RD29A promoter constitutively drives a rice Hd3a expression to promote early-flowering in Saussurea involucrate Kar. et Kir. ex Maxim.

S. involucratae, an endemic and endangered plant, is a valuable and traditional Chinese medicinal herb. In order to control the flowering time of S. involucratae, we used the well-known stress inducible RD29A promoter to drive Hd3a (a FT ortholog from rice) expression in S. involucratae. Unexpectedly, the majority of regenerated buds in RD29A::Hd3a transgenic lines (S-RH) produced flowers in tissue culture stage under normal growth (25 ± 2 °C) condition. Their flowering time was not further influenced by salt treatment. Hd3a in S-RH was strongly expressed in MS media supplemented with or without 50 mM NaCl. RD29A::GUS transgenic experiments further revealed that RD29A constitutively promoted GUS expression in both S. involucrate and halophyte Thellungiella halophile, in contrast to glycophic plants Oryza sativa L. 'Zhonghua 11', in which its expression was up-regulated by cold, salinity, and drought stress. The results supported the hypothesis that RD29A promoter activity is inducible in stress-sensitive plants, but constitutive in stress-tolerant ones. Importantly, S-RH plants produced pollen grains and seeds under normal conditions. Additionally, we found that OsLEA3-1::Hd3a and HSP18.2::Hd3a could not promote S. involucrate to flower under either normal conditions or abiotic stresses. Taken together, we demonstrated the potential of RD29A::Hd3a might be served as a feasible approach in breeding S. involucrate under normal condition.

Nephroprotective Effect of Costus (Saussurea costus) Ethanolic Extract on Oxaliplatin®-induced Nephrotoxicity in Adult Male Wistar Rats.

<b>Background and Objective:</b> Oxaliplatin^® is an antineoplastic platinum-based compound; nephrotoxicity is one of its most serious side effects. This study aimed to explore the nephroprotective potential of Costus Ethanolic Extract (CEE) against Oxaliplatin^®-induced nephrotoxicity. <b>Materials and Methods:</b> Adult male Wistar rats, weighting 140-160 g, were randomly divided into four groups: (1) Normal rats, (2) Rats ingested with CEE (67.08 mg kg¹ day¹), (3) Rats injected (ip) with Oxaliplatin^® (10 mg kg¹ week¹) and (4) rats treated with CEE in combination Oxaliplatin^® injection. <b>Results:</b> After six weeks of treatments, the results revealed that CEE ingestion along with Oxaliplatin^® injection markedly minimized the Oxaliplatin^®-induced renal deterioration; this was evidenced by the significant reduction in serum urea, creatinine, uric acid, Tumor Necrosis Factor Alpha (TNF-α), Interleukin 1Beta (IL¹ß) and Sodium ion (Na⁺) levels as well as kidney Malondialdehyde (MDA), Nitric Oxide (NO) and DNA fragmentation values. Controversially, a marked rise in serum Calcium, Potassium Ion (K⁺) and Cluster of Differentiation 4 (CD4) levels besides renal Glutathione (GSH), Catalase (CAT) and Superoxide Dismutase (SOD) values. Similarly, the histopathological findings confirmed the biochemical ones as the CEE restored the Oxaliplatin^®-induced histological degenerations. <b>Conclusion:</b> In conclusion, CEE exhibited nephron-protection efficiency against Oxaliplatin^®-induced nephrotoxicity; this promising effect may be achieved through the antioxidant and radical scavenging activities of its constituents.

Comparative transcriptomics reveals candidate transcription factors involved in costunolide biosynthesis in medicinal plant-Saussurea lappa.

Costunolides, an important sesquiterpene lactone (STL) isolated from Saussurea lappa, are the major pharmaceutical ingredient of various drug formulations. Identification of the genes and transcriptional regulation of costunolide biosynthesis pathway in S. lappa will propose alternatives for engineering enhanced metabolite biosynthesis in plant. Here, we aimed to unravel the transcription factors (TFs) regulating the costunolide biosynthesis. Comparative transcriptome analysis of root and leaf tissues and transcripts were annotated using various in silico tools. Putative transcription factors were identified using PlantTFDB and TF- gene co-expression network was generated followed by clustering using module based analysis to observe their coordinated behaviour. The module 1 was found to be significant based on its enrichment with major pathway genes. Further, promoter cloning determined the cis acting elements in costunolide synthase (SlCOS1) gene which catalyses the final key step of costunolide biosynthesis. Bioinformatics tools were employed to predict the cis regulatory elements, leading to the identification of MYB family of TFs as an interacting partner of SlCOS1 gene. The present study is the pioneer attempt for TF prediction and elucidation of their regulatory role in costunolide synthesis. This will help in future metabolic engineering of the pharmaceutically important STLs and their yield improvement.

Modulatory effects of Saussurea lappa root aqueous extract against ethephon-induced kidney toxicity in male rats.

Ethephon (2-chloroethyl phosphonic acid) is a plant growth promoter used to control the plant growth process by liberating ethylene and stimulating the production of endogenous ethylene. Medicinal plants are sources of novel drug discovery targets. Costus (Saussurea lappa) has been used as traditional Chinese medicine. The current study was conducted to examine the possible modifying effects of costus (S. lappa) root aqueous extract against kidney toxicity induced by ethephon in male rats. A total of 50 adult male rats were divided into five groups (first, control; second, costus; third, ethephon; fourth, posttreated ethephon with costus; fifth, ethephon self-healing). There is a significant increase in the serum levels of urea, creatinine, potassium ions, chloride ions, kidney injury, DNA damage, and proliferating cell nuclear antigen expressions in treated rats with ethephon when compared to the control group. In contrast, the treated rats with ethephon revealed a significant decrease in the levels of sodium ions and an insignificant decrease in the calcium ions. Saussurea lappa extract modified these alterations when compared to the control group. As a result, costus root extract significantly reduced rat kidney toxicity after ethephon administration. We recommend costus to be included in diet for its valuable effects, and also producers and consumers should become more aware about the toxic effects of ethephon.

Proximate analysis and in vitro biological assays of Saussurea hypoleuca Spreng. root.

Considering the growing interest in medicinal plants having imperative phytoconstituents, a research has been steered to standardize the crude drug from "Saussurea hypoleuca root" by assessing its primary, secondary metabolites and to screened out in vitro biological assays of thrashed plant. Quantitative analysis was done by estimation of the primary and secondary metabolites (total proteins, total carbohydrates, total lipids, total glycosaponins, total alkaloids, total flavonoids, and total polyphenolics) in powder and extracts. The maximum value of total proteins (0.59%), total carbohydrates (53.7%), total lipids (27.12%), total glycosaponins (63.9%), total alkaloids (20.3%), total flavonoids (0.23%) and total polyphenolics (0.919%) were respectively. Antimicrobial assay was done by agar well diffusion method and Minimum Inhibitory Concentration. Haemolytic and DNA protection activity was studied by reported method. Different extracts showed various results however butanol, ethyl acetate, chloroform and methanol give promising results. The results of this present study gives an evidence for the existence of diverse primary and secondary metabolites and thus rationalizes its use in traditional medicines for the cure of different aliments owing to the safety profile on human red blood cells. The conclusions of this research work give an indication that this plant has good potential for antimicrobial activity and has possible pronounced significance as therapeutic agent.

[Regulation of syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid biosynthesis in cell suspension cultures of Saussurea involucrata].

Syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid are three main bioactive ingredients in herbs of Saussurea involucrata with various pharmacological properties, while their contents are very low. In this study, the biosynthesis of syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid in the cell suspension cultures of S. involucrata were regulated by feeding carbon sources and precursors, which resulted in a great increase of the contents and yields of the above three bioactive ingredients. After 16 days of fermentation, the yields of syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid reached 339.0, 225.3, 512.7 mg x L(-1), respectively. Meanwhile, their contents increased up to 67.9, 1.9, 10.6 times of wild medicinal material, respectively. The results provided a solid basis for further studies on application of cell suspension cultures of S. involucrata for large-scale production of bioactive compounds syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid.

Metabolic engineering of the phenylpropanoid pathway enhances the antioxidant capacity of Saussurea involucrata.

The rare wild species of snow lotus Saussurea involucrata is a commonly used medicinal herb with great pharmacological value for human health, resulting from its uniquely high level of phenylpropanoid compound production. To gain information on the phenylpropanid biosynthetic pathway genes in this critically important medicinal plant, global transcriptome sequencing was performed. It revealed that the phenylpropanoid pathway genes were well represented in S. involucrata. In addition, we introduced two key phenylpropanoid pathway inducing transcription factors (PAP1 and Lc) into this medicinal plant. Transgenic S. involucrata co-expressing PAP1 and Lc exhibited purple pigments due to a massive accumulation of anthocyanins. The over-expression of PAP1 and Lc largely activated most of the phenylpropanoid pathway genes, and increased accumulation of several phenylpropanoid compounds significantly, including chlorogenic acid, syringin, cyanrine and rutin. Both ABTS (2,2'-azinobis-3-ethylbenzotiazo-line-6-sulfonic acid) and FRAP (ferric reducing anti-oxidant power) assays revealed that the antioxidant capacity of transgenic S. involucrata lines was greatly enhanced over controls. In addition to providing a deeper understanding of the molecular basis of phenylpropanoid metabolism, our results potentially enable an alternation of bioactive compound production in S. involucrata through metabolic engineering.

Effect of lanthanum on rooting of in vitro regenerated shoots of Saussurea involucrata Kar. et Kir.

In present study, the effect of lanthanum (La) on the rooting of regenerated shoots of Saussurea involucrata Kar. et Kir was analyzed. Rooting occurred from regenerated shoots inoculated on a medium supplemented with La, the plant rooting hormone indole-3-acetic acid (IAA), or both La and IAA together. The highest rooting efficiency (96%), root number/shoot (8.5), and root length (63 mm) were recorded in shoots cultured on medium containing 2.5 µM IAA combined with 100 µM La(3+). In order to elucidate the mechanism of rooting enhancement by La, we examined dynamic changes in antioxidant enzyme activities in plant tissue over time in culture. We found that the activities of peroxidase (POX) and superoxide dismutase (SOD) were significantly higher in plant tissue cultured in IAA plus La than in La or IAA alone. At the same time, the highest H(2)O(2) content was detected in plant tissue in the presence of 2.5 µM IAA plus 100 µM La(3+). In light of these data and previous results, we speculate that La enhanced IAA-induced rooting by acting as a mild abiotic stress to stimulate POX and SOD activities in plant cells. Then, IAA reacted with oxygen and POX to form the ternary complex enzyme-IAA-O(2) that dissociated into IAA radicals and O(2)(-). Subsequently, IAA-induced O(2)(-) readily converted to hydroxyl radical (HO·) via SOD-catalyzed dismutation. Finally, cell wall loosening and cell elongation occurred as a consequence of HO-dependent scission of wall components, leading to root growth. The treatment of IAA combined with La resulted in the highest plantlet survival (80%) compared to single treatments with IAA or La alone. These data suggest that rare earth elements enhance root morphogenesis and the growth of S. involucrata.

Biotransformation of bufadienolides by cell suspension cultures of Saussurea involucrata.

The biotransformation of three bioactive bufadienolides, namely, bufotalin (1), telocinobufagin (2), and gamabufotalin (3) by cell suspension cultures of Saussurea involucrata yielded 11 products. Bufotalin yielded 3-epi-bufotalin (1a), 3-epi-desacetylbufotalin (1b), 3-epi-bufotalin 3-O-ß-D-glucoside (1c), 1ß-hydroxybufotalin (1d), and 5ß-hydroxybufotalin (1e); telocinobufagin yielded 3-dehydroscillarenin (2a), 3-dehydrobufalin (2b), and 3-epi-telocinobufagin (2c); and gamabufotalin yielded 3-epi-gamabufotalin (3a), 3-dehydrogamabufotalin (3b), and 3-dehydro-Δ¹-gamabufotalin (3c), respectively. Among these 11 products, 1a, 1b, 1c, 1d, 3a and 3c are previously unreported. The structures of these metabolites were elucidated based on NMR spectroscopic analyses and mass spectrometry. Most metabolites showed significant cytotoxic activities against human hepatoma (HepG2) and breast cancer (MCF-7) cell lines. In addition, the time course for the biotransformation of 3 was investigated.

Secondary metabolites of plants from the genus Saussurea: chemistry and biological activity.

The Asteraceae family comprises ca. 1000 genera, mainly distributed in Asia and Europe. Saussurea DC., as the largest subgenus of this family, comprises ca. 400 species worldwide, of which ca. 300 species occur in China. Most plants in China grow wild in the alpine zone of the Qingzang Plateau and adjacent regions at elevations of 4000 m. Plants of the genus Saussurea (Asteraceae) are used in both traditional Chinese folk medicine and Tibet folklore medicine, since they are efficacious in relieving internal heat or fever, harmonizing menstruation, invigorating blood circulation, stopping bleeding, alleviating pain, increasing energy, and curing rheumatic arthritis. A large number of biologically active compounds have been isolated from this genus. This review shows the chemotaxonomy of these compounds (215 compounds) such as sesquiterpenoids (101 compounds), flavonoids (19 compounds), phytosterols (15 compounds), triterpenoids (25 compounds), lignans (32 compounds), phenolics (23 compounds), and chlorophylls (11 compounds). Biological activities (anti-inflammatory, anticancer, antitumor, hepatoprotective, anti-ulcer, cholagogic, immunosuppressive, spasmolytic, antimicrobial, antiparasitic, antifeedant, CNS depressant, antioxidant, etc.) of these compounds, including structure-activity relationships, are also discussed.

Saussurea medusa cell suspension cultures for flavonoid production.

Saussurea medusa Maxim. is a valuable traditional Chinese herb. The flavonoids are the main active pharmaceutical compounds in this medicinal plant species and have effective anti-tumor and anti-inflammation properties. This species is now almost extinct in China because of over-exploitation. The establishment of plant cell cultures would be a promising alternative to avoid extinction of this species and establish cultivation for the production of bioactive flavonoids. The callus is induced from leaf explants of S. medusa on Murashiage and Skoog medium supplemented with 0.5 mg/L 6-BA, 2 mg/L NAA, 30 g/L sucrose, and 5 g/L agar. A fine cell suspension is established from the induced light-yellow calluses in the MS liquid medium with 30 g/L sucrose, 0.5 mg/L BA, and 2.0 mg/L NAA for biosynthesis of flavonoids. The kinetics of cell growth and flavonoid accumulation in the cell suspension cultures are investigated. The highest dry weight and flavonoid production reach 17.2 g/L and 607.8 mg/L respectively after 15 d. Significantly high antioxidant activity and flavonoids accumulate in the cell suspension cultures of S. medusa.

A new sesquiterpene lactone from the roots of Saussurea lappa: structure-anticancer activity study.

The dried roots of Saussurea lappa, called costus roots, are used in the traditional system of medicine for the treatment of cancer. In our investigation for the anticancer constituents from the hexane extract of this plant, a new sesquiterpene (1) was isolated along with the known compounds costunolide (2), beta-cyclocostunolide (3), dihydro costunolide (4) and dehydro costuslactone (5). Their structures were established by the extensive spectroscopic analyses. In addition, costunolide and beta-cyclocostunolide derivatives were synthesized using Michael-type addition reaction of NaOMe to the alpha-methylene-gamma-lactone moiety. All the compounds were tested for their in vitro cytotoxic activity. Compound 1 exhibited potent cytotoxic activity and other compounds displayed moderate activity.

Overexpression of the Saussurea medusa chalcone isomerase gene in S. involucrata hairy root cultures enhances their biosynthesis of apigenin.

Saussurea involucrata is a medicinal plant well known for its flavonoids, including apigenin, which has been shown to significantly inhibit tumorigenesis. Since naturally occurring apigenin is in very low abundance, we took a transgenic approach to increase apigenin production by engineering the flavonoid pathway. A construct was made to contain the complete cDNA sequence of the Saussurea medusa chalcone isomerase (CHI) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Using an Agrobacterium rhizogenes-mediated transformation system, the chi overexpression cassette was incorporated into the genome of S. involucrata, and transgenic hairy root lines were established. CHI converts naringenin chalcone into naringenin that is the precursor of apigenin. We observed that transgenic hairy root lines grew faster and produced higher levels of apigenin and total flavonoids than wild-type hairy roots did. Over a culture period of 5 weeks, the best-performing line (C46) accumulated 32.1 mgL(-1) apigenin and 647.8 mgL(-1) total flavonoids, or 12 and 4 times, respectively, higher than wild-type hairy roots did. The enhanced productivity corresponded to elevated CHI activity, confirming the key role that CHI played for total flavonoids and apigenin synthesis and the efficiency of the current metabolic engineering strategy.

A comparison between hairy root cultures and wild plants of Saussurea involucrata in phenylpropanoids production.

Saussurea involucrata is an important medicinal plant that produces a few bioactive secondary metabolites, such as hispidulin, rutin, and syringin. Previously, we established a hairy root culture system for this species through Agrobacterium-mediated transformation. The present study addressed the issue as how hairy root cultures perform in phenylpronoid accumulation. From the ethanolic extract of a hairy root culture established for Saussurea involucrata, syringin, rutin and hispidulin, were isolated and their chemical structures were confirmed by HPLC-ESI-MS. A quantitative study of the compounds showed great levels of syringin and hispidulin (being 43.5+/-1.13 and 0.34+/-0.023 mg g-1 dry weight, respectively), about 40 and 3 times, respectively, higher than those from wild plants. But, the levels of rutin from hairy roots were much lower (0.71+/-0.043 vs. 6.59+/-0.56 mg g-1 dry weight). Compared with untransformed root cultures, syringin and hispidulin levels were also higher. An experiment on culture media showed that MS was superior to others for phenylpropanoids accumulation in hairy roots, a 28-day culture produced 405 mg l-1 syringin.

[Cell suspension culture and flavonoids production in Saussurea involucrate].

OBJECTIVE: To establish a system of cell suspension culture for Saussurea involucrate. METHOD: The effects of different factors on cell growth and flavonoids production of S. involucrate were systematically studied including the media, initial pH values of the medium, carbon sources, inoculum quantity, and plant growth substance. RESULT: The optimum medium was N6, initial pH values of the medium was 5.8, sucrose concentration was 50 g x L(-1), inoculum quantity was 60-80 g x L(-1) FW. Medium supplemented with BA (0.5 mg x L(-1)) and NAA (3 mg x L(-1)) was suitable for cell growth, but medium containing BA (0.2 mg x L(-1)) and NAA (2 mg x L(-1)) was suitable for flavonoids production. CONCLUSION: Cell growth and flavonoids production in the suspention of S. involucrate culture cell should be optimized.

Transformation of Saussurea medusa for hairy roots and jaceosidin production.

Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A. rhizogenes strain R1601 in N6 medium. PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A. rhizogenes into the genome of S. medusa hairy roots. In N6 medium, maximum biomass of the hairy root cultures was achieved [8 g (dry weight) per liter; growth ratio 35-fold] after 21 days of culture. The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture. The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium. In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.