Deciphering triterpenoid saponin biosynthesis by leveraging transcriptome response to methyl jasmonate elicitation in Saponaria vaccaria.

Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional Chinese medicine, containing large quantities of oleanane-type triterpenoid saponins with anticancer properties and structural similarities to the vaccine adjuvant QS-21. Leveraging the MeJA-elicited saponin biosynthesis, we identify multiple enzymes catalyzing the oxidation and glycosylation of triterpenoids in S. vaccaria. This exploration is aided by Pacbio full-length transcriptome sequencing and gene expression analysis. A cellulose synthase-like enzyme can not only glucuronidate triterpenoid aglycones but also alter the product profile of a cytochrome P450 monooxygenase via preference for the aldehyde intermediate. Furthermore, the discovery of a UDP-glucose 4,6-dehydratase and a UDP-4-keto-6-deoxy-glucose reductase reveals the biosynthetic pathway for the rare nucleotide sugar UDP-D-fucose, a likely sugar donor for fucosylation of plant natural products. Our work enables the production and optimization of high-value saponins in microorganisms and plants through synthetic biology approaches.

Sapovaccarin-S1 and -S2, Two Type I RIP Isoforms from the Seeds of Saponaria vaccaria L.

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.

Novel Oleanane-Type Triterpene Glycosides from the Saponaria officinalis L. Seeds and Apoptosis-Inducing Activity via Mitochondria.

Saponaria officinalis L., commonly known as "Soapwort", is a rich source of triterpene glycosides; however, the chemical constituents of S. officinalis seeds have not been fully identified. In this study, we conducted a systematic phytochemical investigation of the seeds of S. officinalis and obtained 17 oleanane-type triterpene glycosides (1-17), including seven new glycosides (1-7). The structures of 1-7 were determined based on a detailed analysis of NMR spectroscopic data and chromatographic and spectroscopic analyses following specific chemical transformation. The cytotoxicities of the isolated compounds were evaluated against HL-60 human promyelocytic leukemia cells, A549 human adenocarcinoma lung cancer cells, and SBC-3 human small-cell lung cancer cells. The cytotoxicities of 1, 4, and 10 toward HL-60 cells and SBC-3 cells were nearly as potent as that of cisplatin. Compound 1, a bisdesmosidic triterpene glycoside obtained in good yield, arrested the cell cycle of SBC-3 cells at the G2/M phase, and induced apoptosis through an intrinsic pathway, accompanied by ROS generation. As a result of the mitochondrial dysfunction induced by 1, mitochondria selective autophagy, termed mitophagy, occurred in SBC-3 cells.

Fatty acid composition, enzyme inhibitory effect, antioxidant and anticancer activity of extract from Saponaria prostrata WILLD. subsp. anatolica HEDGE.

This study attempts to evaluate the antioxidant, enzyme inhibitory, and anticancer properties as well as fatty acid compositions of endemic Saponaria prostrata WILLD. subsp. anatolica HEDGE. The gas chromatography-mass spectrometry (GC-MS) was used to determine the fatty acid content of methanol: dichloromethane extract from S. prostrata subsp. anatolica (SPA). Enzymatic activity was measured against acetylcholinesterase, butyrylcholinesterase and α-glucosidase. DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity and Ferric reducing antioxidant power assay (FRAP) were conducted to antioxidant properties. The anticancer effect of SPA on human MCF-7 breast cancer and human HCT116 colorectal cancer cell line was evaluated by WST-1 cell viability assay, colony formation assay and wound healing assay. In addition, human VEGF Elisa method was used to determine the anti-angiogenic effect, and the quantitative real-time PCR (qRT-PCR) method on p53, Bax and Bcl-2 mRNA levels were used to evaluate apoptosis. While high amounts of palmitic acid (40.8%), linoleic acid (17.75%) and α-linolenic acid (16.84%) were detected in the SPA, the total amount of unsaturated fatty acid (51.34%) was higher than the total amount of saturated fatty acid (48.66%). SPA displayed the most promising acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and α-glycosidase (AG) inhibitory activities (AChE: IC50: 18.03 µg/mL, BuChE: IC50: 44.24 µg/mL and AG: IC50: 210.85 µg/mL). The half maximum inhibitory concentration (IC50) of SPA in MCF-7 and HCT116 cells was determined as 259.79 µg/mL and 97.24 µg/mL, respectively. In addition, it was determined that SPA suppresses colony formation and wound closure, and suppresses angiogenesis as well as triggering apoptosis at a significant level. It is true that endemic S. prostrata subsp. anatolica is a potential source of functional food ingredients, but more analytical and in vivo experiments are needed to explore further secondary metabolite diversity and pharmacological properties.

Use of gamma radiation for inactivating Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes in tahini halva.

Tahini halva is a traditional sweet product that is consumed with bread in different countries. It is a low water activity (aw) product basically made by mixing and cooking tahini, sugar, citric acid and Saponaria officinalis root extract together. Tahini halva maybe contaminated with foodborne pathogens during any stage of production from tahini and other raw ingredients, workers, environment or contact surfaces. The objectives of the study were to i) investigate the efficacy of gamma radiation to inactivate Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes in tahini halva, ii) evaluate the effect of pre-irradiation storage (0, 7 and 30 days at 21 °C) of tahini halva on the sensitivity of these microorganisms toward gamma radiation, and iii) evaluate the effect of post-irradiation storage of tahini halva for up to 6 months on the their survival characteristics. Tahini halva samples were inoculated with Salmonella spp., E. coli O157:H7 and L. monocytogenes separately then stored at 21 °C for 0, 7 and 30 days prior to irradiation at 0-4 KGy and for up to 6 months after irradiation at 4 KGy. Salmonella spp. were the most irradiation resistance among the tested microorganisms. Irradiation (0.8-4.0 KGy) reduced the bacteria in samples stored for 0, 7 and 30 days pre-irradiation in the range of 0.43-2.11, 0.45-2.68 and 0.52-2.7 log10 CFU/g for Salmonella spp., 0.55-3.08, 0.66-3.00 and 0.60-2.80 log10 CFU/g for E. coli O157:H7, and 0.69-2.96, 0.86-4.30, 0.62-3.29 log10 CFU/g for L. monocytogenes, respectively. The D10-value, the irradiation dose needed to inactivate 1 log10 of pathogen, was 1.83, 1.47 and 1.50 KGy for Salmonella spp., 1.28, 1.32 and 1.48 KGy for E. coli O157:H7, and 1.33, 0.94 and 1.27 KGy for L. monocytogenes in pre-irradiation stored samples for 0, 7 and 30 days, respectively. Post-irradiation storage was efficient in decreasing the levels of the microorganisms ca. ≥2 log10 CFU/g in the first month and to undetected level after the second month of storage but enrichment results showed that Salmonella spp. and L. monocytogenes were detected in the samples until of the end of storage period. The study demonstrates that gamma radiation can be applied to inactivate of foodborne pathogens in tahini halva. Irradiation dose at 4 KGy can reduce Salmonella spp., E. coli O157:H7 and L. monocytogenes in tahini halva by 2-3 log10 CFU/g. Storage of tahini halva before or after irradiation may reduce the risk of foodborne pathogens in the product.

Anti-inflammatory and antioxidant effects of Aloe saponaria Haw in a model of UVB-induced paw sunburn in rats.

Ultraviolet B (UVB) irradiation mainly affects biological tissues by inducing an increase in reactive oxygen species (ROS) production which leads to deleterious outcomes for the skin, including pain and inflammation. As a protective strategy, many studies have focused on the use of natural products. The aim of this study was to investigate the effects of Aloe saponaria on nociceptive, inflammatory, and oxidative parameters in a model of UVB-induced sunburn in adult male Wistar rats. Sunburned animals were topically treated with vehicle (base cream), 1% silver sulfadiazine (positive control) or A. saponaria (10%) once a day for 6days. UVB-induced nociception (allodynia and hyperalgesia), inflammation (edema and leukocyte infiltration) and oxidative stress (increases in H2O2, protein carbonyl levels and lipid peroxidation and a decrease in non protein thiol content) were reduced by both A. saponaria and sulfadiazine topical treatment. Furthermore, A. saponaria or its constituents aloin and rutin reduced the oxidative stress induced by H2O2 in skin homogenates in vitro. Our results demonstrate that topical A. saponaria treatment displayed anti-nociceptive and anti-inflammatory effects in a UVB-induced sunburn model, and these effects seem to be related to its antioxidant components.

Signal peptide-regulated toxicity of a plant ribosome-inactivating protein during cell stress.

The fate of the type I ribosome-inactivating protein (RIP) saporin when initially targeted to the endoplasmic reticulum (ER) in tobacco protoplasts has been examined. We find that saporin expression causes a marked decrease in protein synthesis, indicating that a fraction of the toxin reaches the cytosol and inactivates tobacco ribosomes. We determined that saporin is largely secreted but some is retained intracellularly, most likely in a vacuolar compartment, thus behaving very differently from the prototype RIP ricin A chain. We also find that the signal peptide can interfere with the catalytic activity of saporin when the protein fails to be targeted to the ER membrane, and that saporin toxicity undergoes signal sequence-specific regulation when the host cell is subjected to ER stress. Replacement of the saporin signal peptide with that of the ER chaperone BiP reduces saporin toxicity and makes it independent of cell stress. We propose that this stress-induced toxicity may have a role in pathogen defence.

Ribosome inactivating protein saporin induces apoptosis through mitochondrial cascade, independent of translation inhibition.

Ribosome inactivating proteins (RIPs) are toxic translation inhibitors that kill eukaryotic cells by arresting protein synthesis at the translocation step. Saporin-6, expressed in the seeds of Saponaria officinalis plant, is a type I RIP comprising of a single polypeptide chain. Saporin is a specific RNA N-glycosidase and it removes a specific adenine residue from a conserved loop of the large rRNA of eukaryotic cells. Saporin-6 is one of the most potent of several isoforms of saporin, obtained from different tissues of the Saponaria plant. In addition to potently inhibiting translation, saporin has been also shown to induce cell death by apoptosis in different cellular models. To elucidate the mechanism of apoptosis induction by saporin, we have investigated the apoptotic pathway triggered by saporin. We have also analyzed whether the inhibition of protein synthesis by the toxin is the trigger for induction of apoptosis. We demonstrate that saporin-6 induces caspase-dependent apoptosis in U937 cells via the mitochondrial or intrinsic pathway. Unlike many other toxins the catalytic N-glycosidase activity of saporin is not required for apoptosis induction, and the apoptosis onset occurs before any significant inhibition of protein synthesis ensues.

Differences in X-ray absorption due to cadmium treatment in Saponaria officinalis leaves.

A method for detecting cadmium uptake in leaves of Saponaria officinalis doped with a solution of cadmium acetate is described. The technique based on the exposure of dried leaves to X-rays of a wavelength close to that of the metal K-edge could be useful for phytoremediation studies as it could reveal the bioaccumulation in plants due to the treatment either in vivo or in vitro with heavy metals. X-ray microradiography measurements are in agreement with those from peroxidase enzyme assay utilized to follow the oxidative damage induced by heavy metals. At present, as we will see in this report, microradiography has still poorer sensitivity in comparison with enzyme assay, but it has the advantage of being faster, not destructive, and usable even at very high doping levels, where the enzyme assay technique results are fully saturated. Further analysis of the optical density values could lead to a quantitative measurement of the heavy metal in the sample. Thus, the technology developed in this article could be useful for tracing the intake in phytoremediation studies.