysis of the main triterpenoid profile of chloroform-soluble cuticular waxes of heather flowers and leaves by GC–MS revealed the following composition: five triterpene acids – betulinic, oleanolic, ursolic, 3-oxo-olean-12-en-28-oic and 3-oxo-ursan-12-en-28-oic; eight monohydroxyalcohols – α-amyrin, β-amyrin, cycloartanol, 24-methylenecycloartanol, friedelinol, germanicol, lupeol and taraxasterol; three dihydroxyalcohols – betulin, erythrodiol and uvaol; two aldehydes – oleanolic and ursolic; four ketones – α-amyrenone, 4-epi-friedelin, friedelin and taraxerone and seven steroids – campesterol, cholesterol, sitostanol, sitosterol, stigmasterol, stigmasta-3,5-dien-7-one and stigmastane-3,6-dione. Triterpenoids accounted for 20% and 65% by mass of flower and leaf waxes, respectively, which suggest that heather leaves represent a very promising source of these compounds. Ursolic acid was the principal triterpenoid in the cuticular wax of both organs, whereas among the neutral triterpenes, friedelin and uvaol were the most abundant in flowers and leaves, respectively. This report provides the first thorough overview of the triterpenoid composition of cuticular waxes of heather.

Changes in Heligmosomoides polygyrus glycoprotein pattern by saponins impact the BALB/c mice immune response

Saponins of marigold (Calendula officinalis), in particular derivatives of 3-O-monoglucuronide of oleanolic acid, are able to reduce infectivity of Heligmosomoides polygyrus in mice. The purpose of this study was to understand the immune activation provoked by third-stage larvae exposed to marigold glucuronides. We also examined the pattern of glycosylation of larval antigens which appeared to be crucial for induction of cytokine production in BALB/c mice; higher concentrations of IL-6, IFN-γ, IL-10 and TNF-α were observed in serum or intestine one week post infection. Three weeks later, in the chronic phase of infection, cells in culture were able to produce IL-6, IFN-γ, TNF-α and IL-17. Restimulation of cells with H. polygyrus antigen resulted in reduced production of IL-6, and TNF-α. The pattern of cytokine production co-existed with reduced expression of terminal glucose, α-linked mannose, N-acetyl-galactosamine, β-galactose, N-acetyl-glucosamine and α-fucose in several protein bands. Galactose, as a new terminal carbohydrate residue appeared in 20-24kDa protein bands. The number of immunogenic epitopes in parasitic antigens was reduced; only three protein bands of 56, 26 and 12kDa were recognized by IgG1. These studies provide a model system to find the glycosylated molecules expressed on nematodes that improve establishment and survival and characterize cytokine production in mice infected with larvae exposed to saponin. Identification of these molecules is the first step in the recognition of key antigenic epitopes able to induce protective or tolerogenic immune responses.

Characterization of triterpenoid profiles and triterpene synthase expression in the leaves of eight Vitis vinifera cultivars grown in the Upper Rhine Valley

Plant triterpenoids are a diverse group of secondary metabolites with wide distribution, high chemical diversity and interesting pharmacological and antimicrobial properties. The first step in the biosynthesis of all triterpenoids is the cyclization of the 2,3-oxidosqualene precursor, catalyzed by oxidosqualene cyclases (OSCs), which have characteristic product specificities. Biosynthesis and functions of pentacyclic triterpenes have been poorly studied in grapevine. In this study, we first investigated the profile of triterpenoids present in leaf cuticular waxes from eight Vitis vinifera cultivars cultivated in the Upper Rhine Valley. Further quantification of triterpenoids showed that these cultivars can be divided into two groups, characterized by high levels of lupeol (e.g., Pinot noir) or taraxerol (e.g., Gewurztraminer) respectively. We further analyzed the OSC family involved in the synthesis of pentacyclic triterpenes (called VvTTPSs) in the sequenced V. vinifera 40024 genome and found nine genes with similarity to previously characterized triterpene synthases. Phylogenetic analysis further showed that VvTTPS1–VvTTPS3 and VvTTPS5–VvTTPS9 belong to the β-amyrin synthase and multifunctional triterpene synthase clade, whereas VvTTPS10 belongs to the lupeol synthase clade. We studied the expression of several members of the VvTTPS family following biotic and abiotic stresses in V. vinifera 40024 as well as in the eight healthy cultivars. This study further revealed that one candidate gene, VvTTPS5, which does not belong to the lupeol synthase clade, is highly expressed in lupeol-rich cultivars. VvTTPS3, VvTTPS5, VvTTPS6, VvTTPS7 and VvTTPS10 were highly upregulated by UV stress, but only VvTTPS3, VvTTPS5, VvTTPS6 and VvTTPS10 were upregulated following downy mildew and gray mold infections respectively. These results suggest differential roles of VvTTPS against environmental stresses in grape leaves.

Evidence for Separate UDP-Glucose : Spirostanol and UDP-Glucose : Solasodine Glucosyltransferases in Solanum melongena

There are only sparse data on the enzymology of sugar chain synthesis in steroid glycoalkaloids. Earlier papers1,2 have described the ability of crude homogenates of Solanum laciniatum leaves or S. tuberosum tubers to glucosylate solasodine (an alkaloid of the spirosolane type) or solanidine (an alkaloid of the solanidane type), respectively, in the presence of UDP-glucose (UDPG1c). Recently, the glucosyltransferase present in S. tuberosum which catalyzes the formation of solanidine 3-O-monoglucoside (γ-onine) has been partly purified and characterized3-8.